References 16 website: JMR, http://fluoroquinolonethyroid.com

References 16

Links, Abstracts, Articles, etc. These links should work as of 2016; sometimes you have to click on them

several times; if they don’t work, then Google/search the titles

CYPs, mostly FQs with/CYP1A2; Purines

and Purine Metabolism

FQ’s inhibit demethylases and hydroxylases. This is most evident with theophylline.

Theophylline demethylate 3-methylxanthine + 1-methylxanthine. Note: Cipro selectively

blocks these two demethylations.

Theophylline hydroxylate 1,3-dimethyluric acid. Note: Cipro selectively blocked this

hydroxylation only at high concentrations.

https://en.wikipedia.org/wiki/Demethylation Demethylation is the chemical process resulting in the

removal of a methyl group (CH3) from a molecule.[1][2] A common way of demethylation is the

replacement of a methyl group by a hydrogen atom, resulting in a net loss of one carbon and two

hydrogen atoms. The counterpart of demethylation is methylation. In biochemical systems, the

process of demethylation is catalyzed by demethylases. These enzymes oxidize N-methyl groups, which

occur in histones and some forms of DNA. (My note: note that JMHD and TET1 are demethylases).

R2N-CH3 + O → R2N-H + CH2O

One such oxidative enzyme family is the cytochrome P450[3] Alpha-ketoglutarate-dependent nonheme

enzymes are active for demethylation of DNA, operating by similar pathway.

https://en.wikipedia.org/wiki/Hydroxylation Hydroxylation is a chemical process that introduces a

hydroxyl group (-OH) into an organic compound. In biochemistry, hydroxylation reactions are often

facilitated by enzymes called hydroxylases. Hydroxylation is the first step in the oxidative degradation of

organic compounds in air. It is extremely important in detoxification since hydroxylation converts

lipophilic compounds into water-soluble (hydrophilic) products that are more readily removed by the

kidneys or liver and excreted. Some drugs (for example, steroids) are activated or deactivated by

hydroxylation. The principal hydroxylation agent in nature is cytochrome P-450, hundreds of variations

References 16 website: JMR, http://fluoroquinolonethyroid.com

of which are known. Other hydroxylating agents include flavins.[1] The principal residue to be

hydroxylated in proteins is proline. The hydroxylation occurs at the γ-C atom, forming hydroxyproline

(Hyp), an essential element of collagen, in turn a necessary element of connective tissue. Proline

hydroxylation is also a vital component of hypoxia response via hypoxia inducible factors. In some cases,

proline may be hydroxylated instead on its β-C atom. Lysine may also be hydroxylated on its δ-C atom,

forming hydroxylysine (Hyl).

These three reactions are catalyzed by very large, multi-subunit enzymes prolyl 4-hydroxylase, prolyl 3-

hydroxylase and lysyl 5-hydroxylase, respectively. These reactions require iron (as well as molecular

oxygen and α-ketoglutarate) to carry out the oxidation, and use ascorbic acid (vitamin C) to return the

iron to its oxidized state. Deprivation of ascorbate leads to deficiencies in proline hydroxylation, which

leads to less stable collagen, which can manifest itself as the disease scurvy

https://en.wikipedia.org/wiki/Dehydrogenase Dehydrogenases are a subclass of the class of enzymes

labeled “oxidoreductases.” Oxidoreductases, in general, catalyze oxidation and reduction reactions. Any

enzyme that transfers an electron from one molecule to another is considered an oxidoreductase. These

enzymes fall into six categories: oxygenases, reductases, peroxidases, oxidases, hydroxylases, and

dehydrogenases. Most oxidoreductase enzymes are dehydrogenases, although reductases are also

common. Accepted nomenclature for dehydrogenases is "donor dehydrogenase," where the donor is

the molecule giving up an electron.[1]

Oxidation-reduction reactions are essential to growth and survival of organisms, as the oxidation of

carbons produces energy. Energy-producing reactions can drive forward the synthesis of important

energy molecules, such as ATP in glycolysis. For this reason, dehydrogenases have pivotal roles in

metabolism.[2]

http://attic.volgmed.ru/depts/biochem/sources/e-enzyme1.pdf Quick review of enzymes.

http://www.saferpills.org/wp-content/uploads/2012/08/FQ-Feb-2011-Survey-Visualization-3.pdf

From 2011 FQ survey of 131 people: 46/131 = 35% answered “I do not tolerate caffeine”.

http://www.sciencedirect.com/science/article/pii/S0163725813000065 Cytochrome P450 enzymes in

drug metabolism: Regulation of gene expression, enzyme activities, and impact of genetic variation

(2013). “Cytochromes P450 (CYP) are a major source of variability in drug pharmacokinetics and

response. Of 57 putatively functional human CYPs only about a dozen enzymes, belonging to the CYP1, 2,

and 3 families, are responsible for the biotransformation of most foreign substances including 70–80% of

all drugs in clinical use. The highest expressed forms in liver are CYPs 3A4, 2C9, 2C8, 2E1, and 1A2, while

2A6, 2D6, 2B6, 2C19 and 3A5 are less abundant and CYPs 2J2, 1A1, and 1B1 are mainly expressed

extrahepatically. Expression of each CYP is influenced by a unique combination of mechanisms and

References 16 website: JMR, http://fluoroquinolonethyroid.com

factors including genetic polymorphisms, induction by xenobiotics, regulation by cytokines, hormones

and during disease states, as well as sex, age, and others. Multiallelic genetic polymorphisms, which

strongly depend on ethnicity, play a major role for the function of CYPs 2D6, 2C19, 2C9, 2B6, 3A5 and

2A6, and lead to distinct pharmacogenetic phenotypes termed as poor, intermediate, extensive, and

ultrarapid metabolizers. For these CYPs, the evidence for clinical significance regarding adverse drug

reactions (ADRs), drug efficacy and dose requirement is rapidly growing. Polymorphisms in CYPs 1A1,

1A2, 2C8, 2E1, 2J2, and 3A4 are generally less predictive, but new data on CYP3A4 show that predictive

variants exist and that additional variants in regulatory genes or in NADPH:cytochrome P450

oxidoreductase (POR) can have an influence. Here we review the recent progress on drug metabolism

activity profiles, interindividual variability and regulation of expression, and the functional and clinical

impact of genetic variation in drug metabolizing P450s.”

http://www.tandfonline.com/doi/full/10.3109/03602530903286476?scroll=top&needAccess=true

Structure, function, regulation and polymorphism and the clinical significance of human cytochrome

P450 1A2. “Human CYP1A2 is one of the major CYPs in human liver and metabolizes a number of clinical

drugs (e.g., clozapine, tacrine, tizanidine, and theophylline; n > 110), a number of procarcinogens (e.g.,

benzo[a]pyrene and aromatic amines), and several important endogenous compounds (e.g., steroids).

CYP1A2 is subject to reversible and/or irreversible inhibition by a number of drugs, natural substances,

and other compounds. The CYP1A gene cluster has been mapped on to chromosome 15q24.1, with close

link between CYP1A1 and 1A2 sharing a common 5′-flanking region. The human CYP1A2 gene spans

almost 7.8 kb comprising seven exons and six introns and codes a 515-residue protein with a molecular

mass of 58,294 Da. The recently resolved CYP1A2 structure has a relatively compact, planar active site

cavity that is highly adapted for the size and shape of its substrates. The architecture of the active site of

1A2 is characterized by multiple residues on helices F and I that constitutes two parallel substrate

binding platforms on either side of the cavity. A large interindividual variability in the expression and

activity of CYP1A2 has been observed, which is largely caused by genetic, epigenetic and environmental

factors (e.g., smoking). CYP1A2 is primarily regulated by the aromatic hydrocarbon receptor (AhR) and

CYP1A2 is induced through AhR-mediated transactivation following ligand binding and nuclear

translocation. Induction or inhibition of CYP1A2 may provide partial explanation for some clinical drug

interactions. To date, more than 15 variant alleles and a series of subvariants of the CYP1A2 gene have

been identified and some of them have been associated with altered drug clearance and response and

disease susceptibility. Further studies are warranted to explore the clinical and toxicological significance

of altered CYP1A2 expression and activity caused by genetic, epigenetic, and environmental factors.”

https://www.pharmgkb.org/pathway/PA165884757# PharmGKB Caffeine Pathway, Pharmacokinetics

(Note: Good picture of Caffeine Metabolism). “Conversely the CC genotype for ADORA2A rs5751876 is

associated with increased likelihood of being sensitive to caffeine and increased likelihood of insomnia

when exposed to caffeine. [Article:17329997]. See https://www.ncbi.nlm.nih.gov/pubmed/17329997

A genetic variation in the adenosine A2A receptor gene (ADORA2A) contributes to individual

sensitivity to caffeine effects on sleep. “Caffeine is the most widely used stimulant in Western

countries. Some people voluntarily reduce caffeine consumption because it impairs the quality of their

sleep. Studies in mice revealed that the disruption of sleep after caffeine is mediated by blockade of

References 16 website: JMR, http://fluoroquinolonethyroid.com

adenosine A2A receptors. Here we show in humans that (1) habitual caffeine consumption is associated

with reduced sleep quality in self-rated caffeine-sensitive individuals, but not in caffeine-insensitive

individuals; (2) the distribution of distinct c.1083T>C genotypes of the adenosine A2A receptor gene

(ADORA2A) differs between caffeine-sensitive and -insensitive adults; and (3) the ADORA2A c.1083T>C

genotype determines how closely the caffeine-induced changes in brain electrical activity during sleep

resemble the alterations observed in patients with insomnia. These data demonstrate a role of adenosine

A2A receptors for sleep in humans, and suggest that a common variation in ADORA2A contributes to

subjective and objective responses to caffeine on sleep.”

https://www.ncbi.nlm.nih.gov/pubmed/19274061 Allele-specific expression and gene methylation in

the control of CYP1A2 mRNA level in human livers. “The basis for interindividual variation in the

CYP1A2 gene expression is not fully understood and the known genetic polymorphisms in the gene

provide no explanation. We investigated whether the CYP1A2 gene expression is regulated by DNA

methylation and displays allele-specific expression (ASE) using 65 human livers. Forty-eight percent of the

livers displayed ASE not associated to the CYP1A2 mRNA levels. The extent of DNA methylation of a CpG

island including 17 CpG sites, close to the translation start site, inversely correlated with hepatic CYP1A2

mRNA levels (P=0.018). The methylation of two separate core CpG sites was strongly associated with the

CYP1A2 mRNA levels (P=0.005) and ASE phenotype (P=0.01), respectively. The CYP1A2 expression in

hepatoma B16A2 cells was strongly induced by treatment with 5-aza-2'-deoxycytidine. In conclusion, the

CYP1A2 gene expression is influenced by the extent of DNA methylation and displays ASE, mechanisms

contributing to the large interindividual differences in CYP1A2 gene expression.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1365008/ The effect of ciprofloxacin on

theophylline pharmacokinetics in healthy subjects (1995).

https://www.ncbi.nlm.nih.gov/pubmed/1312320 An evaluation of the quinolone-theophylline

interaction using the Food and Drug Administration spontaneous reporting system (1992). “A review

of the Food and Drug Administration's spontaneous reporting system identified 48 reports of adverse

events in patients who received concomitant therapy with ciprofloxacin (n = 39) or norfloxacin (n = 9) and

theophylline. The mean (SD) age of these cases was 68.4 (18.5) years; 25 patients (52%) were female.

The mean percent change in theophylline concentrations was 114%, with a range of 32% to 308%

following the addition of a quinolone to the patient's theophylline regimen. Fourteen (36%) of the 39

patients receiving ciprofloxacin and three (33%) of the nine patients receiving norfloxacin experienced a

seizure. The accumulated evidence suggests that extreme caution should be used when quinolones are

prescribed in conjunction with theophylline, particularly in elderly patients. Further research is required

to identify risk factors that will more specifically predict the magnitude of the interaction.”

https://www.ncbi.nlm.nih.gov/pubmed/1580270 The effects of quinolones on xanthine

pharmacokinetics (1992). “Caffeine, theobromine, and theophylline are among the most widely

consumed compounds in beverages and in pharmaceutical preparations. These methylxanthine alkaloids

are metabolized by similar pathways involving demethylation and hydroxylation that are predominantly

cytochrome P-450 mediated. In vivo and in vitro evidence suggests that the cytochrome P-450 isozymes

involved in the demethylation pathways are distinct from the cytochrome P-450 isozymes involved in the

References 16 website: JMR, http://fluoroquinolonethyroid.com

hydroxylation pathways. Although distinctions can be made between demethylation and hydroxylation

pathways, the evidence suggests that these different cytochrome P-450 isozymes are under common

regulatory control. Any drug inhibiting the family of cytochrome P-450 isozymes involved in the

metabolism of the methylxanthines would, therefore, be expected to have a similar effect on

theophylline, theobromine, and caffeine. A number of quinolones, including enoxacin, pipemidic acid,

ciprofloxacin, norfloxacin, and pefloxacin, have been shown to reduce the clearance of theophylline,

while lomefloxacin has no effect on theophylline or caffeine clearance. It has been hypothesized that only

fluoroquinolones that form a 4-oxo-metabolite inhibit theophylline clearance. Lomefloxacin, which does

not form a 4-oxo-metabolite, would therefore not be expected to inhibit the clearance of theophylline or

caffeine. In contrast, ciprofloxacin, which does form a 4-oxo-metabolite, has been shown to reduce

theophylline and caffeine clearances by about one third. Another hypothesis for the differences among

quinolones suggests that quinolones that have a greater impact on theophylline clearances are more

stereochemically similar to theophylline. Substitutions at position 8 on the quinolone nucleus (as in

lomefloxacin) would result in stearic hindrance and decrease the structural similarity to theophylline.”

http://journal.publications.chestnet.org/data/Journals/CHEST/21568/663.pdf New synthetic

quinolone antibacterial agents and serum concentration of theophylline (1987). “The effect of

pipemidic acid and five new synthetic antibacterial agents--norfloxacin, enoxacin, ofloxacin,

ciprofloxacin, and pefloxacin--on the serum level of theophylline was studied in healthy male adult

volunteers after concomitant oral administration of these agents with a slow release preparation of

theophylline. The results indicated that enoxacin, ciprofloxacin, and pipemidic acid might decrease the

clearance of theophylline in the liver, and the attention should be paid in clinical use when enoxacin or

pipemidic acid is coadministered with theophylline. . . . We have already reported on a study using ENX

at a dose level of 300 mg/day, half the usual dose, in combination with theophylline, 400 mg,’7 in which

a slight increase in the plasma theophylline concentration without any side effects was shown. This fact

suggests that the effect ofENX is dose dependent. . . . However, the concomitant use of a slow release

theophylline, 400 mg, and ENX, 600 mg (daily dose, respectively), is a standard regimen for COPD and

lower respiratory tract infections in Japan.34 Such concomitant use may cause a high incidence of side

effects, and this will be a problem in clinical use. . . . in spite of the fact that the drugs used in this study

belong to the same quinolone derivatives group, their effects on the theophylline level vary greatly.

However, we have no results that will explain these differences, nor are there any earlier reports that can

explain them.”

https://www.ncbi.nlm.nih.gov/pubmed/2344166 In vitro effect of fluoroquinolones on theophylline

metabolism in human liver microsomes (1990). “Some quinolone antibiotics cause increases in levels of

theophylline in plasma that lead to serious adverse effects. We investigated the mechanism of this

interaction by developing an in vitro system of human liver microsomes. Theophylline (1,3-

dimethylxanthine) was incubated with human liver microsomes in the presence of enoxacin,

ciprofloxacin, norfloxacin, or ofloxacin. Theophylline, its demethylated metabolites (3-methylxanthine

and 1-methylxanthine), and its hydroxylated metabolite (1,3-dimethyluric acid) were measured by high-

pressure liquid chromatography, and Km and Vmax values were estimated. Enoxacin and ciprofloxacin

selectively blocked the two N demethylations; they significantly inhibited the hydroxylation only at high

References 16 website: JMR, http://fluoroquinolonethyroid.com

concentrations. Norfloxacin and ofloxacin caused little or no inhibition of the three metabolites at

comparable concentrations. The extent of inhibition was reproducible in five different human livers.

Inhibition enzyme kinetics revealed that enoxacin caused competitive and mixed competitive types of

inhibition. The oxo metabolite of enoxacin caused little inhibition of theophylline metabolism and was

much less potent than the parent compound. Nonspecific inhibition of cytochrome P-450 was ruled out

since erythromycin N demethylation (cytochrome P-450 mediated) was unaffected in the presence of

enoxacin. These in vitro data correlate with the clinical interaction described for these quinolones and

theophylline. We conclude that some quinolones are potent and selective inhibitors of specific isozymes

of human cytochrome P-450 that are responsible for theophylline metabolism. This in vitro system may

be useful as a model to screen similar compounds for early identification of potential drug interactions. . .

. However, theophylline plasma levels become elevated in patients treated simultaneously with certain

quinolones, such as enoxacin or ciprofloxacin (1, 7, 14, 24, 25), and these elevated levels may result in

complaints of nausea, vomiting, tachycardia, or agitation . . . Theophylline is almost entirely (90%)

metabolized in the liver by the hepatic mixed-function oxidase system (13) to 3-methylxanthine (3-MX),

1-methylxanthine (1-MX), and 1,3-dimethyluric acid (1,3-DMU) (Fig. 1). Studies in humans indicate that

certain quinolones cause a dose-dependent inhibition of theophylline metabolism, resulting in decreased

urinary excretion of its metabolites and increased excretion of the parent compound (1, 17). Evidence for

this mechanism is further supported by investigations showing that neither protein binding nor renal

clearance of theophylline are influenced by coadministration of enoxacin (26). Wijnands et al. suggested

(27) that the oxo metabolite of enoxacin, oxo-enoxacin, might be responsible for this inhibition. However,

subsequent in vitro studies with rat hepatocytes indicate that only the parent compound inhibits

theophylline metabolism (9). . . . Studies of humans receiving quinolones which inhibit theophylline

metabolism demonstrate decreased excretion of the two demethylated metabolites of theophylline, little

change in the hydroxylated metabolite, and increased excretion of the parent compound (1, 17).

Enoxacin is the most potent inhibitor of theophylline metabolism in vivo, followed by ciprofloxacin.

Ofloxacin and norfloxacin cause only slight inhibition (12, 23). We investigated an in vitro system of

human microsomes, the results of which correlate well with that observed for in vivo interactions of

quinolones with theophylline. . . . vitro human liver microsomal system, we found that the extent of

inhibition can be graded as enoxacin > ciprofloxacin > norfloxacin. Sano et al. (18) found that the mean

cumulative urine recoveries of 3-MX, 1-MU, and 1,3-DMU in humans were decreased from that of control

by 64, 63, and 28%, respectively, after administration of enoxacin. These results correlate well with those

from our human liver microsomal system (Table 1). We found that enoxacin inhibits 3-MX, 1-MX, and

1,3-DMU formation from control by 76, 68, and 31%, respectively. . . The ratios of theophylline and the

quinolone concentrations in this model are quite similar to those observed in a clinical situation. . .

Immunoinhibition studies in our laboratory have shown that the polycyclic aromatic-hydrocarbon-

inducible cytochrome P-450 is mainly involved in the formation of the two N-demethylated metabolites.

Since enoxacin and ciprofloxacin profoundly inhibited the N demethylations and not the hydroxylation,

one can conclude that quinolones selectively inhibit only certain isozymes in this system. . . . On the basis

of our enzyme kinetics data, we speculate that an inhibitory complex is being formed between certain

quinolones and the enzymes involved in theophylline metabolism. This complexation appears to be

reversible, at least for enoxacin, since the type of inhibition was found to be mixed competitive. Since

these quinolones cause a more potent inhibition to the two N demethylations of theophylline than of the

References 16 website: JMR, http://fluoroquinolonethyroid.com

8-hydroxylation, these data suggest that a similar isozyme may be mediating the theophylline N

demethylation and the metabolism of quinolones.”

https://www.ncbi.nlm.nih.gov/pubmed/3348614 Impact of ciprofloxacin on theophylline clearance

and steadystate concentrations in serum (1988). “The effect of a multiple-dose regimen of oral

ciprofloxacin (750 mg every 12 h for 11 doses) on the clearance and steady-state concentrations of

theophylline in trough (predose) serum was evaluated in nine healthy male subjects, each serving as his

own control. Theophylline was taken as a sustained release tablet per os in a dose of 200 mg every 12 h

for 19 doses. Theophylline concentrations in serum were measured immediately before each theophylline

dose. Ciprofloxacin was administered on study day 4 through the first dose of study day 8. Theophylline

concentrations in serum were also measured on study days 3, 6, 8, and 10 at the following times after

the first dose of each day: 0, 0.25, 0.50, 1, 2, 4, 6, 8, 10, and 12 h. Steady-state theophylline

concentrations in trough serum were significantly higher during ciprofloxacin treatment (day 8) than

before (day 3) or after (day 10) ciprofloxacin administration (P less than 0.01). Likewise, theophylline

clearance was significantly slower (P less than 0.01) during ciprofloxacin treatment (day 8) than before it

(day 3) or after it (day 10). The magnitude of ciprofloxacin-induced changes was approximately 30%.

These results suggest that a multidose regimen of ciprofloxacin significantly slows the clearance of

theophylline and elevates theophylline concentrations in serum. . . . When compared with theophylline

steady-state clearances either before (study day 3) or after ciprofloxacin treatment (study day 10), the

steady-state clearance of theophylline during ciprofloxacin treatment decreased by 31 to 33%. This

decrease is virtually identical to the 30.4% decrease in theophylline clearance reported by Wijnands. . .

On the basis of our data and the data of others, it seems likely that ciprofloxacin prolongs the elimination

of theophylline from serum. Whether an automatic dosage reduction for theophylline would be

warranted is not so certain, since the magnitude of clearance slowing is approximately 30%. It would

clearly be prudent to monitor theophylline concentrations more intensely among patients receiving

ciprofloxacin and to lower theophylline doses by 25 to 33% for those patients who might actually

warrant a dose reduction. Alerting patients to symptoms of theophylline toxicity may be a sufficient

precaution in patients with steady-state theophylline levels that are less than 15 mg/liter.”

https://www.ncbi.nlm.nih.gov/pubmed/2760258 Effect of quinolone antimicrobials on theophylline

pharmacokinetics (1989). “The purpose of the research was to ascertain the comparative differences of

quinolone antibiotics on theophylline pharmacokinetics. Eight healthy male volunteers were randomly

assigned to four treatments. Each was administered norfloxacin (NOR) 800 mg/d, ciprofloxacin (C) 1 g/d,

nalidixic acid (NAL) 2 g/d and placebo (P) for 7 days. On the seventh day of each treatment, theophylline

(5 mg/kg) iv was administered. The elimination half-life (T 1/2), total body clearance (CL) and volume of

distribution at steady state (Vss) of theophylline were calculated using model-independent methods.

ANOVA for repeated measures was used for data comparisons. The mean (SD) theophylline results were:

CL l/kg/h--NOR .038 (.006), C .033 (.006), NAL .045 (.008), P .044 (.007); T 1/2 h--NOR 9.2 (1.8), C 10.6

(1.8), NAL 8.3 (1.8), P 7.5 (1.4). Theophylline Vss differences by treatment were not significant. NOR and

C significantly decreased theophylline's clearance and the clearance change can be of clinical

significance.”

