genetically engineered insulin and its pharmaceutical analogues

ISSN 1990-7508, Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 2008, Vol. 2, No. 4, pp. 356–366. © Pleiades Publishing, Ltd., 2008.Original Russian Text © D.A. Gusarov, V.D. Gusarova, D.I. Bayramashvili, A.F. Mironov, 2008, published in Biomeditsinskaya Khimiya.

356

INTRODUCTION

Insulin is one of the best studied hormones at themoment. Although more than 80 years passed since thediscovery of the fact that insulin produced by pancreasis responsible for the decrease of blood sugar [1] thishormone still attracts much attention of a scientificcommunity. Mortality of diabetes mellitus is on thethird place after mortality of cardiovascular and tumordiseases [2, 3]. In 2000 there were about 150 millionpatients with diabetes mellitus in the world [4] and eachyear their number roughly increases by 2–6% [5, 6].For example, according to prognosis of specialistsnumber of diabetic patients in European countriesincreases up to 33 millions by 2010 [7]; in Russiaalmost 400000 patients need regular administration ofinsulin preparations.

Although all people differ by their habits blood glu-cose is normally maintained within rather narrow rangeand undergoes rather small diurnal variations. Normalglucose concentration in human blood varies from 3 upto 8 mM [5]. Food intake increases blood sugar within15–30 min after meal (Fig. 1) [8]. Diurnal variation ofinsulin content is similar to that of blood glucose (Fig. 2)[8]. Increase in blood insulin concentration representsresponse of an organism to increased glucose level;insulin decreases blood glucose up to the basal levelwithin 2 h after meal [9].

In diabetes mellitus blood glucose undergoes almosta 3–4-fold increase, which is associated with eitherinsufficient insulin production (diabetes mellitus type1) or organism resistance to insulin action (diabetesmellitus type 2). In the case of diabetes mellitus typeone the major goal of replacement therapy consists in

maintenance of basal level of blood glucose and apatient needs constant injections of insulin over thewhole life.

The insulin therapy may be based on classic drugformulations, which usually include so called regularinsulin (soluble rapidly acting insulin) or protaminezinc insulin suspension (long acting insulin NPH, neu-tral protamine Hagedorn) as well as gene engineeredanalogues of insulin, recently appeared in the pharma-ceutical market. The goal of the present review was tosummarize data of application of both traditional prep-

Genetically Engineered Insulin and Its Pharmaceutical Analogues

D. A. Gusarov

a, b

*, V. D. Gusarova

a, b

, D. I. Bayramashvili

a

, and A. F. Mironov

b

a

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Mikloukho-Maklaya 16/10, Moscow, 117997 Russia

b

Lomonosov Moscow State Academy of Fine Chemical Technology, pr. Vernadskogo 86, Moscow, 119571 Russia; tel.: (495) 429-87-40; e-mail: [email protected]

Received August 29, 2007

Abstract

—Studies of replacement therapy of diabetes mellitus resulted not only in introduction of series offorms of insulin available at pharmaceutical market but also in new insulin analogues, which exhibit better con-trol of blood glucose level. The present paper deals with basic tendencies in this field.

Key words

: genetically engineered human insulin, diabetes mellitus, insulin-aspart, insulin-glargin, insulin-detemir, insulin-lyspro.

DOI:

10.1134/S1990750808040057

REVIEWS

*To whom correspondence should be addressed.

6:00 18:0014:0010:00 22:00 2:00 6:00

Blo

od g

luco

se c

once

ntra

tion,

mg/

ml

Bre

akfa

st

Din

ner

Supp

er

Diabetes mellitus

Norm

Time of day, hour:min

0

1000

2000

3000

4000

Fig. 1.

Diurnal changes in blood glucose of a healthy personand a patient with diabetes mellitus (modified from [8, 9]).

BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY

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GENETICALLY ENGINEERED INSULIN AND ITS PHARMACEUTICAL ANALOGUES 357

arations of human insulin and its pharmaceutical ana-logues in endocrinology.

