Title: Glucose dependent
release of insulin from glucose sensing insulin derivatives
United States Patent: 7,316,999
Issued: January 8, 2008
Thomas (Klampenborg, DK), Havelund; Svend (Bagsvaerd, DK), Markussen; Jan
(Herlev, DK), Ostergaard; Soren (Bronshoj, DK), Ridderberg; Signe (Lyngby,
DK), Balschmidt; Per (Espergaerde, DK), Schaffer; Lauge (Copenhagen, DK),
Jonassen; Ib (Valby, DK)
Assignee: Novo Nordisk A/S
Appl. No.: 09/870,884
Filed: May 31, 2001
Executive MBA in Pharmaceutical Management, U. Colorado
Insulin derivatives having a built-in
glucose sensor, capable of delivering insulin from a depot as a function
of the glucose concentration in the surrounding medium (e.g. tissue), such
that the rate of insulin release from the depot increases with an
increased glucose concentration and decreases with a decreased glucose
Description of the
FIELD OF THE INVENTION
The present invention relates to insulin derivatives having a built-in
glucose sensor, capable of delivering insulin from a depot as a function
of the glucose concentration in the surrounding medium (e.g. tissue).
BACKGROUND OF THE INVENTION
Diabetes is a general term for disorders in man having excessive urine
excretion as in diabetes mellitus and diabetes insipidus. Diabetes
mellitus is a metabolic disorder in which the ability to utilize glucose
is partly or completely lost.
Since the discovery of insulin in the 1920's, continuous strides have been
made to improve the treatment of diabetes mellitus. To help avoid extreme
glycaemia levels, diabetic patients often practice multiple injection
therapy, whereby insulin is administered with each meal. Many diabetic
patients are treated with multiple daily insulin injections in a regimen
comprising one or two daily injections of a protracted insulin composition
to cover the basal requirement, supplemented by bolus injections of a
rapid acting insulin to cover the meal-related requirements.
Insulin compositions having a protracted profile of action are well known
in the art. Thus, one main type of such insulin compositions comprises
injectable aqueous suspensions of insulin crystals or amorphous insulin.
Typically, the insulin in these compositions is provided in the form of
protamine insulin, zinc insulin or protamine zinc insulin.
When human or animal insulin is brought to form higher associated forms,
e.g. in the presence of Zn.sup.2+-ions, precipitation in the form of
crystals or amorphous product is the result; see for example pages 20 27
in Jens Brange (editor), Galenics of Insulin, Springer Verlag (1987).
Thus, at pH 7, addition of 6 Zn.sup.2+ ions per insulin hexamer to a
solution of porcine insulin will lead to an almost complete precipitation
of the insulin.
SUMMARY OF THE INVENTION
We have invented new insulin derivatives from which the release of insulin
from an injected or inhaled depot thereof is glucose dependent. In the
depot, the insulin derivative modified with a glucose sensor is either in
the crystalline state or in a highly aggregated soluble state. Both states
bring about a protracted absorption from the site of injection. The
solubility of the crystals and the state of aggregation in the soluble
aggregates are influenced by the glucose concentration in the surrounding
tissue. Increasing the concentration of glucose promotes dissolution of
the crystals and dissociation of the soluble aggregates.
The dose and volume of the subcutaneous or intramuscularly injected depot
is similar to that of the ordinary basal insulin compositions, meant to
cover basal insulin supply by injection once or twice daily. Inhaled
insulin compositions of insulin derivatives having glucose sensor may be
taken several times during the day, typically before or during the meals.
Soluble insulin derivatives featuring lipophilic substituents, capable of
forming high molecular weight aggregates having a higher molecular weight
than aldolase (Mw=158 kDa), have been disclosed in WO 99/21888 (Novo
Nordisk) the contents of which is hereby incorporated in its entirety by
reference. The release of insulin derivative from such aggregates appears
to depend upon diffusion controlled disintegration of the soluble
aggregates. However, some high molecular aggregates, formed from selected
insulin derivatives, disintegrate and form smaller aggregates when glucose
is introduced into a buffer solution containing an aggregated insulin
derivative. The higher the glucose concentration, the more thorough is the
disintegration of the aggregated derivative.