References 16 website: JMR, http://fluoroquinolonethyroid.com

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1401213/ The influence of quinolone derivatives

on theophylline clearance (1986). “Enoxacin decreases the metabolic clearance of the bronchodilator

theophylline not only in severely ill patients, but also in patients with stable chronic obstructive airways

disease. In this comparative study, significantly increased plasma theophylline concentrations were

measured during co-administration of enoxacin (110.9%) and, to a lesser degree, also during co-

administration of pefloxacin (19.6%) and ciprofloxacin (22.8%). Total body clearance of theophylline was

significantly decreased by enoxacin (63.6%), ciprofloxacin (30.4%) and pefloxacin (29.4%). The

pharmacokinetic parameters of theophylline did not change during co-administration of ofloxacin and

nalidixic acid. There is growing evidence that the observed interaction is caused not by the parent drugs,

but by the 4-oxo metabolite of enoxacin, pefloxacin and ciprofloxacin.”

https://www.ncbi.nlm.nih.gov/pubmed/2328197 Comparative effects of ciprofloxacin and

lomefloxacin on the oxidative metabolism of theophylline (1990). “Nine healthy male volunteers were

studied to assess the interaction between theophylline and ciprofloxacin and to assess whether a similar

interaction occurred with lomefloxacin, using a randomised, crossover design. Subjects received

theophylline 125 mg 8 hourly with and without lomefloxacin 400 mg 12 hourly or ciprofloxacin 500 mg

12 hourly for 7 days. Ciprofloxacin treatment lowered total theophylline clearance by 27%, owing to a

decreased clearance via 1-, 3-demethylation and 8-hydroxylation. Lomefloxacin treatment did not alter

theophylline clearance. Ciprofloxacin, at usual clinical doses, could cause a clinically significant

interaction when co-administered with theophylline. . . . Theophylline is metabolised by three major

pathways, 1-demethylation, 3-demethylation and 8-hydroxylation (Birkett et al., 1985) and the effects of

the fluoroquinolones on each of these metabolic pathways has not been established. This study was

designed to identify which metabolic pathways are inhibited by ciprofloxacin and to test the hypothesis

that lomefloxacin, a newer fluoroquinolone without a 4-oxo metabolite, does not inhibit theophylline

metabolism. . . . Ciprofloxacin treatment lowered mean plasma theophylline clearance by 27%,

consistent with the 30% reduction in theophylline clearance reported previously (Wijnands et al., 1986;

Bachmann et al., 1988). Clearance by all three metabolic pathways was lowered, although the decrease

via the 8-hydroxylation pathway (24%) was less than the decrease via the 1-demethylation (37%) and 3-

demethylation (42%) pathways. . . . However, the inhibitory effect of ciprofloxacin pretreatment on the

disposition of theophylline was consistent with previously published in vitro data (Robson et al., 1987,

1988b) and in vivo data (Robson et al., 1988a) suggesting that two isozymes of cytochrome P450 are

involved in the metabolism of theophylline, one isozyme predominantly performing the demethylations

and the other performing the 8-hydroxylation. Lomefloxacin treatment had no effect on theophylline

metabolism consistent with the hypothesis that it is the 4-oxo metabolites of the fluoroquinolones which

inhibit theophylline metabolism (Wijnands et al., 1986). Lomefloxacin, unlike ciprofloxacin, does not form

a 4-oxo metabolite.

https://www.ncbi.nlm.nih.gov/pubmed/1606331 Effect of the addition of ciprofloxacin on

theophylline pharmacokinetics in subjects inhibited by cimetidine (1992). “Although the effect of

individual enzyme inhibitors on hepatic microsomal enzyme activity has been studied extensively, little

data exist on the effects of combinations of inhibiting agents. The purpose of this study was to

investigate the effect of the addition of a second hepatic oxidative enzyme inhibitor on the inhibition of

References 16 website: JMR, http://fluoroquinolonethyroid.com

metabolism in subjects already maximally inhibited by cimetidine. Ciprofloxacin was used as the second

inhibitor. In a randomized crossover sequence, subjects received theophylline 5 mg/kg on day 6 of

therapy with cimetidine 2400 mg/d, ciprofloxacin 1 g/d, both drugs, or while drug-free. Eight normal

volunteers (6 men, 2 women; mean age 25.2 y). Theophylline pharmacokinetic parameters after each

treatment were determined by model independent pharmacokinetic analysis. Statistical analysis of the

data for differences between treatments was assessed by ANOVA for repeated measures. When

administered alone, ciprofloxacin and cimetidine caused a significant increase in theophylline elimination

half-life and a decrease in clearance. Theophylline elimination half-life was significantly longer during

combined therapy compared with either drug alone. Theophylline clearance was lower during combined

treatment, although this relationship did not reach statistical significance. The addition of a second

enzyme inhibitor in subjects receiving maximally inhibiting doses of cimetidine can produce a further

decrease in the hepatic metabolism of drugs that are metabolized by the cytochrome P-450 microsomal

enzyme system. As cimetidine and ciprofloxacin are frequently used together for a variety of common

clinical indications, clinicians should be aware of this drug interaction and should consider that a similar

effect may occur when other enzyme inhibitors are used concomitantly. (My note from Wiki: Cimetidine,

sold under the brand name Tagamet among others, is a histamine H2 receptor antagonist that inhibits

stomach acid production.[2][3][4] It is available over-the-counter and is mainly used in the treatment of

heartburn and peptic ulcers.[2][4][5]. . . . Cimetidine is a potent cytochrome P450 (CYP450) enzyme

inhibitor.[17] It is not a universal inhibitor of the CYP450 oxidative system,[29] but it inhibits a broad

array of CYP450 isoforms, including CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and

CYP3A4.[17][29][30] The drug is said to be most potent in inhibiting CYP1A2, CYP2D6, and CYP3A4,[31] of

which it is described as a moderate inhibitor,[1] and this is notable as these three isoenzymes are

involved in the majority of CYP450-mediated drug biotransformations;[32] also, CYP1A2, CYP2C9,

CYP2C19, CYP2D6, CYP2E1, and CYP3A4 have been identified as involved in the oxidative metabolism of

most commonly used drugs.[33] As such, cimetidine has the potential for a large number of drug

interactions.[17][29][30] Cimetidine is reported to be a competitive, reversible inhibitor of the CYP450

enzymes similarly to certain other inhibitors like azole antifungals and quinidine,[29][24][34][16]

although mechanism-based (suicide), irreversible inhibition has also been identified for at least

CYP2D6.[23] It reversibly inhibits CYP450 enzymes by binding directly with the complexed heme-iron of

the active site via one of its imidazole ring nitrogen atoms, thereby blocking the oxidation of other drugs.

. . . Cimetidine has been found to possess clinically significant albeit weak antiandrogen activity at high

doses.[27][36][37][38] It has been found to directly and competitively displace testosterone and

dihydrotestosterone (DHT) and antagonize the androgen receptor (AR) in animals.[39][40] In addition,

cimetidine has been found to inhibit 2-hydroxylation of estradiol (via inhibition of CYP450 enzymes,

which are involved in the metabolic inactivation of estradiol), resulting in increased levels of

estrogen.[41][42][43][44][45][46]).

https://www.ncbi.nlm.nih.gov/pubmed/9114903 Individual and combined effects of cimetidine and

ciprofloxacin on theophylline metabolism in male nonsmokers (1993). “1. The individual and

combined effects of cimetidine and ciprofloxacin on theophylline metabolism were examined in six young

male nonsmokers. 2. Treatment sequence consisted of 7 days each of cimetidine 400 mg p.o. every 12 h.

ciprofloxacin 500 mg p.o. every 12 h, and the combination of cimetidine and ciprofloxacin. 3. Studies of

References 16 website: JMR, http://fluoroquinolonethyroid.com

theophylline pharmacokinetics were performed at baseline and on the fifth day of each regimen. 4.

Individually, cimetidine and ciprofloxacin decreased the clearance of theophylline by 25% and 32%,

respectively. Therapy with the combined regimen resulted in a 41% reduction in theophylline clearance,

which was greater than that achieved with each drug alone (P < 0.01). 5. Ciprofloxacin, in contrast to

cimetidine, inhibited N-demethylations of theophylline to a significantly greater extent than the

hydroxylation pathway. Combined treatment produced a further decline in formation of 1,3-dimethyluric

acid than each drug alone. 6. These data suggest that coadministration of cimetidine and ciprofloxacin

exerts a greater impairment of theophylline biotransformation than each inhibitor alone. The enhanced

inhibitory effect from the two inhibitors will occur only when sub-maximal doses of each individual agent

are used. . . . We have also shown that cimetidine and ciprofloxacin impair both the demethylation and

the hydroxylation of theophylline (Table 2). This finding is consistent with that reported by Vestal et al.

[17] and provides further evidence that cimetidine is a nonselective inhibitor of theophylline metabolism.

In contrast, the observation that ciprofloxacin has a preferential inhibitory effect on the formation of 3-

methylxanthine and 1-methyluric acid is in keeping with the results of other in vivo and in vitro studies

[18, 19]. The proportionate lowering of theophylline clearance was greater during the combined

treatment phase than with either cimetidine or ciprofloxacin alone (Figure 2). In contrast, Davis et al. [7]

found that ciprofloxacin (500 mg twice daily) and a maximally inhibiting dose of cimetidine (2400 mg

day-') decreased theophylline clearance more than when ciprofloxacin was given alone but not when

cimetidine was given alone. This difference may be attributed to the differences in the daily dose of

cimetidine employed in these studies indicating that when a maximally inhibitory dose of one drug is

used, addition of a second inhibitor may have no further affect on the metabolism of theophylline. The

finding that the combined regimen produced a further decline in the formation of 1,3-dimethyluric acid

when compared with each individual agent suggests that the partially additive inhibitory effects of the

combined regimen on theophylline metabolism can be attributed, at least in part, to an enhanced

inhibition of the hydroxylation pathway. With regard to demethylation, the combined treatment caused

a slight increase in inhibition of the formation of 3-methylxanthine and 1-methyluric acid compared with

ciprofloxacin alone. These data indicate that the inhibitory effects of the combined treatment on

theophylline metabolism is qualitatively similar to that exerted by ciprofloxacin alone (Figure 3). In

conclusion, this study demonstrated that cimetidine and ciprofloxacin, at standard therapeutic doses, are

inhibitors of theophylline elimination. Their combined administration caused a proportionately greater

decrease in theophylline clearance than that achieved with each agent alone. This less than fully additive

effect can be attributed largely to an enhanced inhibition of the formation of 1,3-dimethyluric acid. In

patients receiving theophylline together with several inhibitors, appropriate adjustment of theophylline

dosage should be instituted based on the expected change in theophylline clearance as a result of the

drug interaction and on plasma theophylline concentration measurements.

https://www.ncbi.nlm.nih.gov/pubmed/3481317 Steady-state kinetics of the quinolone derivatives

ofloxacin, enoxacin, ciprofloxacin and pefloxacin during maintenance treatment with theophylline

(1987). “Some of the new quinolone derivatives may be of value in the treatment of respiratory tract

infections. It has been demonstrated that enoxacin, pefloxacin and ciprofloxacin, but not ofloxacin,

decreased the metabolic clearance of the bronchodilator theophylline. This resulted in elevated plasma

theophylline concentrations and, in some of the patients, theophylline toxicity. When the

References 16 website: JMR, http://fluoroquinolonethyroid.com

pharmacokinetic parameters of enoxacin, pefloxacin, ciprofloxacin and ofloxacin obtained in the present

study were compared with those obtained from other studies in healthy volunteers not given

theophylline, there was no evidence of theophylline influencing the clearance of the investigated

quinolones.” (?)

https://www.ncbi.nlm.nih.gov/pubmed/3578320 Ciprofloxacin increases serum levels of theophylline

(1987). “During a clinical trial of orally administered ciprofloxacin in respiratory tract infections,

changes in serum theophylline levels were evaluated in 33 hospitalized patients who also required

theophylline therapy. Patients received intravenous theophylline in standard titrated doses and 750 mg

of oral ciprofloxacin twice daily. Serum theophylline levels in all patients were measured before and

during ciprofloxacin therapy. The mean serum pretreatment theophylline level was 7.8 +/- 4.6

micrograms/ml; during ciprofloxacin therapy, the level increased to 14.6 +/- 7.4 micrograms/ml. Twenty

of the 33 (61 percent) patients evaluated had increases in serum theophylline levels by a mean value of

10.5 micrograms/ml. In 30 percent of patients who experienced increases, theophylline concentrations

were in the toxic range. This occurred more frequently in elderly patients with chronic obstructive

pulmonary disease. In light of the frequency and potential severity of this interaction, careful monitoring

of serum theophylline levels in patients receiving theophylline and ciprofloxacin is recommended.”

https://www.ncbi.nlm.nih.gov/pubmed/3571046 Effect of multiple dose oral ciprofloxacin on the

pharmacokinetics of theophylline and indocyanine green (1987). “Interaction between ciprofloxacin

and theophylline was studied in eight male volunteers, who were randomly divided into two groups. All

subjects were given intravenous theophylline and indocyanine green (ICG) on study days 0, 7 and 14.

Group I subjects received ciprofloxacin 750 mg orally every 12 h on days 1-7. Group II subjects received

ciprofloxacin 750 mg every 12 h on days 6-14. No significant changes in ICG clearance or half-life were

noted. A significant increase in theophylline half-life and volume of distribution was observed (P less than

0.05); however, clearance was not significantly decreased (P = 0.1). A potentially clinically significant

interaction was detected in three subjects whose theophylline clearance decreased by 42-113%. Until

further clinical experience is gained, we advise caution when these agents are coadministered. Some

adjustment in theophylline dosage may be required; therefore, these patients should have serum

theophylline concentration measurements and careful clinical assessment for theophylline toxicity.”

https://www.ncbi.nlm.nih.gov/pubmed/3443151 A clinically significant interaction between

ciprofloxacin and theophylline (1987). “We report a case of theophylline toxicity following the co-

administration of ciprofloxacin. Total theophylline clearance fell from 2.3 l.h-1 to 0.8 l.h-1 when

ciprofloxacin was added to the treatment regimen and returned to 2.1 l.h-1 after ciprofloxacin was

discontinued.”

https://www.ncbi.nlm.nih.gov/pubmed/3678060 Increased theophylline concentrations secondary to

ciprofloxacin (1987). “An 82-year-old man with a history of myasthenia gravis and heart failure was

admitted to the hospital with respiratory failure. Aminophylline and eventually theophylline therapy

were initiated to improve respiratory status. During the hospital stay, the patient developed a resistant

pseudomonal pneumonia. After failure with conventional antibiotics, ciprofloxacin was initiated because

of favorable sensitivity and the planned avoidance of aminoglycoside therapy. Seventy-two hours after

References 16 website: JMR, http://fluoroquinolonethyroid.com

initiation of ciprofloxacin, the patient's theophylline level rose from a steady-state baseline of 9.8

micrograms/ml to 34.7 micrograms/ml. After the theophylline dose was reduced by approximately 67

percent, the patient's theophylline serum concentration returned to baseline (10 micrograms/ml). Until

more data concerning the interaction of theophylline and ciprofloxacin are available, we recommend

close monitoring of theophylline serum concentrations in patients receiving concomitant ciprofloxacin.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2545536/ Drug point: Danger of interaction

between ciprofloxacin and theophylline (1988). “Drs J L BEM and R D MANN (Committee on Safety

ofMedicines, London SW8 5NQ) write: Ciprofloxacin (Ciproxin, Bayer), a new fluoroquinolone antibiotic,

was introduced in 1987 for the treatment of systemic infections. Quinolone antibiotics, including

ciprofloxacin, inhibit theophylline metabolism and both prolong and raise plasma theophylline

concentrations.' A warning about this is included in the ciprofloxacin datasheet. A potentially dangerous

situation occurs when a patient already being treated with theophylline is prescribed ciprofloxacin for a

respiratory tract infection. The early signs of theophylline overdose, nausea and vomiting, may easily be

overlooked or attributed to the side effects of ciprofloxacin. The Committee on Safety of Medicines has

received eight reports of clinically important interaction between ciprofloxacin and theoplylline (table). In

most cases the dose of both drugs was well within the recommended range. The signs and symptoms of

drug interaction appeared rapidly, usually two to three days after the start of ciprofloxacin. One elderly

woman treated with moderate doses of theophylline died with a toxic plasma concentration after a

coadministered short course of ciprofloxacin. Thomson et al observed twofold to threefold increases in

the concentration and 64% reduction in clearance of theophylline in elderly patients who had received

ciprofloxacin 500 mg twice daily for a few days.2 An interaction between ciprofloxacin and theophylline

is not inevitable3 and we have four reports of patients given both drugs who developed adverse

reactions due to other causes. Thus it is difficult to predict which patients are at risk of this interaction,

and we suggest that ciprofloxacin should not normally be used in patients treated with theophylline.

Patients should also be warned against self medication as some cough-cold medicines contain

theophylline. In cases in which ciprofloxacin and theophylline need to be given together plasma

theophylline concentrations should be monitored.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1834921/ Probable fatal interaction between

ciprofloxacin and theophylline (1988). “Dr R HOLDEN (Edinburgh) writes: Thomson et al recently

reported theophylline toxicity in an elderly patient concurrently taking ciprofloxacin.' The Committee on

Safety of Medicines has been notified of several other cases (R D Mann, personal communication). We

report a further probable interaction with a fatal outcome.”

https://www.ncbi.nlm.nih.gov/pubmed/2313005 Theophylline toxicity associated with the

administration of ciprofloxacin in a nursing home patient (1990).

https://www.ncbi.nlm.nih.gov/pubmed/2360338 Seizure with ciprofloxacin and theophylline

combined therapy (1990). “Ciprofloxacin has been reported to cause theophylline toxicity by inhibiting

theophylline metabolism. A 93-year-old woman without a known seizure history, while on ciprofloxacin

and theophylline combined therapy, experienced a grand mal seizure. Her serum theophylline

concentration at the time was 20 micrograms/mL. On previous occasion of theophylline toxicity, she had

References 16 website: JMR, http://fluoroquinolonethyroid.com

a serum theophylline concentration of 27 micrograms/ml but the patient did not experience any seizure.

Several reports suggest that the combination of theophylline and ciprofloxacin has an additive inhibitory

effect on gamma-aminobutyric acid (GABA) sites. Inhibition of the binding of GABA to its receptor sites

has been related to the convulsant effects of other drugs. The seizure in our patient may have been

caused by altered pharmacokinetics and pharmacodynamics brought about by combined therapy of

theophylline and ciprofloxacin.”

https://www.ncbi.nlm.nih.gov/pubmed/1928889 Theophylline toxicity secondary to ciprofloxacin

administration (1991). “We report the case of a 79-year-old woman who presented from a skilled

nursing facility to the emergency department with signs and symptoms of theophylline toxicity and a

serum theophylline concentration of 53.7 mg/L. The patient had been on a regular regimen of

aminophylline for two months, with the addition of ciprofloxacin three days before arrival as the only

identifiable potential cause of theophylline intoxication. She was monitored and treated conservatively

with serial doses of activated charcoal, which resulted in a reduction of her serum theophylline level to a

therapeutic concentration in 15 hours without adverse sequelae. The number of cases of theophylline

intoxication secondary to concurrent ciprofloxacin administration is likely to increase, especially in

nursing home populations, and it should be suspected when these patients present to the ED with the

appropriate signs and symptoms. Management of theophylline intoxication should be based on clinical

presentation as well as concentrations of the drug.”

https://www.ncbi.nlm.nih.gov/pubmed/1541177 Role of ciprofloxacin in fatal seizures (1992).

https://www.ncbi.nlm.nih.gov/pubmed/2867879 Comparison of theophylline and theobromine

metabolism in man (1985). “The total plasma and partial metabolic and renal clearances of

theobromine and theophylline were determined in 13 healthy volunteers. Total plasma clearance for

theobromine was 46% greater than that for theophylline, but the unbound clearances were almost

identical. Theobromine renal clearance was 67% greater than that for theophylline but most of the

difference was due to the lower protein binding of theobromine (free fraction = 0.86 compared to 0.58

for theophylline). Clearance by N-demethylation at the 3-position was 3.7-fold higher (unbound

clearance 2.5-fold higher) for theobromine than for theophylline, showing that the position of the other

methyl substituent (positions 1 or 7) is a major determinant of metabolic rate. There was a high degree

of correlation between theophylline and theobromine plasma clearances (r = 0.86) and also between

partial metabolic clearances both within drugs and across drugs (r = 0.65-0.99). The renal clearances of

theophylline and theobromine were also correlated (r = 0.71). The results support the view that

theophylline and theobromine are metabolized by a common group of cytochromes P-450 under similar

regulatory control. Theobromine is a good model compound for assessing the activity of these enzymes

in man as it has low pharmacological activity and low protein binding, its total and partial metabolic

clearances correlate closely with those of theophylline, and close to 100% of the dose can be recovered

as known metabolites.

https://www.ncbi.nlm.nih.gov/pubmed/1981505 Quinolone inhibition of cytochrome P-450-

dependent caffeine metabolism in human liver microsomes (1990). “Inhibitory effects of the

quinolone antibiotics ofloxacin, lomefloxacin, pipemidic acid, ciprofloxacin, and enoxacin on caffeine

References 16 website: JMR, http://fluoroquinolonethyroid.com

metabolism were examined in vitro with human liver microsomes of four donors. All drugs competitively

inhibited the activity of 3-demethylation, the major pathway of caffeine metabolism. Enoxacin,

ciprofloxacin, and pipemidic acid were strong inhibitors exhibiting Ki values between 0.1 and 0.2 mM.

Lomefloxacin and ofloxacin had moderate effects with Ki values of 1.2 and 3.6 mM, respectively. The rate

of caffeine 7-demethylation (which amounted to about 25% of that for 3-demethylation) was only

slightly affected by the quinolones. Minor, but inconsistent, effects were found on 8-oxidation to 1,3,7-

trimethyluric acid. The results indicate that the reduction of caffeine clearance by concomitant quinolone

application observed in vivo is primarily due to a competitive interaction of the inhibiting quinolones with

the cytochrome P-450 isoenzyme(s) mediating caffeine demethylation.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC298137/pdf/pnas00287-0044.pdf Evidence for the

involvement of several cytochromes P-450 in the first steps of caffeine metabolism by human liver

microsomes (1991). “Caffeine biotransformation and four monooxygenase activities involving

cytochrome P-450IA2, namely ethoxy- and methoxyresorufin O-dealkylases, phenacetin O-deethylase,

and acetanilide 4-hydroxylation were studied in 25 human liver microsomes. All these activities were

highly significantly intercorrelated (r greater than 0.72, p less than 0.001) and correlated with the level of

immunoreactive P-450IA2 content (r greater than 0.65; p less than 0.001). P-450IA content was

measured by immunoblotting with anti-rat P-450 beta-naphthoflavone-B, an antibody that detects only

a single band corresponding to P-450IA2. The formation rate of two caffeine metabolites, namely

paraxathine and theobromine, was correlated with the four monooxygenase activities measured and P-

450IA2-specific content (r greater than 0.75). However, inhibition studies of caffeine metabolism by

phenacetin, a specific substrate of P-450IA2, clearly indicated that only the N-3 demethylation of caffeine

was supported by this enzyme. These in vitro data demonstrate that P-450IA2 is predominantly

responsible for the major metabolic pathway of caffeine and that the formation of other demethylated

metabolites is mediated, at least partly, by other P-450 enzymes.

https://www.ncbi.nlm.nih.gov/pubmed/2813353 Human cytochrome P-450PA (P-450IA2), the

phenacetin O-deethylase, is primarily responsible for the hepatic 3-demethylation of caffeine and N-

oxidation of carcinogenic arylamines (1989). “Aromatic amines are well known as occupational

carcinogens and are found in cooked foods, tobacco smoke, synthetic fuels, and agricultural chemicals.