1. STRUCTURE AND BIOSYNTHESIS OF INSULIN

Insulin is a polypeptide hormone, produced by pan-creatic islet cells. Protein nature of insulin (Fig. 3) wasknown long before elucidation of its primary structurein 1955 [10] (as well as the fact that insulin containsthree disulfide bonds formed by cysteine residues[11

−

14]).

Physiological effects of insulin on glucose contentand lipid metabolism is known for a long time [15]. Itwas found [16] that a single chain polypeptide, proinsu-lin, is a precursor of the two-chain insulin molecule(Fig. 4). Metabolism of proinsulin resulting in forma-tion of a biologically active product of lower molecularmass occurs in

β

-cells of the pancreatic islets ofLangerhans [17–20]. Subsequently it was found that

preproinsulin rather than proinsulin is the insulin pre-cursor; the former differs from proinsulin by the pres-ence of a N-terminal leader fragment [21–23]. It is sug-gested that this leader fragment of 23–24 amino acidresidues is a signal sequence required for proinsulintransfer from polyribosomes to Golgi complex. Aftercleavage of the leader fragment proinsulin moleculeadopts conformation required for correct formation ofdisulfide bonds. Proinsulin cleaved then into insulinand C-peptide is stored in secretory granules [24–26].Subsequently the content of these granules is secretedinto portal blood circulation [24, 27].

In solutions insulin molecules exist as equilibriummixture of monomeric, dimeric, tetrameric and hexam-eric forms [28]. At low concentrations (comparablewith blood concentrations) the monomeric form pre-dominates; this is biologically active form because onlythis form may interact with an insulin receptor [29]. Athigher concentration in both acidic or alkaline media(pH of 8.0 and above) this hormone may exhibit self-association into dimers (in the absence of zinc ions),whereas in neutral or weakly alkaline medium it formshexamers in the presence of zinc ions (Fig. 5). Afterbiosynthesis (and subsequent processing) insulin isaccumulated in the body (in vesicles of islet cells) ascrystal zinc-bound insulin, which is then released inresponse to the increase of blood glucose concentration[30]. Although it is well known that the hexamer existsin three conformations (R6, T3R3, and T6), whichdepend on monomer conformation it still remains unclearwhich conformation is realized during insulin accumula-tion in cells of the islets of Langerhans [31–34].

Insulin molecules may be crystallized at variousconformations [35]. The first crystal structure of insulinwas obtained in 1969; it represented a hexamer, inwhich three dimers were joined together around twozinc ions [36, 37]. Subsequently hexameric structurescontaining even four zinc atoms were also obtained[38]. In the hexamer containing two zinc ions theregions B1–B8 and B20–B30 of the monomeric units

4:00 8:00 12:00 16:00 20:00 24:00 4:00 8:00Time of day, hour:min

0

25

50

Plas

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insu

lin c

once

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tion,

µ

U/m

l

Bre

akfa

st

Din

ner

Supp

erBasal insulin level

Fig. 2.

Physiological profile of insulin in a healthy person(modified from [8, 9]).

GlyIle

Val Glu Gln Cys Cys

Cys

Cys

Cys Cys

ThrSer

Ile

Ser Leu TyrGln

LeuGlu

Asn Tyr Asn

COOH

H

2

N

PheVal

AsnGln His Leu

Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val

GlyGlu

Arg

Gly

Phe

Phe

Tyr

Thr

Pro

Lys

Thr

HOOC

H

2

N

1

6

11

7

20 21

30

197

1

A

-chain

B

-chain

Fig. 3.

Primary structure of human insulin.

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are repulsed by the

α

-helical site B9–19 [39]. This con-formational state is defined as the tense state (T-state).Metal ions coordinate B10 histidine residues in all threedimers. In each dimer A and B chains form a compactmolecule including two A chain

α

-helices and one B-chain

α

-helix (T6 state).

