HK1070589B

HK1070589B - Prolonged delivery of peptides

Info

Publication number: HK1070589B
Application number: HK05103434.3A
Authority: HK
Inventors: 丹尼斯．E．丹利; 罗伯特．A．盖尔芬德; 基兰．F．盖根; 金睿淑; 威廉．J．兰伯特; 戚洪
Original assignee: 西奥斯公司
Priority date: 1993-04-07
Filing date: 2005-04-21
Publication date: 2007-08-10

Description

Extended release of peptides

The application is a divisional application of application No. 94104491.2 filed from 7-mesh 4 of 1994 to the chinese patent office.

The present invention relates to compositions and methods for treating diabetes. More particularly, the invention relates to compositions for the prolonged administration of glucagon-like peptide 1(GLP-1) and derivatives thereof. These compositions are useful for treating non-insulin related diabetes mellitus (NIDDM).

The amino acid sequence of GLP-1 is known as:

His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (sequence ID NO: 1)

In Lopez, l.c. et al p.n.a.s., USA80: 5485-5489 (1983); bell, G.I. et al, Nature302716-718 (1983); heinrich, g. et al, Endocrinol.115: 2176-27: 599 GLP-1 was disclosed in 600 (1984).

On treatment in the pancreas and intestine, GLP-1 is converted into a31 amino acid peptide having amino acids 7-37 of GLP-1, which peptide is hereinafter referred to as GLP-1 (7-37).

It has been shown that this peptide has insulinotropic activity, i.e. it stimulates or causes the stimulation of the synthesis or expression of the hormone insulin. Because of this insulinotropic activity, GLP-1(7-37) can also be referred to as insulinotropic hormone (insulinotropic).

GLP-1(7-37) has the following amino acid sequence:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (sequence ID NO: 2).

GLP-1(7-37), certain derivatives thereof, and the use thereof in treating diabetes in mammals are disclosed in U.S. Pat. Nos. 5118666 ('666 patent) and 5120712 (' 712 patent), which are incorporated herein by reference. GLP-1(7-37) derivatives disclosed in the '666 and' 712 patents include polypeptides that contain or lack one of a variety of amino acids not present in the naturally occurring sequence. Other derivatives of GLP-1(7-37) disclosed in the '666 and' 712 patents include certain C-terminal salts, esters and amides, the salts and esters of which are defined as OM, where M is a pharmaceutically acceptable cation or lower (C)₁-C₆) Branched or straight chain alkyl, amides being defined as-NR²R³Wherein R is²And R³Are identical or different and are selected from hydrogen and lower (C)₁-C₆) Branched or straight chain alkyl.

Certain other polypeptides, alternatively referred to as truncated GLP-1 or truncated insulinotropic hormone, have insulinotropic activity and their derivatives have been disclosed in PCT/US 89/01121(WO 90/11296). These polypeptides, referred to herein as GLP-1(7-36), GLP-1(7-35) and GLP-1(7-34), have the following amino acid sequences, respectively:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg (sequence NO: 3);

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (sequence ID NO: 4); and

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys (sequence ID NO: 5);

in PCT/US 89/011The polypeptide derivatives disclosed in 21 include polypeptides with incoherent amino acid substitutions, or additional amino acids to enhance coupling to a carrier protein, or to enhance their insulinotropic action. Other derivatives of insulinotropic hormone disclosed in PCT/US 89/01121 include certain C-terminal salts, esters and amides, wherein the salts and esters are defined as OM and M is a pharmaceutically acceptable cation or a lower branched or straight chain alkyl group and the amide is defined as-NR²R³Wherein R is²And R³Are the same or different and are selected from hydrogen and lower branched or straight chain alkyl groups.

FIG. 1 shows the effect of prolonged infusion (7 hours) of 4 ng/kg/min insulinotropic hormone on plasma glucose levels in patients with NIDDM.

FIG. 2 shows the effect of a short infusion (60 min) of 10 ng/kg/min insulinotropic hormone on plasma glucose levels in NIDDM patients.

FIG. 3 shows the effect of prolonged infusion (7 hours) of 2 ng/kg/min and 4 ng/kg/min insulinotropic hormone on plasma glucose levels in NIDDM patients.

Figure 4 mean (n-3) plasma concentrations of insulinotropic hormone in mice following subcutaneous administration of a single dose of 0.5mg/0.5ml in different Aqueous Suspensions (AS).

Figure 5 mean (n-3) plasma concentrations of insulinotropic hormone in mice following subcutaneous administration of a single dose of 0.5mg/0.5ml in different Aqueous Suspensions (AS).

Figure 6 mean (n-3) plasma concentrations of insulinotropic hormone in mice following subcutaneous administration of a single dose of 0.5mg/0.5ml in different Aqueous Suspensions (AS).

Figure 7 mean (n-3) plasma concentrations of insulinotropic hormone in mice following subcutaneous administration of a single dose of 0.5mg/0.5ml in different Aqueous Suspensions (AS).