For the primary arylamines, metabolic N-oxidation by hepatic cytochromes P-450 is generally regarded

as an initial activation step leading to carcinogenesis. The metabolic activation of 4-aminobiphenyl, 2-

naphthylamine, and several heterocyclic amines has been shown recently to be catalyzed by rat

cytochrome P-450ISF-G and by its human ortholog, cytochrome P-450PA. We now report that human

hepatic microsomal caffeine 3-demethylation, the initial major step in caffeine biotransformation in

humans, is selectively catalyzed by cytochrome P-450PA. Caffeine 3-demethylation was highly correlated

with 4-aminobiphenyl N-oxidation (r = 0.99; P less than 0.0005) in hepatic microsomal preparations

obtained from 22 human organ donors, and both activities were similarly decreased by the selective

inhibitor, 7,8-benzoflavone. The rates of microsomal caffeine 3-demethylation, 4-aminobiphenyl N-

oxidation, and phenacetin O-deethylation were also significantly correlated with each other and with the

levels of immunoreactive human cytochrome P-450PA. Moreover, a rabbit polyclonal antibody raised to

human cytochrome P-450PA was shown to inhibit strongly all three of these activities and to inhibit the

References 16 website: JMR, http://fluoroquinolonethyroid.com

N-oxidation of the carcinogen 2-naphthylamine and the heterocyclic amines, 2-amino-6-methyldipyrido-

[1,2-a:3',2'-d]imidazole and 2-amino-3-methylimidazo[4,5-f]-quinoline. Human liver cytochrome P-450PA

was also shown to catalyze caffeine 3-demethylation, 4-aminobiphenyl N-oxidation, and phenacetin O-

deethylation. Thus, estimation of caffeine 3-demethylation activity in humans may be useful in the

characterization of arylamine N-oxidation phenotypes and in the assessment of whether or not the

hepatic levels of cytochrome P-450PA, as affected by environmental or genetic factors, contribute to

interindividual differences in susceptibility to arylamine-induced cancers.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC188773/ Inhibitory potency of quinolone

antibacterial agents against cytochrome P450IA2 activity in vivo and in vitro (1992). “Inhibition of

cytochrome P450IA2 activity is an important adverse effect of quinolone antibacterial agents. It results in

a prolonged half-life for some drugs that are coadministered with quinolones, such as theophylline. The

objective of the study described here was to define the parameters for quantifying the inhibitory

potencies of quinolones against cytochrome P450IA2 in vivo and in vitro and to investigate the

relationship between the results of both approaches. Cytochrome P450IA2 activity in vitro was measured

by using the 3-demethylation rate of caffeine (500 microM) in human liver microsomes. The inhibitory

potency of a quinolone in vitro was determined by calculating the decrease in the activity of cytochrome

P450IA2 caused by addition of the quinolone (500 microM) into the incubation medium. The mean values

(percent reduction of activity without quinolone) were as follows: enoxacin, 74.9%; ciprofloxacin, 70.4%;

nalidixic acid, 66.6%; pipemidic acid, 59.3%; norfloxacin, 55.7%; lomefloxacin, 23.4%; pefloxacin, 22.0%;

amifloxacin, 21.4%; difloxacin, 21.3%; ofloxacin, 11.8%; temafloxacin, 10.0%; fleroxacin, no effect. The

inhibitory potency of a quinolone in vivo was defined by a dose- and bioavailability-normalized

parameter calculated from changes of the elimination half-life of theophylline and/or caffeine reported

in previously published studies. Taking the pharmacokinetics of the quinolones into account, it was

possible to differentiate between substances with and without clinically relevant inhibitory effects by

using results of in vitro investigations. The in vitro test described here may help to qualitatively predict

the relevant drug interactions between quinolones and methylxanthines that occur during therapy.. . .

Another important biological effect of this drug class concerns the metabolism of several unrelated

pharmaceutical agents, resulting in occasional adverse reactions. It has been reported that some of the

quinolone antibacterial agents cause a reduced velocity of caffeine and theophylline degradation in vivo

(see below) (for a review, see reference 10) and in vitro (14, 40). Furthermore, reductions in the

antipyrine metabolism rate and the R-warfarin oxidation rate were observed in humans when quinolones

were coadministered with these drugs (23, 24, 46). . . . In investigations with human liver microsomes,

the mechanism of this interaction was determined for ofloxacin, ciprofloxacin, enoxacin, lomefloxacin,

and pipemidic acid. A competitive-type inhibition of P450LA2 activity by all of these compounds was

found (14). This cytochrome P450 isoform is primarily responsible for the first steps of both caffeine and

theophylline metabolism in the liver, as shown in investigations with specific antibodies (5, 37) and

genetically engineered cells that express single cytochrome P450 isoforms (13). The main metabolic

pathway of caffeine in all systems tested, i.e., 3-demethylation, is mediated almost exclusively by

cytochrome P450IA2 (5) and, therefore, may be used as a specific probe for P450LA2 activity. Serious

incidents (theophylline intoxication), including death, have occurred because of this drug interaction (3,

25). Thus, the objective of the present study was to define the parameters for quantifying the inhibitory

References 16 website: JMR, http://fluoroquinolonethyroid.com

potencies of quinolones against cytochrome P450IA2 in vivo and in vitro and to investigate the

relationship between in vitro and in vivo effects. This is of considerable interest in light of the abundance

of new quinolones. The probability of encountering these adverse drug reactions should be considered

when comparing the therapeutic values of otherwise similar quinolones. . . . All quinolones tested (with

the exception of fleroxacin) showed an inhibitory effect on caffeine 3-demethylation under the in vitro

conditions used in this study. This effect was most pronounced for enoxacin and ciprofloxacin. A possible

modification of the in vitro effects because of degradation of the quinolones has been tested for

ciprofloxacin (12). Reduction in the concentration of intact ciprofloxacin was not detectable as a result of

incubation. The concentration of the Ml metabolite (cleavage of piperazinyl substituent) of ciprofloxacin

was less than 0.3% of the ciprofloxacin concentration, and other metabolites were below the limit of

detection. It has been reported that some quinolones (i.e., enoxacin, ciprofloxacin, ofloxacin,

lomefloxacin, and pipemidic acid [14, 29]) exert a competitive type of inhibition on caffeine 3-

demethylation. The structures of the substances tested previously (14, 29) cover the range of structures

of those of the compounds included in the present investigation. Therefore, it is safe to conclude that all

congeners used in this study exerted their inhibitory effects by binding to the active site of the enzyme,

resulting in a competitive type of inhibition. . . . The low standard deviation between donors provides

further evidence that P450 isozymes other than P450IA2 (or isoforms coordinately regulated with

P450IA2) that are not susceptible to inhibition do not take part in caffeine metabolism (5). . .

.Cytochrome P450IA2 has been reported to be responsible for caffeine 3-demethylation, which is the

major caffeine degradation step in humans (5). There is evidence that this isozyme also takes part in 1-

and 7-demethylation of caffeine and mediates the main portions of the theophylline demethylation and

hydroxylation pathways (although other cytochrome P450 isoforms may also play a minor part in

primary theophylline metabolism [37]). Additionally, similar Km values for all primary metabolic steps of

theophylline and caffeine have been reported (6, 14, 15, 21). Thus, it is not surprising that inhibition of

P450IA2 activity by the concomitant application of quinolones results in a similar degree of half-life

prolongation and/or clearance reduction for both caffeine and theophylline (10). This enables the pooling

of in vivo inhibition data for both methylxanthines. The association between the increasing half-lives of

the methylxanthines with higher doses of the inhibitory quinolones (16, 38) may readily be explained by

the competitive type of inhibition that has been observed in vitro. . . . . The inhibitory effects of

quinolones on methylxanthine metabolism in vivo are postulated to be the result of a mutually exclusive

concurrence at the cytochrome P450IA2 binding site. The extent of inhibition depends not only on the

affinity of a quinolone to the site but also on the concentrations of this drug and possible active

metabolites at the cytochrome binding site. This may account for the lack of a complete correlation

between in vivo and in vitro data.”

https://www.ncbi.nlm.nih.gov/pubmed/8429824 Quinolone antibacterial agents: relationship

between structure and in vitro inhibition of the human cytochrome P450 isoform CYP1A2 (1993).

“The inhibitory effect of 44 quinolone antibacterials and derivatives (common structure, 4-oxoquinoline-

3-carboxylic acid) on cytochrome P450 isoform CYP1A2 activity was tested using human liver microsomes

and caffeine 3-demethylation as a specific test system for this enzyme. By direct comparison of molecules

differing structurally in only one position, the following structure-activity relationships were found. 3'-

Oxo derivatives had a reduced or similar activity and M1 metabolites (cleavage of piperazinyl

References 16 website: JMR, http://fluoroquinolonethyroid.com

substituent) had a greater inhibitory activity, compared with the parent molecule. Alkylation of the 7-

piperazinyl substituent resulted in a reduced inhibitory potency. Naphthyridines with an unsubstituted

piperazinyl group at position 7 displayed a greater inhibitory potency than did corresponding quinoline

derivatives. Derivatives with a fluorine substitution at position 8 had only a minor effect. Molecular

modeling studies with inhibitors and caffeine showed that it is possible to explain the potency of the

quinolones to inhibit CYP1A2 on a molecular level. The keto group, the carboxylate group, and the core

nitrogen at position 1 are likely to be the most important groups for binding to the active site of CYP1A2,

because the molecular electrostatic potential of all inhibitors is very similar to that of caffeine in these

regions. The presence of a piperazinyl substituent, however, seems to be no prerequisite for inhibitory

potency. Finally, an equation to estimate the potency to inhibit CYP1A2 was developed by quantitative

structure-activity relationship analysis.

https://www.ncbi.nlm.nih.gov/pubmed/3663445 Characterisation of theophylline metabolism in

human liver microsomes (1987). “1. A radiometric high performance liquid chromatographic method is

described for the assay of theophylline metabolism in vitro by the microsomal fraction of human liver. 2.

Formation of the three metabolites of theophylline (3-methylxanthine, 1-methylxanthine and 1,3-

dimethyluric acid) were linear with protein concentrations to 4 mg ml-1 and with incubation times up to

180 min. 3. The coefficients of variation for the formation of 3-methylxanthine, 1-methylxanthine and

1,3-dimethyluric acid were 1.2%, 1% and 1.6%, respectively. 4. Theophylline is metabolised by

microsomal enzymes with a requirement for NADPH. 5. The mean (n = 7) Km values for 1-demethylation,

3-demethylation and 8-hydroxylation were 545, 630 and 788 microM, respectively, and the mean Vmax

values were 2.65, 2.84 and 11.23 pmol min-1 mg-1, respectively. 6. There was a high correlation between

the Km and Vmax values for the two demethylation pathways suggesting that the demethylations are

performed by the same enzyme. 7. Overall the in vitro studies are consistent with the in vivo results

which suggest the involvement of two cytochrome P-450 isozymes in the metabolism of theophylline. . .

The metabolic pathways for theophylline in man have been confirmed in a number of studies (Grygiel &

Birkett, 1981; Grygiel et al., 1984; Ogilvie, 1978). The major route is the 8-hydroxylation to 1,3-

dimethyluric acid (DMU) which accounts for 45-55% of the total theophylline clearance. The other

metabolic products are 3- methylxanthine (3MX) and 1-methyluric acid (1MU) which represent 13-16%

and 20-25% of theophylline clearance, respectively. Subsequently it has been shown that 1MU is formed

from 1-methylxanthine (1MX) by a reaction mediated by xanthine oxidase (Birkett et al., 1983). Although

the metabolic pathways involved in theophylline metabolism have been identified and quantified,

relatively little information exists about the enzymes involved. In 1976 Lohman & Miech reported that

theophylline was metabolized to DMU and 1MU by rat liver slices and that this activity was blocked by

classical cytochrome P-450 inhibitors and stimulated by pretreatment with P-450 inducing agents.

Further, the subcellular localisation of this enzymatic activity supported the involvement of the

cytochrome P-450 system as theophylline metabolism was located in the microsomal fraction but not in

the mitochondrial or cytosolic fractions. No 3MX was formed in the rat liver slice preparation and this is

consistent with the hypothesis that unlike man and rabbits, rats cannot demethylate theophylline in the

1 position.. . . In man, the enzymes involved in theophylline metabolism have been investigated by in vivo

studies using known inducers and inhibitors of the P-450 system (Grygiel & Birkett, 1981; Grygiel et al.,

1984; Robson et al., 1984; Miners et al., 1985; Grygiel et al., 1979). These studies provide inferential

References 16 website: JMR, http://fluoroquinolonethyroid.com

evidence that cytochrome P-450 system is the enzyme involved in the metabolism of theophylline in man

and are consistent with the more direct evidence in rat liver slices and rat microsomes (Lohman & Miech,

1976). We have postulated on the basis of data from in vivo studies (Birkett et al., 1985) that there are at

least two isozymes of cytochrome P-450 involved in the metabolism of theophylline, one isozyme

predominantly involved in the 1- and 3-demethylations and another isozyme predominantly performing

the 8-hydroxylation. Although the involvement of two isozymes of cytochrome P-450 in these pathways

can to some extent be differentiated, particularly by inhibitors, their regulation must be closely linked as

there is a high degree of correlation between all three pathways in healthy control subjects (Birkett et al.,

1985). To gain further information on the metabolism of theophylline in man, an in vitro assay has been

developed and used to study thekinetics of theophylline metabolism in human liver microsomes. . . .

Although the in vitro metabolism of theophylline has been demonstrated in rat liver microsomes

(Lohman & Miech, 1976) this is the first report of the in vitro metabolism of theophylline by human liver

microsomes. In the absence of a NADPH generating system no products were formed. (My note: In vivo,

the cytosolic pentose pathway primarily supplies P450 enzymes with NADPH for oxidative

transformation. In vitro microsomal assays, which are devoid of the cytosolic fraction, are routinely

supplied NADPH by either direct addition of NADPH or use of an NADPH-regenerating system (NRS),

consisting of β-NADP+, glucose-6-phosphaste (G6P), and G6P dehydrogenase (G6PDH). This is

consistent with the in vitro animal data (Lohman & Miech, 1976) and the in vivo human data supporting

the involvement of the cytochrome P-450 enzyme system in the metabolism of theophylline to 3MX, 1MX

and DMU. The metabolic profile in vitro is similar to that in vivo where 50-60% is metabolised by 8-

hydroxylation, 15-20% by 3-demethylation and 20-25% by 1-demethylation. No 1-methyluric acid (1MU)

was detected which is consistent with the proposal that, in vivo, 1MU is formed from 1MX by xanthine

oxidase which is not located in the microsomal fraction used in these in vitro studies. . . . the 1- and 3-

demethylation are predominantly performed by the same P-450 isozyme and that the 8-hydroxylation is

performed by a different P-450 isozyme. . . . The data presented are consistent with the involvement of

two isozymes of cytochrome P-450 in the metabolism of theophylline, but definitive evidence awaits the

isolation and characterisation of these cytochrome P-450 isozymes.”

https://www.ncbi.nlm.nih.gov/pubmed/8236273 Biotransformation of methylxanthines in

mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms.

Allocation of metabolic pathways to isoforms and inhibitory effects of quinolones. (1993). “V79

Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes

P450IA1, P450IA2, P450IIB1, human P450IA2, and rat liver epithelial cells expressing murine P450IA2

were used to allocate metabolic pathways of methylxanthines to specific isoforms and to test the

suitability of such cell lines for investigations on drug interactions occurring at the cytochrome expressed.

The cell lines were exposed to caffeine and/or theophylline and concentrations of metabolites formed in

the medium were determined by HPLC. Caffeine was metabolized by human, rat and murine P450IA2,

resulting in the formation of four primary demethylated and hydroxylated metabolites. However, there

were differences in the relative amounts of the metabolites. The human and the mouse P450IA2 isoforms

predominantly mediated 3-demethylation of caffeine. The rat cytochrome P450IA2 mediated both 3-

demethylation and 1-demethylation of caffeine to a similar extent. The results support the hypothesis

that caffeine plasma clearance is a specific in vivo probe for determining human P450IA2 activity.

References 16 website: JMR, http://fluoroquinolonethyroid.com

Addition of the quinolone antibiotic agents pipemidic acid or pefloxacin, both known to inhibit caffeine

metabolism in vivo and in human liver microsomes, reduced formation rates of all metabolites of caffeine

in cells expressing rat and human P450IA2. Theophylline was mainly metabolized via 8-hydroxylation. All

cell lines tested were able to carry out this reaction, with highest activities in cell lines expressing rat or

human P450IA2, or rat P450IA1.”

https://www.ncbi.nlm.nih.gov/pubmed/8966447 Clinico-pharmacological case (4). Epileptic seizure as

an unwanted drug effect on theophylline poisoning. (1994) “In spite of the better understanding of the

pharmacokinetics and optimized galenics of oral theophylline formulations, therapy with this

bronchodilator still bears risks because of its narrow therapeutic window combined with substantial

inter- and intra-individual variability of theophylline metabolism. In particular, the comedication with a

variety of drugs inhibiting theophylline metabolism requires consideration as a potential source of

toxicity. Besides mild, self-limited adverse effects, potentially life-threatening toxic manifestations such

as ventricular tachyarrhythmias, shock, and seizures can occur especially with high plasma

concentrations. We report the case of a 72-year-old patient with chronic obstructive pulmonary disease

who was admitted for surgical treatment of an ulcer of the foot. During combined therapy with

theophylline and ciprofloxacin he developed signs of theophylline toxicity with a single episode of partial

seizures. These symptoms rapidly improved with repetitive application of activated charcoal and sorbitol.

Clinically relevant drug-drug interactions with theophylline and the role and mechanism of action of

activated charcoal in intoxicated patients are discussed.”

https://www.ncbi.nlm.nih.gov/pubmed/8963004 A patient with theophylline-quinolone interaction.

Not all quinolones are equal. 1996. “

https://www.ncbi.nlm.nih.gov/pubmed/8843297 Structure-related inhibitory effect of antimicrobial

enoxacin and derivatives on theophylline metabolism by rat liver microsomes. (1996). “Enoxacin, an

antimicrobial fluoroquinolone with a 7-piperazinyl-1, 8-naphthyridine skeleton, is a potent inhibitor of

cytochrome P-450-mediated theophylline metabolism. The present study was designed to clarify, using

seven enoxacin derivatives, the molecular characteristics of the fluoroquinolone responsible for the

inhibition. Three derivatives with methyl-substituted 7-piperazine rings inhibited rat liver microsomal

theophylline metabolism to 1,3-dimethyluric acid to an extent similar to that of enoxacin (50% inhibitory

concentrations [IC50s] = 0.39 to 0.48 mM). 7-Piperazinyl-quinoline derivatives, 8-hydroenoxacin (8-Hy)

and 1-cyclopropyl-8-fluoroenoxacin (8-F1), which have a hydrogen and a fluorine at position 8,

respectively, more weakly inhibited metabolite formation (IC50s = 0.88 and 1.29 mM, respectively). Little

inhibition (IC50 > 2 mM) was observed in those with 3'-carbonyl and 4'-N-acetyl groups on the piperazine

rings. The substrate-induced difference spectra demonstrated that the affinities of enoxacin, 8-Hy, and 8-

F1 to cytochrome P-450 were parallel with their inhibitory activities. The substituent at position 8 was

found to determine the molecular conformations of the fluoroquinolones, and the planarity in molecular

shape decreased in the same order as the inhibitory activity (enoxacin > 8-Hy > 8-F1). Moreover, the 3'-

carbonyl and 4'-N-acetyl groups decreased the basicity of their vicinal 4'-nitrogen atoms when judged

from their electrostatic potentials, which showed a remarkably broadened negative charge around the

nitrogens. As a result, the planarity of the whole molecule and the basicity of the 4'-nitrogen atom of

References 16 website: JMR, http://fluoroquinolonethyroid.com

enoxacin are likely to be dominant factors in the inhibition of theophylline metabolism by cytochrome P-

450.”

https://www.ncbi.nlm.nih.gov/pubmed/8737124 Pharmacokinetic interactions related to the

chemical structures of fluoroquinolones. (1996). “Fluoroquinolone derivatives interact with

methylxanthines (theophylline, caffeine) and metallic ion-containing drugs to different degrees. The rat

appears to be a suitable model for predicting such interactions in man. It has been possible to determine

the relationship between the chemical structure of the fluoroquinolone and the magnitude of the

interaction. Fluoroquinolones with a bulky substituent at the position 8, such as sparfloxacin,

lomefloxacin and fieroxacin, are less prone to interact with theophylline than those without an 8-

substituent, such as enoxacin. This substituent determines the planarity of the whole fluoroquinolone

molecule and the interaction tends to be more significant for planar fluoroquinolones. Furthermore, a 4'-

nitrogen atom in the 7-piperazinyl group is essential for the interaction to occur. The nitrogen atom is

possibly the site that binds cytochrome P-450, which catalyses theophylline metabolism. The reduction in

bioavailability of fluoroquinolones by concurrent administration of aluminium hydroxide is more striking

for derivatives with fewer substituents on the essential structure and on the piperazinyl group, such as

norfloxacin, ciprofloxacin and enoxacin. Substitution at the 5-position diminishes the interaction, which

suggests that the 5-substituent may affect the formation and/or stability of unabsorbable chelate

complex which is the probable cause of the interaction. These findings are potentially useful in designing

fluoroquinolones less prone to drug interactions.”

https://www.ncbi.nlm.nih.gov/pubmed/9364414 Interaction between pefloxacin and aminophylline

in genetically epilepsy-prone rats. (1997). “The effects of a chronic treatment with pefloxacin on

aminophylline-induced seizures in genetically epilepsy-prone rat have been investigated. Two series of

experiments were performed. In the first, animals received pefloxacin orally twice a day for five days,

then were administered aminophylline intraperitoneally and the occurrence of seizures was evaluated. In

the second series of experiments, theophylline serum concentration was evaluated in rats subject to the

same experimental protocol. Pefloxacin significantly, and in a dose-dependent manner, increased the

occurrence of seizure phases induced by aminophylline, but did not influence theophylline serum levels

measured at different times after the injection of aminophylline. We suggest that additive neurotoxic

effects of both pefloxacin and aminophylline might contribute to the increased severity of seizure score.

The possible role of GABA-benzodiazepine, excitatory amino acid and purinergic mechanism, and the role

of pharmacokinetic factors are discussed.”

https://www.ncbi.nlm.nih.gov/pubmed/9378847 Absence of a pharmacokinetic interaction between

intravenous theophylline and orally administered levofloxacin. (1997). “A randomized, placebo-

controlled, two-way crossover study in 16 healthy men was performed to determine the effect of orally

administered levofloxacin at steady-state conditions, given at 500 mg every 12 hours, on the

pharmacokinetics of theophylline given as a single 4.5-mg/kg intravenous infusion. Participants were

assigned randomly to receive theophylline with levofloxacin in one study period and theophylline with

placebo in the other period. Fourteen individuals completed the study. Mean (+/-SD) values for

theophylline pharmacokinetic parameters for the levofloxacin and placebo treatments, respectively,

were peak plasma concentrations (Cmax) of 11.4 (1.8) micrograms/mL and 10.7 (1.3) micrograms/mL;

References 16 website: JMR, http://fluoroquinolonethyroid.com

areas under the concentration time curve from time 0 extrapolated to infinity (AUCzero-infinity) of 124

(32) micrograms.hr/mL and 126 (30) micrograms.hr/mL; volumes of distribution at steady state (Vdss)

31.7 (3.5) L and 32.0 (3.9) L; clearances (Cl) of 48.6 (11.6) mL/min and 47.4 (10.3) mL/min; and half-lives

(t1/2) of 8.1 (1.9) hours and 8.2 (1.8) hours. There were no statistically significant differences between

treatments for any of these parameters. There was no pharmacokinetic interaction between levofloxacin

administered orally at steady-state conditions and intravenously administered theophylline.”

https://www.ncbi.nlm.nih.gov/pubmed/9222075 Effect of trovafloxacin, a new fluoroquinolone

antibiotic, on the steady-state pharmacokinetics of theophylline in healthy volunteers. (1997).

