CHAPTER 3

PIZOTIFEN

80

3.1. DRUG PROFILE

The main use for Pizotifen is for the prevention of vascular

headache including migraine and cluster headache. Pizotifen is one of a range

ofmedicationsusedforthispurpose.Otheroptionsinclude propranolol, topiramate, valproic

acid and amitriptyline. While Pizotifen is reasonably effective, its use is limited by side

effects, principally drowsiness and weight gain, and it is usually not the first choice

medicine for preventing migraines, instead being used as an alternative when other drugs

have failed to be effective. [1-3]

Other applications for which Pizotifen may be used include as an antidepressant,

or for the treatment of anxiety or social phobia. Animal studies also suggest that

pizotyline could be used in the treatment of serotonin syndrome or MDMA overdose in a

similar manner to the closely related antihistamine/antiserotonin drug cyproheptadine. [4-5]

Side effects include sedation, dry mouth, drowsiness, increased appetite and

weight gain. Occasionally it may cause nausea, headaches, or dizziness. In rare cases,

anxiety, aggression and depression may also occur. Pizotifen is contraindicated in

patients who suffer from hypersensitivity to any of its components, also Pizotifen is

contraindicated in gastric outlet obstruction, pregnancy, angle-closure glaucoma and

difficulty urinating [6-8]

.

Pizotifen is a serotonin antagonist acting mainly at the 5-HT2A and 5HT2C

receptors. It also has some activity as an antihistamine as well as some anticholinergic

activity.

Figure-3.A: Structure of Pizotifen

81

IUPAC NAME : 4-(1-methyl-4-piperidylidine)-9, 10-dihydro -4H-benzo-

[4, 5]cyclohepta[1,2]-thiophene

FORMULA : C19H21NS

MOLECULAR WEIGH

: 295.443 g/mol

SOLUBILITY : Soluble in Methanol and water

List of brand names of PIZOTIFEN

Table 3.1

BRAND NAME

FORMULATION COMBINATION AVAILABLE

STRENGTH

MANUFACTURER

MIGRANIL TABLET ----- 0.725mg LNGA

TABLET ----- 2.175mg

SANOMIGRAN TABLET ----- 0.5 mg NOVARTIS

TABLET ----- 1.5mg

PIZOTIFEN TABLET ----- 500 mcg ACTAVIS UK LTD

TABLET ----- 1.5 mg

82

3.2. LITERATURE SURVEY

Several analytical methods have been reported for the determination of Pizotifen

in pure drug, pharmaceutical dosage forms and in biological samples using

spcetrophotometry liquid chromatography, electro kinetic chromatography high

performance thin layer chromatography either in single or in combined forms

Nielsen JA et al [9]

emphasized the effects of the 5-hydroxytryptamine receptor

antagonists metergoline, pirenperone and Pizotifen on 5-hydroxytryptamine and

dopamine synthesis were determined by measuring the rate of accumulation of 5-

hydroxytryptamine and 3, 4-dihydroxyphenylalanine, respectively, after administering l-

tryptophan and m-hydroxybenzylhydrazine, an inhibitor of aromatic-l-amino acid

decarboxylase. 5-Hydroxytryptophan, 3,4-dihydroxyphenylalanine and l-tryptophan were

measured in four forebrain regions, the caudate putamen, nucleus accumbens, nucleus

septi lateralis, and nucleus amygdaloideus centralis, which contain terminals of 5-

hydroxytryptamine and dopamine neurons. Metergoline increased 5-hydroxytryptophan

and 3, 4-dihydroxyphenylalanine accumulation, and decreased l-tryptophan concentration

in a dose- and time-dependent manner. Pirenperone increased 5-hydroxytryptophan and

3, 4-dihydroxyphenylalanine accumulation, but had no effect on l-tryptophan levels.

These effects of pirenperone were time-and dose-related. Finally, Pizotifen increased 5-

hydroxytryptophan accumulation in a dose-related and time-dependent manner, but did

not alter 3, 4-dihydroxyphenylalanine or l-tryptophan concentrations. All the drug effects

generally occurred in all four nuclei. These results suggest that 5-hydroxytryptamine

receptor antagonists may affect synthesis in 5-hydroxytryptamine and/or dopamine

neurons after l-tryptophan treatment and aromatic-l-amino acid decarboxylase inhibition.