In the conformation containing four zinc ions threeN-termini of B-chains (B1–B8 residues) are associatedinto

α

-helix in the presence of high concentrations ofchloride ion. Such conformation is known as R6(relaxed) [40]. Transition of T6 into R6 is possible dur-ing binding of such ligands as phenol, cresol, methylpa-raben, resorcine, etc. [29]. Such binding involves thesites of insulin molecule known as hydrophobic pock-ets. In T3R6 (asymmetric structure in which 4 ions arecoordinated by B10 histidines) there are three suchpockets, whereas in R6 there are 6 pockets. It is knownthat phenol and its derivatives may be used in the insu-lin dosage as either antibacterial preservatives [41] oradditives preventing deamidation reactions [42].Recently it has been demonstrated that phenolic com-pound stabilize T3R3 and R6 hexamers and thusincrease resistance of insulin molecule to thermal dena-turation and polymerization [29].

2. INSULIN DOSAGE PREPARATIONS

Production of human insulin was started in 1979(using the transamination reaction for substitution of C-terminal alanine residue in pig insulin for threonine res-idue) [43–46].

Simultaneously, using the development of methodsof gene engineering recombinant human insulin wasproduced by means of

E. coli

[47–54] and (since 1987)by yeast cultures

Saccharomyces cerevisiae

,

Pichiapastories

[55–57]. Increasing standards requirementsfor insulin preparations stimulated constant improve-ments of methods of insulin production over the lasttwenty years [6, 58, 59]. The methodology for produc-tion of APS insulin is well studied and reviewed in theliterature. However, it should be noted that various drugpreparations differed by duration of hypoglycemiceffect may be obtained from the final substance(including the most popular short acting and long act-ing insulins).

Besides traditionally injection dosage forms of insu-lin the newest developments include an inhalation formas well as actively developed peroral, transdermal andother preparations.

As it has been mentioned above for maintenance ofnormal level of blood glucose diabetic patients needrepeated injections of insulin preparations. Modernclassic therapy of diabetes mellitus is mainly based onapplication of soluble insulin and a suspension of prot-amine zinc insulin (NPH insulin).

The soluble drug dosage form of insulin exhibitsrapid and short lasting hypoglycemic effect. Such prep-arations begin to act within 30–40 min after injection,maximum of their action is observed after 2–4 h and thehypoglycemic effect lasts for about 8 h [60]. The mostknown short acting insulins are: Humulin Regular (EliLilly, USA), Actrapid HM (NovoNordisk, Denmark),Insuman Rapid (Sanofi-Aventis, France-Germany). In

Lys

AsnCysTyrAsnGlu

LeuGln

TyrLeuSer

Cys

Ile

SerThrCysCysGlnGluVal

IleGly

Arg

Lys

Gln

Leu

Ser

Gly

Gln

Leu

Ala

LeuPro

GlnLeu

Ser Gly Ala Gly Pro Gly Gly Gly Leu Glu ValGln

Gly Val GlnLeu

AspGlu

Ala

Glu

Arg

Arg

Thr

Pro

Thr

Tyr

Phe

Phe

Gly

Arg

GluGly

CysValLeuTyrLeuAlaGluValLeuHisSerGlyCysLeuHis

GlnAsn

ValPhe

COOHH

2

N

A

-chain

B

-chain

1

6

7

11

19

20 21

30

7

1

Fig. 4.

Schematic presentation of human proinsulin (white and grey colors show amino acid residues corresponding to insulin andC-peptide, respectively).


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Russia the following insulins are produced: Insuran R(Institute of Bioorganic Chemistry, RAS), Biosulin R(Pharmstandard-Ufavita), Rinsulin R (National Bio-technology).