The state of aggregation and the power of glucose to diminish this state
can be demonstrated by gel filtration of the aggregated insulin
derivatives in buffers containing varying concentrations of glucose in the
The increased release of insulin derivative from subcutaneous depots can
be demonstrated by the different levels of the insulin derivative in the
plasma of pigs clamped at various blood glucose levels, e.g. 5 and 10 mM,
after injection of the same dose of the insulin derivative.
This new concept of glucose dependent insulin release complies with the
convenience of the state of the art injection regimens of insulin therapy,
and requires neither surgery nor the danger associated with storage of
large implanted depots in the body.
DETAILED DESCRIPTION OF THE INVENTION
The expression "insulin derivative" as used herein (and related
expressions) refers to human insulin or an analogue thereof in which at
least one organic substituent is bound to one or more of the amino acids.
By "analogue of human insulin" as used herein (and related expressions) is
meant human insulin in which one or more amino acid residues have been
deleted and/or replaced by other amino acid residues, including non-codeable
amino acid residues, or human insulin comprising additional amino acid
residues, i.e. more than 51 in total. The amino acid sequence of human
insulin is given i.a. in The Merck Index, 11th Edition, published in 1989
by Merck & Co., Inc., page 4888. The sequence of human insulin, A and B
chains, is as follows: Human insulin, A chain:
G-I-V-E-Q-C-C-T-S-I-C-S-L-Y-Q-L-E-N-Y-C-N (SEQ ID NO: 1); Human insulin, B
chain: F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-P-K-T (SEQ ID
NO: 2). Analogs of human insulin exemplified in the instant invention
include, but are not limited to the following: Human insulin, Asp.sup.B28
chain analog: F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-D-K-T
(SEQ ID NO: 3); Human insulin, Lys.sup.B28, Pro.sup.B29 B chain analog:
F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-K-P-T (SEQ ID NO:
4); Human insulin, Gly.sup.A21 A chain analog:
G-I-V-E-Q-C-C-T-S-I-C-S-L-Y-Q-L-E-N-Y-C-G (SEQ ID NO: 5); Human insulin,
Lys.sup.B3, Ile.sup.B28 B chain analog:
F-V-K-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-I-K-T (SEQ ID NO:
6); Human insulin, Asp.sup.A21 A chain analog:
G-I-V-E-Q-C-C-T-S-I-C-S-L-Y-Q-L-E-N-Y-C-D (SEQ ID NO: 7); Human insulin,
des.sup.B30 B chain analog:
F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-P-K (SEQ ID NO: 8);
Human insulin, Phe.sup.B26 B chain analog:
F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-F-T-P-K-T (SEQ ID NO:
9); Human insulin, Orn.sup.B30 B chain analog:
F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-P-K-X (SEQ ID NO:
X=ornithine; Human insulin, Dap.sup.B30 B chain analog:
F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-P-K-X (SEQ ID NO:
X=diaminopropionic acid; Human insulin, Lys.sup.B30 B chain analog:
F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-P-K-K (SEQ ID NO:
12); Human insulin, Pro.sup.B30 B chain analog:
P-F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-P-K-T (SEQ ID NO:
13); Human insulin, Asp.sup.B30 B chain analog:
F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-P-K-D (SEQ ID NO:
14); Human insulin, Glu.sup.B30 B chain analog:
F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-P-K-E (SEQ ID NO:
15); Human insulin, Ams(BOC).sup.B30 B chain analog:
F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-P-K-X (SEQ ID NO:
X=O-aminoserine(BOC); and Human insulin, Dab.sup.B30 B chain analog:
F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-P-K-X (SEQ ID NO:
By "depot" is meant the amount of subcutaneous or intramuscularly injected
or inhaled insulin composition, either in the form of crystalline
compositions, such as NPH insulin and Lente insulin, or as solutions, such
as albumin binding or soluble aggregating or acid solutions of
neutral-precipitating, of insulin analogues or insulin derivatives.
