WO2009033588A2 - Self-controlled insulin delivery system - Google Patents

Abstract

The present invention relates to a glucose sensitive vesicle (preferably a multi-lamellar liposome) comprising an insulin-independent glucose transporter integrated into the membrane of the liposome, which further comprises glucokinase, insulin, Mg++ and ATP. Glucose diffuses into the liposome depending on its concentration and will be converted to glucose-6-phosphate. The accumulation of glucose-6-phosphate eventually causes the vesicle to burst. Thus, these liposomes allow an automatic adjustment of insulin levels in a glucose-dependent manner and are, therefore, useful for the treatment of diabetic patients.

Description

SELF-CONTROLLED INSULIN DELIVERY SYSTEM

The present invention relates to a self-controlled insulin delivery system.

Type 1 diabetics need to control their glucose-level continually to keep it within the range of 80-120 mg/dl. Depending on the glucose level, measures have to be taken, e.g. intake of carbohydrates in the case of hypoglycemia, or injection of insulin in the case of hyperglycemia. This means that in order to maintain blood glucose it is necessary to adjust them.

At present there is no method to automatically adjust insulin release depending on blood glucose levels.

In the prior art, delivery of insulin enclosed in liposomes has been described. For example, Tragi et al. (Wien Klin Wochenschr 1979, 91(13), 448-51) describe the use of liposomes comprising egglecithin, dicetylphosphate and cholesterol in which insulin is encapsulated. This reference also suggests the use of multi-lamellar liposomes. Spangler (Diabetes Carel 1990, 13(9), 911-22) describes the targeting of insulin to the liver via liposomes. Dapergolas and Gregoriadis (Lancet 1976, 2 (7990), 824-7) also disclose the administration of insulin via liposomes, but mentions that administration of free insulin is more effective than administration of liposome- entrapped insulin.

The present invention provides liposomes which can be used in a method to automatically adjust insulin levels in a glucose dependent manner.

It is known that in pancreatic beta-islet cells of the healthy individual, stored insulin is released in a glucose-dependent manner from vesicles. Glucose diffuses into the cells via the glucose transporter GLUT2. Thus, GLUT2 is considered to be a "glucose sensor". Within the cells, the enzyme glucokinase (GCK) converts glucose to glucose-6-phosphate. This mediates the release of insulin from vesicles by exocytosis. In the pancreas, glucokinase is the glucose sensor for insulin release.

The present invention relates in particular to vesicles comprising a glucose sensor integrated into the membrane of said vesicle, which further comprises glucokinase, insulin or analogs thereof, Mg++ and ATP. The term insulin as used herein refers to both insulin and to analogs of insulin. Preferably, said vesicle is a liposome.

The term vesicle, as used herein, relates to a small and enclosed compartment, which comprises at least one membrane enclosing the compartment. The term compartment relates to the core of the vesicle. The membrane separates the contents of the core from the outside environment of the vesicle. The term membrane as used herein refers to a lipid bilayer enclosing a compartment.

The term liposome as used herein relates to a spherical vesicle with a membrane comprising a phospholipid and a cholesterol bilayer. The term lipid as used herein relates to an amphiphilic class of hydrocarbon-containing organic compounds.

In the present invention, insulin is encapsulated in a vesicle comprising insulin independent glucose sensors. Preferably, said insulin is human insulin (Seq ID No. 4) or porcine insulin (Seq ID No. 5) or an analog thereof. Preferred analogs are Glulysine insulin (human insulin Lys(B3)-Glu(B29)); Lispro insulin (human insulin Lys(B28)-Pro(B29)); Aspart insulin (human insulin Asp(B28)); Glargine insulin (human insulin Gly(A21)-L- Arg(B31, B32)) or Detemir insulin (Thr(B30) is deleted, Lys(B29) is ε-myristoylated) or combinations thereof.

Preferably, said glucose sensor which is integrated into the membrane of the liposome is GLUTl, GLUT2 or GLUT3. More preferably, said GLUTl, GLUT2 or GLUT3 are human GLUTl, human GLUT2 or human GLUT3. Human Glucose transporter

GLUT2 is defined by Seq ID No. 6. Human Glucose transporter GLUT2 is defined by Seq ID No. 1. Human Glucose transporter GLUT3 is defined by Seq ID No. 2.