Shaik Mukidur rahman et al [10]

reported the following method, the aim of this

study was to develop and validate an isocratic reversed phase high-performance liquid

chromatographic method for quantification of Pizotifen malate in pharmaceutical solid

dosage formulations. Good chromatographic separation of Pizotifen malate was achieved

by using an analytical column, C18 ODS column. The system was operated at 40°C oven

temperature using a mobile phase consisting of acetonitrile and acetate buffer pH 7.0

(60:40) at a flow rate of 2 ml/min. The method showed high sensitivity with good

linearity (r2= 0.99997) over the tested concentration range of 0.0020 - 0.0300 mg/ml for

83

Pizotifen malate. Detection was carried out at 231 nm and retention time was 2.838 min.

Placebo and blank studies were performed and no peak was observed at the retention time

of Pizotifen malate. The intermediate precision and accuracy results (mean ± RSD, n=3)

were (99.11±0.21) % and (99.19±0.55) % respectively with tailing factor (1.26±0.19).

The proposed method was validated in terms of selectivity, linearity, accuracy, precision,

range, detection and quantization limit, system suitability and solution stability. This

method can be successfully employed for simultaneous quantitative analysis of Pizotifen

malate in pharmaceutical solid dosage formulations.

A.S. Sidhu et al emphasized

[11], a reliable and simple method for the routine

analysis of pharmaceutical dosage forms by high-performance liquid chromatography

using a C18 Bondapak reversed-phase column with a binary solvent system consisting of

acetonitrile and 0.05 M potassium dihydrogen phosphate has been developed.

Standardized extraction procedures for drugs in various dosage forms have been

developed and successfully applied to a wide range of current pharmaceutical

formulations.

Zhou Wenxia et al [12]

discribed this method An HPLC method is developed for t

he determination of Pizotifen and for the standardization of Pizotifen reference substance.

The chromatographic system consists of Chromosil C 18 column (250×5 6mm, 5μm) an

d mobile phase of acetonitrile-water-triethylamine (55∶45∶0 2) with flow rate of 1 0ml·mi

n -1 . The detection wavelength is 256nm. The linear range was 6 5-208μg·min -1, A=64

185 74+81992 3C. The method was simple, sensitive and accurate.

M.A.Abounassif et al[13]

proposed the photostability of selected benzocycloheptane

antihistaminic agents, namely, loratadine (I), Pizotifen (II), ketotifen fumarate (III) and c

yproheptatidine (IV), was investigated. Both I and II were photolabile while III and IV w

ere photo stable. To perform stability studies on the photolabile compounds (I and II), spe

cific stability-indicating high performance liquid chromatographic (HPLC) methods were

established. The accuracy, precision and reliability of the developed HPLC methods for t

he assay of I and II in their pharmaceutical dosage forms were reported. Assay results for

both drugs were within R.S.D. values <2%. The stability-indicating power of the develop

ed methods was validated through study of UV-degraded solutions of I and II contained i

n quartz cells. The photostability of both drugs was studied under UV-irradiation at 254 n

84

m. The photo degradation kinetics of both drugs, studied in different solvents, are also re

ported. TLC fractionation of photodegraded solutions of both drugs, revealed two fluores

cent photodegradates of drug I. The use of UV-absorbers (ascorbic acid and p-amino ben

zoic acid (PABA)) enhanced the photostability of both drugs possibly through a spectral-

overlay effect.

J. S. Hong et al [14]

described the purpose of this study was to correlate the

chlordecone-elicited tremor activity with alterations of brain neurotransmitters. A single

injection of chlordecone (80 mg/kg, ip) significantly increased the brain levels of 5-

hydroxyindoleacetic acid (5-HIAA) but did not affect the concentrations of dopamine,

dihydroxphenylacetic acid, aspartate, taurine, glutamate, glycine, and γ-aminobutyric

acid (GABA). There was a dose- and time-related correlation between the increases in

striatal 5-HIAA levels and tremor after chlordecone treatment. A subsequent study with

pargyline indicated that the increase in striatal 5-HIAA level represented an increase in

the turnover of serotonin. This study plus the previous finding that Pizotifen (BC-105), a

serotonin receptor blocker, attenuated chlordecone-elicited tremor strongly suggests a

possible involvement of the serotonin system in mediating the tremor elicited by this

insecticide.