By "absorption" is meant the process by which the insulin in the depot is
transferred to the circulation.
By "glucose sensor" is meant a chemical group, capable of binding to or
reacting with glucose. The glucose sensor is part of the insulin molecule.
For reversible binding, the dissociation constant, K.sub.d, of the
sensor-glucose complex is usually in the range from 0.01 .mu.M to 100 mM,
for example from 1 .mu.M to 20 mM or from 1 mM to 20 mM or from 1 mM to
100 mM. Examples of reversible glucose sensors are organic borates,
preferably aryl boronates or other borates, where the attachment to an
insulin derivative is via a carbon-boron bond. Alkyl boronates are
oxidatively labile and often unstable (Snyder, Kuck and Johnson, J. Am.
Chem. Soc 1938, 60, 105). Boronate sensors that bind glucose under
physiological conditions are preferred. Simple aryl boronates, such as
phenyl boronate, binds glucose only at relatively high pH, >9 (Shinkai and
Takeuchi, Trends Anal. Chem. 1996, 15, 188). Acidic boronates, which bind
glucose at physiological pH, are preferred. Examples of such boronate
glucose sensors are aminomethyl-aryl-2-boronates (Bielecki, Eggert and
Norrild, J. Chem. Soc., Perkin Trans 2 1999, 449), other boronates with
amino groups in the vicinity (Shiino et al, J. Controlled Release 1995,
37, 269), or aryl boronates substituted with electron-withdrawing groups (Eggert
et al., J. Org. Chem. 1999, 64, 3846), e.g. sulfo-, carboxy-, nitro-,
cyano-, fluoro-phenyl boronates, pyridine boronates, pyridinium boronates
or their combinations. Diboronates may be employed to insure
glucose-selectivity over for instance fructose.
Such acidic boronates assume a tetrahedral configuration in aqueous
solvent at physiological pH, thereby allowing binding of glucose.
Reversible glucose sensors may also be peptides or pseudopeptides,
optionally containing boronates. Examples of irreversible glucose binders
are oxyamines and hydrazines, which react with glucose to form oximes and
hydrazones (Veprek and Jezek, J. Peptide Sci. 1999, 5, 203; Peri, Dumy and
Mutter, Tetrahedron 1998, 54, 12269). Examples of useful oxyamine
functions are aminoxyacetic acid, AOA (Vilaseca et al. Bioconjugate Chem.
1993, 4, 515), and O-aminoserine, Ams (Spetzler and Hoeg-Jensen, J. Pept.
Sci. 1999, 5, 582).
In theory, one way to obtain tight glucose control would be to couple a
glucose sensor, positioned in the tissue of the patient, to a computer
that controls an insulin pump. The pump is via a catheter connected to a
needle inserted under the skin. However, it appears as if such a feed back
control system has not yet been implemented, possibly because of lack of
stable and reliable of glucose sensors. Glucose sensors inserted in the
tissue appears to get overgrown with fibrin, and it appears that
non-invasive sensors, e.g. based on infrared optics, remain to be invented
Attempts to develop systems for glucose dependent release of insulin from
a depot has previously been described. A carbohydrate binding lectin, such
as concanavalin A, immobilized to a solid matrix, such as hollow fibres,
binds an insulin derivative substituted with a carbohydrate moiety, such
as maltotriose, maltose or dextran. The matrix allows diffusion of
dissolved glucose and insulin derivative. As the systemic glucose
concentration rises, glucose displaces increasing amounts of the insulin
derivative from the matrix, thus making more insulin available to the
circulation, and thereby to the insulin receptors, when it is needed. It
appears as if none of these lectin based systems have been implemented
clinically, probably due to the inconvenience of implanting the insulin
containing matrix in the body, and to the danger of carrying a large
insulin depot within the body.