“Some fluoroquinolone antibiotics interfere with theophylline clearance, thereby raising concentrations

of circulating theophylline and increasing the potential for toxicity. The effect of steady-state serum

concentrations of the new fluoroquinolone trovafloxacin on the steady-state pharmacokinetics of

theophylline was examined in 12 healthy male volunteers. For 7 days, the subjects received morning and

evening theophylline doses adjusted to achieve steady-state plasma concentrations of 8-15 mg/L, the

lower end of the therapeutic range. From day 8 to day 15, six volunteers received, in addition to

theophylline, 200 mg of trovafloxacin in the morning and placebo in the evening (group A) and six

received placebo twice daily (group B). Serial plasma samples obtained over 12 h and 60 h after the

morning theophylline dose on days 7 and 14, respectively, were analysed for theophylline by HPLC with

UV detection. There were no significant differences in mean Cmax or AUC(0-12) between the two groups

on day 7 or on day 14, nor were there significant within-group differences on the two days. On day 14,

mean Cmax, AUC(0-12) and T(1/2) (measured on day 14 only) in group A were 10.15 mg/L, 107.32 mg x

h/L and 9.0 h, respectively. In group B, the values were 10.81 mg/L, 113.73 mg x h/L and 8.3 h,

respectively. The study drugs were well tolerated, and no clinically significant changes in vital signs or

laboratory test values were noted. We conclude that steady-state concentrations of trovafloxacin have

no clinically significant effect on the steady-state concentrations of theophylline within the therapeutic

range in healthy subjects..”

https://www.ncbi.nlm.nih.gov/pubmed/9089427 Phase I pilot study of the effects of trovafloxacin

(CP-99,219) on the pharmacokinetics of theophylline in healthy men. (1997). “This study examined

the effect of trovafloxacin (CP-99,219) on the pharmacokinetics and pharmacodynamics of a single dose

of theophylline, when administered to steady-state concentrations. Twelve healthy, nonsmoking male

volunteers participated. A 450-mg dose of theophylline was administered at 7:00 AM on day 1. On day 4,

volunteers received 300 mg of trovafloxacin (CP-99,219) daily in the morning for 7 days. The 450-mg

dose of theophylline was repeated on day 8 at 7:00 AM concomitantly with 300 mg of trovafloxacin.

Theophylline concentrations in plasma and trovafloxacin in serum were determined using reverse-phase

high-performance liquid chromatography. There was no significant difference between the geometric

mean values for Cmax of theophylline, 6.42 micrograms/mL and 6.00 micrograms mL on days 1 and 8,

respectively. A change (P = 0.032) in the geometric mean of the area under the concentration-time curve

extrapolated to infinity (AUC0-infinity) for theophylline was noted for trovafloxacin was administered.

Mean terminal phase elimination rate constants (Kes) were reduced (P = 0.001) by 13% after

administration of trovafloxacin from day 1 to day 8. In general, changes in theophylline clearance of less

than 20% are unlikely to be of clinical significance. In this study, oral administration of trovafloxacin in

References 16 website: JMR, http://fluoroquinolonethyroid.com

300 mg doses to achieve steady-state concentration resulted in an 8.4% increase in the extent of

systemic exposure (AUC0-infinity) to theophylline. Assuming that this AUC change is based on oral

clearance and not absorption, one would not expect to see clinically significant changes in the

pharmacokinetics of theophylline. No pharmacodynamic changes resulted from the pharmacokinetic

changes of theophylline.”

https://www.ncbi.nlm.nih.gov/pubmed/9023273 Aging and drug interactions. III. Individual and

combined effects of cimetidine and cimetidine and ciprofloxacin on theophylline metabolism in

healthy male and female nonsmokers. (1997). “The individual and combined effects of cimetidine and

ciprofloxacin on theophylline metabolism were examined in healthy young and elderly male and female

nonsmokers. Single-dose studies of theophylline pharmacokinetics were performed at base line and on

the fifth day of each of three treatment regimens consisting of 400 mg cimetidine every 12 hr, 500 mg

ciprofloxacin every 12 hr and the combination of cimetidine and ciprofloxacin. Base-line theophylline

plasma clearance and formation clearance of theophylline metabolites decreased with age in both

gender groups to a similar extent (20% less in elderly men than in young men; 24% less in elderly women

than in young women). Individually, cimetidine and ciprofloxacin produced proportionate declines in

plasma theophylline clearance that were similar among the four groups (range, 23.4-32.7% decrease).

The combined regimen yielded further impairment in theophylline elimination compared with each agent

alone (range, 35.9-42.6% decrease). Cimetidine was a nonselective inhibitor of theophylline metabolic

pathways in young men, but it exerted a greater inhibitory effect on N-demethylation pathways in the

other groups. Ciprofloxacin inhibited N-demethylations of theophylline to a greater extent than the

hydroxylation pathway. Coadministration of these two inhibitors further reduced the formation of

theophylline metabolites. The proportionate reduction in formation clearance of theophylline

metabolites was similar among the four groups. Thus, the response to inhibition of theophylline

metabolism by cimetidine and ciprofloxacin is not influenced by age or gender.”

https://www.ncbi.nlm.nih.gov/pubmed/9433655 Theophylline and warfarin interaction studies with

grepafloxacin. (1997). “Two phase I trials, each involving 16 healthy adult volunteers, were performed

to investigate possible interactions between grepafloxacin and theophylline or warfarin. In the

theophylline study, grepafloxacin 600 mg was administered once daily for 10 days to 12 volunteers who

were receiving a maintenance dose of theophylline. This dose of theophylline was designed to produce

mean serum theophylline concentrations of 7.5 mg/L; 4 volunteers received theophylline plus placebo.

Pharmacokinetic parameters of theophylline were determined before grepafloxacin treatment and on

day 10 of grepafloxacin or placebo administration. Peak theophylline concentrations and the area under

the concentration-time curve increased significantly during grepafloxacin treatment, and apparent total

clearance of theophylline was reduced by approximately 50%. No changes were observed in the placebo

group and theophylline appeared to have no effect on the pharmacokinetics of grepafloxacin. In the

warfarin study, grepafloxacin 600 mg was given once daily for 14 days to volunteers receiving a

maintenance dose of warfarin. Warfarin was discontinued during the last 4 days of grepafloxacin

administration. The pharmacodynamics of warfarin did not change after administration of grepafloxacin.

Similarly, warfarin had no significant effect on the pharmacokinetics of grepafloxacin. We conclude that

during treatment with grepafloxacin maintenance, doses of theophylline should be reduced by 50%, and

References 16 website: JMR, http://fluoroquinolonethyroid.com

we recommend that serum concentrations of theophylline be monitored during treatment with

grepafloxacin. However, no dose adjustment is necessary for grepafloxacin when it is coadministered

with theophylline, and dose adjustment does not seem to be required in concomitant treatment with

grepafloxacin and warfarin.”

https://www.ncbi.nlm.nih.gov/pubmed/9736563 Absence of effect of rufloxacin on theophylline

pharmacokinetics in steady state. (1997). “Several quinolone antibacterial agents are known to inhibit

the metabolism of theophylline, with the potential to cause adverse events due to raised theophylline

concentrations during coadministration. A randomized crossover study was therefore conducted with 12

healthy male volunteers (ages, 23 to 34 years; body weight, 64 to 101 kg) to evaluate a possible

interaction between rufloxacin and theophylline. Both drugs were administered at steady state. . . . In

conclusion, rufloxacin did not affect theophylline pharmacokinetics at steady state. Therefore,

therapeutic coadministration of rufloxacin and theophylline is not expected to cause an increased

incidence of theophylline-related adverse events.”

https://www.ncbi.nlm.nih.gov/pubmed/9688062 Development of a new quantitative approach for

the isobolographic assessment of the convulsant interaction between pefloxacin and theophylline in

rats. (1997). “A new mathematical approach was developed to quantify convulsant interaction

between pefloxacin and theophylline in rats.”

https://www.ncbi.nlm.nih.gov/pubmed/9661016 Effects of DU-6859a, a new quinolone antimicrobial,

on theophylline metabolism in in vitro and in vivo studies. (1998). “In vitro and in vivo studies were

conducted to investigate the drug interaction between a new quinolone antimicrobial, DU-6859a, and

theophylline (TP). The effect of DU-6859a on TP metabolism was evaluated in vitro by measuring the rate

of TP metabolite formation by using human liver microsomes. DU-6859a inhibited the metabolism of TP,

especially the formation of 1-methylxanthine, in vitro, but to a lesser extent than other drugs that are

known to interact with TP. TP was administered alone (200 mg twice a day [b.i.d.] for 9 days) or in

combination with DU-6859a (50 or 100 mg b.i.d. for 5 days) to six healthy subjects. DU-6859a

administered at a dose of 50 mg resulted in no changes in serum TP concentrations, and slight increases

in serum TP concentrations were observed at a dose of 100 mg. Moreover, the administration of 100 mg

of DU-6859a resulted in decreases in all urinary TP metabolites, with significant differences. It appears

that although DU-6859a has a weak inhibitory effect on TP metabolism in vitro, its concomitant use with

TP at clinical dosage levels does not cause any adverse effects, showing only a slight increase in blood TP

concentrations and a decrease in urinary metabolites.”

http://ndt.oxfordjournals.org/content/13/4/1006.long Interactions with ciprofloxacin and

erythromycin leading to aminophylline toxicity. (1998). “A number of commonly prescribed drugs are

known to interact with the metabolism of aminophylline, many to increase plasma levels to within the

toxic range. Two cases of serious aminophylline toxicity are described, one fatal, which were precipitated

by the co-prescription of antibiotics. The management of aminophylline overdose is discussed, with

particular respect to the input from the renal and intensive care units.”

References 16 website: JMR, http://fluoroquinolonethyroid.com

https://www.ncbi.nlm.nih.gov/pubmed/12212302 Effect of enoxacin on pharmacokinetics of

theophylline in rats. (1999). “In order to obtain an experimental evidence for Enoxacin(ENX) to be

correctly used in clinical treatment, we studied the effect of ENX on the pharmacokinetic parameters of

theophylline(TP). A single oral dose of TP 20 mg/kg was given to rats and ENX(300 mg/kg, 450 mg/kg)

was co-administered orally three times to those rats. The plasma concentrations of TP were determined

by HPLC after TP was administered 1, 2, 3, 5, 7, 12 and 24 hrs. The results showed that TP was eliminated

by one compartment model. TP plasma concentrations and AUC were significantly increased. T1/2 beta

of TP was prolonged. The total clearance of TP was decreased when compared with the control. This

interaction was dose-dependent. It was concluded that the interaction between ENX and TP existed.

Concomitant use of ENX with TP should be avoided.”

https://www.ncbi.nlm.nih.gov/pubmed/10567778 Lack of effect of gemifloxacin on the steady-state

pharmacokinetics of theophylline in healthy volunteers. (1999). “In conclusion, theophylline and

gemifloxacin may be co-administered without any adjustment in theophylline dose.”

https://www.ncbi.nlm.nih.gov/pubmed/10382036 Pharmacokinetic interactions between

fluoroquinolones and methylxanthines. (1999).

https://www.ncbi.nlm.nih.gov/pubmed/10348779 Effect of HSR-903, a new fluoroquinolone, on the

concentration of theophylline in serum. (1999). “It has been reported that some quinolones, such as

enoxacin, lead to a marked increase in the concentration of theophylline in serum when they are

coadministered with theophylline, resulting in an increased risk of serious side effects (2, 8). It is,

therefore, clinically important to evaluate whether a quinolone increases the theophylline concentrations

in serum when it is administered concomitantly. Accordingly, we studied the effect of HSR-903 on the

concentrations of theophylline in serum in healthy male adult volunteers. . . . In conclusion, HSR-903

proved to slightly increase the theophylline concentrations in serum and was classified as a class II

quinolone, indicating that the theophylline concentration in serum should be monitored and the

theophylline dose should be adjusted if concomitant administration of theophylline and HSR-903 is

necessary.”

https://www.ncbi.nlm.nih.gov/pubmed/10096258 Interaction of pefloxacin and enoxacin with the

human cytochrome P450 enzyme CYP1A2. (1999). “Pefloxacin is reported to cause clinically relevant

inhibition of theophylline metabolism in vivo, but in vitro pefloxacin was only a weak inhibitor of the

cytochrome P450 CYP1A2, mediating main theophylline biotransformation. We therefore further

characterized the interaction between pefloxacin and CYP1A2. . . . Enoxacin and to a lesser extent

pefloxacin may cause clinically relevant interactions with further CYP1A2 substrates. The data suggest

that the pefloxacin interaction is partly mediated by its major metabolite norfloxacin.”

https://www.ncbi.nlm.nih.gov/pubmed/11806624 Safety and effectiveness of lomefloxacin in

patients with acute exacerbation of chronic bronchitis (AECB) chronically treated with oral

theophyllines. (2001). “Theophylline plasma levels determined in 103 patients at baseline, during and

at the end of the lomefloxacin treatment did not significantly change. We conclude that orally

administered lomefloxacin at standard recommended dosage is well tolerated and effective in elderly

References 16 website: JMR, http://fluoroquinolonethyroid.com

patients with AECB. No dose adjustment is required even when it is co-administered with

methylxanthines.”

https://www.ncbi.nlm.nih.gov/pubmed/11502527 Drug interactions with clinafloxacin. (2001).

“Concomitant administration of 200 or 400 mg of clinafloxacin reduces mean theophylline clearance by

approximately 50 and 70%, respectively, and reduces mean caffeine clearance by 84%.”

https://www.ncbi.nlm.nih.gov/pubmed/11408986 Effect of levofloxacin on theophylline clearance

during theophylline and clarithromycin combination therapy. (2001). “To report a case of decreased

theophylline clearance by the addition of levofloxacin in a patient receiving theophylline and

clarithromycin. A 59-year-old Japanese man who was receiving theophylline for emphysema

experienced stimulation, insomnia, and tachycardia due to theophylline toxicity after clarithromycin and

levofloxacin were added to the regimen. The combination of these agents resulted in a decrease in

theophylline clearance to approximately 60% of the initial value obtained while the patient was receiving

theophylline alone. The adverse effects disappeared after the dosage was reduced and the theophylline

serum concentration decreased; however, there was no change in theophylline clearance. After

discontinuation of levofloxacin, the theophylline serum concentration decreased, and theophylline

clearance returned to the initial level even though clarithromycin was continued. Levofloxacin is believed

not to influence the clearance of theophylline, although some new fluoroquinolones have been reported

to do so. This case indicates that levofloxacin and clarithromycin inhibited theophylline metabolic

pathways catalyzed by both CYP1A2 and CYP3A4 and resulted in the decrease in theophylline clearance.

The clearance of theophylline, therefore, is not influenced by clarithromycin alone. Careful monitoring is

required when levofloxacin is prescribed for patients who are taking clarithromycin with theophylline.”

https://www.ncbi.nlm.nih.gov/pubmed/11352444 Lack of pharmacokinetic interaction between

moxifloxacin, a novel 8-methoxyfluoroquinolone, and theophylline. (2001). “To investigate the

plasma and urinary pharmacokinetics, safety and tolerability of theophylline and moxifloxacin after

single and repeated doses of either compound administered alone or concomitantly with the other . . .

Moxifloxacin - in contrast to some older quinolones - does not interact pharmacokinetically with

theophylline, confirming preclinical results on the absence of cytochrome P450-mediated metabolism.”

https://www.ncbi.nlm.nih.gov/pubmed/11249829 Profile of moxifloxacin drug interactions.(2001).

“We report a brief description of the interaction profile of moxifloxacin. After oral administration, the

absorption of moxifloxacin was unaffected by ranitidine or by food consumption. Drugs containing

multivalent cations (e.g., Mg(++), Al(+++), and Fe(++), but not Ca(++)) impaired absorption. No clinically

relevant effect of moxifloxacin was seen on the pharmacokinetics of digoxin under combination steady

state conditions. Also, moxifloxacin did not affect the pharmacokinetics of theophylline or vice versa. This

result, plus further data proving lack of interaction with glyburide, warfarin, and oral contraceptives,

confirms the absence of metabolic interactions involving the cytochrome P-450 system, as previously

reported. Concomitant administration of probenecid did not affect the elimination of moxifloxacin.

Moxifloxacin thus has a unique drug interaction profile that is advantageous for its safe use.”

References 16 website: JMR, http://fluoroquinolonethyroid.com

https://www.ncbi.nlm.nih.gov/pubmed/11172695 Adverse reactions to fluoroquinolones. an

overview on mechanistic aspects. (2001). “This review focuses on the most recent research findings on

adverse reactions caused by quinolone antibiotics. Reactions of the gastrointestinal tract, the central

nervous system (CNS) and the skin are the most often observed adverse effects. Occasionally major

events such as phototoxicity, cardiotoxicity, arthropathy and tendinitis occur, leading to significant

tolerability problems. Over the years, several structure-activity and side-effect relationships have been

developed, in an effort to improve overall antimicrobial efficacy while reducing undesirable side-effects.

In this article we review the toxicity of fluoroquinolones, including the newer derivatives such

levofloxacin, sparfloxacin, graepafloxacin and the 7-azabicyclo derivatives, trovafloxacin and

moxifloxacin. A special attention is given to new data on mechanistic aspects, particularly those

regarding CNS effects. In recent years extensive in vivo and in vitro experiments have been performed in

an attempt to explain the neurotoxic effects of quinolones sometimes observed under therapeutic

conditions. However, the molecular target or receptor for such effects is still not exactly known. Several

mechanisms are thought to be responsible. The involvement of gamma-aminobutyric acid (GABA) and

excitatory amino acid (EAA) neurotransmission and the kinetics of quinolones distribution in brain tissue

are discussed. In addition, quinolones may interact with other drugs--theophylline and nonsteroidal

antiflammatory drugs (NSAID(s))--in producing CNS effects This article provides information about the

different mechanisms responsible of quinolones interaction with NSAID(s), methylxanthines, warfarin

and antiacids.”

https://www.ncbi.nlm.nih.gov/pubmed/11957117 Effect of pazufloxacin mesilate on the serum

concentration of theophylline. (2002). “We studied the effect of pazufloxacin mesilate (T-3762), a new

fluoroquinolone for intravenous administration, on the serum concentration of theophylline. Evaluation

consisted of comparisons of serum levels of theophylline (when it was given alone, as a control), with

serum levels of theophylline when T-3762 was given concomitantly. We measured the serum

concentrations and the urinary excretion rates of theophylline in healthy adult male volunteers who were

given theophylline for 5 days, followed by an i.v. infusion of T-3762. Blood and urine samples were

investigated on the third and fifth days after the concomitant dosing with T-3762, to compare the serum

levels and urinary concentrations of theophylline with the control values. We found that the serum

concentration and the urinary excretion rates of theophylline on the fifth day after concomitant dosing

with T-3762 were significantly increased compared with the levels when the volunteers had been given

theophylline alone.”

https://www.ncbi.nlm.nih.gov/pubmed/11883649 Ignoring pharmacokinetics may lead to isoboles

misinterpretation: illustration with the norfloxacin-theophylline convulsant interaction in rats. (2002).

“To investigate the norfloxacin-theophylline convulsant interaction in vivo, with an experimental

approach distinguishing between pharmacodynamics and pharmacokinetics contributions to the

observed effect. Male Sprague Dawley rats (n = 38) were infused each compound separately or in

different combination ratios. Infusion was maintained until the onset of maximal seizures. Cerebrospinal

fluid and plasma samples were collected for high performance liquid chromatography drug

determination. The nature and intensity of the pharmacodynamics interaction between drugs was

quantified with an isobolographic approach. Isobolograms suggested a relatively marked antagonism

References 16 website: JMR, http://fluoroquinolonethyroid.com

between norfloxacin and theophylline at the cerebrospinal fluid (previously shown to be part of the

biophase) and dose levels, but not at the plasma (free and total concentrations) levels. These apparent

discrepancies could be explained by nonlinear distribution or/and distribution desequilibrium

phenomenon. These findings showed that the quantitative isobolographic approach is appropriate to

assess the nature and intensity of the pharmacodynamic interaction between two drugs when data are

collected within the biophase, but that data interpretation outside the biophase can be risky due to

further pharmacokinetic complexities, in particular slow or/and nonlinear diffusion into the biophase.”

https://www.ncbi.nlm.nih.gov/pubmed/14567511 The effect of orally administered marbofloxacin on

the pharmacokinetics of theophylline. (2003). “As certain quinolones can interfere with the

metabolism of theophylline by competitive inhibition of the hepatic microsomal cytochrome P450

system, concomitant use of these drugs with theophylline could result in theophylline toxicity. This study

investigated the effect of orally administered marbofloxacin (2 and 5 mg/kg each once daily) on steady-

state plasma pharmacokinetics of theophylline after concomitant oral administration of a sustained

release theophylline preparation in dogs. Marbofloxacin caused some alteration in theophylline

metabolism. A 2 mg/kg dose of marbofloxacin did not clearly result in an increased area under the

concentration--time curve (AUC) or decreased clearance of theophylline, but at a dose of 5 mg/kg, a

statistically significant increase in AUC and a decrease in the total clearance of theophylline was found.

The 26% reduction in theophylline clearance is probably not clinically significant in healthy dogs, but for

dogs with renal impairment, there might be a chance of theophylline accumulation when dosed

concomitantly with marbofloxacin.”

https://www.ncbi.nlm.nih.gov/pubmed/?term=fluoroquinolones+caffeine+2003 Influence of sex on

the pharmacokinetic interaction of fleroxacin and ciprofloxacin with caffeine. (2003). “Previous

pharmacokinetic studies have shown that a number of the quinolones inhibit the metabolism of caffeine.

To evaluate the effect of sex on the interaction between two quinolones and caffeine. Multiple-dose,

double-blind, randomised, three-period crossover study. Twelve male and twelve female healthy

volunteers. Subjects received by mouth either fleroxacin 400 mg once daily and caffeine 100 mg three

times daily, ciprofloxacin 500 mg twice daily and caffeine 100 mg three times daily, or caffeine alone, for

3 days. Subjects received each of the other regimens after 12-day washout periods. Plasma and urine

concentrations were determined by validated high-performance liquid chromatography procedures and

the data were analysed by noncompartmental linear pharmacokinetic methods. Analysis of the

interaction by sex revealed that females showed a significant difference in caffeine pharmacokinetics in

the presence of ciprofloxacin (area under the concentration-time curve [AUC], peak plasma

concentration [C(max)], time to C(max) [t(max)] and apparent total body clearance [CL/F]) and fleroxacin

(AUC and CL/F) when compared with males. Significant differences between sexes were also observed in

the pharmacokinetics of ciprofloxacin (AUC, elimination rate constant [beta] and CL/F) and fleroxacin

(C(max) and beta) in the presence of caffeine. However, these significant differences disappeared when

AUC and C(max) were normalised to 70 kg bodyweight and CL/F was expressed as per kg bodyweight.

The effect of quinolones on the pharmacokinetics of caffeine, and the reciprocal effect, are different

between the sexes, due in part to different bodyweights.”