J. W. Gorrod et al [15]

described the formation of iminium ions from the

condensation of chiral secondary or primary amines with a, b-unsaturated aldehydes or

ketones can be used as an effective platform for the acceleration of a wide variety of

catalytic asymmetric cycloaddition and conjugate addition reactions. The reversible

formation of the active iminium ion species simulates the π-electronics and equilibrium

dynamics traditionally associated with Lewis acid activation of a,b-unsaturated carbonyl

compounds lowering the energy level of the LUMO associated with the π-system and

activating subsequent reaction. Importantly, these iminium ion catalyzed processes offer

the opportunity to conduct reactions in the presence of both moisture and air greatly

adding to the practicality and general applicability of the chemistry described. Proposed

catalytic cycles and transition state models for the induction of asymmetry provide

reliable and robust predictive tools for the outcome of reactions and high functional

group tolerance suggests this class of transformation will have broad application in the

arena of synthetic organic chemistry as the area matures. This review describes the rapid

85

expansion of iminium ion catalysis over recent years from its conceptual introduction to

the development of a whole new arsenal of highly practical and effective methods with

which to approach challenging and fundamental bond construction processes.

86

3.3. EXPERIMENTAL

3.3.1. Instrumentation

Peak HPLC containing LC 20 AT pump, variable wavelength programmable

uv/vis detector and Rheodyne injector was employed for the investigation. The

chromatographic analysis was performed on a Chromosil C18 column (250 mm x 4.6

mm, 5μm) column. Degassing of the mobile phase was done by using a Loba ultrasonic

bath sonicator. A Denwar balance was used for weighing the materials.

3.3.2. Chemicals and Solvents

The reference sample of Pizotifen (API) was obtained from V.V MED,

Hyderabad. The Formulation was procured from the local market. Acetonitrile, Methanol,

ammonium Di hydrogen phosphate, Tri ethyl amine and orthophosphoric acid used were

of HPLC grade and purchased from Merck Specialties’ Private Limited, Mumbai, India.

3.3.3. The buffer solution

About 1.0 mL of orthophosphoric acid was diluted to 1000 mL with water. This

solution was mixed and pH was adjusted to 5.8 with ortho phosphoric acid and filtered

through 0.45μ nylon filter.

3.3.4. The mobile phase

A mixture of Methanol: Acetonitrile in the ratio of 10:90 v/v was prepared and

used as mobile phase.

3.3.5. Standard solution of the drug

For analysis we 100 ppm standard solution was prepared in mobile phase.

Required concentrations were obtained from 100 ppm standard solution by proper

dilution.

87

3.3.6. Sample (tablet) solution

The formulation tablets of Pizotifin (Migralin - 0.725mg) were crushed to give

finely powdered material. With Powder we prepared 70 ppm solution in mobile phase

and then filtered through Ultipor N66 Nylon 6, 6 membrane sample filter paper.

88

3.4. METHOD DEVELOPMENT

For developing the method (as described in chapter 1 and 2), a systematic study of

the effect of various factors was undertaken by varying one parameter at a time and

keeping all other conditions constant. Method development consists of selecting the

appropriate wave length and choice of stationary and mobile phases. The following

studies were conducted for this purpose.

3.4.1. Detection wavelength

The spectrum of diluted solutions of the Pizotifen in mobile phase was recorded

separately on UV spectrophotometer. The peak of maximum absorbance wavelength was

observed. The spectra of the both Pizotifen were showed that a wavelength was found to

be 230 nm.

3.4.2. Choice of stationary phase

Preliminary development trials have performed with octadecyl columns with

different types, configurations and from different manufacturers. Finally the expected

separation and shapes of peak was succeeded Chromosil C18 column.

3.4.3. Selection of the mobile phase

In order to get sharp peak, low tailing factor and base line separation of the

components, we carried out a number of experiments by varying the composition of

various solvents and its flow rate. To effect ideal separation of the drug under isocratic

conditions, mixtures of solvents like methanol, water and Acetonitrile with or without

different buffers indifferent combinations were tested as mobile phases on a C18

stationary phase. A mixture of Methanol: Acetonitrile in the ratio of 10:90 v/v was

proved to be the most suitable of all the combinations since the chromatographic peak

obtained was better defined and resolved and almost free from tailing.

3.4.4. Flow rate

Flow rates of the mobile phase were changed from 0.6 – 1.6 mL/min for optimum

separation. A minimum flow rate as well as minimum run time gives the maximum

89

saving on the usage of solvents. It was found from the experiments that 1.0 mL/min flow

rate was ideal for the successful elution of the analyte.