Another suggested glucose-controlled insulin release system is based on
the glucose oxidase catalysed conversion of glucose to gluconic acid. The
glucose oxidase is immobilized to a matrix, e.g. of ethylene/vinyl acetate
copolymer, and the insulin or insulin derivative is trapped in the matrix
in the solid state. As the pH is lowered locally due to the production of
gluconic acid the solubility of insulin increases. Thus, the rate of
release of soluble insulin from the solid state reflects the glucose
concentration. Likewise, it appears as if none of these glucose oxidase
based systems have been implemented clinically, possibly for the same
Furthermore, attempts to provide glucose controlled insulin release from a
depot in which the glucose sensing molecular structure is part of a
matrix, i.e. a soluble or solid polymer have been made.
Another type of protracted insulin compositions is a solution having a pH
value below physiological pH from which the insulin analogue will
precipitate when the solution is injected because of the rise in the pH
value to physiological pH when the solution has been injected. This
principle may be combined with the present invention by incorporation of
the glucose-sensor in the insulin analogue. In addition to the glucose
sensor these analogues have an amino acid residue in position A21 which is
stable at pH values as low as practically useful in solutions to be
injected. Examples of suitable amino acid residues at position A21 are
glycine, serine and alanine. Also, the insulins have mutations to increase
the net charge of the molecule by about 2 units, e.g. Thr in position B27
can be substituted with Arg and Thr-OH in position B30 can be substituted
with Thr-NH.sub.2 or basic residues can be added, e.g. B31 B32 Arg-Arg.
Soluble insulin derivatives having a lipophilic substituent linked to the
.epsilon.-amino group of a lysine residue in any of the positions B26 to
B30 have been described in the literature. Such derivatives have a
protracted profile of action after subcutaneous injection as compared to
soluble human insulin, and this protracted action has been explained by a
reversible binding to albumin in subcutis, blood and peripheral tissue.
An additional mechanism of prolonging the action of some of the soluble
insulin derivatives featuring a lipophilic substituent has been disclosed,
i.e. derivatives capable of forming high molecular weight aggregates,
having a higher molecular weight than aldolase (Mw=158 kDa) when analysed
in a defined gel filtration system.
In healthy persons, the blood glucose concentration is about 5 mM, rising
to about 7 mM after the meals. Today, even when applying the most advanced
insulin treatment, using rapid acting insulins for meal-related injections
and soluble depot insulin for basal insulin based on frequent monitoring
of blood glucose, diabetics often experience glucose concentrations out of
control. If too much insulin is administered, so that glucose
concentrations get below about 3 mM, hypoglycaemic events might occur,
leading to unconsciousness. When too little insulin is administered and
glucose concentrations rises to about 20 mM, acetone appears in the blood
and gives rise to diabetic ketoacidosis and, eventually, diabetic coma.
However, it is desirable to control the blood glucose concentration of
diabetics more tightly, as close to the 5 mM as possible, in order to
diminish diabetic late complications. The DCCT (Diabetes Complication
Clinical Trial) study from 1993 in USA examined the development of
diabetic complications in type 1 diabetics during 9 years (N Engl J Med
1993, 329, 977 986). The UKPDS (United Kingdom Prospective Diabetes Study)
studied the development of complications in type 2 diabetics during 15
years (Lancet 1998, 352, 854 865). Even though the pattern of
complications differs between these two types of diabetics both
investigations conclude that a tight control of blood glucose results in a
marked reduction of complications. Thus, there is an unmet medical need
for means to obtain glucose control in diabetics closer to the normal
value of 5 mM.
In one preferred embodiment the present invention is based on the
discovery of soluble and aggregated forms of insulin derivatives, wherein
the state of aggregation is being influenced by glucose. The aggregate is
preferably soluble in water at neutral pH, in the range of 6.8 to 8.5. The
soluble, aggregated forms of insulin derivatives dissociates slowly after
subcutaneous injection, making them suitable for a long-acting insulin
composition, the advantage being that the composition contains no
precipitate. The higher the concentration of glucose is in the tissue the
higher the rate of dissociation and of the subsequent absorption. The
advantages of soluble rather than suspended compositions are higher
precision in dosing, avoidance of shaking of the vial or pen, allowance
for a thinner needle meaning less pain during injection, easier filling of
vials or cartridge and avoidance of a ball in the cartridge used to
suspend the precipitate in the absence of air.