Among long-acting insulins NPH insulins are themost popular in the world: their hypoglycemic effect ismore predictable and they are easily mixed with short-acting insulins [61]. Zinc-insulins are of rarer use: pro-longation of their effect is achieved by the presence ofvarious zinc crystals. Such insulins are usually pre-scribed as subcutaneous injections; this is determinedby necessity of formation of drug “store”, which pro-longs hypoglycemic effect. The long-acting insulinsbegin to act only 2–4 h after administration, peak oftheir effect is observed within 4–10 h and usually dura-tion of their effect does not exceed 12–18 h [60]. TheNPH dosage insulins are usually formed by co-precip-itation of insulin with protamine peptides at neutralpH values in the presence of zinc ions, phenol, and/orphenol derivatives [28]. This results in formation of astable complex, which includes negatively chargedinsulin molecules and polycationic molecules of prot-amine peptides. This complex stabilized by zinc ionsand aromatic phenol compounds preferentially con-sists of tetragonal crystals [62]. In NPH insulins insu-lin and protamine sulfate are linked at an isophaneratio (i.e. in the absence of excess of one of compo-nents) [62].

Protamines are commercial name of strongly basicpeptides found in sturgeon milt [63]. Production ofNPH insulins usually employs protamine sulfateobtained from salmon milt [64]. Prolongation of insulineffect is determined by slow degradation of the prota-mine complex by fibrinolytic enzymes [28].

The most known insulins of this class include:Humulin NPH (Eli Lilly), Protaphan HM (NovoNord-isk), Insuman Basal (Sanofi-Aventis, France-Ger-many). In Russia the following insulins of this class areproduced: Insuran NPH (Institute of Bioorganic Chem-istry, RAS), Biosulin H (Pharmstandard-Ufavita), Rin-sulin H (National Biotechnology).

Now special attention is also paid not only to theinjection dosage forms of insulin, but also to peroral,intranasal and other drug dosage forms, which wouldimprove life of diabetic patients.

The peroral drug dosage form of insulin is the mostconvenient form for the treatment of diabetes mellitus,if insulin molecule would not be degraded in gas-trointestinal tract by proteolytic enzymes [65]. Seriousattempts are now undertaken to solve this problem. Forexample, some nonacylated amino acids have beeninvestigated as insulin carriers, which form a noncova-lent complex inducing conformational changes in thisprotein (conformation unfolding); this facilitates pas-sive diffusion of insulin molecules through lipid bilay-ers before these molecules dissociate passing throughgut cell membranes [66]. There are peroral liposomal

capsules for peroral insulin delivery [67]. Carboxyme-thylcellulose conjugates of insulin have also beentested; they can be absorbed by gut epithelium withoutdenaturation of hormone molecules [68]. At themoment the most promising is hexyl-insulin-monocon-jugate-2 (HIM2) representing human recombinant insu-lin containing polyethylene glycol-7-hexyl attached tolysine B29 [69, 70]. HIM2 may provide insulin absorp-tion by epithelial cells without degradation of insulinmolecule. Moreover this peroral preparation is able toreproduce physiological pathway of insulin, which issecreted in human body by pancreas and reaches liver(via portal system) before it enters general blood circu-lation [71].

Comparing injection and peroral insulin dosageforms one can come to the following conclusions. Ofcourse repeated daily injection of insulin are inconve-

Monomer

Dimer

Hexamer T6

Fig. 5.

Schematic presentation of possible tertiary structuresof human insulin (PDB Protein Database), (modified from[35]).

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nient for patients and seriously complicate their lives.Peroral forms of insulin lack such shortcomings. How-ever, costs of the peroral therapy may significantlyexceed costs of the injection therapy. The peroral ther-apy is based on a spray delivery system, which consistsof insulin preparation itself and a device, which createsaerosol particles of certain and the same size andadministered aerosol into oral cavity at the rate160 kg/h [72, 73]. Detailed study of the peroral formshas shown that such system is especially convenient forpatients after low food intake; this corresponds to 6–8 daily doses [72].

Intranasal application of insulin is the other poten-tial route to avoid inconvenience associated with insu-lin injections. Nasal mucosa provides rather largeabsorption and has large number of blood capillaries[65]. Bioavailability of insulin administered intohuman organism during its intranasal delivery may beincreased by means of various additions (bile acid salts,1–4% sodium glycocholate and sodium deoxycholate),surfactants (0.8% lauret-9), and also phospholipids (2%didecanoyl phosphatidylcholine) [65, 74]. Spray anddrops dosage forms of insulin have been proposed forintranasal delivery.

