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.

REVIEWS

Genetically Engineered Insulin and Its Pharmaceutical Analogues

D. A. Gusarova, b*, V. D. Gusarovaa, b, D. I. Bayramashvilia, and A. F. Mironovb
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: gusarovda@mail.ru
Received August 29, 2007

Abstract—Studies of replacement therapy of diabetes mellitus resulted not only in introduction of series of
forms 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

INTRODUCTION maintenance of basal level of blood glucose and a

Insulin is one of the best studied hormones at the
moment. Although more than 80 years passed since the
discovery of the fact that insulin produced by pancreas
is responsible for the decrease of blood sugar [1] this
hormone still attracts much attention of a scientific
community. Mortality of diabetes mellitus is on the
third place after mortality of cardiovascular and tumor
diseases [2, 3]. In 2000 there were about 150 million
patients with diabetes mellitus in the world [4] and each
year their number roughly increases by 2–6% [5, 6].
For example, according to prognosis of specialists
number of diabetic patients in European countries
increases up to 33 millions by 2010 [7]; in Russia
almost 400000 patients need regular administration of
patient needs constant injections of insulin over the
whole life.
The insulin therapy may be based on classic drug
formulations, which usually include so called regular
insulin (soluble rapidly acting insulin) or protamine
zinc insulin suspension (long acting insulin NPH, neu-
tral protamine Hagedorn) as well as gene engineered
analogues of insulin, recently appeared in the pharma-
ceutical market. The goal of the present review was to
summarize data of application of both traditional prep-
Breakfast

Supper
Dinner

insulin preparations.
Blood glucose concentration, mg/ml

Although all people differ by their habits blood glu-

cose is normally maintained within rather narrow range
and undergoes rather small diurnal variations. Normal
glucose concentration in human blood varies from 3 up 4000
to 8 mM [5]. Food intake increases blood sugar within Diabetes mellitus
3000
15–30 min after meal (Fig. 1) [8]. Diurnal variation of
insulin content is similar to that of blood glucose (Fig. 2) 2000
[8]. Increase in blood insulin concentration represents
response of an organism to increased glucose level; 1000
insulin decreases blood glucose up to the basal level Norm
within 2 h after meal [9]. 0
In diabetes mellitus blood glucose undergoes almost
a 3–4-fold increase, which is associated with either
insufficient insulin production (diabetes mellitus type
1) or organism resistance to insulin action (diabetes 6:00 10:00 14:00 18:00 22:00 2:00 6:00
mellitus type 2). In the case of diabetes mellitus type Time of day, hour:min
one the major goal of replacement therapy consists in
Fig. 1. Diurnal changes in blood glucose of a healthy person
*To whom correspondence should be addressed. and a patient with diabetes mellitus (modified from [8, 9]).

356
 GENETICALLY ENGINEERED INSULIN AND ITS PHARMACEUTICAL ANALOGUES 357

preproinsulin rather than proinsulin is the insulin pre-

Breakfast

Supper
Dinner
cursor; the former differs from proinsulin by the pres-
Plasma insulin concentration, µU/ml

ence of a N-terminal leader fragment [21–23]. It is sug-

gested that this leader fragment of 23–24 amino acid
50 residues is a signal sequence required for proinsulin
transfer from polyribosomes to Golgi complex. After
cleavage of the leader fragment proinsulin molecule
adopts conformation required for correct formation of
25 disulfide bonds. Proinsulin cleaved then into insulin
and C-peptide is stored in secretory granules [24–26].
Subsequently the content of these granules is secreted
into portal blood circulation [24, 27].
Basal insulin level In solutions insulin molecules exist as equilibrium
0 mixture of monomeric, dimeric, tetrameric and hexam-
4:00 8:00 12:00 16:00 20:00 24:00 4:00 8:00 eric forms [28]. At low concentrations (comparable
Time of day, hour:min with blood concentrations) the monomeric form pre-
dominates; this is biologically active form because only
Fig. 2. Physiological profile of insulin in a healthy person this form may interact with an insulin receptor [29]. At
(modified from [8, 9]).
higher concentration in both acidic or alkaline media
(pH of 8.0 and above) this hormone may exhibit self-
arations of human insulin and its pharmaceutical ana- association into dimers (in the absence of zinc ions),
logues in endocrinology. whereas in neutral or weakly alkaline medium it forms
hexamers in the presence of zinc ions (Fig. 5). After
biosynthesis (and subsequent processing) insulin is
1. STRUCTURE AND BIOSYNTHESIS OF INSULIN accumulated in the body (in vesicles of islet cells) as
crystal zinc-bound insulin, which is then released in
Insulin is a polypeptide hormone, produced by pan- response to the increase of blood glucose concentration
creatic islet cells. Protein nature of insulin (Fig. 3) was [30]. Although it is well known that the hexamer exists
known long before elucidation of its primary structure in three conformations (R6, T3R3, and T6), which
in 1955 [10] (as well as the fact that insulin contains depend on monomer conformation it still remains unclear
three disulfide bonds formed by cysteine residues which conformation is realized during insulin accumula-
[11−14]). tion in cells of the islets of Langerhans [31–34].
Physiological effects of insulin on glucose content Insulin molecules may be crystallized at various
and lipid metabolism is known for a long time [15]. It conformations [35]. The first crystal structure of insulin
was found [16] that a single chain polypeptide, proinsu- was obtained in 1969; it represented a hexamer, in
lin, is a precursor of the two-chain insulin molecule which three dimers were joined together around two
(Fig. 4). Metabolism of proinsulin resulting in forma- zinc ions [36, 37]. Subsequently hexameric structures
tion of a biologically active product of lower molecular containing even four zinc atoms were also obtained
mass occurs in β-cells of the pancreatic islets of [38]. In the hexamer containing two zinc ions the
Langerhans [17–20]. Subsequently it was found that regions B1–B8 and B20–B30 of the monomeric units

HOOC 30
Thr
Lys
Pro
A-chain H Thr
Ser Leu Tyr
11
2N Tyr
1 Cys Gln
Gly Leu Phe
6
Ile Glu 20 21 OH
H
2N
Ile
Val Ser Asn CO Phe
1 Glu Gln Cys Tyr Cys Asn
Cys Thr Gly
Phe
B-chain Val 7 Arg
Asn 19 Glu
Gln His 7 Gly
Leu Cys Cys
Gly Ser
His Leu Val Glu Ala Leu Tyr Leu Val

Fig. 3. Primary structure of human insulin.

BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol. 2 No. 4 2008

358 GUSAROV et al.

Gly Gly Gly Leu

Gly Pro Glu Val
Gly Ala Gln
Ser Gly Val
Leu Gln
Gln Leu
Pro Asp
Leu Glu

Ala Ala
Glu
Leu
Arg
Gln
Arg
Gly
Thr 30
Ser
Lys
Leu
Pro
Gln
Leu Thr
Lys
1 11 Ser TyrGln Tyr
Arg Cys
A-chain Gly Leu Phe
H Ile 6
Ile Glu 20 21 OOH
2N 1 Val Glu Gln Ser Asn Tyr Cys Asn C Phe
Cys Cys Thr
Gly
Phe
B-chain Val 7 Arg
Asn 19 Glu
Gln
His Leu
7 Gly
Cys Gly Cys
Ser His Leu Val
Val Glu Ala Leu Tyr Leu

Fig. 4. Schematic presentation of human proinsulin (white and grey colors show amino acid residues corresponding to insulin and
C-peptide, respectively).

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 three
dimers. In each dimer A and B chains form a compact
molecule including two A chain α-helices and one B-
chain α-helix (T6 state).
In the conformation containing four zinc ions three
N-termini of B-chains (B1–B8 residues) are associated
into α-helix in the presence of high concentrations of
chloride 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 the
sites of insulin molecule known as hydrophobic pock-
ets. In T3R6 (asymmetric structure in which 4 ions are
coordinated by B10 histidines) there are three such
pockets, whereas in R6 there are 6 pockets. It is known
that phenol and its derivatives may be used in the insu-
lin dosage as either antibacterial preservatives [41] or
additives preventing deamidation reactions [42].
Recently it has been demonstrated that phenolic com-
pound stabilize T3R3 and R6 hexamers and thus
increase 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 methods
of gene engineering recombinant human insulin was
produced by means of E. coli [47–54] and (since 1987)
by yeast cultures Saccharomyces cerevisiae, Pichia
pastories [55–57]. Increasing standards requirements
for insulin preparations stimulated constant improve-
ments of methods of insulin production over the last
twenty years [6, 58, 59]. The methodology for produc-
tion of APS insulin is well studied and reviewed in the
literature. However, it should be noted that various drug
preparations differed by duration of hypoglycemic
effect 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 form
as well as actively developed peroral, transdermal and
other preparations.
As it has been mentioned above for maintenance of
normal level of blood glucose diabetic patients need
repeated injections of insulin preparations. Modern
classic therapy of diabetes mellitus is mainly based on
application of soluble insulin and a suspension of prot-
amine zinc insulin (NPH insulin).
The soluble drug dosage form of insulin exhibits
rapid 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 the
hypoglycemic effect lasts for about 8 h [60]. The most
known short acting insulins are: Humulin Regular (Eli
Lilly, USA), Actrapid HM (NovoNordisk, Denmark),
Insuman Rapid (Sanofi-Aventis, France-Germany). In

BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol. 2 No. 4 2008

 GENETICALLY ENGINEERED INSULIN AND ITS PHARMACEUTICAL ANALOGUES 359

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 the
most popular in the world: their hypoglycemic effect is
more 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 of Monomer
various zinc crystals. Such insulins are usually pre-
scribed as subcutaneous injections; this is determined
by necessity of formation of drug “store”, which pro-
longs hypoglycemic effect. The long-acting insulins
begin to act only 2–4 h after administration, peak of
their effect is observed within 4–10 h and usually dura-
tion of their effect does not exceed 12–18 h [60]. The
NPH dosage insulins are usually formed by co-precip-
itation of insulin with protamine peptides at neutral
pH values in the presence of zinc ions, phenol, and/or Dimer
phenol derivatives [28]. This results in formation of a
stable complex, which includes negatively charged
insulin molecules and polycationic molecules of prot-
amine peptides. This complex stabilized by zinc ions
and aromatic phenol compounds preferentially con-
sists of tetragonal crystals [62]. In NPH insulins insu-
lin and protamine sulfate are linked at an isophane
ratio (i.e. in the absence of excess of one of compo-
nents) [62].
Protamines are commercial name of strongly basic
peptides found in sturgeon milt [63]. Production of
NPH insulins usually employs protamine sulfate
obtained from salmon milt [64]. Prolongation of insulin
effect is determined by slow degradation of the prota-
mine complex by fibrinolytic enzymes [28].
The most known insulins of this class include: Hexamer T6
Humulin NPH (Eli Lilly), Protaphan HM (NovoNord-
isk), Insuman Basal (Sanofi-Aventis, France-Ger- Fig. 5. Schematic presentation of possible tertiary structures
many). In Russia the following insulins of this class are of human insulin (PDB Protein Database), (modified from
produced: Insuran NPH (Institute of Bioorganic Chem- [35]).
istry, RAS), Biosulin H (Pharmstandard-Ufavita), Rin-
sulin H (National Biotechnology).
capsules for peroral insulin delivery [67]. Carboxyme-
Now special attention is also paid not only to the thylcellulose conjugates of insulin have also been
injection dosage forms of insulin, but also to peroral, tested; they can be absorbed by gut epithelium without
intranasal and other drug dosage forms, which would denaturation of hormone molecules [68]. At the
improve life of diabetic patients. moment the most promising is hexyl-insulin-monocon-
The peroral drug dosage form of insulin is the most jugate-2 (HIM2) representing human recombinant insu-
convenient form for the treatment of diabetes mellitus, lin containing polyethylene glycol-7-hexyl attached to
if insulin molecule would not be degraded in gas- lysine B29 [69, 70]. HIM2 may provide insulin absorp-
trointestinal tract by proteolytic enzymes [65]. Serious tion by epithelial cells without degradation of insulin
attempts are now undertaken to solve this problem. For molecule. Moreover this peroral preparation is able to
example, some nonacylated amino acids have been reproduce physiological pathway of insulin, which is
investigated as insulin carriers, which form a noncova- secreted in human body by pancreas and reaches liver
lent complex inducing conformational changes in this (via portal system) before it enters general blood circu-
protein (conformation unfolding); this facilitates pas- lation [71].
sive diffusion of insulin molecules through lipid bilay- Comparing injection and peroral insulin dosage
ers before these molecules dissociate passing through forms one can come to the following conclusions. Of
gut cell membranes [66]. There are peroral liposomal course repeated daily injection of insulin are inconve-

BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol. 2 No. 4 2008

360 GUSAROV et al.
Table 1. Comparison of physiological properties of prepara-
tions of insulin and its analogues
Insulin Onset Maximum Duration
preparation of effect, h effect, h of 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 20–24
manifested
Detemir 1–2 6–8 10–18
nient for patients and seriously complicate their lives.
Peroral forms of insulin lack such shortcomings. How-
ever, costs of the peroral therapy may significantly
exceed costs of the injection therapy. The peroral ther-
apy is based on a spray delivery system, which consists
of insulin preparation itself and a device, which creates
aerosol particles of certain and the same size and
administered aerosol into oral cavity at the rate
160 kg/h [72, 73]. Detailed study of the peroral forms
has shown that such system is especially convenient for
patients 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 large
absorption and has large number of blood capillaries
[65]. Bioavailability of insulin administered into
human organism during its intranasal delivery may be
increased 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 and
drops dosage forms of insulin have been proposed for
intranasal delivery.
Special attention is paid to so-called lung insulin
crystals (a powder system for inhalation from 3 to 9 U
of insulin). Although inhalation of insulin crystals was
proposed in 1925 [75] only 46 years later it became
possible to decrease blood glucose in a diabetic patient
by means of the lung crystals of mixed pig and cattle
insulins [76]. This way of delivery has several advan-
tages: use of pulmonary crystals is painless; pulmones
have huge absorption surface and numerous blood ves-
sels; lungs lack any peptidases which could destroy
polypeptide molecules [65, 77]. Thus, insulin mole-
cules may be easily absorbed, penetrate through thin
alveolar capillary barriers into blood circulation and
cause requested hypoglycemic effect. Nevertheless,
prescription of lung crystals has several shortcomings
as well. First, such way of insulin delivery is doubtful
in diabetic patients suffering with asthma, chronic
bronchitis; its employment is also complicated in
BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY
smokers (if diabetic patients refuse to stop smoking)
[65]. Second, it has been shown that lung crystals
cause more frequent antibody production against for-
eign (for patient’s organism) insulin than the injection
insulin dosage forms [78].
Peroral and intranasal ways of insulin delivery to
human organism as well as lung crystals may be used
for 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 the
other noninvasive pathways of insulin delivery have
been proposed for achievement of requested hypogly-
cemic effect. These include methods of transdermal
delivery based on increased permeability of skin sur-
face for macromolecules by sonication [79] or due to
insulin 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 investigated
mainly in experiments.
3. PHARMACEUTICAL INSULIN ANALOGUES
There is evidence that intensive insulin therapy of
diabetic patients is associated with increased risk of the
development of hypoglycemic shock in both day and
night 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 of
healthy individuals [83, 84]. For example, absorption of
short-acting insulin may be decreased due to self-asso-
ciation of insulin molecule into dimers, tetramers, and
hexamers [85, 86]. On the contrary, the effect of long-
acting preparations of insulin (e.g. NPH insulin) may
be too short to exhibit necessary glycemic control over-
night [83, 87]. It should be also noted, that long-acting
preparations of insulin are suspensions and insufficient
mixing of such preparation by patient before adminis-
tration cannot be ruled out [88, 89]. For solution of such
problems several analogues of human insulin have been
developed; some of them (e. g. Insulin Aspart and Insu-
lin Lispro) are analogues of short-acting insulin,
whereas some others (insulin Glargin and insulin
Detemir) 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 was
more close to the basal one. For example, it was
reported about accelerated absorption of injected prep-
aration of insulin with artificial amino acid substitu-
tions at B9–B12 and B26–B28 [90]. Self-association of
insulin molecules was suppressed in the same way.
Vol. 2 No. 4 2008
 GENETICALLY ENGINEERED INSULIN AND ITS PHARMACEUTICAL ANALOGUES 361

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
Insulin
G J V E Q C C T S J C S L Y Q L E
A chain
Glargin
G J V E Q C C T S J C S L Y Q L E
A chain
Insulin
F V N Q H L C G S H L V E A L Y L
B chain
Glargin
F V N Q H L C G S H L V E A L Y L
B chain
Lispro
F V N Q H L C G S H L V E A L Y L
B chain
Aspart
F V N Q H L C G S H L V E A L Y L
B chain
Glulisine
F V K Q H L C G S H L V E A L Y L
B chain
Detemir
F V N Q H L C G S H L V E A L Y L
B chain
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
Insulin
N Y C N OH
A chain
Glargin
N Y C G OH
A chain
Insulin
V C G E R G F F Y T P K T OH
B chain
Glargin
V C G E R G F F Y T P K T R R OH
B chain
Lispro
V C G E R G F F Y T K P T OH
B chain
Aspart
V C G E R G F F Y T D K T OH
B chain
Glulisine
V C G E R G F F Y T P E T OH
B chain
Detemir
V C G E R G F F Y T P Ka OH
B chain

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 Lys Extra rapid absorption, short-acting effect
was substituted for Glu

However, first insulin derivatives of extra rapid action
exhibited low biological activity [91]. Studies of the
effects of amino acid substitutions on interaction of
insulin molecules with receptors revealed the most
essential contribution of substitutions at B22–B29 and
the increase of activity of insulin analogue would be
achieved in the case of correct change of amino acid
residues 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

BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol. 2 No. 4 2008

362 GUSAROV et al.
Blood hormone concentration, ng/ml
5
Insulin lispro
Insulin aspart
4 Insulin R
3
2 action mainly employed modification of amino acid
1 reported about insulin analogues with substitutions of
0 and arginine exhibited similar properties [111]. The
100 200 300 400 500 600
Time, min
Fig. 6. Pharmacodynamic characteristics of insulin R,
lispro, and aspart (modified from [65, 96, 97, 104, 128]).
decrease in biological activity [90]. Table 3 lists the
most 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 is
involved into formation and stabilization of hexamer
due to binding with zinc ion. Thus, substitution of this
amino acid residue for Asp would result in increased
absorption ability of such modified insulin. Indeed, it
was demonstrated that Asp10 absorption was two times
faster than absorption of native insulin [94]. Although
this 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 insulin
analogue of extra rapid action; it is characterized by
amino acid substitutions at B28 (lysine) and B29 (pro-
line) [96–99].
Prescription of daily subcutaneous injections of
insulin lispro (instead of commonly used insulin) to
patients with diabetes mellitus type I resulted in signif-
icant decrease of risk of hypoglycemia [100]. After
intramuscular injection insulin lispro enters blood cir-
culation faster than unmodified insulin and the effect of
insulin 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 monomeric
form) [9, 101]. In contrast to common short-acting
preparation insulin lispro may be injected just before
meal or (even better) after meal in dependence of
patient’s preferences and other conditions [102, 103].
Pharmacokinetics of insulin lispro exhibited less
dependence on dose [104] or site of injection [105] than
kinetics of common insulin. Some studies investigated
common application of insulin lispro together with
insulin NPH. It was noted that this provided better
BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY
maintenance of necessary glucose level both before and
after meal compared with combined use of soluble
insulin and insulin NPH [106]. Other clinical studies
demonstrated the employment of insulin lispro together
with long-acting form of insulin lispro known as insu-
lin NPL (protamine lispro) improved time course of
insulin absorption compared with insulin lispro only
[107–109].
Search for insulin derivative exhibiting extra short
sequences of insulin molecule within B22–B29. It was
phenylalanine at B25 for histidine or tyrosine [110].
Derivatives obtained by amino acid at B28 for lysine
major 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 insulin
lispro (also known under the commercial name of
Humalog).
In 2001 Novo Nordisk A/S introduced to the market
a rapidly acting insulin analogue (the trademarks
NovoRapid and NovoLog) which was equivalent to
insulin lispro. Interestingly, this modified insulin
appeared earlier than insulin lispro [112, 113]. This
analogue also known as insulin aspart was formed by
substitution B28 proline for as partic acid [86, 114].
Clinical trials provided convincing evidence that
employment of insulin aspart provided marked
improvement 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 reported
that due to lack (or decreased) hypersensitivity of these
patients to insulin aspart it would be better to use this
insulin preparation instead of ordinary insulin [121,
122]. Interestingly, in contrast to insulin lispro prepara-
tions [100] subcutaneous injections of insulin aspart
not only decreased blood glucose level but also the
level of acetylated hemoglobin; this provides maximal
modeling of physiological secretion of insulin in
human organism [123–125]. Most clinical studies have
shown insignificant difference between insulin aspart
and insulin lispro in their absorption profiles and rela-
tive effects on blood glucose [124–126]. Nevertheless,
some researchers indicate faster absorption of insulin
aspart; this results in higher concentration of blood
insulin 40–120 min after its administration (maximal
efficiency) compared with insulin lispro [127, 128].
Figures 6 and 7 show comparative pharmacokinetic
and pharmacodynamic profiles of insulin aspart, insu-
lin lispro, and insulin.
A new insulin analogue, known as Glulisine (the
trademark Apidra, produced by Aventis), has recently
appeared in the market of pharmacological prepara-
tions; it has been developed as the insulin analogue of
extra rapid action [65, 129, 130]. This analogue differs
Vol. 2 No. 4 2008
 GENETICALLY ENGINEERED INSULIN AND ITS PHARMACEUTICAL ANALOGUES
from human insulin by substitution of B3 Asn residue
for lysine and also B29 Lys for Glu [131, 132]. The new
pharmaceutical product exhibited clear advantages in
decreasing acetylated hemoglobin up to basal level and
it was recommended for therapy in combination with
such 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 of
insulin [107, 108, 133, 134]. However, such approach
has certain shortcomings. First, resuspension of insulin