The liposome may further comprise Zn++.

In a preferred embodiment of the vesicles hereinbefore described, said glucokinase is human glucokinase (hexokinase 4, EC 2.7.1.2) (Seq ID No. 3).

The concentration of Mg++ is equal or higher than the dissociation constant of glucokinase. Preferably, said concentration is between 0.5 to 15 mM, more preferably between 2 to 10 mM.

ATP (adenosine triphosphate) is required for the conversion of glucose to glucose-6- phosphate by glucokinase inside the vesicles. The ATP concentration is preferably >10 mM, more preferably >30 mM.

The vesicles hereinbefore described may further comprise multiple layers, each of which comprises the ingredients listed above. Such liposomes are also known as multi- lamellar vesicles (MLVs). Such vesicles in which protein drugs are encapsulated are e.g. disclosed in Anderson et al. 1994, CYTOKINE 6, p.92-101. The term multi-lamellar liposome (MLV) as used herein relates to a liposome with a multiple layer structure wherein said layers are separated by aqueous medium.

The vesicles may comprise a phospholipid and cholesterol bilayer.

Said phospholipid is selected from one or more phospholipids of the group comprising DMPC, DOTAP, DOPC, MOPC, egg phosphatidylethanolamine, DOPE, egglecithin, dicetylphosphate, dipalmitoyl lecithin, digalactosyl diglyceride, sphingosine, lecithin, egg phosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylcholine and/or long-chain or intermediate-chain phosphatidylcholine.

The vesicles may, as a non-limiting example, comprise DMPC, DOTAP, DOPC, MOPC, egg phosphatidylethanolamine,DOPE and/or cholesterol. As used herein, DMPC is dimyristoyl phosphatidyl choline, DOTAP is l,2-Dioleoyl-3-Trimethylammonium- Propane, DOPC is l^-di-oleoyl-sn-glycero-S-phosphocholine, DOPE is dioleylphosphatidylethanolamine, MOPC is l-myristoyl-2-oleyl-phosphatidylcholine and Cholesterol is also known as 10,13-dimethyl-17-(6-methylheptan-2-yl)- 2,3,4,7,8,9,1 l^.U.lSJό.^-dodecahydrolH-cyclopentataJphenanthren-S-ol.

The vesicles may also comprise a mixture of lipids. In one embodiment, the mixture comprises egglecithin, dicetylphosphate and cholesterol. In another embodiment, the mixture comprises cholesterol, dipalmitoyl lecithin, digalactosyl diglyceride and sphingosine. In another embodiment, the vesicle comprises lecithin and cholesterol, preferably in a molar ratio of 9:2. In another embodiment, the vesicle comprises egg phosphatidylcholine and cholesterol or dipalmitoylphosphatidylcholine and cholesterol, preferably dipalmitoylphosphatidylcholine and cholesterol. In a further embodiment, the vesicle comprises phospholipid, cholesterol and triglyceride. Furthermore, the vesicle may comprise long-chain phosphatidylcholine or intermediate- chain phosphatidylcholine.

The vesicles may additionally also comprise a neutral lipid. Said neutral lipid may be a triglyceride. Such a triglyceride may be a short-chain triglyceride.

Furthermore, all embodiments of the vesicles described above may additionally comprise PEG (polyethylene glycol) on the surface of the liposome.

Expression and purification of insulin, preferably human insulin, is well known in the art (see e.g. Kjeldsen et al., Biotechnol. Appl. Biochem. 1999, 29, 79-86). Expression and purification of glucokinase is also well known in the art (e.g. in Lange et al, Biochem. J. (1991), 277, 159-163).

GLUTl maybe isolated from erythrocyte membranes or expressed in a suitable expression system as described below for GLUT2.

GLUT2 can be expressed in a suitable expression system, e.g. in RINm5F cells (Eisner et al., Diabetologia), and purified, e.g. by affinity chromatography using a GLUT2-specific antibody.

GLUT3 may be isolated from neuron membranes or expressed in a suitable expression system as described above for GLUT2.

The use of liposomes for encapsulation of drugs is well known (Katre et al., Am J

Drug Deliv 2004, 2 (4), p 213-227).