3.4.5. Optimized chromatographic conditions

Chromatographic conditions as optimized above were shown in Table 3.2 These

optimized conditions were followed for the determination of Pizotifen in bulk samples

and its combined tablet Formulations. The chromatograms of standard and sample were

shown in Figure 3.B

Mobile phase Methanol: Acetonitrile 10:90 v/v

Pump mode Isocratic

Mobile phase PH 5.8

Diluent The mobile phase

Column Chromosil C18 column (250 mm x

4.6 mm, 5μ)

Column Temp Ambient

Wavelength 230 nm

Injection Volume 20 μl

Flow rate 1.0 mL/min

Run time 6 min

Retention Time 2.019 min

Table 3.2: Optimized chromatographic conditions for estimation PIZOTIFEN

90

Figure 3.B: RP - HPLC Chromatogram of standard solution

91

3.5. VALIDATION OF THE PROPOSED METHOD

The proposed method was validated as per ICH guidelines. As described in

chapter 1 and 2 the parameters studied for validation were specificity, linearity, precision,

accuracy, robustness, system suitability, limit of detection, limit of quantification, and

solution stability.

3.5.1. Specificity

The specificity of method was performed by comparing the chromatograms of

blank, standard and sample. It was found that there is no interference due to excipients in

the tablet formulation and also found good correlation between the retention times of

standard and sample. The specificity results are shown in Table 3.3.

NAME OF THE SOLUTION Retention Time in Minutes

BLANK NO PEAKS

PIZOTIFEN 2.01

Table 3.3: Specificity study

3.5.2 Linearity

Linearity was performed by preparing mixed standard solutions of Pizotifen at

different concentration levels including working concentration mentioned in experimental

condition i.e. 25 ppm. Twenty micro liters of each concentration was injected in duplicate

into the HPLC system. The response was read at 230 nm and the corresponding

chromatograms were recorded. From these chromatograms, the mean peak areas were

calculated and linearity plots of concentration over the mean peak areas were constructed

individually. The regressions of the plots were computed by least square regression

method. Linearity results were presented in Table 3.4.

92

Table 3.4: Linearity results of Pizotifen

Figure 3.C: On X axis concentration of sample solution, On Y axis peak area response

-50000

0

50000

100000

150000

200000

0 5 10 15 20 25 30 35

A

R

E

A

Conc (ppm)

Level Concentration of PIZOTIFEN In ppm Mean peak area

Level -1 5.0 30333.1

Level -2 10.0 61990.7

Level -3 15.0 88759.2

Level -4 20.0 121043.1

Level -5 25.0 148510.7

Level -6 30.0 186164.2

Slope

Intercept

Correlation coefficient

6119.996

966.4

0.998

93

3.5.3. Precision

Precision is the degree of repeatability of an analytical method under normal

Operational conditions. Precision of the method was performed as intraday precision,

Inter day precision.

3.5.3.1. Intraday precision

To study the intraday precision, six replicate standard solution of Pizotifen was

injected. The percent relative standard deviation (% RSD) was calculated and it was

found to be 1.4, which are well within the acceptable criteria of not more than 2.0.

Results of system precision studies are shown in Table 3.5.

SAMPLE

CONC(PPM) INJECTION No. PEAKS

AREA

R.S.D

(Acceptance

criteria 2.0)

Pizotifen

25 ppm

1 152526.6

1.40 2 151615.4

3 152127.8

4 153481.7

5 150705.3

6 156830.7

Table 3.5: System Precision

3.5.3.2. Inter Day precision

To study the inter day precision, six replicate standard solution of PIZOTIFEN

was injected on third day of sample preparation. The percent relative standard deviation

(% RSD) was calculated and it was found to be 1.07, which are well within the

acceptable criteria of not more than 2.0. Results of system precision studies are shown in

Table 3.6.

94

SAMPLE

CONC(PPM) INJECTION No. PEAKS

AREA

R.S.D

(Acceptance

criteria 2.0)

Pizotifen

25

1 146180.5

1.07

2 142312.7

3 143153.8

4 145268.1

5 143858.6

6 145253.9

Table 3.6: (Inter day)

3.5.4. Accuracy

The accuracy of the method was determined by standard addition method. A

known amount of standard drug was added to the fixed amount of pre-analyzed tablet

solution. Percent recovery was calculated by comparing the area before and after the

addition of the standard drug. The standard addition method was performed at 25%, 50%

and 75% level. The solutions were analyzed in triplicate at each level as per the proposed

method. The percent recovery and % RSD was calculated and results are presented in

Table 3.7. Satisfactory recoveries ranging from 99.93 to 100.4 were obtained by the

proposed method. This indicates that the proposed method was accurate.