Special attention is paid to so-called lung insulincrystals (a powder system for inhalation from 3 to 9 Uof insulin). Although inhalation of insulin crystals wasproposed in 1925 [75] only 46 years later it becamepossible to decrease blood glucose in a diabetic patientby means of the lung crystals of mixed pig and cattleinsulins [76]. This way of delivery has several advan-tages: use of pulmonary crystals is painless; pulmoneshave huge absorption surface and numerous blood ves-sels; lungs lack any peptidases which could destroypolypeptide molecules [65, 77]. Thus, insulin mole-cules may be easily absorbed, penetrate through thinalveolar capillary barriers into blood circulation andcause requested hypoglycemic effect. Nevertheless,prescription of lung crystals has several shortcomingsas well. First, such way of insulin delivery is doubtfulin diabetic patients suffering with asthma, chronicbronchitis; its employment is also complicated in

smokers (if diabetic patients refuse to stop smoking)[65]. Second, it has been shown that lung crystalscause more frequent antibody production against for-eign (for patient’s organism) insulin than the injectioninsulin dosage forms [78].

Peroral and intranasal ways of insulin delivery tohuman organism as well as lung crystals may be usedfor the decrease of blood glucose after mean. However,it is also important to reduce glucose up to basal level.Besides subcutaneous injections of NPH insulin theother noninvasive pathways of insulin delivery havebeen proposed for achievement of requested hypogly-cemic effect. These include methods of transdermaldelivery based on increased permeability of skin sur-face for macromolecules by sonication [79] or due toinsulin incorporation into lipid transfersomes [80].However, information accumulated in the modern liter-ature is still insufficient for proper evaluation of trans-dermal insulin delivery.

Although such modes of insulin delivery are poten-tially perspective they are still carefully investigatedmainly in experiments.

3. PHARMACEUTICAL INSULIN ANALOGUES

There is evidence that intensive insulin therapy ofdiabetic patients is associated with increased risk of thedevelopment of hypoglycemic shock in both day andnight time [81, 82].

This is mainly associated with the fact that pharma-codynamic and pharmacokinetic properties of tradi-tional insulin preparations non-ideally mimic proper-ties of this hormone produced in the organisms ofhealthy individuals [83, 84]. For example, absorption ofshort-acting insulin may be decreased due to self-asso-ciation of insulin molecule into dimers, tetramers, andhexamers [85, 86]. On the contrary, the effect of long-acting preparations of insulin (e.g. NPH insulin) maybe too short to exhibit necessary glycemic control over-night [83, 87]. It should be also noted, that long-actingpreparations of insulin are suspensions and insufficientmixing of such preparation by patient before adminis-tration cannot be ruled out [88, 89]. For solution of suchproblems several analogues of human insulin have beendeveloped; some of them (e. g. Insulin Aspart and Insu-lin Lispro) are analogues of short-acting insulin,whereas some others (insulin Glargin and insulinDetemir) exhibit properties of long-acting insulins(Tables 1, 2).

It was earlier demonstrated that application of injec-tion dosage of insulins with special amino acid substi-tutions exhibited hypoglycemic profiling which wasmore close to the basal one. For example, it wasreported about accelerated absorption of injected prep-aration of insulin with artificial amino acid substitu-tions at B9–B12 and B26–B28 [90]. Self-association ofinsulin molecules was suppressed in the same way.

Table 1.

Comparison of physiological properties of prepara-tions of insulin and its analogues

Insulinpreparation

Onsetof effect, h

Maximum effect, h

Durationof effect, h

Insulin R 0.5–1 2–4 5–8

Lispro 0.1–0.25 1–2 4–5

Aspart 0.1–0.25 1–2 4–5

Insulin NPH 2–4 4–10 12–18

Glargin 1–2 4–5, Poorly manifested

20–24

Detemir 1–2 6–8 10–18


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However, first insulin derivatives of extra rapid actionexhibited low biological activity [91]. Studies of theeffects of amino acid substitutions on interaction ofinsulin molecules with receptors revealed the mostessential contribution of substitutions at B22–B29 and

the increase of activity of insulin analogue would beachieved in the case of correct change of amino acidresidues within this particular region of the sequence[92, 93]. It was also found that substitutions of pheny-lalanine residues at B24 or B25 cause the most dramatic

Table 2.