Methods of preparing liposomes comprising drugs are well known in the art. The present invention also relates to a method of preparing the vesicles hereinbefore described comprising: adding an aqueous solution comprising a glucose sensor integrated into the membrane of a vesicle, glucokinase, insulin or analogs thereof, Mg++ and ATP to a lipid- organic phase to obtain an emulsion followed by removing the organic.

The present invention also relates to a method of preparing a vesicle comprising the steps of (a) sterilizing powdered lipid, (b) adding sterile powdered lipid to aqueous protein, (c) rapidly mixing the suspension of b), (d) bath sonicating said mixture, (e) freezing said mixture, (f) thawing said mixture, (g) repeating steps c) to f) two times.

For sterilization of the lipid powder or solution, 15000 to 150 000 rads of gamma irradiation are used, preferably using a 137Cs source. The protein mixture is present in a 0.9 % saline buffer comprising Mg++, Zn++ and ATP as described above. To this solution, the sterile lipid is added. A lipid to aqueous ratio of 10 to 1000 mg lipid / ml aqueous protein solution, preferably 100 to 500, more preferably 200 to 400, containing 0.1 to 10 mg protein (insulin, insulin-independent glucose transporter, prepferably GLUTl, GLUT2 or GLUT3, and glucokinase) per ml, preferably 0.5 to 2 mg is used. The suspension is mixed rapidly, bath sonicated, frozen, thawed, and this cycle is repeated twice.

A method of preparing a self-controlled delivery system using multivesicular liposomes is also disclosed. An insulin-containing aqueous phase is emulsified into a lipid- organic phase to form a water-in-oil emulsion. The organic phase (solvent) preferably comprises chloroform or methylene chloride. The lipid solution preferably comprises one or two phosphatidylcholines (more preferably mono-unsaturated), a phosphatidylglycerol, cholesterol and one or two triglycerides.Then, this water-in-oil emulsion is further emulsified into an aqueous solution to obtain a water-in-oil-in-water emulsion. The solvent is then removed to convert the water-in-oil-in-water emulsion into a multivesicular liposome particle. Preferably, the emulsion is flushed with nitrogen to remove the solvent. The particles are concentrated. The potency is then adjusted to the final product concentration. The resulting multivesicular liposomes will comprise insulin, Mg++, Zn++, insulin-independent glucose transporter, preferably GLUTl, GLUT2 or GLUT3 and glucokinase as disclosed above (Katre et al., Am J Drug Deliv 2004, 2 (4), p 213-227).

Furthermore, the invention also relates to a pharmaceutical composition comprising a vesicle as hereinbefore described and a pharmaceutically acceptable excipient.

The invention also relates to a kit comprising a pharmaceutical composition hereinbefore described.

The liposome described above is administered to a patient suffering from Type 1 diabetes or a patient suffering from Type 2 diabetes who is treated with insulin. Thus, the present invention also relates to the use of the liposomes described above for the preparation of a medicament for treatment of diabetic patients. Preferably, said patients are Type I diabetes patients.

Once administered, e.g. subcutaneously, interstitial glucose diffusion into the vesicles can occur mediated by an insulin-dependent glucose transporter depending on glucose concentration. Inside the vesicles, the glucose is converted to glucose-6-phosphate by glucokinase. Glucose-6-phosphate can no longer diffuse out of the liposome. As a consequence, the osmotic pressure increases in a glucose-dependent manner, leading to an influx of water from the interstitium. As a consequence, the liposomes are swelling and leaks will occur which allow a release of ingredients, including insulin. An increase of the surface of 2% to 3% may lead to such leaks. Finally, the liposomes will burst if the osmotic pressure rises further, and the ingredients are released completely.

When the liposome comprises multiple layers, the outermost layer will become unstable first, optionally followed by the next layer depending on glucose concentration. The depot function of multi-layer liposomes is well known (Katre et al., Am J Drug Deliv 2004, 2 (4), p 213-227).

Unused liposomes will be phagocytosed after 12 to 24 hours. Thus, no accumulation of unactivated liposomes occurs. Phagocytosis may be controlled by lipid composition (Nanobiotechnologie II - Anwendung in der Medizin und Pharmazie, 2004, V. Wagner and D. Wechsler, Vol. 50, Ed: VDI Technologiezentrum GmbH im Auftrag des BMBF) especially by pegylation of the surface of the liposomes, which can increase the half life to about 20 h.