preparation by patients may result in wrong dosage.
Second, injections of suspension preparations are
rather 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 the
market a long-acting analogue, insulin glargin, known
under the trademark Lantus [135–137]. Prolongation of
the biological effect was achieved by substitution of
A21 Asn for Gly and elongation of B-chain by two
amino acid residues [138–140]. Such manipulations
resulted 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 biological
activity). On the other hand, solubility of this prepara-
tion is lower due to similar values of pH of biological
media and pI of this preparation. It has been demon-
strated that in contrast to insulin NPH subcutaneous
administration of insulin glargin causes long-lasting
hypoglycemic effect, which is characterized by almost
total lack of maximum [139], this reduce risk of
hypoglycemic shock especially during nighttime [138,
141]. However, this preparation has serious shortcom-
ings. First, almost neutral pI value of insulin glargin
excludes possibility of its mixing with short-acting
insulin preparations [65, 97]. Second, this preparation
has pH of 4 and so its injection is rather painful [142,
143]. There are some questions on mutagenicity of
insulin glargin. Its affinity for receptor of IGF-1 (insu-
lin like growth factor) is six times higher than that of
human insulin; since this receptor is responsible for
Table 4. Long-acting insulin analogs
Clinician name Modification in insulin molecule
Glargin HOE 901 A21 Asn was substituted for Gly, two Arg were added to B30
Detemir NN304 B30 Thr was removed, B29 Lys was acylated with miristic acid (C14 : 0) Slow absorption, low affin-
BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY
363
The rate of hypoglycemic effect, mg glucose/min
6
Insulin lispro
5 Insulin aspart
Insulin R
4
3
2
1
0
100 200 300 400 500 600 700
Time, min
Fig. 7. Pharmacokinetic characteristics of insulin R, lispro,
and aspart (modified from [65, 96, 97, 104, 128]).
regulation of ocular revascularization it is suggested
that insulin glargin may accelerate diabetic retinopathy
[114, 136, 144].
Insulin Detemir (also known under the trademark
Levemir) developed by Novo Nordisk A/S is one of the
most modern insulin derivatives exhibiting long-lasting
effect [83, 145, 146]. This is acylated insulin (lysine
(B29)-tetradecanoyl destreonyl (B30) insulin). It
exhibits long-lasting effect, first of all due to increased
self-association, and secondly, due to its binding with
albumin [147]. Clinical studies have shown advantages
of the use of insulin detemir compared with insulin
NPH, because using this analogue it is easier to control
and predict glucose level; this minimizes risk of
hypoglycemia [148, 149]. In contrast to insulin glargin
and insulin NPH insulin detemir is soluble at neutral
pH and so its injections are painless. However, insulin
detemir exhibits significantly lower affinity to insulin
receptor than insulin NPH and so the dose of the former
required for achievement of the same effect should be
2.35 times higher than that of insulin NPH [150]. Nev-
ertheless, it has been reported that the increase of the
dose does not increase of probability of hypersensitiv-
ity to this preparation.
It should be noted that the most accurate imitation of
normal level and time course of blood glucose is
achieved by simultaneous employment of insulin
Detemir and insulin Aspart [83].
Properties of preparation
Slow absorption
ity for insulin receptor
Vol. 2 No. 4 2008
364 GUSAROV et al.
CONCLUSIONS
Modern insulin replacement therapy of diabetes
mellitus still requires improvement and search for new
more perspective approaches improving quality of life
of diabetic patients goes on. The main directions of
such search include attempts to avoid uncomfortable
and often painful injections by replacing them for per-
oral, intranasal and other noninvasive dosage forms,
and also development of pharmaceutical analogues of
insulin characterized by better modeling of glycemic
profile. Studies conducted in Russia and other countries
resulted in appearance of new dosage forms of insulin
and its analogues in the pharmaceutical market. These
insulin analogues demonstrate either better modeling of
physiological changes in the organism related to food
intake (short-acting analogues) or reproduce normal
basal diurnal insulinemia (long-acting analogues).
REFERENCES
1. Banting, F.G. and Best, C.H., J. Lab. Clin. Med., 1922,
vol. 7, p. 464.
2. Barfoed, H.C., Chem. Eng. Prog., 1987, vol. 83, pp. 49–
54.
3. Ladish, M.R. and Kohlmann, K.L., Biotechnol. Prog.,
1992, vol. 461, pp. 469–478.
4. Kjeldsen, T., Ludvigsen, S., Diers, I., Balschmidt, P.,
Sorensen, A.R., and Kaarsholm, N.C., J. Biolog. Chem.,
2002, vol. 277, pp. 18245–18248.
5. Klyushnichenko, V., Brush, R., and Bulychev, A., Bio-
process Int., 2004, vol. 2, pp. 45–59.
6. Bairamashvili, D.I., Ros. Khim. Zh., 2005, vol. XLIX,
no. 1, pp. 34–45.
7. Zimmet, P. and Alberti, K., Nature, 2001, vol. 414,
pp. 782–787.
8. Ten, S. and McLaren, N.K., Endocrinology Web Text-