LEVEL

Amount of

PIZOTIFEN

spiked

(ppm)

Amount of

PIZOTIFEN

recovered

(ppm)

%

Recovery

MEAN

%

Recovery

%R.S.D MEAN

%

R.S.D

25 %

5 4.97 99.4

0.53

5 5.02 100.4

5 4.98 99.6

50%

10 10.01 100.1

0.15

10 9.99 99.9

10 9.98 99.8

75%

15 14.99 99.93

0.101

15 15.02 100.13

15 15.01 100.06

99.92994 0.2603

Table 3.7: Percentage recovery and %RSD

95

3.5.5. Robustness

The robustness study was performed by slight modification in flow rate of the

mobile phase, pH of the buffer and composition of the mobile phase. Pizotifen at 6 ppm

concentration was analyzed under these changed experimental conditions. It was

observed that there were no marked changes in chromatograms, which demonstrated that

the developed method was robust in nature. The results of robustness study are shown in

Table 3.8.

Condition Mean area % assay % difference

Unaltered 144805.5 100.0 0.0

Flow rate at 0.8 mL/min

Flow rate at 1.2mL/min

144720.3

143951.2

99.94

99.41

0.06

0.59

Mobile phase:

ACN: Methanol

92 % 08%

88% 12%

144902.5

144763.4

100.06

99.97

0.06

0.03

pH of mobile phase at 6.0 144275.2 99.63 0.37

pH of mobile phase at 5.6 144358.7 99.69 0.31

Table 3.8: Robustness

3.5.6. System suitability

System suitability was studied under each validation parameters by injecting six

replicates of the standard solution. The system suitability parameters are given in

Table 3.9.

96

Parameter Tailing factor Theoretical plates

Specificity study 1.34 4964.61

Linearity study 1.17 4590.18

Precision study 1.48 4875.34

Table 3.9: System Suitability

3.5.7. Limit of detection and Limit of quantification

Limit of detection (LOD) is defined as the lowest concentration of analyte that

gives a detectable response. Limit of quantification (LOQ) is defined as the lowest

Concentration that can be quantified reliably with a specified level of accuracy and

Precision. For this study six replicates of the analyte at lowest concentration were

Measured and quantified. The LOD and LOQ of Pizotifen are given in Table 3.10.

Parameter Measured volume

Limit of Quantification 65 ng/ml

Limit of Detection 20 ng/ml

Table 3.10: LOQ and LOD

97

3.6. RESULTS AND DISCUSSION

Proper selection of the stationary phase depends up on the nature of the sample,

molecular weight and solubility. Mixture of orthophosphosphoric acid, acetonitrile and

methanol was selected as mobile phase and the effect of composition of mobile phase on

the retention time of Pizotifen was thoroughly investigated. The concentration of the

orthophosphoric acid, acetonitrile and methanol were optimized to give symmetric peak

with short run time. A system suitability test was applied to representative

chromatograms for various parameters. The results obtained were within acceptable

limits and are represented in Table 3.2. Thus, the system meets suitable criteria.

Ten points graphs was constructed covering a concentration range 5-30 ppm

(Three independent determinations were performed at each concentration). Linear

relationships between the peak area signal of Pizotifen the corresponding drug

concentration was observed. The standard deviation of the slope and intercept were low.

The calibration curve was obtained for a series of concentration in the range of 05-30

ppm and it was found to be linear. The data of regression analysis of the calibration

curves are shown in Table 3.4.

The validated method was applied for the assay of commercial tablets containing

Pizotifen. Sample was analyzed for five times after extracting the drug as mentioned in

assay sample preparation of the experimental section. The results presented good

agreement with the labeled content. Low values of standard deviation denoted very good

repeatability of the measurement. Thus it was showing that the equipment used for the

study was correctly and hence the developed analytical method is highly repetitive. For

the intermediate precision a study carried out by the same author working on the same

day on two consecutive days indicated a RSD of 1.235. This indicates good method

precision.