Examples

Sterile preparation of lipids

Lipid powder or solution is irradiated with 100000 rads of gamma irradiation (e.g. using a 137Cs source).

Sterile filtration of protein solutions can be done using any suitable method. Such methods are well known to the skilled person.

Incorporation of proteins into liposomes

Aqueous carrier free protein in 0.9 % saline with 7.5 mM MgCl2 and 5 mM ATP is added to sterile, powdered lipid.

A lipid to aqueous ratio of 300 mg lipid / ml aqueous protein solution, containing 2 mg protein (insulin, GLUTl, GLUT2 or GLUT3 and glucokinase) per ml. The suspension is mixed rapidly, bath sonicated, frozen, thawed, and this cycle is repeated twice.

Claims

Claims

1. A vesicle comprising a glucose sensor integrated into the membrane of said vesicle, which further comprises glucokinase, insulin or analogs thereof and Mg++.

2. A vesicle according to claim 1 which further comprises ATP.

3. A vesicle according to any of the previous claims wherein said glucose sensor is GLUTl, GLUT2 or GLUT3.

4. A vesicle according to any of the previous claims which additionally comprises Zn++.

5. A vesicle according to any of the previous claims wherein said vesicle is a liposome.

6. A vesicle according to claim 5, wherein the liposome is a multi-lamellar liposome.

7. A vesicle according to claim 6 comprising GLUTl, GLUT2 or GLUT3 integrated into the membrane of the liposome, which further comprises glucokinase, insulin, Zn++ and Mg++.

8. A vesicle according to any of the preceding claims, wherein said liposome comprises a phospholipid and a cholesterol bilayer.

9. A vesicle according to claim 8, wherein the phospholipid is selected from one or more phospholipids of the group comprising DMPC, DOTAP, DOPC, MOPC, egg phosphatidylethanolamine, DOPE, egglecithin, dicetylphosphate, dipalmitoyl lecithin, digalactosyl diglyceride, sphingosine, lecithin, egg phosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylcholine and/or long-chain or intermediate-chain phosphatidylcholine.

10. A vesicle according to any of the preceding claims wherein said vesicle additionally comprises a triglyceride.

11. A vesicle according to any of the preceding claims, wherein insulin is human insulin.

12. A vesicle according to any of the preceding claims, wherein said GLUTl, GLUT2 or GLUT3 is human GLUTl, human GLUT2 or human GLUT3.

13. A vesicle according to any of claims 3 to 11, wherein said glucokinase is human glucokinase.

14. A pharmaceutical composition comprising a vesicle according to any of claims 1 to 13 and a pharmaceutically acceptable excipient.

15. A method of preparing a vesicle according to any one of claims 1 to 13 comprising adding an aqueous solution comprising a glucose sensor integrated into the membrane of a vesicle, glucokinase, insulin or analogs thereof, Mg++ and ATP to a lipid-organic phase to obtain an emulsion followed by removing the organic solvent.

16. A method of preparing a vesicle according to any one of claims 1 to 13 comprising the steps of

a) sterilizing powdered lipid;

b) adding sterile powdered lipid to aqueous protein;

c) rapidly mixing the suspension of b);

d) bath sonicating said mixture;

e) freezing said mixture;

f) thawing said mixture;

g) repeating steps c) to f) two times.

17. A method of preparing a self-controlled delivery system using multivesicular liposomes comprising the steps of

a) emulsifying an insulin-containing aqueous phase onto a lipid-organic phase to form a water-in-oil emulsion;

b) emulsifying the emulsion of step a) into an aqueous solution to obtain a water- in-oil-in-water emulsion;

c) removing the solvent to convert the water-in-oil-in-water emulsion of step b) into a multivesicular liposome particle;

d) concentrating the particle obtained in step c);

e) adjusting the potency to the final product concentration.

18. Use of a vesicle according to any one of claims 1 to 13 for the preparation of a medicament for treatment of patients suffering from diabetes.

19. The use of claim 18, wherein said patients are Type I diabetes patients.

20. A kit comprising a pharmaceutical composition according to claim 14.

21. The liposome, compositions, methods and use hereinbefore described, especially with reference to the foregoing examples.

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