book, 2004.
9. Bhatnagar, S., Srivastava, D., Jayadev, M.S.K., and
Dubey, A.K., Progress in Biophysics and Molecular
Biology, 2006, vol. 91, pp. 199–228.
10. Brown, H., Sanger, F., and Kitai, R., Biochem J., 1955,
vol. 60, pp. 556–565.
11. Stern, K.G. and White, A., J. Biol. Chem., 1936, vol.
117, pp. 95–110.
12. Freudenberg, K. and Wegmann, T., Z. Physiol. Chem.,
1930, vol. 187, p. 89.
13. Vigneaud, V. du, Fitch, A., Pekarek, E., and Lock-
wood, W.W., Proc. Soc. Exp. Biol. Med., 1935, vol. 33,
p. 371.
14. Jensen, H., Evans, E.A., Pennington, W.D., and Schock,
E.D., J. Biol. Chem., 1936, vol. 114, pp. 199–208.
15. Steiner, D.F., Diabetes, 1977, vol. 26, pp. 322–340.
16. Grant, P.T., Essays in Biochemistry, 1971, vol. 6,
pp. 69–92.
17. Howell, S.L., Kostianovsky, M., and Lacy, P.E., J. Cells
Biol., 1969, vol. 42, pp. 695–705.
18. Kemmler, W., Steiner, D.F., and Borg, J., J. Biol. Chem.,
1973, vol. 248, pp. 4544–4551.
BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY
19. Steiner, D.F., Cunningham, D., Spigelman, L., and
Aten, B., Science, 1967, vol. 157, pp. 697–700.
20. Docherty, K., Carrol, R.J., and Steiner, D.F., Proc. Natl.
Acad. Sci. USA, 1982, vol. 79, pp. 4613–4617.
21. Tager, H.S., Emdin, S.O., Clark, J.L., and Steiner, D.F.,
J. Biol. Chem., 1973, vol. 248, pp. 3476–3482.
22. Kemmler, W., Peterson, J.D., and Steiner, D.F., J. Biol.
Chem., 1971, vol. 246, pp. 6786–6791.
23. Given, B.D., Cohen, R.M., Shoelson, R.M., Frank, B.H.,
Rubenstrein, A.H., and Tager, H.S., J. Clin. Invest.,
1985, vol. 76, pp. 1398–1405.
24. Murray, R., Granner, D., Mayes, P., and Rodwell, V.,
Biokhimiya cheloveka (Harper’s Biochemistry) [Rus-
sian translation] Moscow: Mir., 1993.
25. Mackin, R.B., CLMS Cellular and Molecular Life Sci-
ences, 1998, vol. 54, pp. 696–702.
26. Cowley, D.J. and Mackin, R.B., FEBS Lett., 1997,
vol. 402, pp. 124–130.
27. Munte, C.E., Vilela, L., Kalbitzer, H.R., and Garratt, R.C.,
FEBS J., 2005, vol. 272, pp. 4284–4293.
28. Brange, J., Galenics of Insulin, Berlin: Springer-Verlag,
1987, pp. 20–29.
29. Huus, K., Havelund, S., Olsen, H.B., Weet, M.van de,
and Frokjaer, S., Biochemistry, 2006, vol. 45, pp. 4014–
4024.
30. Blundell, T.L., Diabetes, 1972, vol. 21, pp. 492–505.
31. Grant, P.T. and Frank, B.H., Biochem. J., 1972,
vol. 126, pp. 433–440.
32. Goldman, J. and Carpenter, F.H., Biochemistry, 1974,
vol. 13, pp. 4566–4574.
33. Summerell, J.M., Osmand, A., and Smith, G.H., Bio-
chem. J., 1965, vol. 95, p. 31.
34. Emdin, S.O., Dodson, G.G., Gutfield, J.M., and Gut-
field, S.M., Diabetologia, 1980, vol. 19, pp. 174–182.
35. Whittingham, J.L., Scott, D.J., Chance, K., Wilson, A.,
Finch, J., Brange, J., and Dodson, G.G., J. Mol. Biol.,
2002, vol. 318, pp. 479–490.
36. Adams, M.J., Nature, 1969, vol. 224, pp. 491–495.
37. Peking Insulin Structure Group, Peking Rev., 1971,
vol. 40, pp. 10–16.
38. Shlichtkrull, J., Brange, J., Christiansen, A.H., Hal-
lund, O., Heding, L.G., Jorgensen, K.H., Munkgaard,
S., Rasmussen, M., Sorensen, E., and Volund, A.A.,
Horm. Metab. Res., 1974, vol. 5, pp. 134–143.
39. Ciszak, E. and Smith, G.D., Biochemistry, 1994,
vol. 33, pp. 1512–1517.
40. Ciszak, E., Beals, J.M., Frank, B.H., Baker, J.C.,
Carter, N.D., and Smith, G.D., Structure, 1995, vol. 3,
pp. 615–622.
41. Derewenda, U., Derewenda, Z., Dodson, E.J., Dod-
son, G.G., Reynolds, C.D., Smith, G.D., Sparks, C., and
Swenson, D., Nature, 1989, vol. 338, pp. 594–596.
42. Brange, J. and Langkjaer, L., Acta Pharm. Nord., 1992,
vol. 4, pp. 149–158.
43. Ovchinnikov, Yu.A., Bioorganicheskaya khimiya
(Bioorganic Chemistry), Moscow: Prosveshchenie,
1987, pp. 151–153.
44. US Patent #4183849, 1980.
45. Petrides, D., Biotechnol. Bioeng., 1995, vol. 48,
pp. 529–541.
Vol. 2 No. 4 2008
 GENETICALLY ENGINEERED INSULIN AND ITS PHARMACEUTICAL ANALOGUES
46. US Patent # 4029642, 1977.
47. Goeddel, D.V., Kleid, D.G., Bolivar, F., Heyneker, H.L.,
Yansura, D.G., Crea, R., Hirose, T., Kraszewski, A.,
Itakura, K., and Riggs, A.D., Proc. Natl. Acad. Sci.
USA, 1979, vol. 76, pp. 106–110.
48. Miller, W.L. and Baxter, J.D., Diabetologia, 1980,
vol. 18, pp. 431–436.
49. US Patent #4421685, 1983.
50. Williams, D.C., Van Frank, R.M., Muth, W.L., and Bur-
nett, J.P., Science, 1982, vol. 215, pp. 687–689.
51. Johnson, I., Nature, 1983, vol. 219, pp. 632–637.
52. Burnett, J., Experimental Manipulation of Gene Expres-
sion, New York: Academic Press, 1983, pp. 259–277.
53. Chance, R.E., Peptides: Synthesis-Structure-Function,
Pierce Chemical Co., 1981, pp. 721–728.
54. Kroeff, E.P., Owens, R.A., Campbell, E.L., Johnson, R.D.,
and Marks, H.I., J. Chromatogr., 1989, vol. 461, pp. 45–
61.
55. Kjeldsen, T., Appl. Microbiol. Biotechnol., 2000,
vol. 54, pp. 277–286.
56. Cereghino, G.P.L. and Cregg, J.M., Curr. Opin. Bio-
tech., 1999, vol. 10, pp. 422–427.
57. US Patent Application #20030104607, 2003.
58. Gusarov, D.A., Vostrikov, V.V., Ruchko, E.A., Las-
man, V.A., Mikhalev, A.V., and Bairamashvili, D.I.,
Biotekhnologiya, 2006, vol. 2, pp. 44–49.
59. Gusarov, D., Lasman, V., and Bayramashvili, D.,
J. Chromatogr. B, 2007, vol. 853, pp. 354–359.