The stability of Pizotifen in standard and sample solutions containing determined

by storing the solutions at ambient temperature (20±100C). The solutions were checked in

triplicate after three successive days of storage and the data were compared with freshly

prepared samples. In each case, it could be noticed that solutions were stable for 48 hrs,

98

as during this time the results did not decrease below 98%. This denotes that Pizotifen is

stable and standard and sample solutions for at least 2 days at ambient temperature.

The system suitability parameter like capacity factor, asymmetry factor, tailing

factor and number of theoretical plates were also calculated. It was observed that all the

values are within the limits. The statistical evaluation of the proposed method was

revealed its good linearity, reproducibility and its validation for different parameters and

let us to the conclusion that it could be used for the rapid and reliable determination of

Pizotifen in tablet formulation.

A validated RP-HPLC method has been developed for the determination of

Pizotifen in tablet dosage form. The proposed method is simple, rapid, accurate, precise

and specific. Its chromatographic run time of 6 min allows the analysis of a large number

of samples in short period of time. Therefore, it is suitable for the routine analysis of

Pizotifen in pharmaceutical dosage form.

99

3.7. BIBILOGRAPHY

1. Stark RJ, Valenti L, Miller GC; “Management of migraine in Australian general

practice”; Medical Journal of Australia; 2007; 187(3): 142-6.

2. Barnes N, Millman G. Do; “Pizotifen or propranolol reduce the frequency of

migraine headache Archives of Disease in Childhood”; 2004; 89(7): 684-5.

3. Pierangeli G, Cevoli S, Sancisi E, Grimaldi D, Zanigni S, Montagna P, Cortelli P;

“Which therapy for which patient?”; Neurological Sciences; 2006; Suppl 2:

S153-8.

4. Standal JE; “Pizotifen as an antidepressant”; Acta Psychiatrica Scandinavica;

1977; 56(4): 276-9.

5. Banki CM; “Clinical observations with Pizotifene (Sandomigran) in the treatment

of nonmigrainous depressed women”; Archive fur Psychiatrie and

Nervenkrankheiten: 1978; 225(1): 67-72.

6. Young R, Khorana N, Bondareva T, Glennon RA; “Pizotyline effectively

attenuates the stimulus effects of N-methyl-3, 4-methylenedioxyamphetamine

(MDMA)”; Pharmacology, Biochemistry and Behavior; 2005; 82(2): 404-10.

7. Crowder D, Maclay WP; “Pizotifen once daily in the prophylaxis of migraine:

results of a multi-centre general practice study”; Current Medical Research and

Opinion; 1984; 9(4): 280-5.

8. Dixon AK, Hill RC, Roemer D, Scholtysik G; “Pharmacological properties of 4-

(1-methyl-4-piperidylidine)-9,10-dihydro-4H-benzo-[4,5]cyclohepta[1,2]-

thiophene hydrogen maleate (Pizotifen)”; Arzneimittelforschung; 1977;

27(10):1968-79.

9. Neurochemistry International; 1986; 9(3): 423-429.

10. Shaikh M. Rahman, Abul kalam lutful kabir md. et al; “Validation and

application of reversed phase high-performance liquidchromatographic method

for quantification of Pizotifen malage in pharmaceutical solid dosage

formulations”; Pak J.Pharm.Sci; 2010; 23(4); 435-41.

11. S. Sidhu, J.M. Kennedy and S. Deeble; “General method for the analysis of

pharmaceutical dosage forms by high-performance liquid chromatographya”;

Journal of Chromatography; 1987; 391: 233-242.

100

12. Determination of Pizptifen by HPLC,zhou wenxiao,wang jucai 1 and zhang

jiange 2(Guangxi institute for drug, control, http://en.cnki.com.cn/article_en/

cjfdtotal-zgys200307023.htm.

13. “Stability studies on some benzocycloheptane antihistaminic agents”; Journal of

Pharmaceutical and Biomedical Analysis; 2005; 36(5): 1011-1018.

14. “Toxicology and applied pharmacology effects of chlordecone exposure on brain

neurotransmitters: possible involvement of the serotonin system in chlordecone”,

1984; 73(2): 336-344.

15. J. W. Gorrod and g. Aislaitner; “The metabolism of alicyclic amines to reactive

iminium ion intermediates”; European Journal of Drug Metabolism and

Pharmacokinetics; 1994; 19(3): 209-17.