Amino acid sequence of A and B chains of insulin and its analogues

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

InsulinA chain G J V E Q C C T S J C S L Y Q L E

GlarginA chain G J V E Q C C T S J C S L Y Q L E

InsulinB chain F V N Q H L C G S H L V E A L Y L

GlarginB chain F V N Q H L C G S H L V E A L Y L

LisproB chain F V N Q H L C G S H L V E A L Y L

AspartB chain F V N Q H L C G S H L V E A L Y L

GlulisineB chain F V

K

Q H L C G S H L V E A L Y L

DetemirB chain F V N Q H L C G S H L V E A L Y L

18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

InsulinA chain N Y C N OH

GlarginA chain N Y C

G

OH

InsulinB chain V C G E R G F F Y T P K T OH

GlarginB chain V C G E R G F F Y T P K T

R R

OH

LisproB chain V C G E R G F F Y T

K P

T OH

AspartB chain V C G E R G F F Y T

D

K T OH

GlulisineB chain V C G E R G F F Y T P

E

T OH

DetemirB chain V C G E R G F F Y T P

K

a

OH

Table 3.

Rapidly acting analogues of human insulin

Clinician name Modification in insulin molecule Properties of preparation

“AspB10” B10 Histidine was substituted for Asp Increased affinity for insulin receptor, rapid absorption

Lispro B28 Pro and B29 Lys were interchanged Reduced self-association ability, rapid absorption, short acting effect

Aspart B28 Pro was substituted for Asp Reduced self-association ability

Glulisine B3 Asn was substituted for Lys, B29 Lyswas substituted for Glu

Extra rapid absorption, short-acting effect

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decrease in biological activity [90]. Table 3 lists themost known insulin analogues of extra rapid action.

Insulin known as AspB10 was the first rapidly act-ing analogue of insulin [9]. It has already been men-tioned that in human insulin histidine residue at B10 isinvolved into formation and stabilization of hexamerdue to binding with zinc ion. Thus, substitution of thisamino acid residue for Asp would result in increasedabsorption ability of such modified insulin. Indeed, itwas demonstrated that Asp10 absorption was two timesfaster than absorption of native insulin [94]. Althoughthis analogue demonstrated potential advantages com-pared with commonly used insulin its clinical applica-tion became impossible due to its carcinogenic proper-ties in patients [95].

Insulin Lispro became the first successful insulinanalogue of extra rapid action; it is characterized byamino acid substitutions at B28 (lysine) and B29 (pro-line) [96–99].

Prescription of daily subcutaneous injections ofinsulin lispro (instead of commonly used insulin) topatients with diabetes mellitus type I resulted in signif-icant decrease of risk of hypoglycemia [100]. Afterintramuscular injection insulin lispro enters blood cir-culation faster than unmodified insulin and the effect ofinsulin lispro is shorter than that of unmodified insulin.This is associated with lack of self-association (in solu-tion insulin lispro molecules exist only in monomericform) [9, 101]. In contrast to common short-actingpreparation insulin lispro may be injected just beforemeal or (even better) after meal in dependence ofpatient’s preferences and other conditions [102, 103].Pharmacokinetics of insulin lispro exhibited lessdependence on dose [104] or site of injection [105] thankinetics of common insulin. Some studies investigatedcommon application of insulin lispro together withinsulin NPH. It was noted that this provided better

maintenance of necessary glucose level both before andafter meal compared with combined use of solubleinsulin and insulin NPH [106]. Other clinical studiesdemonstrated the employment of insulin lispro togetherwith long-acting form of insulin lispro known as insu-lin NPL (protamine lispro) improved time course ofinsulin absorption compared with insulin lispro only[107–109].