References 16 website: JMR, http://fluoroquinolonethyroid.com

https://www.ncbi.nlm.nih.gov/pubmed/18315779 Prevalence and predictors of potential drug-drug

interactions in Regione Emilia-Romagna, Italy. (2004). “Drug-drug interactions (DDIs) are preventable

medication errors associated with potentially serious adverse events and death . . . The most commonly

identified potentially interacting medication pairs were . . . theophylline/aminophylline and

ciprofloxacin/fluvoxamine. . . . Awareness of the most prevalent potential DDIs can help practitioners

prevent concomitant use of these dangerous medication combinations.”

https://www.ncbi.nlm.nih.gov/pubmed/15120718 A new respiratory fluoroquinolone, oral

gemifloxacin: a safety profile in context. (2004). “Adverse experiences (AEs) were observed in 44.7% of

gemifloxacin-treated patients and 47.5% of those who received comparator drugs.”

https://www.ncbi.nlm.nih.gov/pubmed/16455179 Complexation of norfloxacin with DNA in the

presence of caffeine. (2006). “1)H NMR spectroscopy (500 MHz) has been used to quantify the

complexation of the antibacterial antibiotic Norfloxacin (NOR) with DNA in the presence of Caffeine

(CAF). Separate studies have been made for the self-association of NOR, its hetero-association with CAF

and complexation with a model self-complementary DNA tetramer, 5'-d(TpGpCpA), in order to determine

the equilibrium parameters (induced chemical shifts, association constants, enthalpy and entropy) of the

two-component mixtures to aid the analysis of the three-component systems. Investigations of the self-

association of NOR and its hetero-association with CAF show that the aggregation of NOR molecules and

association with CAF in solution are driven by the stacking of aromatic chromophores. The complexation

of NOR with d(TGCA) has been analysed in terms of intercalation with the double-stranded form and

non-intercalative binding with the single-stranded form of DNA. Investigations of the competitive binding

of NOR and CAF with DNA show that at physiological concentrations of NOR (muM) and CAF (mM) the

dominant mechanism influencing the affinity of NOR with DNA is the displacement of bound NOR

molecules from DNA due to CAF-DNA complexation (i.e. the protector action of Caffeine).”

https://www.ncbi.nlm.nih.gov/pubmed/11592692 Hetero-association of caffeine and aromatic drugs

and their competitive binding with a DNA oligomer. (2001).

https://www.ncbi.nlm.nih.gov/pubmed/12084457 Self-association and unique DNA binding

properties of the anti-cancer agent TAS-103, a dual inhibitor of topoisomerases I and II. (2003).

https://www.ncbi.nlm.nih.gov/pubmed/16958785 Effect of ofloxacin on theophylline

pharmacokinetics at clinical dosage in dogs. (2006). “We examined the effects of ofloxacin (OFX) and

norfloxacin (NFX) on theophylline (TP) pharmacokinetics in dogs. OFX, as a noncompetitive and

mechanism-based inhibitor, and NFX, as a noncompetitive inhibitor, were orally administered (5 mg/kg)

for a single dose or multiple doses (12 hourly for 3 days). TP (5 mg/kg, i.v) was injected at 2 h after the

final dose of the fluoroquinolones (FQs). The same dose of TP was injected (i.v) 3 weeks before the start

of FQs treatment for control. Multiple doses of OFX significantly reduced the total body clearance (Cl(B))

of TP from 0.117 to 0.085 L/h/kg, although a single dose did not change it. Neither a single dose nor

multiple doses of NFX changed the TP pharmacokinetics. Plasma NFX concentrations increased after

multiple doses. Those of OFX also increased but were still two orders of magnitude below the K(i) for

noncompetitive inhibition of CYP1A in dogs. Time-dependent reduction in Cl(B) of TP suggests that

References 16 website: JMR, http://fluoroquinolonethyroid.com

mechanism-based inhibition of OFX was the major mode to decrease Cl(B) of TP. The mechanism-based

inhibition may result in substantial inhibition of CYP1A activities in clinical conditions.”

https://www.ncbi.nlm.nih.gov/pubmed/17516267 Visual hallucinations secondary to ciprofloxacin

treatment. (2007). “We describe a case of a 74-year-old woman who experienced visual hallucinations

after ciprofloxacin administration, when she was also taking theophylline, which resolved on cessation of

the ciprofloxacin. Although uncommon, all ophthalmologists should be aware of this potential problem

and be familiar with the adverse visual effects which may occur in patients simultaneously administered

quinolones and theophylline.”

https://www.ncbi.nlm.nih.gov/pubmed/17499154 The effect of chronic cadmium exposure on the

pharmacokinetics of theophylline and ciprofloxacin in rats. (2007). “Cadmium has been associated

with a number of adverse health effects but the impact of those effects on the pharmacokinetics of

different drugs has not been investigated. Therefore, the pharmacokinetics of theophylline and

ciprofloxacin were studied in cadmium-exposed and control rats (72 rats) following i.p. (6.5mg/kg) and

p.o. (10mg/kg) administration, respectively. The third-generation offsprings of rats exposed to 100

microg/mL of cadmium chloride in drinking water were used in this study . . . The current investigation

showed that chronic exposure to cadmium could have a very significant impact on altering the

pharmacokinetic parameters of various drugs. Therefore, in cadmium-polluted areas, dose adjustments

and drug monitoring, especially for drugs with a narrow therapeutic window, should be carried out.”

https://www.ncbi.nlm.nih.gov/pubmed/18543578 The influence of a newly developed quinolone:

antofloxacin, on CYP activity in rats. (2008). “To investigate a newly developed quinolone antibiotics,

the effect of antofloxacin hydrochloride on cytochrome P450 isoforms in rats was examined. A cocktail

approach was adopted. Theophylline (CYP1A2), midazolam (CYP3A), chlorzoxazone (CYP2E1),

dextromethorphan (CYP2D6), omeprazole (CYP2C19) and diclofenac (CYP2C9) were used as probes in the

study, and own control was adopted. In Protocol 1, probes were given to rats simultaneously by co-

administration with antofloxacin. The blood samples were obtained at designated time, and plasma

concentrations of the six probes were determined by LC-MS. The pharmacokinetic parameters were

calculated and compared in experimental groups in the absence and presence of antofloxacin. The result

showed that the presence of antofloxacin resulted in a significant increase in theophylline values of

AUC0-T and t1/2 (PAUC0-T = 0.0004 vs control Pt1/2 = 0.005 vs control), indicating that antofloxacin

delayed the clearance of theophylline. In Protocol 2, the probes' pharmacokinetic parameters were

compared in rats that received six probes before and after 14.5 days of consecutive administration of

antofloxacin (15 mg x kg(-1), given orally, twice daily). The results suggested that the AUC0-T of

chlorzoxazone was significantly decreased (P = 0.024), while that of dextromethorphan was significantly

increased (P = 0.027). In conclusion, these results indicated that antofloxacin may inhibit the activity of

CYP1A2, thus delaying the clearance of its substrates, and may have a slight inhibitory effect on CYP2D6

as well as an inductive effect on CYP2E1 following chronic administration.”

https://www.ncbi.nlm.nih.gov/pubmed/19707748 Seizures associated with levofloxacin: case

presentation and literature review. (2009). “Clinicians are exhorted to pay close attention when

References 16 website: JMR, http://fluoroquinolonethyroid.com

initiating levofloxacin therapy in patients taking medications with epileptogenic properties that are

CYP1A2 substrates.”

https://www.ncbi.nlm.nih.gov/pubmed/21234553 Ciprofloxacin-induced theophylline toxicity: a

population-based study. (2011). “Ciprofloxacin can inhibit the cytochrome P450-mediated metabolism

of theophylline, but the clinical relevance of this drug interaction is uncertain. We studied the risk of

theophylline toxicity associated with the co-prescription of ciprofloxacin and theophylline. . . . Treatment

with ciprofloxacin is associated with a significant increase in the risk of theophylline toxicity. When

clinically appropriate, alternate antibiotics should be considered for elderly patients receiving

theophylline.”

https://www.ncbi.nlm.nih.gov/pubmed/20580800 Effects of fluoroquinolones on CYP4501A and 3A in

male broilers. (2011). “The inhibitory effects of fluoroquinolones on the enzyme activity, protein levels

and mRNA expression of liver cytochrome P450 (CYP) 1A and 3A were investigated in male broiler chicks.

Enrofloxacin (20 mg/kg), sarafloxacin (8 mg/kg) and marbofloxacin (5.5 mg/kg) were administrated in

drinking water for 7 consecutive days. A cocktail of the probe drugs caffeine and dapsone was used to

determine CYP1A and 3A activity. Western blot analysis and real-time PCR were used to determine the

effects on protein levels of CYP1A and 3A, and on CYP1A4, 1A5, 3A37 mRNA levels. Enrofloxacin

increased the half-life of elimination for both caffeine and dapsone, and decreased expression of CYP1A

and 3A protein. Marbofloxacin decreased the metabolism of caffeine and expression of CYP1A protein.

However, no change in mRNA expression was observed for any treatment group. This suggested that

high doses of enrofloxacin and marbofloxacin, but not sarafloxacin, inhibit CYP in chick liver raising the

possibility of drug-drug interaction when using these compounds.”

https://www.ncbi.nlm.nih.gov/pubmed/?term=fluoroquinolone+theophylline+2010 Comparative use

of isolated hepatocytes and hepatic microsomes for cytochrome P450 inhibition studies: transporter-

enzyme interplay. (2010). “Accurate assignment of the concentration of victim drug/inhibitor available

at the enzyme active site, both in vivo and within an in vitro incubation, is an essential requirement in

rationalizing and predicting drug-drug interactions. Inhibitor accumulation within the liver, whether as a

result of active transport processes or intracellular binding, may best be accounted for using hepatocytes

rather than hepatic microsomes to estimate in vitro inhibitory potency. The aims of this study were to

compare K(i) values determined in rat liver microsomes and freshly isolated rat hepatocytes of four

cytochrome P450 (P450) inhibitors (clarithromycin, enoxacin, nelfinavir, and saquinavir) with known

hepatic transporter involvement and a range of uptake (cell/medium concentration ratios 20-3000) and

clearance (10-1200 μl/min/10(6) cells) properties. Inhibition studies were performed using two well

established P450 probe substrates (theophylline and midazolam). Comparison of unbound K(i) values

showed marked differences between the two in vitro systems for inhibition of metabolism. In two cases

(clarithromycin and enoxacin, both low-clearance drugs), inhibitory potency in hepatocytes markedly

exceeded that in microsomes (10- to 20-fold), and this result was consistent with their high cell/medium

concentration ratios. For nelfinavir and saquinavir (high-clearance, extensively metabolized drugs), the

opposite trend was seen in the K(i) values: despite very high cell/medium concentration ratios, stronger

inhibition was evident within microsomal preparations. Hence, the consequences of hepatic

accumulation resulting from uptake transporters vary according to the clearance of the inhibitor. This

References 16 website: JMR, http://fluoroquinolonethyroid.com

study demonstrates that transporter-enzyme interplay can result in differences in inhibitory potency

between microsomes and hepatocytes and hence drug-drug interaction predictions that are not always

intuitive.”

https://www.ncbi.nlm.nih.gov/pubmed/21978534 Simultaneous analysis of fluoroquinolones and

xanthine derivatives in serum by molecularly imprinted matrix solid-phase dispersion coupled with

liquid chromatography. (2011).

https://www.ncbi.nlm.nih.gov/pubmed/21882652 Effect of caffeine, norfloxacin and nimesulide on

heartbeat and VEGF expression of zebrafish larvae. (2011). “The use of pharmaceuticals during

pregnancy may causes abnormalities to the embryo. Sometime the drug also effect to the new born if the

drug transferred through lactation. We have used zebrafish model to see the effect of some

pharmaceuticals on embryos and larvae. Three drugs, caffeine, norfloxacin and nimesulide, were used for

this study to see the effect mainly the hatching rate of eggs, heart beat rate and the vascular endothelial

growth factor (VEGF) expression of the larvae. VEGF is an important signaling protein that involved

generating the new blood vessels during embryonic development. We have used 10, 20, 50, 100 microg

ml(-1) concentrations of all the drugs to see the effect. No significant mortality or malformations were

observed in zebrafish embryos. Hatching was stared from 60 hr. In control group, 91% hatching rate was

observed. Lowest hatching rate was observed using highest concentration of norfloxacin (100 microg

ml(-1)) and nimesulide (100 microg ml(-1)) i.e. 55 and 56% respectively. In control group, 110 to 115

heart beat rate was counted per minute. Significantly higher heart beat was observed in caffeine treated

group which is 125 to 140 min(-1) Lower heart beat was noted in nimesulide treated group which is 100

min(-1). We have tried to observe the possible effect of VEGF of the larvae by these three drugs.

Expression of VEGF was very low in caffeine treated group. Almost no VGF expression was observe in 100

microg ml(-1) caffeine treated group. These studies suggest that there is a possibility that high dosage of

caffeine can harm the unborn baby or new born babies, if the mothers use caffeine.”

https://www.ncbi.nlm.nih.gov/pubmed/20662110 A simple chromatographic method for

determining norfloxacin and enoxacin in pharmacokinetic study assessing CYP1A2 inhibition. (2011).

https://www.ncbi.nlm.nih.gov/pubmed/21892200 Modulation of pharmacokinetics of theophylline

by antofloxacin, a novel 8-amino-fluoroquinolone, in humans. (2011). “The 5-day treatment with

antofloxacin significantly increased the area of the plasma concentration-time curve and peak plasma

concentration of theophylline, accompanied by a decrease in the excretion of theophylline metabolites.

On the contrary, theophylline did not affect the pharmacokinetics of antofloxacin. In vitro studies using

pooled human hepatic microsomes demonstrated that antofloxacin was a weak reversible and

mechanism-based inhibitor of CYP1A2. The clinical interaction between theophylline and antofloxacin

was further validated by the in vitro results. The results showed that antofloxacin increases the plasma

theophylline concentration, partly by acting as a mechanism-based inhibitor of CYP1A2. . . . In 1984,

Wijnands et al first reported severe clinical adverse effects with the concomitant use of TP and enoxacin,

and they found that co-administration of enoxacin markedly increased plasma TP concentrations11.

Consequently, a series of fluoroquinolones, including ciprofloxacin, tosufloxacin, clinafloxacin,

grepafloxacin, and pefloxacin, have been reported to interfere with TP metabolism by inhibiting CYP1A2

References 16 website: JMR, http://fluoroquinolonethyroid.com

activity. Therefore, it is important to evaluate the interaction between ATFX and TP to understand the

pharmacokinetics and safety of these drugs. . . . A recent report has demonstrated that treatment with

ciprofloxacin is associated with a significant increase in the risk of TP toxicity in elderly patients. Our

study confirmed the idea that the co-administration of ATFX increases the plasma levels of TP in young

populations. These results indicate that ATFX probably decreases TP clearance and increases the risk of

TP toxicity in elderly patients. Further studies are required to investigate the pharmacokinetic

interactions between ATFX and TP in elderly populations. Based on the results of our study, it was

concluded that ATFX appears to be a clinical mechanism-based inhibitor of CYP1A2. Co-administration of

ATFX may increase TP concentrations in plasma, which would raise the risk of TP toxicity in patients. We

suggest that when patients receiving TP require treatment with antibiotics, avoidance of ATFX may be

clinically appropriate. Alternatively, close monitoring for TP concentrations and toxicity is warranted in

cases where the use of ATFX is required.”

https://www.ncbi.nlm.nih.gov/pubmed/21512260 A physiologically based pharmacokinetic model

characterizing mechanism-based inhibition of CYP1A2 for predicting theophylline/antofloxacin

interaction in both rats and humans. (2011). “Clinical studies have revealed that some

fluoroquinolones may cause severe adverse effects when co-administered with substrates of CYP1A2.

Our previous study showed antofloxacin (ATFX) was responsible for mechanism-based inhibition (MBI) of

the metabolism of phenacetin in rats. In the clinical setting, ATFX is likely to be administrated with

theophylline (TP), which is mainly metabolized by CYP1A2. The aim of the present study was to

investigate the possible mechanism of TP/ATFX interaction. In vitro studies showed that the inhibitory

effect of ATFX on the formation of three TP metabolites depended on NADPH, the pre-inhibition time,

and ATFX concentration, i.e., factors which characterize MBI. In vivo studies demonstrated that single-

dose ATFX (20 mg/kg) did not affect the pharmacokinetic behavior of TP, but multidose ATFX (20 mg/kg

b.i.d. for 7.5 days) significantly increased the AUC of TP, decreased the amount of three TP metabolites in

urine, and suppressed hepatic microsomal activity. A physiologically based pharmacokinetic (PBPK)

model characterizing MBI of the three TP metabolites was developed for predicting TP/ATFX interaction

in rats; this model was further extrapolated to humans. The predicted results were in good agreement

with observed data. All the results indicated that ATFX was responsible for MBI of the metabolism of TP,

and the PBPK model characterizing MBI may give good prediction of TP/ATFX interaction.”

https://www.ncbi.nlm.nih.gov/pubmed/22868963 Fluoroquinolones and theophylline can also lower

the seizure threshold. (2012).

https://www.ncbi.nlm.nih.gov/pubmed/26933518 Application of a Physiologically Based

Pharmacokinetic Model to Study Theophylline Metabolism and Its Interactions With Ciprofloxacin and

Caffeine. (2016). “Theophylline is a commonly used bronchodilator. However, due to its narrow

therapeutic range, moderate elevation of serum concentration can result in adverse drug reactions

(ADRs). ADRs occur because of interhuman pharmacokinetic variability and interactions with

coprescribed medicines. We developed a physiologically based pharmacokinetic (PBPK) model of

theophylline, caffeine, and ciprofloxacin metabolisms to: examine theophylline pharmacokinetic

variability, and predict population-level outcomes of drug-drug interactions (DDIs). A simulation-based

equation for personalized dosing of theophylline was derived. Simulations of DDI show that calculated

References 16 website: JMR, http://fluoroquinolonethyroid.com

personalized doses are safe even after cotreatment with large doses of strong inhibitors. Simulations of

adult populations indicate that the elderly are most susceptible to ADRs stemming from theophylline-

ciprofloxacin and theophylline-caffeine interactions. Females, especially Asians, due to their smaller

average size, are more susceptible to DDI-induced ADRs following typical dosing practices. Our

simulations also show that the higher adipose and lower muscle fractions in females significantly alter

the pharmacokinetics of theophylline or ciprofloxacin.”

https://www.ncbi.nlm.nih.gov/pubmed/26838075 Chronic administration of caderofloxacin, a new

fluoroquinolone, increases hepatic CYP2E1 expression and activity in rats. (2016). “Caderofloxacin is a

new fluoroquinolone that is under phase III clinical trials in China. Here we examined the effects of

caderofloxacin on rat hepatic cytochrome P450 (CYP450) isoforms as well as the potential of

caderofloxacin interacting with co-administered drugs . . . Fourteen-day administration of caderofloxacin

can induce the expression and activity of hepatic CYP2E1 in rats. When caderofloxacin is administered, a

potential drug-drug interaction mediated by CYP2E1 induction should be considered.”

https://www.ncbi.nlm.nih.gov/pubmed/25893329 Effects of norfloxacin on hepatic genes expression

of P450 isoforms (CYP1A and CYP3A), GST and P-glycoprotein (P-gp) in Swordtail fish (Xiphophorus

Helleri) (2015). “The presence of antibiotics including norfloxacin in the aquatic environment may cause

adverse effects in non-target organisms. But the toxic mechanisms of fluoroquinolone to fish species are

still not completely elucidated. Thus, it is essential to investigate the response of fish to the exposure of

fluoroquinolone at molecular or cellular level for better and earlier prediction of these environmental

pollutants toxicity. The sub-chronic toxic effects of norfloxacin (NOR) on swordtail fish (Xiphophoru s

helleri) were investigated by measuring mRNA expression of cytochrome P450 1A (CYP1A), cytochrome

P450 3A (CYP3A), glutathione S-transferase (GST) and P-glycoprotein (P-gp) and their corresponding

enzyme activities (including ethoxyresorufin O-deethylase, erythromycin N-demethylase and GST. Results

showed that NOR significantly affected the expression of CYP1A, CYP3A, GST and P-gp genes in

swordtails. The gene expressions were more responsive to NOR exposure than their corresponding

enzyme activities. Moreover, sexual differences were found in gene expression and enzyme activities of

swordtails exposed to NOR. Females displayed more dramatic changes than males. The study further

demonstrated that the combined biochemical and molecular parameters were considered as useful

biomarkers to improve our understanding of potential ecotoxicological risks of NOR exposure to aquatic

organisms.”

http://journal.waocp.org/article_31092_ed2aea50b610e26b2ea3a3d446d98584.pdf High Efficacy of

Levofloxacin-Dexlansoprazole-Based Quadruple Therapy as a First Line Treatment for Helicobacter

pylori Eradication in Thailand (2015). “During the endoscopy, 4 biopsy samples from gastric antrum

were obtained for rapid urease test, H. pylori culture and Epsilometer test (E-test) or

GenoType®HelicoDR, histological examination and CYP2C19 genotype. The results of CYP2C19 genotype

testing were expressed as: rapid metabolizer (RM), intermediate metabolizer (IM) or poor metabolizer

(PM). . . . The CYP2C19 genotype tests revealed 54.1% RM, 34.7% IM and 11.2% PM . . . Our study

supported this idea and also demonstrated high eradication rate (grade A) of H. pylori infection from 14-

day levofloxacin-dexlansoprazole quadruple therapy as a first line treatment regardless of CYP2C19

genotype PM.”

References 16 website: JMR, http://fluoroquinolonethyroid.com

https://www.ncbi.nlm.nih.gov/pubmed/24580816 A multi-biomarker assessment of single and

combined effects of norfloxacin and sulfamethoxazole on male goldfish (Carassius auratus)(2014). “In

the present study, the sublethal effects of norfloxacin alone and in combination with sulfamethoxazole in

goldfish (Carassius auratus) were investigated, the biomarkers including acetylcholinesterase (AChE) in

brain, 7-ethoxyresorufin O-deethylase (EROD), glutathione S-transferase (GST), and superoxides

dismutase (SOD) activities in liver, vitellogenin (Vtg) in serum and DNA damage in gonad were

determined after 1, 2, 4 and 7 days of exposure. Brain AChE activity was significantly inhibited by

norfloxacin (≥0.4 mg/L) after 4 and 7 days and the mixtures with sulfamethoxazole (≥0.24 mg/L) after 4

days of exposure, and significant concentration-response relationships were obtained. Liver EROD, GST

and SOD activities were significantly increased by the individual and mixed pharmaceuticals in most

cases and exhibited analogously bell-shaped concentration-response curves. Serum Vtg was increased by

the highest concentration of norfloxacin and two higher concentrations of the mixtures. Higher

concentrations of the test antibiotics induced significant DNA damage in a concentration- and time-

dependent manner. The results indicated that selected antibiotics possesses cytotoxic and genotoxic

potential against the non-target organism C. auratus.”

https://www.ncbi.nlm.nih.gov/pubmed/24717959 Evaluation of the best method to assess antibiotic

potentiation by phytochemicals against Staphylococcus aureus (2014). “The increasing occurrence of

bacterial resistance to antibiotics has now reached a critical level. Finding antibiotic coadjuvants capable

to inhibit the bacterial resistance mechanisms would be a valuable mid-term solution, until new classes

of antibiotics are discovered. Selected plant alkaloids were combined with 5 antibiotics against 10

Staphylococcus aureus strains, including strains expressing distinct efflux pumps and methicillin-resistant

S. aureus strains. The efficacy of each combination was assessed using the microdilution checkerboard,

time-kill, Etest, and disc diffusion methods. The cytotoxicity of the alkaloids was evaluated in a mouse

fibroblast cell line. Potentiation was obtained in 6% of all 190 combinations, especially with the

combination of: ciprofloxacin with reserpine (RES), pyrrolidine (PYR), and quinine (QUIN); tetracycline

with RES; and erythromycin with PYR. The highest cytotoxicity values were found for QUIN (half maximal

inhibitory concentration [IC50] = 25 ± 2.2 mg/L) and theophylline (IC50 = 100 ± 4.7 mg/L).” (My note:

From Wiki “Alkaloid”: theophylline is a purine-like alkaloid, a pseudoalkaloid – alkaloid-like compounds

that do not originate from amino acids ).

https://www.ncbi.nlm.nih.gov/pubmed/24555232 In vivo evaluation of the metabolic ratio of CYP2C9

and CYP1A2 drug markers after administration of afobazole in comparison to standard inducers and

inhibitors of cytochromes (2013). “The effect of subchronic peroral administration in effective doses of

afobazole (5 mg/kg), and cytochrome P450 inductors (rifampicin, 13.4 mg/kg; phenytoin, 10.4 mg/kg)

and inhibitors (fluconazole, 35.7 mg/kg; ciprofloxacin, 44.0 mg/kg) on the metabolic ratio (MR) of drugs-

markers of CYP2C9 and CYP1A2 activity was studied in rats. Afobazole did not change the MR of

compounds metabolized by the P450 isoforms studied. After peroral administration of standard P450

inductors and inhibitors, statistically significant bidirectional effects were identified, which demonstrated

the expedience of administering a complex of selected compounds, markers, and CYP2C9 and CYP1A2

activity modificators for comparative evaluation of the effects of new drugs in rats. It is recommended to

References 16 website: JMR, http://fluoroquinolonethyroid.com

evaluate the activity of CYP1A2 by determining the MR for one of two caffeine metabolites, paraxanthine

or theobromine, and the activity of CYP2C9 by determining the MR of metabolite Exp-3174 to losartan.”

https://www.ncbi.nlm.nih.gov/pubmed/23587793 Detection of norfloxacin and monitoring its effect

on caffeine catabolism in urine samples (2013). “A multi-walled carbon nano tube (MWCNT) modified

pyrolytic graphite (MPG) electrode is prepared and applied to detect norfloxacin (NFX) based on its

electrochemical reduction. The experimental parameters affecting the NFX determination were

optimized in terms of MWCNT amount, pH, reaction time, and square wave frequency. The dynamic

range for the NFX analysis ranged between 1.2 and 1000µM with a detection limit of 40.6±3.3nM. The

effect of NFX on the catabolism of caffeine has been studied by determining its concentration in the urine

samples after the prolonged administration of NFX using the MPG electrode. The results show that the

catabolism of caffeine is inhibited by ~65% after five days of NFX administration, consequently the

caffeine concentration in the urine sample is increased, which is reflected in terms of ~2.5 times increase

in the peak current of caffeine. The determinations of NFX and caffeine were selective and the method

was successfully applied in biological fluids and pharmaceutical tablets for the test compound analysis. In

future this method can be useful for the selective determination of NFX and studying its effect on caffeine

catabolism.”

https://www.ncbi.nlm.nih.gov/pubmed/24399735 Pharmacokinetics of sunitinib in combination with

fluoroquinolones in rabbit model (2013). “Fluoroquinolones are widely prescribed antibiotics.