60. Thompson, R., Christie, D., and Hindmarsh, P.C., Cur-
rent Paediatrics, 2006, vol. 16, pp. 117–122.
61. Balabolkin, M.I. and Klebanova, E.M., Lechashchii
vrach, 2006, vol. 2, pp. 117–122.
62. Hvaas, A. and Skelbaek-Pedersen, B., J. Pharmaceuti-
cal Biomedical Analysis, 2005, vol. 37, pp. 551–557.
63. Ando, T., Yamasaki, M., and Suzuki, K., Protamines,
Isolation, Characterization Structure and Function,
Berlin: Springer-Verlag 1973.
64. Hoffman, J.A., Chance, R.E., and Johnson, M.G., Prot.
Exp. Purif., 1990, vol. 1, pp. 127–133.
65. Gomez-Perez, F.J. and Rull, J.A., Archives of Medical
Research, 2005, vol. 36, pp. 258–272.
66. Vila, A., Sanchez, A., Tobio, M., Calvo, P., and
Alonso, M.J., J. Control Release, 2002, vol. 78, pp. 15–
24.
67. Ramadas, M., Paul, W., Dileep, K.J., Anitha, Y., and
Sharma, C.P., J. Microencapsul., 2000, vol. 17,
pp. 405–411.
68. Marschutz, M.K., Caliceti, P., and Bernkop-Shnurch,
A., Pharm. Res., 2000, vol. 17, pp. 1468–1474.
69. Prego, C., Garcia, M., Torres, D., and Alonso, M.J.,
J. Control Release, 2005, vol. 101, pp. 151–162.
70. Wajsberg, E., Miyazaki, Y., Triplitt, C., Cersosimo, E.,
and DeFonzo, R.A., Diabetes Care, 2004, vol. 27,
pp. 2868–2873.
71. Clement, S., Still, J.G., Kosutic, G., and McAllister, R.G.,
Diabetes Technol. Ther., 2002, vol. 4, pp. 459–466.
72. Cernea, S., Kidron, M., Wohlgernter, J., Modi, P., and
Raz, I., Clin. Ther., 2004, vol. 12, pp. 2084–2091.
73. Modi, P., Mihic, M., and Lewin, A., Diabetes Metab.
Res. Rev., 2002, vol. 18, pp. 838–842.
74. Laley-Bennis, D., Boillot, J., Bardein, C., Zirinis, P.,
Coste, A., Escudier, E., Chast, F., Peynegre, R.,
BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY
365
Slama, G., and Selam, J.L., Diabetes Metab., 2001,
vol. 27, pp. 372–377.
75. Gänslen, M., Klin. Wochenschr, 1925, vol. 4, pp. 71.
76. Wigley, W.F., Londono, J.H., Wood, S.H., Shipp, J.C.,
and Waldman, R.H., Diabetes, 1971, vol. 20, pp. 552–
556.
77. Cefalu, W.T., Skyler, J.S., Kourides, I.A., Lands-
chulz, W.H., Balgtas, C.C., Cheng, S.-L., and
Gelfand, R.A., Ann. Intern. Med., 2001, vol. 134,
pp. 203–207.
78. Stoever, J.A. and Palmer, J.P., Diab. Technol. Ther.,
2002, vol. 4, pp. 157–161.
79. Mitragotri, S., Blankschtein, D., and Langer, R., Sci-
ence, 1995, vol. 269, pp. 850–853.
80. Owens, D.R., Nature, 2002, vol. 1, pp. 529–540.
81. Reichard, O., Nilsson, B.-Y., and Rosenqvist, U.,
N. Engl. J. Med., 1993, vol. 329, pp. 304–309.
82. Wang, P.H., Lau, J., and Chalmers, T.C., Lancet, 1993,
vol. 341, pp. 1306–1309.
83. Hermansen, K., Fontaine, P., Kukolja, K.K., Peter-
kova, V., Leth, G., and Gall, M.A., Diabetologia, 2004,
vol. 47, pp. 622–629.
84. The Diabetes Control and Complications Trial
Research Group, Diabetes, 1997, vol. 46, pp. 271–286.
85. Kang, S., Brange, J., Burch, A., Volund, A., and
Owens, D.R., Diabetes Care, 1991, vol. 14, pp. 1057–
1065.
86. Østerberg, O., Erichsen, L., Ingwersen, S.H., Plum, A.,
Poulsen, H.E., and Vicini, P., J. Pharmacodynamics,
2003, vol. 30, pp. 221–235.
87. Starke, A.A., Heinemann, L., Hohmann, A., and
Berger, M., Diabet. Med., 1989, vol. 6, pp. 239–244.
88. Jehle, P.M., Micheler, C., Jehle, D.R., Breitig, D., and
Boehm, B.O., Lancet, 1999, vol. 354, pp. 1604–1607.
89. Kølendorf, K., Bojsen, J., and Deckert, T., Horm.
Metab. Res., 1983, vol. 15, pp. 274–278.
90. European Patent Application #214,826, 1989.
91. Mrke, E.A., Hoppe-Seyler’s Z. Physiol. Chem., 1979,
vol. 260, pp. 1619–1632.
92. Shoelson, S., Fickova, M., Haneda, M., Nahum, A.,
Musso, G., Kaiser, E.T., Rubenstern, A.H., and
Tager, H., Proc. Natl. Acad. Sci. USA, 1983, vol. 80,
pp. 7390–7394.
93. Kobayashi, Y., Biomed. Res., 1984, vol. 5, pp. 267–272.
94. Schwartz, G.P., Burke, G.T., and Katsoyannis, P.G.,
Proc. Natl. Acad. Sci. USA, 1987, vol. 84, pp. 6408–
6411.
95. Dreier, K., Diabetes Metab. Rev., 1992, vol. 8, pp. 259–
285.
96. Howey, D.C., Bowsher, R.R., Brunelle, R.L., and
Woodworth, J.R., Diabetes, 1994, vol. 43, pp. 396–402.
97. Lindholm, A., Best Practice & Research Clinical Gas-
troenterology, 2002, vol. 3, pp. 475–492.
98. Milicevic, Z., Profozic, V., Wyatt, J., Ristic, S., Wood-
worth, J.R., Seger, M., Kaliterna, D., Bates, P., and
Metelko, Z., Diabet. Med., 2001, vol. 18, pp. 562–566.
99. Tsui, E., Barnie, A., Ross, S., Parkes, R., and Zinman, B.,
Diabetes Care, 2001, vol. 24, pp. 1722–1727.
100. Lunt, H., Kendall, D., Moore, M.P., Scott, R.S., Cole, D.,
Frampton, C.M., and Cullens, M., Intern. Med. J., 2004,
vol. 34, pp. 320–323.
101. Blundell, T., Dodson, G., Hodgkin, D., and Merkola, D.,
Adv. Protein Chem., 1972, vol. 26, pp. 279–402.
Vol. 2 No. 4 2008
366 GUSAROV et al.
102. Fujiwara, M., Baba, T., Neugebauer, S., Hasegawa, K.,
Hosoya, E., Tanaka, K., Shimada, K., Yamada, D., and
Watanabe, T., Diabet. Med., 2004, vol. 21, pp. 285–297.
103. Schernthaner, G., Wein, W., Shnawa, N., Bates, P.C.,
and Birkett, M.A., Diabet. Med., 2004, vol. 21, pp. 279–
284.
104. Woodworth, J., Howey, D., and Bowsher, R., Diabetes,
1993, vol. 42, p. 54A.
105. Ter Braak, E.W., Woodworth, J.R., Bianchi, R., Cer-
imele, B., Erkelens, D.W., Thijssen, J.H., and Kurtz, D.,