Search for insulin derivative exhibiting extra shortaction mainly employed modification of amino acidsequences of insulin molecule within B22–B29. It wasreported about insulin analogues with substitutions ofphenylalanine at B25 for histidine or tyrosine [110].Derivatives obtained by amino acid at B28 for lysineand arginine exhibited similar properties [111]. Themajor goal of all such manipulations undertaken was to“bypass” patent rights of Eli Lilly and Co for produc-tion of such extra rapidly acting analogue as insulinlispro (also known under the commercial name ofHumalog).

In 2001 Novo Nordisk A/S introduced to the marketa rapidly acting insulin analogue (the trademarksNovoRapid and NovoLog) which was equivalent toinsulin lispro. Interestingly, this modified insulinappeared earlier than insulin lispro [112, 113]. Thisanalogue also known as insulin aspart was formed bysubstitution B28 proline for as partic acid [86, 114].Clinical trials provided convincing evidence thatemployment of insulin aspart provided markedimprovement in control of blood glucose level in dia-betic patients after meal [86, 115–117]. In patients’viewpoint the quality of their lives improved after sub-stitution of commonly used rapidly acting insulin par-ticularly for insulin aspart [118–120]. It was reportedthat due to lack (or decreased) hypersensitivity of thesepatients to insulin aspart it would be better to use thisinsulin preparation instead of ordinary insulin [121,122]. Interestingly, in contrast to insulin lispro prepara-tions [100] subcutaneous injections of insulin aspartnot only decreased blood glucose level but also thelevel of acetylated hemoglobin; this provides maximalmodeling of physiological secretion of insulin inhuman organism [123–125]. Most clinical studies haveshown insignificant difference between insulin aspartand insulin lispro in their absorption profiles and rela-tive effects on blood glucose [124–126]. Nevertheless,some researchers indicate faster absorption of insulinaspart; this results in higher concentration of bloodinsulin 40–120 min after its administration (maximalefficiency) compared with insulin lispro [127, 128].Figures 6 and 7 show comparative pharmacokineticand pharmacodynamic profiles of insulin aspart, insu-lin lispro, and insulin.

A new insulin analogue, known as Glulisine (thetrademark Apidra, produced by Aventis), has recentlyappeared in the market of pharmacological prepara-tions; it has been developed as the insulin analogue ofextra rapid action [65, 129, 130]. This analogue differs

600500400300200100Time, min

Blood hormone concentration, ng/ml

Insulin lisproInsulin aspartInsulin R

0

1

2

3

4

5

Fig. 6.

Pharmacodynamic characteristics of insulin R,lispro, and aspart (modified from [65, 96, 97, 104, 128]).


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from human insulin by substitution of B3 Asn residuefor lysine and also B29 Lys for Glu [131, 132]. The newpharmaceutical product exhibited clear advantages indecreasing acetylated hemoglobin up to basal level andit was recommended for therapy in combination withsuch long-acting insulin preparations as insulin NPH[132].

As it has been mentioned above maintenance of nec-essary glycemic control (especially during nighttime)requires employment of long-acting insulin prepara-tions. The long lasting effect is achieved due to subcu-taneous administration of insulin suspensions (prota-mine insulin). Such suspension may include both crys-tals of insulin and crystals of short-acting analogues ofinsulin [107, 108, 133, 134]. However, such approachhas certain shortcomings. First, resuspension of insulinpreparation by patients may result in wrong dosage.Second, injections of suspension preparations arerather painful. The other (more preferential) way con-sists in employment of special analogues such as insu-lin glargin and insulin detemir (Table 4).