Ciprofloxacin is a well-known inhibitor of cytochrome P450 CYP3A4 and causes numerous drug

interactions that are not found for levofloxacin and moxifloxacin. CYP3A4 is involved in the metabolism

of the new oral multikinase inhibitor sunitinib which is indicated for the treatment of gastrointestinal

stromal tumor (GIST) and advanced renal cell carcinoma (RCC). This study investigated the effects of

single intravenous dose of ciprofloxacin, levofloxacin or moxifloxacin on the pharmacokinetics of

sunitinib. . . . The study proved a significant effect of the coadministration of ciprofloxacin and

levofloxacin on the pharmacokinetics of sunitinib in rabbits. The influence of moxifloxacin on the

pharmacokinetics of sunitinib was insignificant. Therefore, this fluoroquinolone seems to be the most

appropriate in combination with this tyrosine kinase inhibitor.”

https://www.ncbi.nlm.nih.gov/pubmed/23356842 Effect of ciprofloxacin and grapefruit juice on oral

pharmacokinetics of riluzole in Wistar rats (2013). “The objective of this study was to explore potential

drug-drug/food interactions of ciprofloxacin and grapefruit juice, known hepatic cytochrome P450 (CYP)

1A2 inhibitors, on single-dose oral pharmacokinetics of riluzole, a substrate of CYP 1A2 enzymes.

Pharmacokinetic parameters of riluzole were determined in Wistar rats after single-dose co-

administration with ciprofloxacin and grapefruit juice. In-vitro metabolic inhibition studies using rat and

human liver microsomes and intestinal absorption studies of riluzole in a rat everted gut-sac model were

conducted to elucidate the mechanism of interaction. A validated HPLC method was employed to

quantify riluzole in the samples obtained in various studies. Co-administration of ciprofloxacin with

riluzole caused significant increase in systemic exposure of riluzole (area under the curve, maximum

plasma concentration and mean residence time were found to increase). Co-administration of grapefruit

juice with riluzole did not cause any significant difference in the pharmacokinetic parameters of riluzole.

In-vitro metabolism studies demonstrated significant inhibition of riluzole metabolism when it was co-

References 16 website: JMR, http://fluoroquinolonethyroid.com

incubated with ciprofloxacin or grapefruit juice. No significant change was observed in apparent

permeability of riluzole. Co-administration of ciprofloxacin with riluzole increases the systemic levels of

riluzole and thereby the oral pharmacokinetic properties of riluzole while co-administration of grapefruit

juice with riluzole has no significant effect.”

https://www.ncbi.nlm.nih.gov/pubmed/23073666 Effect of the fluoroquinolone antibacterial agent

DX-619 on the apparent formation and renal clearances of 6β-hydroxycortisol, an endogenous probe

for CYP3A4 inhibition, in healthy subjects (2013). “To examine the effect of the fluoroquinolone DX-

619 on CYP3A4 and urinary excretion of 6β-hydroxycortisol, an endogenous probe of hepatic CYP3A4

activity, in healthy subjects. The effect of DX-619 on CYP3A4 was examined in human liver microsomes.

The apparent formation and renal clearance of 6β-hydroxycortisol (CL(6β-OHF) and CL(renal,6β-OHF),

respectively) were determined in placebo- and DX-619-treated subjects. 6β-hydroxycortisol uptake was

determined in HEK293 cells expressing OAT1, OAT3, OCT2, MATE1, and MATE2-K. DX-619 was a

mechanism-based inhibitor of CYP3A4, with K(I) and k(inact) of 67.9 ± 7.3 μmol/l and 0.0730 ± 0.0033

min(-1), respectively. Pharmacokinetic simulation suggested in vivo relevance of CYP3A4 inhibition by

DX-619. CL(6β-OHF) and CL(renal,6β-OHF) were decreased 72% and 70%, respectively, on day 15 in DX-

619-treated group compared with placebo (P < 0.05). 6β-hydroxycortisol was a substrate of OAT3

(K(m) = 183 ± 25 μmol/l), OCT2, MATE1, and MATE2-K. Maximum unbound concentration of DX-619

(9.1 ± 0.4 μmol/l) was above K(i) of DX-619 for MATE1 (4.32 ± 0.79 μmol/l). DX-619 caused a moderate

inhibition of hepatic CYP3A4-mediated formation and significant inhibition of MATE-mediated efflux of

6β-hydroxycortisol into urine. Caution is needed in applying CL(6β-OHF) as an index of hepatic CYP3A4

activity without evaluating CL(renal,6β-OHF).”

Ibuprofen:

https://www.ncbi.nlm.nih.gov/pubmed/15289789 Interindividual variability in ibuprofen

pharmacokinetics is related to interaction of cytochrome P450 2C8 and 2C9 amino acid

polymorphisms. “Low ibuprofen clearance occurs in a substantial proportion of healthy subjects, is not

enantiospecific, and is strongly linked to CYP2C8 and CYP2C9 polymorphisms.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2778050/ Cytochrome P450 2C8 pharmacogenetics:

a review of clinical studies.

https://en.wikipedia.org/wiki/CYP2C9 Wiki: “CYP2C9 is an important cytochrome P450 enzyme with a

major role in the oxidation of both xenobiotic and endogenous compounds. CYP2C9 makes up about 18%

of the cytochrome P450 protein in liver microsomes (data only for antifungal). Some 100 therapeutic

drugs are metabolized by CYP2C9, including drugs with a narrow therapeutic index such as warfarin and

phenytoin and other routinely prescribed drugs such as acenocoumarol, tolbutamide, losartan, glipizide,

and some nonsteroidal anti-inflammatory drugs. By contrast, the known extrahepatic CYP2C9 often

metabolizes important endogenous compound such as 5-hydroxytryptamine and, owing to its

epoxygenase activity, various polyunsaturated fatty acids, converting these fatty acids to a wide range of

References 16 website: JMR, http://fluoroquinolonethyroid.com

biological active products.[5][6] In particular, CYP2C9 metabolizes arachidonic acid to the following

eicosatrienoic acid epoxide (termed EETs).”

Chlorpheniramine: CYP2D6

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1874352/ The roles of CYP2D6 and stereoselectivity in

the clinical pharmacokinetics of chlorpheniramine. “Stereoselective elimination of chlorpheniramine

occurs in humans, with the most pharmacologically active (S)-(+)-enantiomer cleared more slowly than

the (R)-(−)-enantiomer. CYP2D6 plays a role in the metabolism of chlorpheniramine in humans.”

http://www.tandfonline.com/doi/abs/10.3109/00498259109039454 Metabolism of chlorpheniramine

in rat and human by use of stable isotopes (1991). “1. The metabolism of chlorpheniramine (I) was

examined in vivo in rats and a human volunteer; in the rats a stable isotope was used. 2. In addition to

the unchanged drug (I) and the N-demethylated metabolites (II and III), nine further metabolites were

identified in rat urine, four of which were also found in human urine. Chlorpheniramine N-oxide (IV), 3-(p-

chlorophenyl)-3-(2-pyridyl)propanol (V), 3-(p-chlorophenyl)-3-(2-pyridyl)-N-acetylaminopropane (VII) and

3-(p-chlorophenyl)-3-(2-pyridyl)-propionic acid (XIII) were identified in rat and human urine. 3. The

hydroxylated metabolites of the pyridyl ring of the unchanged drug, II, V and VII, and the glucuronide of

XIII were identified only in rat urine. XIII was found in rat urine as long as 6 days after the last dose.”

https://www.ncbi.nlm.nih.gov/pubmed/9231341 Stereoselective N-demethylation of

chlorpheniramine by rat-liver microsomes and the involvement of cytochrome P450 isozymes (1997).

“Previous studies have suggested that degradation of the two stereoisomers of chlorpheniramine in the

liver might be catalysed by different types of cytochrome P450. Stereoselective N-demethylation of

chlorpheniramine and the involvement of cytochrome P450 (CYP) isozymes have, therefore, been

investigated in the liver microsomes of eight-week-old male rats . . . The difference between the intrinsic

clearance of the two enantiomers by N-demethylation was because of differences in affinity for the

catalysing enzyme. This is indicative of stereoselective involvement of the main enzyme concerned in the

N-demethylation of the enantiomers, considered to be CYP 2C11. Anti-CYP 2C11 also partially inhibited

the N-demethylation of racemic chlorpheniramine in rat-liver microsomes exposed to phenobarbitone

and 3-methylcholanthrene. That CYP 2B1 was involved in the N-demethylation of both enantiomers was

also supported by results from an experiment using phenobarbitone-inducible rat-liver microsomes.

CYP1A1 did not, however, catalyse the N-demethylation of either enantiomer. These results indicate that

N-demethylation of the S-(+)-enantiomer of chlorpheniramine occurs preferentially in the microsomes,

demonstrating the stereoselective contribution of CYP2C11. Immunoinhibition studies suggest,

moreover, that the N-demethylation of both chlorpheniramine enantiomers is catalysed by CYP2B1, but

not by CYP1A1.”

https://www.ncbi.nlm.nih.gov/pubmed/9616188 In vitro characterization of cytochrome P450 2D6

inhibition by classic histamine H1 receptor antagonists (1998). “Classic antihistamines, namely

References 16 website: JMR, http://fluoroquinolonethyroid.com

diphenhydramine, chlorpheniramine, clemastine, perphenazine, hydroxyzine, and tripelennamine, share

structural features with substrates and inhibitors of the polymorphic cytochrome P450 (CYP) isozyme

CYP2D6. Therefore, the current study was undertaken to characterize the in vitro inhibition of CYP2D6 by

these commonly used, histamine H1 receptor antagonists. . . . These data demonstrate that classic

histamine H1 receptor antagonists, available in over-the-counter preparations, inhibit CYP2D6 in vitro.

Furthermore, the CYP2D6-inhibitory concentrations of these antihistamines are in the range of their

expected hepatic blood concentrations, suggesting that, under specific circumstances, clinically relevant

interactions between classic antihistamines and CYP2D6 substrates might occur.”

Pseudoephedrine

http://en.cnki.com.cn/Article_en/CJFDTOTAL-BXYY201108045.htm Effect of pseudoephedrine and

ephedrine on the activities of cytochrome P450 enzymes in rat liver microsomes. (2011). “Objective:

To study the effect of ephedrine and pseudoephedrine on the activities of cytochrome P450

enzymes(CYP450s) in rat liver microsomes. Method: The probe substrate of CYP450 enzymes: diclofenac

for CYP2C6,omeprazole for CYP2C,phenacetin for CYPlA2,chlorzoxazone for CYP2E1,testosterone for

CYP3A,was individually co-incubated with different concentration of ephedrine and pseudoephedrine in

rat liver microsomes. Then the relative metabolic clearance rate(RMCR) of each probe substrate was

determined by HPLC method. Results: The IC50 values of pseudoephedrine for the activities of CYP1A1/2

and CYP2E1 were 54.0 and 206.7μmol/L respectively. Whereas at concentration up to 400μmol/L,

ephedrine promoted the RMCR of phenacetin and diclofenac up to 2.3-fold and 1.6-fold respectively.

Conclusion: Pseudoephedrine has inhibitory action on the activities of CYP1A1/2 and CYP2E1,whereas

ephedrine has induction on the activities o f CYP2C and CYP1A1/2 in some extent.”

Purines and Purine Metabolism

http://molecularautism.biomedcentral.com/articles/10.1186/s13229-016-0109-5 Urinary

metabolomics of young Italian autistic children supports abnormal tryptophan and purine

metabolism. “Urinary metabolites displaying the largest differences between young ASD and control

children belonged to the tryptophan and purine metabolic pathways. Also, vitamin B6, riboflavin,

phenylalanine-tyrosine-tryptophan biosynthesis, pantothenate and CoA, and pyrimidine metabolism

differed significantly. ASD children preferentially transform tryptophan into xanthurenic acid and

quinolinic acid (two catabolites of the kynurenine pathway), at the expense of kynurenic acid and

especially of melatonin. Also, the gut microbiome contributes to altered tryptophan metabolism, yielding

increased levels of indolyl 3-acetic acid and indolyl lactate.”

https://en.wikipedia.org/wiki/Xanthurenic_acid

https://en.wikipedia.org/wiki/Hypoxanthine

References 16 website: JMR, http://fluoroquinolonethyroid.com

https://en.wikipedia.org/wiki/Xanthosine

https://en.wikipedia.org/wiki/Inosine

https://en.wikipedia.org/wiki/Purinergic_receptor

https://en.wikipedia.org/wiki/Nucleotide_salvage A salvage pathway is a pathway in which nucleotides

(purine and pyrimidine) are synthesized from intermediates in the degradative pathway for nucleotides.

Salvage pathways are used to recover bases and nucleosides that are formed during degradation of RNA

and DNA. This is important in some organs because some tissues cannot undergo de novo synthesis.

The salvaged bases and nucleosides can then be converted back into nucleotides.

Phosphoribosyltransferases add activated ribose-5-phosphate (Phosphoribosyl pyrophosphate, PRPP) to

bases, creating nucleoside monophosphates. There are two types of phosphoribosyltransferases:

adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase

(HGPRT). It is an important enzyme in Purine pathway metabolism.[1] It is also involved in Lesch-Nyhan

syndrome associated with a deficiency of HGPRT

https://en.wikipedia.org/wiki/Lesch%E2%80%93Nyhan_syndrome Lesch–Nyhan syndrome.

https://en.wikipedia.org/wiki/Hypoxanthine-guanine_phosphoribosyltransferase HPRT expression on

the mRNA and protein level is induced by hypoxia inducible factor 1 (HIF1A). HIF-1 is a transcription

factor that directs an array of cellular responses that are used for adaptation during oxygen deprivation.

This finding implies that HPRT is a critical pathway that helps preserve the cell's purine nucleotide

resources under hypoxic conditions as found in pathology such as myocardial ischemia.[9]

References 16 website: JMR, http://fluoroquinolonethyroid.com

https://wp.nyu.edu/biochemistry_2/wp-content/uploads/sites/1136/2015/04/Purine-Metabolism-de-

novo-synthesis-and-salvage-pathway-2015.pdf Purine Metabolism Info. Major site of purine synthesis

in liver. Protozoa: no de novo synthesis, must get purines from host.

http://onlinelibrary.wiley.com/doi/10.1111/j.1471-4159.2008.05275.x/full HIF-1 alpha is an essential

effector for purine nucleoside-mediated neuroprotection against hypoxia in PC12 cells and primary

cerebellar granule neurons. “Hypoxia-inducible factor-1 alpha (HIF-1α) and purine nucleosides

adenosine and inosine are critical mediators of physiological responses to acute and chronic hypoxia. The

specific aim of this paper was to evaluate the potential role of HIF-1α in purine-mediated

neuroprotection. We show that adenosine and inosine efficiently rescued clonal rat pheochromocytoma

(PC12) cells (up to 43.6%) as well as primary cerebellar granule neurons (up to 25.1%) from hypoxic

insult, and furthermore, that HIF-1α is critical for purine-mediated neuroprotection. Next, we studied

hypoxia or purine nucleoside increased nuclear accumulation of HIF-1α in PC12 cells. As a possible result

of increased protein stabilization or synthesis an up to 2.5-fold induction of HIF-1α accumulation was

detected. In cerebellar granule neurons, purine nucleosides induced an up to 3.1-fold HIF-1α

accumulation in cell lysates. Concomitant with these results, small interfering RNA-mediated reduction of

HIF-1α completely abolished adenosine- and inosine-mediated protection in PC12 cells and severely

hampered purine nucleoside-mediated protection in primary neurons (up to 94.2%). Data presented in

this paper thus clearly demonstrate that HIF-1α is a key regulator of purine nucleoside-mediated rescue

of hypoxic neuronal cells. . . . Adenosine is the final metabolite in the stepwise dephosphorylation of ATP

and it is produced and released in response to ischemia and hypoxia in the CNS. It acts as a powerful

endogenous neuroprotectant during ischemia-induced energy failure by decreasing neuronal

metabolism, increasing cerebral blood flow, and by playing a variety of different roles as an intercellular

messenger. These effects are mediated through the interaction of adenosine with specific receptors

(Rudolphi et al. 1992; Sweeney 1997; Kobayashi et al. 1998; von Lubitz 1999), and this stimulation was

hypothesized to result in an effective treatment for stroke (Dunwiddie and Masino 2001; review).

Likewise, the adenosine derivative inosine was shown to have protective effects against insults related to

ischemia and reperfusion (Shen et al. 2005), to induce neurite outgrowth (Benowitz et al. 1998), and to

stimulate the extension of new neuronal projections into denervated areas in adult rats with unilateral

cortical infarcts (Chen et al. 2002). Our own data showed that purine nucleosides protected neuronal

cells from rotenone-induced cell death (Bocklinger et al. 2004; Heftberger et al. 2005). The discovery that

purine nucleosides play a role in endogenous neuroprotection has given rise to extensive efforts towards

developing future ischemia/reperfusion drug therapies. To achieve such a goal, however, a complete

understanding of the intracellular signaling mechanisms involved in purine-mediated neuroprotection is

required. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that plays an essential role in

cellular and systemic homeostatic responses to hypoxia (Semenza 2000). Under hypoxia, the HIF-1α

protein is stabilized and translocation into the nucleus is increased. In the nucleus, HIF-1α associates with

HIF-1β to form the active transcription factor complex. The target genes of the HIF-1 complex are

involved in energy metabolism and cell viability. The HIF-dependent hypoxic response pathway plays a

prominent role in mediating the consequences of many disease states, including cerebral ischemia (for

review, see Semenza 2000). Along these lines, it was recently demonstrated (Baranova et al. 2007) that

HIF-1-mediated responses have an overall beneficial role in the ischemic brain. . . . The aim of this study

References 16 website: JMR, http://fluoroquinolonethyroid.com

was to determine (i) whether adenosine and its derivative inosine might regulate the cellular response to

hypoxia in neuronal cells and (ii) whether HIF-1α might contribute to adenosine-mediated

neuroprotectionAs a consequence of acute reduction in oxygen tension we observed increased cell death

of PC12 cells and primary cerebellar granule neurons. To determine whether purine nucleosides may

attenuate hypoxic cell death, cells were exposed to hypoxia in the presence of adenosine and inosine. In

agreement with results by others (Huffaker et al. 1984; Braumann et al. 1986; Gysbers and Rathbone

1996; Kobayashi and Millhorn 1999; Muroi et al. 2004; Ribeiro 2005; Tomaselli et al. 2005b), the data

presented here demonstrate that adenosine and inosine efficiently rescue the viability of hypoxic PC12

cells and primary cerebellar granule neurons. Earlier data suggested that the effects of adenosine were

apparently the result of its conversion to inosine by adenosine deaminase (Haun et al. 1996). In contrast,

a more recent study (Benowitz et al. 1998) showed that the addition of adenosine induced goldfish

retinal ganglion cells to extend lengthy neurites and that these effects were highly specific and did not

reflect conversion of the nucleosides to their derivatives. The same authors also showed that the action

of inosine was not due to its hydrolysis to hypoxanthine, as hypoxanthine was inactive in retinal ganglion

cells. Along these lines, our previous results in primary neurons showed that the activity of adenosine is

not blocked by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an inhibitor of adenosine deaminase,

suggesting an independent effect of adenosine (Heftberger et al. 2005). Although adenosine is a full

agonist of all four human adenosine receptors, inosine may activate A1 but is apparently ineffective on

the two A2R (Fredholm et al. 2001a). This may explain the observation that inosine was less effective

than adenosine in this study at least in PC12 cells. On the other hand, Jin et al. (1997) showed that

inosine is able to bind and activate adenosine A3R, and Haskóet al. (2000) found that A1R- and A2R-

antagonists partially blocked the suppressive effect of inosine on proinflammatory cytokine production.

In addition to adenosine receptors, neurons have nucleoside transport systems that play an important

role in regulating the concentrations and effects of purine nucleosides (Benowitz et al. 1998; Heftberger

et al. 2005; and for a review, see: Rathbone et al. 1999). Along these lines other authors reported that

exogenously applied inosine acted directly on an intracellular target, which may coincide with a serine-

threonine kinase, protein kinase N (Greene and Tischler 1976; Volontéet al. 1989; Batistatou et al. 1992),

and is linked to the response elements of genes associated with axon growth, including growth

associated protein (GAP)-43, L1, and alpha-1 tubulin (Benowitz et al. 1998; Petrausch et al. 2000). There

is a strong link between adenosine and hypoxia-related signaling. The expression levels of adenosine and

adenosine receptors are regulated in conditions of cellular stress, and signal transduction increases via

one or more of the adenosine receptors. Hypoxia apparently induces a program that shifts the tissue

phenotype toward an increase in extracellular adenosine. In turn, adenosine receptor activation tends to

limit the potential damage incurred by hypoxia (for review, see Fredholm 2007). . . . Apart from

adenosine receptor-mediated signaling, a number of metabolic pathways have been identified that

regulate gene expression during hypoxia (Bickler and Donohoe 2002; Seta et al. 2002). Among these, the

activation of HIF-1 transcription factors is the best characterized. HIF-1 is considered one of the master

regulators that orchestrate physiological responses to acute and chronic hypoxia (Semenza 2000;

Baranova et al. 2007). Therefore, we studied HIF-1α modulation in hypoxia in PC12 cells as well as in

cerebellar granule cells. As a consequence of cellular exposure to reduced oxygen, we observed an

increased stability of HIF-1α protein in cellular extracts of PC12 cells as well as in primary cerebellar

granule neurons. In addition, our results suggested that hypoxia led to an increased nuclear

References 16 website: JMR, http://fluoroquinolonethyroid.com

accumulation of HIF-1α as result of possible increased stabilization or synthesis and an increased

transcriptional activity. Our data are consistent with results reported by others (Masuda et al. 1994;

Krieg et al. 1998; Ruscher et al. 1998) that showed hypoxia stimulated an increase in HIF-1 binding

activity in purified neurons and altered the regulation of HIF-1 transcriptional targets in neuronal cell

lines and glial cultures. We also tested the potential effect of purine nucleosides. Our results showed that

hypoxia-induced HIF-1α was significantly enhanced by purine nucleosides. Our data are consistent with

previous findings (De Ponti et al. 2007) that demonstrated that the activation of the A2AR by adenosine

treatment induced HIF-1 DNA-binding activity, nuclear accumulation, and transactivation capacity in

J774A.1 mouse macrophages. To further study the non-redundant role of HIF-1α in the rescue of hypoxic

PC12 cells and cerebellar granule neurons we employed a siRNA approach to specifically knockdown HIF-

1α expression. Following partial siRNA-mediated knockdown of the HIF-1α transcription factor, we

observed a significant increase in the hypoxia-induced cell death of PC12 cells and cerebellar granule

neurons. Compared with cultured PC12 cells, primary cerebellar granule neurons showed a higher

spontaneous loss of viability. Consequently, we observed that hypoxic insult led only to a further 1.5-fold

increase in neuronal cell death compared with the 3.3-fold increase in PC12 cell death. This fact may be

one of the reasons for the observation that the HIF siRNA knockdown had less striking effects on the cell

viability of primary neurons compared with PC12 cells. We found even more exciting the observation that

purine nucleoside-mediated rescue was completely abrogated upon siRNA-mediated knockdown of HIF-

1α. . . . The future challenge will be to link molecular/genetic events with physiological mechanisms.