The apparent volume of elution of aggregates, as estimated by the
distribution coefficient, K.sub.AV, changes to a higher value when the
glucose concentration is increased from 0 to 20 mM or to 100 mM, as
determined by gel filtration using a Bio-Gel P300 (BIO-RAD). In order to
achieve an optimal effect of glucose on the state of aggregation in this
experiment, the concentration of sodium chloride should be decreased just
to obtain an aggregation about the size of aldolase (i.e. the K.sub.AV
value of 0.10).
The aggregated form can be observed for insulin derivatives under
conditions where the hexameric unit is known to exist for most insulins.
Thus, in a preferred embodiment, the aggregated form is composed of
hexameric subunits, preferably of at least 4, more preferably 5 to 500,
hexameric subunits. Any hexameric subunit of the aggregated forms of the
compounds of this invention may have any of the known R.sub.6,
R.sub.3T.sub.3, or T.sub.6 structures, T.sub.6 being the preferred form (Kaarsholm,
Biochemistry 28, 4427 4435, 1989).
Substances like Zn.sup.2+ known to stabilise the hexameric unit are also
found to stabilise the aggregated form of some insulin derivatives. The
building blocks forming the aggregates may be the hexameric units known
from the X-ray crystallographic determined structure of insulin (Blundell,
Diabetes 21 (Suppl. 2), 492 505, 1972). Ions like Zn.sup.2+, known to
stabilise the hexameric unit as 2 or 4 Zn.sup.2+/hexamer complexes
(Blundell, Diabetes 21 (Suppl. 2), 492 505, 1972), are essential for the
formation of aggregates for most insulin analogues and derivatives. Thus,
compositions of glucose dependent aggregating insulin derivatives
according to this invention preferably comprises at least 2 zinc ions,
more preferably 2 to 5 zinc ions, still more preferably 2 to 3 zinc ions,
per 6 molecules of insulin derivative. Moreover, the compositions
advantageously comprise at least 3 molecules of a phenolic compound per 6
molecules of insulin derivative. In the central cavity of the 2 Zn.sup.2+/hexamer
structure 6 residues of Glu.sup.B13 provide binding sites for up to 3
Ca.sup.2+ ions (Sudmeier et al., Science 212, 560 562, 1981). Thus,
addition of Ca.sup.2+ ions stabilises the hexamer and may be added to the
pharmaceutical compositions, on the condition that the insulin derivative
remains in solution.
The disappearance half-time of the aggregate of the invention after
subcutaneous injection in healthy human subjects, having normal blood
glucose concentrations about 5 mM, is preferably as long as or longer than
that of a human insulin NPH composition.
In a particularly preferred embodiment of the present invention, the
aggregate is composed of insulin derivatives, which have an albumin
binding which is lower than that of Lys.sup.B29(N.sup..epsilon.-tetradecanoyl)
des(B30) human insulin.
The substituent at the lysine residue of the insulin derivative of the
aggregate according to the invention is preferably a lipophilic group
containing from 6 to 40 carbon atoms.
Examples of suitable lipophilic substituents (groups) are the acid
residues of lithocholic acid, cholic acid, hyocholic acid, deoxycholic
acid, chenodeoxycholic acid, ursodeoxycholic acid, hyodeoxycholic acid or
In another preferred embodiment, the lipophilic substituent is connected
to the .epsilon.-amino group of a lysine residue using an amino acid
linker. According to this embodiment the lipophilic substituent is
advantageously connected to a lysine residue via a .gamma.- or an .alpha.-glutamyl
linker or via a .beta.- or an .alpha.-aspartyl linker.
In yet another preferred embodiment the lipophilic substituent comprises
the glucose sensor in the form of a borate group, an aryl boronate, an
amino aryl boronate or a glucose binding peptide.