In 2000 Aventis Pharmaceuticals introduced to themarket a long-acting analogue, insulin glargin, knownunder the trademark Lantus [135–137]. Prolongation ofthe biological effect was achieved by substitution ofA21 Asn for Gly and elongation of B-chain by twoamino acid residues [138–140]. Such manipulationsresulted in: (i) formation of rather stable hexamers; (ii)the increase of isoelectric point up to 6.7 (human insu-lin pI is 5.4), which is closer to blood pH (about 7.3).On the one hand, after administration of such prepara-tion certain time interval is needed for hexamer conver-sion into the monomeric form (exhibiting biologicalactivity). On the other hand, solubility of this prepara-tion is lower due to similar values of pH of biologicalmedia and pI of this preparation. It has been demon-strated that in contrast to insulin NPH subcutaneousadministration of insulin glargin causes long-lastinghypoglycemic effect, which is characterized by almosttotal lack of maximum [139], this reduce risk ofhypoglycemic shock especially during nighttime [138,141]. However, this preparation has serious shortcom-ings. First, almost neutral pI value of insulin glarginexcludes possibility of its mixing with short-actinginsulin preparations [65, 97]. Second, this preparationhas pH of 4 and so its injection is rather painful [142,143]. There are some questions on mutagenicity ofinsulin glargin. Its affinity for receptor of IGF-1 (insu-lin like growth factor) is six times higher than that ofhuman insulin; since this receptor is responsible for

regulation of ocular revascularization it is suggestedthat insulin glargin may accelerate diabetic retinopathy[114, 136, 144].

Insulin Detemir (also known under the trademarkLevemir) developed by Novo Nordisk A/S is one of themost modern insulin derivatives exhibiting long-lastingeffect [83, 145, 146]. This is acylated insulin (lysine(B29)-tetradecanoyl destreonyl (B30) insulin). Itexhibits long-lasting effect, first of all due to increasedself-association, and secondly, due to its binding withalbumin [147]. Clinical studies have shown advantagesof the use of insulin detemir compared with insulinNPH, because using this analogue it is easier to controland predict glucose level; this minimizes risk ofhypoglycemia [148, 149]. In contrast to insulin glarginand insulin NPH insulin detemir is soluble at neutralpH and so its injections are painless. However, insulindetemir exhibits significantly lower affinity to insulinreceptor than insulin NPH and so the dose of the formerrequired for achievement of the same effect should be2.35 times higher than that of insulin NPH [150]. Nev-ertheless, it has been reported that the increase of thedose does not increase of probability of hypersensitiv-ity to this preparation.

It should be noted that the most accurate imitation ofnormal level and time course of blood glucose isachieved by simultaneous employment of insulinDetemir and insulin Aspart [83].

700500400300200100Time, min

The rate of hypoglycemic effect, mg glucose/min

Insulin lisproInsulin aspartInsulin R

1

2

3

4

5

600

6

0

Fig. 7.

Pharmacokinetic characteristics of insulin R, lispro,and aspart (modified from [65, 96, 97, 104, 128]).

Table 4.

Long-acting insulin analogs

Clinician name Modification in insulin molecule Properties of preparation

Glargin HOE 901 A21 Asn was substituted for Gly, two Arg were added to B30 Slow absorption

Detemir NN304 B30 Thr was removed, B29 Lys was acylated with miristic acid (C

14 : 0

) Slow absorption, low affin-ity for insulin receptor

364


Vol. 2

No. 4

2008

GUSAROV et al.

CONCLUSIONS

Modern insulin replacement therapy of diabetesmellitus still requires improvement and search for newmore perspective approaches improving quality of lifeof diabetic patients goes on. The main directions ofsuch search include attempts to avoid uncomfortableand often painful injections by replacing them for per-oral, intranasal and other noninvasive dosage forms,and also development of pharmaceutical analogues ofinsulin characterized by better modeling of glycemicprofile. Studies conducted in Russia and other countriesresulted in appearance of new dosage forms of insulinand its analogues in the pharmaceutical market. Theseinsulin analogues demonstrate either better modeling ofphysiological changes in the organism related to foodintake (short-acting analogues) or reproduce normalbasal diurnal insulinemia (long-acting analogues).

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genetically engineered insulin and its pharmaceutical analogues

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