Various authors have already published work along these lines. One group (De Ponti et al. 2007),

reported that HIF-1α activation induced by the A2A receptor-specific agonist CGS21680 required the

phosphoinositide-3 kinase and protein kinase C pathways, but was not mediated by changes in iron

levels. Another study (Sodhi et al. 2001) provided evidence that MAPK and phosphoinositide-3 kinase-Akt

pathways may interact synergistically in the activation of HIF-1α. A large literature exists on the role of

the HIF-1 transcription factor in the expression of genes, including erythropoietin, that enhance oxygen

delivery to tissues (Kallio et al. 1998; Semenza 1999). Hypoxia also increases the expression of genes

whose products facilitate alterations in metabolism that optimize cell function during hypoxia (for

review, see Bickler and Donohoe 2002). For example, hypoxic pre-conditioning, a treatment known to

protect the newborn rat brain against hypoxic–ischemic injury, markedly increased HIF-1α (Bergeron et

al. 1999). The unique feature of HIF-1α is the regulation of its concentration. During normoxia, HIF-1α is

rapidly degraded by the ubiquitin proteasome system, and exposure to hypoxic conditions prevents its

degradation (Semenza 2000). The amino-terminal half of each subunit contains basic helix-loop-helix and

Per-Arnt-Sim (PAS) motifs that are required for dimerization and DNA binding. The carboxyl-terminal half

of HIF-1α contains domains that mediate hypoxia-inducible nuclear localization, protein stabilization,

and transactivation (for review, see Semenza 2000). Among the potential mechanisms that might

regulate transactivation, phosphorylation seems to play an important role. Indeed, several studies

showed that phosphorylation via the MAPK is necessary for activation of HIF-1α transcriptional activity

but not for its stabilization in hypoxic conditions (Salceda and Caro 1997; Minet et al. 2000; Hur et al.

2001). Other studies (Conrad et al. 1999), suggested that hypoxia caused specific regulation of the stress

activated protein kinase (SAPK) and p38 MAPK or the p42/44 MAPK signaling pathways, depending on

the cell type. Despite these differences, HIF-1 is activated by hypoxia in all cell types (reviewed by Mottet

et al. 2003). . . . In conclusion, the data presented here showed that (i) adenosine and inosine, which

References 16 website: JMR, http://fluoroquinolonethyroid.com

have increasingly been recognized as powerful endogenous neuroprotectants, efficiently rescued hypoxic

PC12 cells and cerebellar granule neurons and (ii) HIF-1α plays a non-redundant role as a key regulator in

the purine nucleoside-mediated rescue of hypoxic neuronal cells.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4289108/ Genetic Variation in Melatonin Pathway

Enzymes in Children with Autism Spectrum Disorder and Comorbid Sleep Onset Delay. “ Sleep

disruption is common in individuals with autism spectrum disorder (ASD). Genes whose products regulate

endogenous melatonin modify sleep patterns and have been implicated in ASD. Genetic factors likely

contribute to comorbid expression of sleep disorders in ASD. We studied a clinically unique ASD

subgroup, consisting solely of children with comorbid expression of sleep onset delay. We evaluated

variation in two melatonin pathway genes, acetylserotonin O-methyltransferase (ASMT) and cytochrome

P450 1A2 (CYP1A2). We observed higher frequencies than currently reported (p < 0.04) for variants

evidenced to decrease ASMT expression and related to decreased CYP1A2 enzyme activity (p ≤ 0.0007).

We detected a relationship between genotypes in ASMT and CYP1A2 (r2 = 0.63). Our results indicate that

expression of sleep onset delay relates to melatonin pathway genes.” (Check out the genes again against

my data).

CYP1A2 polymorphisms in slow melatonin metabolisers: a possible relationship with autism spectrum

disorder? (Need to google link). There are wide interindividual differences (10- to 200-fold) in CYP1A2

activity (Gunes & Dahl 2008). Several reports indicate that single nucleotide polymorphisms (SNPs) in the

CYP1A2 gene are associated with increased inducibility, decreased activity or inducibility or even loss of

activity of the CYP1A2 enzyme as compared with the wild-type CYP1A2*1A (Nakajima et al. 1999; Sachse

et al. 1999; Chevalier et al. 2001; Zhou et al. 2009a,b, 2010).The (sub) variant alleles associated with

decreased or absent activity of CYP1A2 are *1C, *1K, *3, *4, *5, *6 and *7.The proportion of individuals

with the slow phenotype narrowly ranges from 12% to 14% (Butler et al. 1992; Nakajima et al. 1994), but

varies among ethnic populations (Zhou et al. 2010).The *1F variant was found in 31.8% of 495 healthy

Caucasian volunteers (Skarke et al. 2005). Caffeine clearance is considered as the gold standard for

assessment of CYP1A2 activity, because more than 90% of the primary metabolism of caffeine depends

on CYP1A2 (Härtter et al. 2006). However, as melatonin is metabolised more exclusively by CYP1A2,

melatonin has been proposed as an alternative probe drug to assess CYP1A2 activity (Härtter et al.

2001).

http://biorxiv.org/content/early/2016/04/02/046722 Low parental melatonin levels increases autism

spectrum disorder risk in children. “Background: Low melatonin levels are a frequent finding in autism

spectrum disorder (ASD) patients. Melatonin is also important for normal neurodevelopment and

embryonic growth. As a free radical scavenger and antioxidant melatonin is highly effective in protecting

DNA from oxidative damage. Melatonin deficiency, possibly due to low CYP1A2 activity, could be a major

factor, and well a common heritable variation. ASD is already present at birth. As the fetus does not

References 16 website: JMR, http://fluoroquinolonethyroid.com

produce melatonin, low maternal melatonin levels should be involved. Methods: We measured 6-

sulfatoxymelatonin in urine of mothers of a child with ASD that attended our sleep clinic for people with

an intellectual disability (ID), and asked for parental coffee consumption habits, as these are known to be

related to CYP1A2 activity. Results: 6-Sulfatoxymelatonin levels were significantly lower in mothers than

in controls (p = 0.005), as well as evening coffee consumption (p = 0.034). In mothers with a second child

with ASD and/or ID, 6-sulfatoxymelatonin levels were lower compared to mothers with one child with

ASD (p = 0.084), Conclusions: Low parental melatonin levels, likely caused by low CYP1A2 activity, seem

to be a major contributor to ASD and possibly ID etiology.”

http://phoenixrising.me/research-2/glutathione-depletionmethylation-blockades-in-chronic-fatigue-

syndrome/why-is-the-prevalence-of-chronic-fatigue-syndrome-higher-in-women-than-in-men

Plausible hypothesis involving CYP1A2.

http://molpharm.aspetjournals.org/content/79/3/549.full Organization of NADPH-Cytochrome P450

Reductase and CYP1A2 in the Endoplasmic Reticulum—Microdomain Localization Affects

Monooxygenase Function. http://www.medschool.lsuhsc.edu/pharmacology/reed.aspx “Cytochromes

P450 (P450) constitute a family of heme-containing enzymes that are important in oxidative metabolism

of a multitude of endogenous and exogenous compounds (Nelson, 2003). P450s catalyze these reactions

by interacting with their redox partner, NADPH-cytochrome P450 reductase (CPR) in a 1:1 molar ratio

(Miwa et al., 1979). During substrate metabolism, electrons are transferred from NADPH to CPR, which

can then transfer electrons to the P450 (Gigon et al., 1969). Although a 1:1 molar complex between CPR

and P450 is needed for metabolism, the concentration of P450 enzymes greatly outnumber the level of

CPR, approximately 20:1 in liver microsomes (Peterson et al., 1976). The subsaturating levels of CPR

create a situation in which a single CPR molecule must supply electrons to a number of P450 enzymes,

rendering metabolically silent those P450s that are unable to complex with CPR. Such a system must be

highly organized to maintain efficient substrate metabolism, and one potential means of organization is

through the lipid bilayer. The P450s, along with their redox partners, are embedded in the endoplasmic

reticulum (ER) membrane (Peterson et al., 1976), and it has been well established that phospholipid is a

required component of an active P450 system (Strobel et al., 1970). Most in vitro studies for the

reconstitution of P450 activities use dilauroylphosphatidylcholine as the lipid milieu, but other lipids have

been used for these systems, including phosphatidylcholine (PC), phosphatidylethanolamine,

phosphatidylserine, and phosphatidic acid (Ingelman-Sundberg et al., 1981; Kim et al., 2003; Cho et al.,

2008; Reed et al., 2008). The alteration of phospholipid components of reconstituted systems (RCS) can

lead to variations in the rate of substrate metabolism, P450 incorporation into the membrane, and

stability of the enzyme (Blanck et al., 1984; Ingelman-Sundberg et al., 1996; Reed et al., 2006; Jang et al.,

2010). Such differences attributable to lipid composition prompt questions as to how the P450 system is

organized in the ER lipid bilayer. Members of our laboratory have initiated studies to analyze and

characterize the lipid environment of ER and to determine whether the P450 system resides in discrete

lipid microdomains, which may influence CPR-P450 and P450-P450 interactions. Early structural

perceptions of the of lipid bilayer were established by the fluid mosaic model (Singer and Nicolson, 1972),

which described the bulk of the phospholipids as being organized discontinuously, a small fraction of the

lipid specifically interacting with integral proteins. Studies with the plasma and Golgi membranes have

References 16 website: JMR, http://fluoroquinolonethyroid.com

greatly enhanced our views on the organization of the lipid membrane, which has been proven to play a

fundamental role in protein-protein and protein-lipid interactions (Brown and London, 1998).

Sphingolipids and sterols create a liquid-ordered phase of the membrane as a result of their high melting

temperatures, and these domains are involved in the sorting, transmembrane signaling, and

transporting of lipids and proteins (Brown and London, 1998). These ordered lipid phases prevent the

domains from being solubilized by nonionic detergents (Brown and London, 1998), imparting the term

detergent-resistant membranes (DRMs). Such domains were initially characterized by their low density

and insolubility in ice-cold 1% Triton X-100 (Brown and London, 2000), but more recently, a number of

other nonionic detergents have been used including Brij 98. Relative to the plasma membrane, the roles

of lipid microdomains in the structure of the ER membrane and the function of ER-resident proteins has

not been fully investigated. This is probably because there are relatively low levels of sphingolipids and

cholesterol at the ER membrane (Glaumann and Dallner, 1968). These lipids are two components of the

classic DRM located in the plasma membrane (Pike, 2004). Lipid microdomains in the ER that are

analogous to those in the plasma membrane have been described (Bae et al., 2004; Pielsticker et al.,

2005; Browman et al., 2006; Hayashi and Fujimoto, 2010). Given the specificity of lipid effects on P450

activity (discussed above), it is possible that the function of these enzymes may be affected by lipid

microdomain formation in the ER (Bösterling et al., 1979). In this article, we demonstrate the existence of

lipid microdomains in the ER and the presence of the P450 system within these regions. . . . Lipid vesicles

were prepared using purified lipids to approximate the lipid composition found in the ER and DRM. CPR

and CYP1A2 were incorporated into these vesicles to assess the effect of lipid composition on substrate

metabolism and CPR-CYP1A2 binding affinity . . . Recent studies have characterized “lipid raft-like”

domains at the ER membrane (Pielsticker et al., 2005; Browman et al., 2006; Hayashi and Fujimoto,

2010), and a number of researchers have suggested that specific ER lipids may form organized domains

affecting P450 function (Stier and Sackmann, 1973; Kim et al., 2007; Jang et al., 2010). These studies

raise the following questions: 1) do the enzymes of the P450 system reside in organized raft-like

domains? and 2) does P450 localization in these membrane regions affect P450 function? To address

these questions, we attempted to isolate these organized domains from microsomal samples and

determine whether the components of the P450 system were localized to these regions. Highly organized

lipid domains display different solubility characteristics from disordered domains when treated with

nonionic detergents . . . Densitometric analysis of the blots showed that approximately 73% of CYP1A2

(Fig. 1C) and 68% of CPR (Fig. 1D) were found to reside in the DRM fractions. In contrast, only 33% of the

total protein was located in the buoyant DRM fractions. . . . Having demonstrated the presence of

CYP1A2 and CPR in DRM fractions using a typical Brij 98 treatment protocol, the next step was to

determine whether the lipid composition of these fractions possessed classic detergent-resistant

membrane characteristics. As mentioned previously, the classic detergent-resistant membranes found

within the plasma membrane and other intracellular organelles are enriched in cholesterol and

sphingolipids. Upon analysis of each fraction, cholesterol was found to be enriched in the DRM fractions

(Fig. 2). The lipid composition of the total microsomal membrane, DRM, and non-DRM fractions was then

analyzed by thin-layer chromatography. Analysis of the DRM fractions illustrated a significant

enrichment of sphingomyelin (Fig. 3), which made up approximately 12% of the lipid of these fractions, in

contrast to 4% in the total microsomal membrane and less than 1% in the non-DRM fractions. There

were significantly lower levels of phosphatidylcholine and phosphatidylinositol in the DRM fractions

References 16 website: JMR, http://fluoroquinolonethyroid.com

compared with the total microsomal membrane. Cholesterol accounted for approximately 23% of the

DRM fraction lipids. The level of this component is high in contrast to the 5 and 2% composition in the

total and non-DRM fractions, respectively. The lipid-to-protein ratio was roughly 3.7 times higher in the

DRM fraction compared with the non-DRM fractions (data not shown). It is noteworthy that this specific

lipid composition is similar to that of the DRMs found in the plasma membrane and other intracellular

organelles (Brown and London, 2000; Pike, 2004; Hayashi and Fujimoto, 2010). It should be noted that

although cholesterol content is highest in fraction 3 of the sucrose gradient (Fig. 2), the highest amounts

of CYP1A2 and CPR are present in fraction 4 (Fig. 1). These results demonstrate the heterogeneity in DRM

fractions and that not all cholesterol-containing DRMs contain CYP1A2 and CPR. Nonetheless, there are

significant quantities of cholesterol in the fractions exhibiting enrichment of CPR and CYP1A2 . . . There

was a significant shift of CYP1A2 and CPR into the more dense regions of the gradient after treatment

with MβC. Whereas intact DRM fractions contained more than 73% of microsomal CYP1A2 and 68% of

CPR, only 6% of CYP1A2 and 2.5% of CPR was detected in DRM fractions after MβC treatment and

detergent solubilization . . . It is noteworthy that when the cholesterol-depleted microsomes were

reconstituted with cholesterol by treatment with the MβC-cholesterol complex, both CYP1A2 and CPR

migrated back into the DRM fractions of the sucrose gradient. Cholesterol repletion also allowed for

partial recovery of activity in the microsomes for both substrates tested. Collectively, these results

demonstrate that cholesterol is an important structural component of the DRM and affects the catalytic

efficiency of microsomal substrate metabolism . . . To determine whether lipid composition affected

substrate binding, spectral substrate binding was examined in three different vesicle systems . . . These

results suggest that there is a differential sensitivity of CPR binding to CYP1A2 depending on the

substrate present. These results clearly demonstrate that the lipid components found in detergent-

resistant membranes stimulate CYP1A2 activities, primarily by increasing the efficiency of the CPR-

CYP1A2 complex . . . The study corroborates previous findings indicating that DRMs exist in the ER

membrane (Pielsticker et al., 2005; Browman et al., 2006; Hayashi and Fujimoto, 2010) and

demonstrates that CYP1A2 and CPR reside primarily in these domains. We then tested the effects of lipid

composition on CYP1A2 function by comparing its activity in lipid vesicles that were representative of the

total ER and ordered (detergent-resistant) lipid domains to that in standard phosphatidylcholine vesicles.

This is the first investigation to examine the effects of these specific lipid compositions on P450 activity

and its interaction with CPR with lipids at physiologically relevant concentrations. . . Plasma membrane

DRMs have been described as heterogeneous domains typically enriched in sterols and sphingomyelin

(Brown and London, 1998; Pike, 2004). In agreement with other reports, the current study illustrated that

the overall ER lipid composition was low in cholesterol and sphingomyelin (Glaumann and Dallner, 1968),

which may explain the small number of investigations attempting to characterize ER-DRMs. However, by

using a standard technique to isolate these domains from rabbit liver microsomes with Brij 98 at 37°C

(Drevot et al., 2002), detergent-resistant regions were found to be enriched in cholesterol and

sphingomyelin. There were major differences in the lipid composition of the total ER membrane

compared with the lipid composition in the DRM fractions. These results demonstrate lipid domain

formation within the ER bilayer. Lipid analysis demonstrated that ER microdomains could be isolated at

physiological temperatures and had a composition similar to DRMs found in the plasma membrane and

other intracellular organelles. CYP1A2 and CPR were found to be enriched in the DRM fractions after Brij

98 solubilization, in contrast to the total microsomal protein. Our data are consistent with a previous

References 16 website: JMR, http://fluoroquinolonethyroid.com

large proteomic study identifying 39 proteins including CYP1A2 and CPR in ER microdomains (Bae et al.,

2004). It is noteworthy that our results are in contrast to those found by Hayashi and Fujimoto (2010),

who reported that CPR did not reside in DRMs. This discrepancy can be explained by the difference in the

tissue source. Their experiments were done using Chinese hamster ovary cells. The ER of these cells has

different lipid and protein compositions, which could significantly affect the localization of individual

proteins. Our study further investigated the nature of the CYP1A2- and CPR-containing DRMs by

demonstrating that the domains were cholesterol-dependent. Cholesterol sequestration by MβC

rendered the DRMs sensitive to detergent treatment, which led to the solubilization of CYP1A2 and CPR.

Analogous to the plasma membrane, the cholesterol component seems necessary for the structural

integrity of these domains. These results are similar to those of other studies in which MβC-mediated

cholesterol depletion led to solubilization of proteins residing in ER-DRMs (Browman et al., 2006; Hayashi

and Fujimoto, 2010). It is noteworthy that when MβC-depleted microsomes were reconstituted with

cholesterol, both CYP1A2 and CPR migrated to the DRM fractions, and catalytic activities were restored .

. . Several conclusions can be drawn from the current study. First, detergent-resistant lipid microdomains

can be found in the endoplasmic reticulum and, similar to that found in the plasma membrane, the ER

DRMs are enriched with both sphingomyelin and cholesterol. Second, CYP1A2 and CPR are found to

preferentially localize to these resistant membranes, and the removal of cholesterol by treatment with

methyl-β-cyclodextrin leads to a significant shift of CYP1A2 and CPR out of the DRM fractions to more

dense regions of the gradient. Furthermore, the DRM phospholipids seem to modulate P450 function.

Reconstituted systems having phospholipid content similar to that found in the ER membrane cause

alterations in CYP1A2 metabolic activities. Although the Vmax values obtained from the CPR titration

curves were not significantly changed, there was a substantial decrease in the Kmapp for CPR compared

with phosphatidylcholine alone. It is noteworthy that elevation of sphingomyelin and cholesterol to levels

seen in detergent-resistant microdomains caused a further decrease in the Kmapp for CPR. All told, the

Kmapp for CPR in reconstituted systems having phospholipid content similar to that found in DRMs is

between 21 and 29 times smaller than that found in PC vesicles, suggesting that the unique lipid content

of these vesicles substantially increases CYP1A2-dependent metabolism by increasing the efficiency of

CPR-CYP1A2 interactions. These results demonstrate that lipid microdomains can have a significant

influence on the localization of proteins of the P450 electron transport chain and that residence in these

DRMs can have a significant influence on P450 function.”

https://en.wikipedia.org/wiki/Cytochrome_P450_reductase Cytochrome P450 reductase [3] (EC

1.6.2.4; also known as NADPH:ferrihemoprotein oxidoreductase, NADPH:hemoprotein oxidoreductase,

NADPH:P450 oxidoreductase, P450 reductase, POR, CPR, CYPOR) is a membrane-bound enzyme

required for electron transfer from NADPH to cytochrome P450 in the endoplasmic reticulum[4] of the

eukaryotic cell. Since all microsomal P450 enzymes require POR for catalysis, it is expected that

disruption of POR would have devastating consequences. The reduction of cytochrome P450 is not the

only physiological function of POR. The final step of heme oxidation by mammalian heme oxygenase

requires POR and O2.

http://www.jnsci.org/files/html/e125.htm Melatonin in Children with Autism Spectrum Disorders:

How Does the Evidence Fit Together? “In reference to defects in melatonin degradation, there are

References 16 website: JMR, http://fluoroquinolonethyroid.com

numerous polymorphisms located either within the CYP1A2 gene, or in intronic regions, that are reported

to influence subsequent enzymatic activity.[47-53] A potential relationship has also been implicated

between presence of predicted slow-metabolizing alleles in CYP1A2 and susceptibility to ASD with

comorbid sleep problem.[35, 36] Interestingly, all of the individuals included in the Braam et al., 2013

study (n=11) were diagnosed as slow melatonin metabolizers and it was observed for these children that

the efficacy of supplemental melatonin exhibited disappearing effectiveness over the course of 4-8

weeks. We also evaluated slow-metabolizing alleles in CYP1A2 and the relationship to expression of

sleep onset insomnia in a small population of children with ASD and comorbid sleep onset insomnia

(n=15).[40] While we were only able to evaluate a small sample of children, we observed increased

frequencies for variants in the CYP1A2 gene related to decreased enzyme activity (p≤0.0007). Some

patients evaluated in our genetic study were also included in the study of overnight endogenous and

pharmacokinetic melatonin profiles. There was no evidence indicating individuals with slow-metabolizing

alleles in the CYP1A2 gene were actually slow melatonin metabolizers (T1/2 > 2 hours).[30] We also did

not observe disappearing effectiveness of melatonin treatment in our patients over the course of 17

weeks. However, we observed that expression of insomnia in ASD, and response to supplemental

melatonin treatment, was potentially related to dysfunctional variation in both the ASMT and CYP1A2

melatonin pathway genes. A relationship was observed between genotypes at SNP rs6644635 in the 5’-

untranslated region of ASMT and genotypes at SNP rs2069514 (my note: not in 23andme data) in the

promoter region of CYP1A2 (r2=0.63). This implicates a potential mechanism connecting lower levels of

ASMT transcript production with reduced CYP1A2 metabolic activity in some children with ASD and

comorbid sleep onset insomnia;[40] the net result may be normal nocturnal blood melatonin levels in

these children. The Braam et al. study did not assess effects related to polymorphisms in the ASMT gene.