Diabetes Care, 1996, vol. 19, pp. 1437–1440.
106. Altuntas, Y., Ozen, B., Ozturk, B., Sengul, A., Ucak, S.,
Ersoy, O., and Karul, S., Diabetes Obes.Metab., 2003,
vol. 5, pp. 371–378.
107. Roach, P., Woodworth, J., Gudat, U., Cerimele, B., Die-
bler, F., Pein, M., and Dreyer, M., Diabet. Med., 2003,
vol. 20, pp. 946–952.
108. Herz, M., Diabet. Med., 2002, vol. 19, pp. 917–923.
109. DeFellipis, M.R., Bakaysa, D.L., Bell, M.A.,
Heady, M.A., Li, S., Pye, S., Youngman, K.M., Rad-
ziuk, J., and Frank, B.H., J. Pharm. Sci., 1998, vol. 87,
pp. 170–176.
110. US Patent #5149777, 1992.
111. US Patent Application #0020013269, 2002.
112. Brange, J., Ribel, U., Hansen, J.F., Dodson, G.,
Hansen, M.T., Havelund, S., Malberg, S.G., Norris, F.,
Norris, K., and Snel, L., Nature, 1988, vol. 333,
pp. 679–682.
113. Brange, J., Diabetologia, 1997, vol. 40, pp. S48–S55.
114. Owens, D.R., Zinman, B., and Bolli, G.B., Lancet,
2001, vol. 358, pp. 739–746.
115. Heinemann, L., Kapitza, C., Starke, A.A., and Heise, T.,
Diabet. Med., 1996, vol. 13, pp. 683–684.
116. Lindholm, A., McEwen, J., and Riis, A.P., Diabetes
Care, 1999, vol. 22, pp. 801–805.
117. Home, P.D., Lindholm, A., Hylleberg, B., and Round,
P., Diabetes Care, 1998, vol. 21, pp. 1904–1909.
118. Bott, U., Ebrahim, S., Hirschberger, S., and Skovlund,
S.E., Diabet. Med., 2003, vol. 20, pp. 626–634.
119. Robinson, R.T.C.E., Harris, N.D., Ireland, R.H., Lind-
holm, A., and Heller, S.R., Br. J. Clin. Pharmacol.,
2003, vol. 55, pp. 246–251.
120. Pettitt, D.J., Ospina, P., Kolaczynski, J.W., and
Jovanovic, L., Diabetes Care, 2003, vol. 26, pp. 183–
186.
121. Airaghi, L., Lorini, M., and Tedeschi, A., Diabetes
Care, 2001, vol. 24, pp. 2000.
122. Yasuda, H., Nagata, M., Moriyama, H., Fujihira, K.,
Kotani, R., Yamada, K., Ueda, H., and Yokono, K., Dia-
betes Care, 2001, vol. 24, pp. 2008–2009.
123. Ushakova, O.V. and Shapiro, I.A., Probl. Endokrinol.,
2006, vol. 52, pp. 9–12.
124. Bode, B., Weinstein, R., Bell, D., McGill, J., Nadeau, D.,
Raskin, P., Davidson, J., Henry, R., Huang, W.-C., and
Reinhardt, R.R., Diabetes Care, 2002, vol. 25, pp. 439–
444.
125. Plank, J., Wutte, A., Brunner, G., Siebenhofer, A., Sem-
litsch, B., Sommer, R., Hirschberger, S., and Pieber, T.R.,
Diabetes Care, 2002, vol. 25, pp. 2053–2057.
126. Homko, C., Deluzio, A., Jimenez, C., Kolaczynski, J.W.,
and Boden, G., Diabetes Care, 2003, vol. 26, pp. 2027–
2031.
BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY
127. Von Mach, M.A., Brinkmann, C., Hansen, T., Weile-
mann, L.S., and Beyer, J., Exp. Clin. Endocrinol. Dia-
betes, 2002, vol. 110, pp. 416–419.
128. Bartolo, P.Di, Pellicano, F., Scaramuzza, A., Fabbri, T.,
Malandri, P., Miselli, V., Casetti, T., and Cannata, F.,
Diabetes Research Clinical Practice, 2006, vol. 74,
pp. S119–S122.
129. Rakatzi, I., Ramrath, S., Ledwig, D., Dransfeld, O.,
Bartels, T., Seipke, G., and Eckel, J., Diabetes, 2003,
vol. 52, pp. 2227–2238.
130. Rakatzi, I., Seipke, G., and Eckel, J., Biochem. Biophys.
Res. Commun., 2003, vol. 310, pp. 852–859.
131. Becker, R., Frick, A., Wessels, D., and Scholtz, H., Dia-
betes, 2003, vol. 52, p. A110.
132. Dailey, G., Rosenstock, J., Moses, R.G., and Ways, K.,
Diabetes Care, 2004, vol. 27, pp. 2363–2368.
133. US Patent #5547929, 1996.
134. US Patent #5970973, 1999.
135. Plakogiannis, R., Nathan, J.P., and Rosenberg, J.M.,
Drug Topics, 2000, vol. 144, pp. 41.
136. Bolli, G.B. and Owens, D.R., Lancet, 2000, vol. 356,
pp. 443–445.
137. Bolli, G.B., Di Marchi, R.D., Park, G.D., Pramming, S.,
and Koivisto, V.A., Diabetologia, 1999, vol. 42,
pp. 1151–1167.
138. Tan, C.Y., Wilson, D.M., and Buckingham, B., Pediat-
ric Diabetes, 2004, vol. 5, pp. 80–86.
139. Yki-Jarvinen, H., Eur. J. Clin. Invest., 2004, vol. 34,
pp. 410–416.
140. Alemzadeh, R., Ellis, J.N., Holzum, M.K., Parton, E.A.,
and Wyatt, D.T., Pediatrics, 2004, vol. 114, pp. e91–
e95.
141. Schober, E., Schoenle, E., Van Dyk, J., and Wernicke-
Panten, K., Diabetes Care, 2001, vol. 24, pp. 2005–
2006.
142. Raskin, P., Klaff, L., Bergenstal, R., Halle, J.P., Don-
ley, D., and Mecca, T., Diabetes Care, 2000, vol. 23,
pp. 1666–1671.
143. McKeage, K. and Goa, K.L., Drugs, 2001, vol. 61,
pp. 1599–1624.
144. Berger, M., Lancet, 2000, vol. 356, pp. 2013–2014.
145. Pieber, T.R., Draeger, E., Kristensen, A., and Grill, V.,
Diabet. Met., 2005, vol. 22, pp. 850–857.
146. Hermansen, K., Madsbad, S., Perrils, H., Kristensen, A.,
and Axelsen, M., Diabetes Care, 2001, vol. 24, pp. 296–
301.
147. Vague, P., Selam, J.-L., Skeie, S., Leeuw, I.D.,
Elte, J.W.F., Haahr, H., Kristensen, A., and Draeger, E.,
Diabetes Care, 2003, vol. 26, pp. 590–596.
148. Russell-Jones, D., Simpson, R., Hylleberg, B., Drae-
ger, E., and Bolinder, J., Clin. Ther., 2004, vol. 26,
pp. 724–736.
149. Kiess, W., Raile, K., Galler, A., and Kapellen, T., Dia-
betes Care, 2004, vol. 27, pp. 2567–2568.
150. Brunner, G.A., Sendhofer, G., Wutte, A., Ellmerer, M.,
Sogaard, B., Siebenhofer, A., Hirschberger, S., Krejs, G.J.,
and Pieber, T.R., Exp. Clin. Endocrinol. Diabetes, 2000,
vol. 108, pp. 100–105.
Vol. 2 No. 4 2008