The present invention furthermore provides novel insulin derivatives
capable of forming aggregates, in which the degree of aggregation is
inversely correlated to the glucose concentration. These insulin
derivatives may be provided in the form of aggregates in pharmaceutical
compositions or, alternatively, they may be provided in a non-aggregated
form in pharmaceutical compositions, in which case the aggregates form
after subcutaneous injection of said compositions.
Accordingly, the present invention furthermore is concerned with
pharmaceutical compositions comprising an aggregate of insulin derivatives
or non-aggregated insulin derivatives, which form aggregates after
subcutaneous injection, the degree of aggregation being inversely
correlated to the glucose concentration. The dissociation of the soluble
insulin polymers into soluble insulin hexamers by the action of glucose
molecules can be described by the following equation: insulin polymer+n
glucose .revreaction.insulin hexamer where n is the number of glucose
molecules required to break the polymeric insulin network, releasing the
insulin hexamers from the network. The advantage of n being larger than 1
is apparent from FIG. 1 (see Original Patent), which shows that increasing
n from 1 to 6 increases the steepness of the curve for the fraction of
free insulin hexamers over polymer, bound insulin hexamers. Thus, a faster
release of insulin at a high glucose concentration, and a slower release
at a low glucose concentration, is possible by the multiple interactions
between insulin hexamers than by a mechanism involving just one bond.
Preferably, the pharmaceutical composition according to the present
invention comprises aggregates, a substantial fraction of which have a
higher molecular weight than aldolase as determined by gel filtration
using the medium of the composition as eluent.
In another embodiment, a pharmaceutical composition comprises both
aggregating and rapid acting insulin analogues, the latter preferably
being human insulin or one of the insulin analogues Asp.sup.B28 human
insulin, Lys.sup.B28Pro.sup.B29 human insulin,
Gly.sup.A21,Lys.sup.B3,Ile.sup.B28 human insulin,
Asp.sup.A21,Lys.sup.B3,Ile.sup.B28 human insulin or des(B30) human
insulin. Such a composition will provide both a rapid onset of action and
a prolonged profile of action, the latter being influenced by the blood
glucose concentration of the diabetic patient. In case the two insulins of
the mixture form mixed hexamers both will be under influence of the blood
In this embodiment, the pharmaceutical composition preferably comprises
aggregating insulin and rapid acting insulin in a molar ratio of from
90:10 to 10:90.
The slow dissociation of the aggregated forms may be further slowed down
in vivo by the addition of physiological acceptable agents that increase
the viscosity of the pharmaceutical composition. Thus, the pharmaceutical
composition according to the invention may furthermore comprise an agent
that increases the viscosity, preferably polyethylene glycol,
polypropylene glycol, copolymers thereof, dextrans and/or polylactides.
In yet another embodiment, the insulin derivative containing a glucose
sensing group is prepared as a crystalline NPH composition, using
protamine to form the crystals, or as a crystalline Lente composition,
using Zn.sup.2+-ions in the crystals. In these cases the rate of
dissolution of the crystals is enhanced by the interaction between glucose
and the glucose sensing group.
In yet another embodiment, the protracted insulin compositions are
solutions having a pH value below physiological pH from which the insulin
analogue will precipitate because of the rise in the pH value to
physiological pH when the solution has been injected. Such analogues are
described in EP 0 254 516 B1 (Novo Nordisk) and EP 0 368 187 B1 (Hoechst).
These analogues have an amino acid residue in position A21 which is stable
at pH values as low as practically useful in solutions to be injected.
Examples of suitable amino acid residues at position A21 are glycine,
serine or alanine. Also, the insulins have mutations to increase the net
charge of the molecule by about 2, e.g. Thr in position B27 can be
substituted with Arg and Thr-OH in position B30 can be substituted with
Thr-NH.sub.2 or have additional basic residues, e.g. B31 B32 Arg-Arg. When
this principle is combined with the present invention by incorporation of
the glucose-sensor in these insulin analogues, the solubility of the
crystals is enhanced by the interaction between glucose and the glucose
sensing group, facilitating the absorption.