Therefore, it is possible the patients with a slow melatonin metabolism only had dysfunctional alleles in

CYP1A2 and not in ASMT. However, to fully understand this underlying relationship to the etiology of ASD

with comorbid sleep onset insomnia, it will be necessary to evaluate larger ASD datasets focusing on

children with comorbid sleep onset insomnia and incorporating assessment of melatonin

pharmacokinetic data.

http://www.popline.org/node/202892 The effect of oral contraceptives on the pharmacokinetics of

melatonin in healthy subjects with CYP1A2 g.-163C>A polymorphism. “The effect of oral contraceptives

(OCs) on melatonin metabolism was studied in 29 subjects genotyped for CYP1A2 SNP g.-163C>A

polymorphism. Plasma melatonin and 6-OH-melatonin concentrations were measured after a 6-mg dose

of melatonin using a validated liquid chromatography/mass spectrometry method. The mean melatonin

AUC and C(max) values were 4- to 5-fold higher in OC users than in non-OC users (P < .0001), whereas

the weight-adjusted clearance was significantly lower in OC users (P < .0001). No significant difference in

melatonin pharmacokinetics between the genotypes and no additional effect by the genotype on the OC-

induced increase in melatonin exposure were evident. Melatonin exposure had no significant effect on

the subjects' state of alertness. In conclusion, a significant inhibitory effect of OCs on the CYP1A2-

catalyzed melatonin metabolism was seen; thereby, OC use can alter CYP1A2-phenotyping results.”

http://www.ebmconsult.com/articles/pharmacogenetics-cyp1a2-genetic-polymorphisms-table Table of

CYP1A2 polymorphisms

References 16 website: JMR, http://fluoroquinolonethyroid.com

http://www.mayomedicallaboratories.com/test-catalog/Clinical+and+Interpretive/89401 Mayo Clinic

CYP1A2 Table for polymorphisms

http://molpharm.aspetjournals.org/content/64/3/659 Genetic Polymorphism of CYP1A2 in Ethiopians

Affecting Induction and Expression: Characterization of Novel Haplotypes with Single-Nucleotide

Polymorphisms in Intron 1. “We developed SNP-specific polymerase chain reaction-restriction

fragment length polymorphism genotyping and molecular haplotyping methods for the intron 1 SNPs,

and four different haplotypes were identified: CYP1A2*1A (wild-type for all SNPs), CYP1A2*1F (–164A),

CYP1A2*1J (–740G and –164A), and CYP1A2*1K (–730T, –740G, and –164A), having frequencies of 39.9,

49.6, 7.5, and 3.0%, respectively. The frequency of CYP1A2*1J and CYP1A2*1K among Saudi Arabians (n

= 136) was 5.9% and 3.6%, and among Spaniards (n = 117) 1.3% and 0.5%, respectively. Functional

significance of the different intron 1 haplotypes was analyzed. Subjects with CYP1A2*1K had significantly

decreased CYP1A2 activity in vivo, and reporter constructs with this haplotype had significantly less

inducibility with 2,3,7,8-tetrachlorodibenzo-p-dioxin in human B16A2 hepatoma cells. Electrophoretic

mobility shift assay using nuclear extracts from B16A2 cells revealed a specific DNA binding protein

complex to an Ets element. Efficient competition was obtained using oligonucleotide probes carrying the

wt sequence and Ets consensus probe, whereas competition was abolished using probes with the –

730C>T SNP alone or in combination with –740T>G (CYP1A2*1K). The results indicate a novel

polymorphism in intron 1 of importance for Ets-dependent CYP1A2 expression in vivo and inducibility of

the enzyme, which might be of critical importance for determination of interindividual differences in

drug metabolism and sensitivity to carcinogens activated by CYP1A2. CYP1A2, a hepatic enzyme

inducible by smoking, metabolizes various chemical procarcinogens, such as food-derived heterocyclic

and aromatic mutagens, N-heterocyclics found in tobacco smoke, and difuranocoumarins, to reactive

carcinogens (McManus et al., 1990). It is also involved in the metabolism of several drugs such as

paracetamol, theophylline, caffeine, and clozapine (Bertilsson et al., 1994). Endogenous substrates of

CYP1A2 include estradiol and uroporphyrinogen. The enzyme has a significant role in chemical

carcinogenesis (Eaton et al., 1995) and is induced by its substrates, and a polymorphism in its capacity to

activate procarcinogens has been indicated . . . . Several studies have indicated the presence of wide

interindividual and ethnic differences in CYP1A2 activity when caffeine has been used as a probe drug.

Striking differences (greater than 15-fold) in levels of CYP1A2 mRNA expression from human liver (Ikeya

et al., 1989) and polymorphic metabolism of procarcinogens by human liver microsomes have been

reported (Minchin et al., 1985). Unlike other drug-metabolizing cytochromes P450, such as CYP2D6 and

CYP2C19, no nucleotide differences that could clearly explain the phenotypic variability in CYP1A2 gene

expression or inducibility have been identified . . . In the present investigation we have evaluated

interindividual variability in CYP1A2 activity in an African population and compared the activity between

Ethiopians living in Sweden and Ethiopia to investigate any environmental influence. We have found

new CYP1A2 haplotypes with SNPs in intron 1, which affect binding of nuclear proteins and inducibility

in reporter gene systems, and correlate to the CYP1A2 activity monitored in vivo using caffeine as a

probe drug. . . . The only additional SNP in CYP1A2*1K, as compared with the others, is –730C>T, and,

thus, the presence of this SNP is apparently critical for a decreased CYP1A2 activity in vivo . . . However,

there was 40% less induction of cells transfected with CYP1A2*1K, and this difference was significant

compared with cells treated with CYP1A2*1A, *1F,or *1J (p < 0.01 using independent t test), indicating

References 16 website: JMR, http://fluoroquinolonethyroid.com

that the CYP1A2*1K haplotype is less inducible . . . The results indicate that polymorphism in intron 1 of

the CYP1A2 gene might be critical for CYP1A2 inducibility and that transcriptional factors of the Ets

family are of importance in this respect. By contrast, the data do not provide evidence for an important

influence of environmental factors different between Sweden and Ethiopia for the control of CYP1A2

expression, in contrast to what has been seen for CYP2D6. Three different SNPs were found in intron 1,

and we identified four haplotypes present in Ethiopians, of which two are novel, CYP1A2*1J and

CYP1A2*1K. Subjects with the CYP1A2*1K allele had significantly reduced CYP1A2 activity as compared

with those carrying CYP1A2*1A, CYP1A2*1F,or CYP1A2*1J. Thus, the –164C>A alone (CYP1A2*1F) or in

combination with –740T>G (CYP1A2*1J) has apparently no influence on CYP1A2 activity in vivo, in

accordance with other studies showing no significance of the –164 SNP on CYP1A2 activity . . . Thus, we

assume that environmental factors such as dietary habits have small effects on CYP1A2 activity and

cannot primarily explain interethnic differences in activity. This is in accordance with a recent study on

twins, which indicated that the CYP1A2 activity is mainly governed by genetic factors . . . Although

CYP1A2 is only involved in the metabolism of about 5% of commonly prescribed drugs, it apparently

participates in the metabolism of 75% of drugs associated with adverse drug reactions metabolized by

enzymes having variant alleles (Phillips et al., 2001). Interindividual differences in its activity might thus

be of substantial importance for the determination of the outcome of drug treatment, and knowledge

about the basis for such interindividual differences, both genetic and environmental, might be useful to

avoid adverse drug reactions. In combination with the well established role of CYP1A2 for the metabolic

activation of procarcinogens, the polymorphism here described in intron 1 might, thus, also be of critical

importance for determination of the individual's susceptibility to liver cancer risk following long-term

exposure to several dietary procarcinogens.”

https://www.ncbi.nlm.nih.gov/pubmed/16609368 Search for an association between the human

CYP1A2 genotype and CYP1A2 metabolic phenotype (2006) By same author above. “The genotype

responsible for more than 60-fold interindividual differences in human hepatic CYP1A2 constitutive

expression is not understood. Resequencing the human CYP1A1_CYP1A2 locus (39.6 kb) in five major

geographically isolated subgroups recently led to the identification of 85 single nucleotide

polymorphisms (SNPs), 57 of which were double-hit SNPs. Here, we attempted to correlate the CYP1A2

genotype with a metabolic phenotype. We chose 16 SNPs (all having a minor allele frequency > or =0.05

in Caucasians) to genotype 32 DNA samples (26 Caucasians, six Ethiopians) in which CYP1A2 metabolism

had previously been determined. From 280 subjects (five locations worldwide) that had been CYP1A2-

phenotyped, we genotyped the 10 highest, 14 lowest and eight intermediate DNA samples. Although no

SNP was significant (P<0.05), possibly due to the small sample size, we found a trend for several of the

six SNPs across the CYP1A2 linkage disequilibrium block associated with the trait. Five CYP1A2

haplotypes were inferred, two of which had not previously been reported; haplotype 1A2H10 showed the

greatest association with CYP1A2 activity. Regulatory sequences responsible for the large interindividual

differences in hepatic CYP1A2 gene basal expression might reside, in part, with some of these CYP1A2

SNPS but, in large part, might be located either cis (in nearby sequences not yet haplotyped) or trans in

that they are not linked to the gene. We conclude that no SNP or haplotype in the CYP1A2 gene has yet

been identified that can unequivocally be used to predict the metabolic phenotype in any individual

patient.”

References 16 website: JMR, http://fluoroquinolonethyroid.com

http://smpdb.ca/view/SMP00017 Vitamin B6 pathways, note important in aromatic and sphingolipid

https://www.ncbi.nlm.nih.gov/pubmed/15646820 Metabolism of 2-phenylethylamine to phenylacetic

acid, via the intermediate phenylacetaldehyde, by freshly prepared and cryopreserved guinea pig liver

slices. “BACKGROUND: 2-Phenylethylamine is an endogenous amine, which acts as a neuromodulator

of dopaminergic responses. Exogenous 2-phenylethylamine is found in certain foodstuffs and may cause

toxic side-effects in susceptible individuals. MATERIALS AND METHODS: The present investigation

examined the metabolism of 2-phenylethylamine to phenylacetic acid, via phenylacetaldehyde, in

freshly prepared and cryopreserved liver slices. Additionally, it compared the relative contribution of

aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase by using specific inhibitors for each

oxidizing enzyme. RESULTS: In freshly prepared and cryopreserved liver slices, phenylacetic acid was

the main metabolite of 2-phenylethalamine. In freshly prepared liver slices, phenylacetic acid was

completely inhibited by disulfiram (inhibitor of aldehyde dehydrogenase), whereas isovanillin (inhibitor

of aldehyde oxidase) inhibited acid formation to a lesser extent and allopurinol (inhibitor of xanthine

oxidase) had no effect. In cryopreserved liver slices, isovanillin inhibited phenylacetic acid by 85%,

whereas disulfiram inhibited acid formation to a lesser extent and allopurinol had no effect.

CONCLUSION: In liver slices, 2-phenylethylamine is rapidly oxidized to phenylacetic acid, via

phenylacetaldehyde, by aldehyde dehydrogenase and aldehyde oxidase with no contribution from

xanthine oxidase.

https://www.ncbi.nlm.nih.gov/pubmed/25776752 Molecular dissection of a Borrelia burgdorferi in

vivo essential purine transport system. “The Lyme disease spirochete Borrelia burgdorferi is dependent

on purine salvage from the host environment for survival. The genes bbb22 and bbb23 encode purine

permeases that are essential for B. burgdorferi mouse infectivity. We now demonstrate the unique

contributions of each of these genes to purine transport and murine infection. The affinities of

spirochetes carrying bbb22 alone for hypoxanthine and adenine were similar to those of spirochetes

carrying both genes. Spirochetes carrying bbb22 alone were able to achieve wild-type levels of adenine

saturation but not hypoxanthine saturation, suggesting that maximal hypoxanthine uptake requires the

presence of bbb23. Moreover, the purine transport activity conferred by bbb22 was dependent on an

additional distal transcriptional start site located within the bbb23 open reading frame. The initial rates

of uptake of hypoxanthine and adenine by spirochetes carrying bbb23 alone were below the level of

detection. However, these spirochetes demonstrated a measurable increase in hypoxanthine uptake over

a 30-min time course. Our findings indicate that bbb22-dependent adenine transport is essential for B.

burgdorferi survival in mice. The bbb23 gene was dispensable for B. burgdorferi mouse infectivity, yet its

presence was required along with that of bbb22 for B. burgdorferi to achieve maximal spirochete loads in

infected mouse tissues. These data demonstrate that both genes, bbb22 and bbb23, are critical for B.

burgdorferi to achieve wild-type infection of mice and that the differences in the capabilities of the two

transporters may reflect distinct purine salvage needs that the spirochete encounters throughout its

natural infectious cycle.”

References 16 website: JMR, http://fluoroquinolonethyroid.com

https://www.ncbi.nlm.nih.gov/pubmed/22710875 Borrelia burgdorferi harbors a transport system

essential for purine salvage and mammalian infection. “Borrelia burgdorferi is the tick-borne

bacterium that causes the multistage inflammatory disease Lyme disease. B. burgdorferi has a reduced

genome and lacks the enzymes required for de novo synthesis of purines for synthesis of RNA and DNA.

Therefore, this obligate pathogen is dependent upon the tick vector and mammalian host environments

for salvage of purine bases for nucleic acid biosynthesis. This pathway is vital for B. burgdorferi survival

throughout its infectious cycle, as key enzymes in the purine salvage pathway are essential for the ability

of the spirochete to infect mice and critical for spirochete replication in the tick. The transport of

preformed purines into the spirochete is the first step in the purine salvage pathway and may represent a

novel therapeutic target and/or means to deliver antispirochete molecules to the pathogen. However,

the transport systems critical for purine salvage by B. burgdorferi have yet to be identified. Herein, we

demonstrate that the genes bbb22 and bbb23, present on B. burgdorferi's essential plasmid circular

plasmid 26 (cp26), encode key purine transport proteins. BBB22 and/or BBB23 is essential for

hypoxanthine transport and contributes to the transport of adenine and guanine. Furthermore, B.

burgdorferi lacking bbb22-23 was noninfectious in mice up to a dose of 1 × 10(7) spirochetes. Together,

our data establish that bbb22-23 encode purine permeases critical for B. burgdorferi mammalian

infectivity, suggesting that this transport system may serve as a novel antimicrobial target for the

treatment of Lyme disease.”

https://www.ncbi.nlm.nih.gov/pubmed/19666713 GuaA and GuaB are essential for Borrelia

burgdorferi survival in the tick-mouse infection cycle. “Pathogens lacking the enzymatic pathways for

de novo purine biosynthesis are required to salvage purines and pyrimidines from the host environment

for synthesis of DNA and RNA. Two key enzymes in purine salvage pathways are IMP dehydrogenase

(GuaB) and GMP synthase (GuaA), encoded by the guaB and guaA genes, respectively. While these genes

are typically found on the chromosome in most bacterial pathogens, the guaAB operon of Borrelia

burgdorferi is present on plasmid cp26, which also harbors a number of genes critical for B. burgdorferi

viability. Using molecular genetics and an experimental model of the tick-mouse infection cycle, we

demonstrate that the enzymatic activities encoded by the guaAB operon are essential for B. burgdorferi

mouse infectivity and provide a growth advantage to spirochetes in the tick. These data indicate that the

GuaA and GuaB proteins are critical for the survival of B. burgdorferi in the infection cycle and highlight a

potential difference in the requirements for purine salvage in the disparate mammalian and tick

environments.”

https://www.ncbi.nlm.nih.gov/pubmed/25424653 Purine import into malaria parasites as a target for

antimalarial drug development. “Infection with Plasmodium species parasites causes malaria.

Plasmodium parasites are purine auxotrophs. In all life cycle stages, they require purines for RNA and

DNA synthesis and other cellular metabolic processes. Purines are imported from the host erythrocyte by

equilibrative nucleoside transporters (ENTs). They are processed via purine salvage pathway enzymes to

form the required purine nucleotides. The Plasmodium falciparum genome encodes four putative ENTs

(PfENT1-4). Genetic, biochemical, and physiologic evidence suggest that PfENT1 is the primary purine

transporter supplying the purine salvage pathway. Protein mass spectrometry shows that PfENT1 is

expressed in all parasite stages. PfENT1 knockout parasites are not viable in culture at purine

References 16 website: JMR, http://fluoroquinolonethyroid.com

concentrations found in human blood (<10 μM). Thus, PfENT1 is a potential target for novel antimalarial

drugs, but no PfENT1 inhibitors have been identified to test the hypothesis. Identifying inhibitors of

PfENT1 is an essential step to validate PfENT1 as a potential antimalarial drug target.”

http://jb.asm.org/content/195/19/4387.full Helicobacter pylori Salvages Purines from Extracellular

Host Cell DNA Utilizing the Outer Membrane-Associated Nuclease NucT. “Helicobacter pylori is a

bacterial pathogen that establishes life-long infections in humans, and its presence in the gastric

epithelium is strongly associated with gastritis, peptic ulcer disease, and gastric cancer. Having evolved

in this specific gastric niche for hundreds of thousands of years, this microbe has become dependent on

its human host. Bioinformatic analysis reveals that H. pylori has lost several genes involved in the de

novo synthesis of purine nucleotides, and without this pathway present, H. pylori must salvage purines

from its environment in order to grow. While the presence and abundance of free purines in various

mammalian tissues has been loosely quantified, the concentration of purines present within the gastric

mucosa remains unknown. There is evidence, however, that a significant amount of extracellular DNA is

present in the human gastric mucosal layer as a result of epithelial cell turnover, and this DNA has the

potential to serve as an adequate purine source for gastric purine auxotrophs

https://www.ncbi.nlm.nih.gov/pubmed/12735109 Intrinsic hepatic phenotype associated with the

Cyp1a2 gene as shown by cDNA expression microarray analysis of the knockout mouse. “Several

forms of cytochrome P450 (CYP) appear to metabolize principally pharmaceutical agents, as well as

other dietary and plant chemicals. Other CYP forms have major roles in steroid, sterol, and bile acid

metabolism. CYP1A2 expression is constitutively high in mouse liver and is well known for metabolizing

several drugs and many procarcinogens to reactive intermediates that can cause toxicity or cancer.

CYP1A2 is also known to carry out several endogenous functions such as uroporphyrinogen and

melatonin oxidation and the 2- and 4-hydroxylations of estradiol. We have used cDNA microarray

analysis of the untreated Cyp1a2(-/-) knockout mouse to search for changes in gene expression that

might indicate important intrinsic roles for this enzyme. For 15 of the up- or downregulated genes, these

increases or decreases were corroborated by reverse-transcription real-time polymerase chain reaction.

Other than upregulation of the Hprt gene (used in the selection procedure for disrupting the Cyp1a2

gene), we found several genes upregulated that are associated with cell-cycle regulation and lipid

metabolism. Besides Cyp1a2, the gene exhibiting the greatest downregulation was Igfbp1 (insulin-like

growth factor binding protein-1), showing only 12% expression of that in the Cyp1a2(+/+) wild-type liver.

Recurrent themes between both up- and downregulated genes include cell-cycle control, insulin action,

lipogenesis, and fatty acid and cholesterol biosynthetic pathways. Histologically, the Cyp1a2(-/-) mouse

exhibited an approximately 50% decrease in lipid stored in hepatocytes, and 50% increase in lipid present

in interstitial fat-storing cells compared with that in the Cyp1a2(+/+) wild-type. These data suggest that

the CYP1A2 enzyme might perform additional hepatic endogenous functions heretofore not

appreciated.”

https://www.ncbi.nlm.nih.gov/pubmed/12464254 Study of P450 function using gene knockout and

transgenic mice. “The xenobiotic-metabolizing P450s have been extensively studied for their ability to

metabolize endogenous and exogenous chemicals. The latter include drugs and dietary and

environmentally derived toxicants and carcinogens. These enzymes also metabolize endogenous steroids

References 16 website: JMR, http://fluoroquinolonethyroid.com

and fatty acids. P450s are thought to be required for efficient removal of most xenobiotics from the body

and to be responsible for the hazardous effects of toxicants and carcinogens based on their ability to

convert chemicals to electrophilic metabolites that can cause cellular damage and gene mutations. P450

catalytic activities have been extensively studied in vitro and in cell culture, yielding considerable

information on their mechanisms of catalysis, substrate specificities, and metabolic products. Targeted

gene disruption has been used to determine the roles of P450s in intact animals and their contributions

to the mechanisms of toxicity and carcinogenesis. The P450s chosen for study, CYP1A1, CYP1B1, CYP1A2,

and CYP2E1, are conserved in mammals and are known to metabolize most toxicants and chemical

carcinogens. Mice lacking expression of these enzymes do not differ from wild-type mice, indicating that

these P450s are not required for development and physiological homeostasis. However, the P450 null

mice have altered responses to the toxic and carcinogenic effects of chemicals as compared with wild-

type mice. These studies establish that P450s mediate the adverse effects of drugs and dietary,

environmental, and industrial chemicals and serve to validate molecular epidemiology studies that seek

to determine links between P450 polymorphisms and susceptibility to chemically associated diseases.

More recently, P450 humanized mice have been produced.”

https://www.ncbi.nlm.nih.gov/pubmed/15970798 Theophylline pharmacokinetics: comparison of

Cyp1a1(-/-) and Cyp1a2(-/-) knockout mice, humanized hCYP1A1_1A2 knock-in mice lacking either the

mouse Cyp1a1 or Cyp1a2 gene, and Cyp1(+/+) wild-type mice. “OBJECTIVES: Pharmacokinetics of

theophylline was investigated in Cyp1(+/+) wild-type mice, Cyp1a1(-/-) and Cyp1a2(-/-) knockout mice,

and humanized hCYP1A1_1A2 mice lacking either the mouse Cyp1a1 or Cyp1a2 gene. METHODS AND

RESULTS: Animals received a single dose of theophylline (8 mg/kg i.p.), either alone or pretreated with

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 microg/kg i.p.) 24 h prior to theophylline. We found that

mouse or human CYP1A2 is the predominant enzyme for theophylline metabolism, the contribution of

mouse or human CYP1A1 to theophylline metabolism is negligible, and another TCDD-inducible enzyme

plays a minor role in 1-methyluric acid and 1,3-dimethyluric acid formation as well as enhanced

theophylline clearance from the body. The half-life of elimination from plasma was more than four times

longer in Cyp1a2(-/-) than Cyp1(+/+) mice and more than 10 times different after TCDD pretreatment. In

humanized hCYP1A1_1A2 mice lacking the mouse Cyp1a2 gene, the half-life of elimination from plasma

was two to three times longer than that in Cyp1(+/+) mice and four to five times different after TCDD

pretreatment. CONCLUSION: Replacement of mouse Cyp1a2 with a functional human CYP1A2 gene

restored the ability to metabolize theophylline, and the metabolism changed to a humanized profile (i.e.

3-methylxanthine formation, not seen in the wild-type mouse). TCDD-pretreated hCYP1A1_1A2 Cyp1a2(-

/-) mice exhibited enhanced theophylline metabolism and clearance, due to induction of the human

CYP1A2 enzyme. Comparing the hCYP1A1_1A2 Cyp1a2(-/-) and wild-type mice with published clinical

studies, we found theophylline clearance to be about 5 times and 12 times, respectively, greater than

that reported in humans.”

https://getd.libs.uga.edu/pdfs/miller_erica_f_201308_phd.pdf PURINE SALVAGE IN HELICOBACTER

PYLORI. “To fill this gap in knowledge, we asked whether H. pylori can carry out de novo purine

biosynthesis, and whether its purine salvage network is complete. Based on genomic data from the fully

sequenced H. pylori genomes, we combined mutant analysis with physiological studies to determine that

References 16 website: JMR, http://fluoroquinolonethyroid.com

H. pylori, by necessity, must acquire purines from its human host. Furthermore, we found the purine

salvage network to be complete, allowing this organism to use any single purine nucleobase or

nucleoside for growth.”

http://journal.frontiersin.org/article/10.3389/fpls.2014.00153/full Transport proteins of parasitic

protists and their role in nutrient salvage. “The loss of key biosynthetic pathways is a common feature

of important parasitic protists, making them heavily dependent on scavenging nutrients from their hosts.

This is often mediated by specialized transporter proteins that ensure the nutritional requirements of the

parasite are met. Over the past decade, the completion of several parasite genome projects has

facilitated the identification of parasite transporter proteins. This has been complemented by functional

characterization of individual transporters along with investigations into their importance for parasite

survival. In this review, we summarize the current knowledge on transporters from parasitic protists and

highlight commonalities and differences in the transporter repertoires of different parasitic species, with

particular focus on characterized transporters that act at the host-pathogen interface.”

https://repositorio-aberto.up.pt/bitstream/10216/69104/2/92437.pdf Macrophage nutriprive

antimicrobial mechanisms. “In addition to oxidative and antibiotic mechanisms of antimicrobial

activity, macrophages are able to deprive intracellular pathogens of required nutrients. Thus, microbial

killing may not rely only in the toxic environment the microbe reaches but also may result from the

scarcity of nutrients in the cellular compartment it occupies. Here, we analyze evidence for such

nutriprive (from the latin privare, to deprive of nutrients), antimicrobial mechanisms.”

http://erj.ersjournals.com/content/31/5/949 Extracellular purines are biomarkers of neutrophilic

airway inflammation.