Sites enabling the attachment of a glucose sensor are the N-terminal amino
groups of glycine A1 and phenylalanine B1 and the .epsilon.-amino group of
lysine B29. One or more additional or alternative lysine residues may be
incorporated for this purpose, e.g. in position B3 or B28. Furthermore the
glucose sensor may be incorporated as part of the peptide chain,
preferably in the C-terminal part of the B-chain.
The pharmaceutical composition preferably further comprises a buffer
substance, such as a phosphate, for example sodium phosphate, glycine or
glycylglycine buffer, an isotonicity agent, such as sodium chloride or
glycerol, and phenol and/or m-cresol as a preservative. Optionally,
mannitol or sorbitol can be added as isotonicity agents and the resulting
interaction with the glucose sensor can be utilized to adjust stability
and the release profile of the composition. Among the auxiliary substances
of a pharmaceutical composition according to the present invention, the
sodium chloride, used as isotonic agent, the zinc- and optionally calcium
ions, which promote and stabilize the hexamer formation, are particularly
important since they facilitate the aggregation of the insulin derivative
in the composition and thereby effectively prolong the time of
disappearance from the site of injection. A pharmaceutical composition
according to the invention preferably comprises chloride ions in a
concentration of 5 to 150 mM.
In pharmaceutical compositions, the concentration of the glucose-sensing
insulins of the present invention is generally in the range from 0.1 to 15
mM for example from 0.1 to 2 mM. The amount of zinc contained in the
compositions is 0.3 0.9% by weight relative to the insulin derivative.
Phenolic compounds like phenol or m-cresol or mixtures thereof are
suitably applied in a total concentration of from 5 to 50 mM, and chloride
ions in a concentration of from 10 mM to 100 mM.
The present invention furthermore relates to a method of treating diabetes
mellitus comprising administering to a person in need of such treatment an
effective amount of water-soluble aggregates of insulin derivatives
according to the invention or effective amount an insulin derivative
according to the invention, capable of forming water-soluble aggregates
upon subcutaneous injection, aggregate size depending on the glucose
The optimal dose level for any patient will depend on a variety of factors
including the efficacy of the specific human insulin derivative employed,
the age, body weight, physical activity, and diet of the patient, on a
possible combination with other drugs, and on the severity of the case of
diabetes. It is recommended that the daily-dosage of the human insulin
derivative of this invention be determined for each individual patient by
those skilled in the art in a similar way as for known insulin
The glucose sensor building blocks used in preparation of the
glucose-sensing insulins can be prepared as described in the included
examples. The insulin derivatives of the invention can be prepared by the
general methods disclosed in WO 95/07931 (Novo Nordisk A/S), WO 96/00107
(Novo Nordisk A/S), WO 97/31022 (Novo Nordisk A/S), WO 98/02460 (Novo
Nordisk A/S), EP 511 600 (Kuraray Co. Ltd.) and EP 712 862 (Eli Lilly).
Some of the derivatives listed in the aforementioned patent applications,
and described in the publications of Markussen, Diabetologia 39, 281 288,
1996; Kurzhals, Biochem J. 312, 725 731, 1995; Kurzhals, J. Pharm Sciences
85, 304 308, 1996; and Whittingham, Biochemistry 36, 2826 2831, 1997 as
being protracted due to the albumin binding mechanism, do also posses the
ability to form high molecular weight soluble aggregates. Lys.sup.B29 (N.sup..epsilon.-lithocholyl-.gamma.-glutamyl)
des(B30) human insulin from WO 95/07931 and Lys.sup.B29(N.sup..epsilon.
.omega.-carboxyheptadecanoyl) des(B30) human insulin from WO 97/31022 are
examples of insulin derivatives capable of forming high molecular weight
soluble aggregates at neutral pH.
Claim 1 of 26 Claims
1. A crystalline, or soluble
aggregate or aggregate-forming insulin derivative, wherein the insulin
derivative comprises a lipophilic substituent, and a glucose-sensing group,
wherein the glucose-sensing group is an aryl boronate group, wherein the
glucose-sensing group is built into a substituent capable of effecting the
formation of high molecular aggregates, and wherein the substituent causing
aggregation is a lipophilic group.
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