Figure 8 mean (n-3) plasma concentrations of insulinotropic hormone in mice following subcutaneous administration of a single dose of 0.5mg/0.13ml in different Aqueous Suspensions (AS).

Figure 9 mean (n-3) plasma concentrations of insulinotropic hormone in mice following subcutaneous administration of a single dose of 0.5mg/0.13ml in different Aqueous Suspensions (AS).

Figure 10 shows a pharmacokinetic study of the insulin stimulating hormone zinc precipitate.

In one embodiment, the invention is directed to a method of treating non-insulin related diabetes in a mammal in need thereof, which comprises repeatedly administering over an extended period of time a compound having an extended duration of action after each administration, said extended duration of action being necessary to achieve sustained glycemic control in said mammal, said compound being selected from the group consisting of:

(a) a peptide having the following amino acid sequence:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly; (sequence ID NO: 2)

(b) A peptide having the following amino acid sequence:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-X (sequence ID NO: 7)

Wherein X is selected from the following compounds:

(A)Lys，

(B)Lys-GLy，

(C)Lys-GLy-Arg；

(c) a polypeptide derivative containing the following basic structure

H₂N-W-COOH

Wherein W is an amino acid sequence selected from the group consisting of:

His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (sequence ID NO: 1) to know

His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg (sequence ID No.6)

When processed in a mammal, such derivatives form polypeptide derivatives having insulinotropic activity;

(d) polypeptide derivatives comprising the following basic structure,

H₂N-R-COOH

wherein R is an amino acid sequence selected from the group consisting of:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg; (sequence ID NO: 3)

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly; (sequence ID NO: 4) and

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys; (sequence ID NO: 5)

And (e) derivatives of said peptides from (a) to (d), wherein said derivatives are selected from the group consisting of:

(1) a pharmaceutically acceptable acid addition salt of said peptide;

(2) a pharmaceutically acceptable carboxylate salt of said peptide;

(3) a pharmaceutically acceptable base addition salt of said peptide;

(4) a pharmaceutically acceptable lower alkyl ester of said peptide;

(5) a pharmaceutically acceptable amide of said peptide, wherein said pharmaceutically acceptable amide is selected from the group consisting of an amide, a lower alkyl amide, and a lower dialkyl amide.

Preferably the method of administration is subcutaneous administration.

Another preferred method of administration is intramuscular administration.

Another preferred method of administration is transdermal.

A particularly preferred method of administration is administration by infusion pump.

Another preferred method of administration is by oral inhalation.

Another preferred method of administration is by nasal inhalation.

Another preferred method of administration is gastrointestinal administration.

In a further embodiment, the invention is directed to a composition of matter comprising the following compounds:

(i) a compound selected from the group consisting of:

(a) a peptide having the following amino acid sequence:

(b) A peptide having the following amino acid sequence:

Wherein X is selected from the following:

(A)Lys，

(B)Lys-GLy，

(C)Lys-GLy-Arg；

(c) a polypeptide derivative comprising the following basic structure:

H₂N-W-COOH

wherein W is an amino acid sequence selected from the group consisting of:

His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly

(sequence ID NO: 1) and

His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg (sequence)

ID NO：6)

The derivative forms a polypeptide derivative having insulinotropic activity when treated in a mammal;

(d) a polypeptide derivative comprising the following basic structure:

H₂N-R-COOH

wherein R is an amino acid sequence selected from the group consisting of:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (sequence ID NO: 2)

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg (sequence ID NO: 3)

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (sequence ID NO: 4) and

and derivatives of said peptides from (a) to (d), wherein said derivatives are selected from the group consisting of:

(1) a pharmaceutically acceptable acid addition salt of said peptide;

(2) a pharmaceutically acceptable carboxylate salt of said peptide;

(3) a pharmaceutically acceptable base addition salt of said peptide;

(4) a pharmaceutically acceptable lower alkyl ester of said peptide;

(ii) A polymer capable of prolonging the action of said compound in order to achieve sustained glycemic control.

Particularly preferred compositions are those wherein the polymer is a low molecular weight polymer.

Further particularly preferred are compositions wherein the polymer is selected from: polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyoxyethylene-polyoxypropylene copolymer, polysaccharide selected from cellulose, cellulose derivatives, chitosan, gum arabic, karaya resin, guar gum, xanthan resin, tragacanth gum, alginic acid, carrageenan, agarose and furce-llanas, dextran, starch derivatives, hyaluronic acid, polyesters, polyamides, polyanhydrides and polyorthoesters, and particularly preferred polymers are selected from polyethylene glycol and polyvinylpyrrolidone.

In another embodiment, the invention is directed to a composition of matter comprising:

(i) a compound selected from the group consisting of:

(a) a peptide having the following amino acid sequence:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (sequence ID NO: 2);

(b) a peptide having the following amino acid sequence:

Wherein a compound selected from:

(A)Lys，

(B)Lys-GLy，

(C)Lys-Gly-Arg；

(c) a polypeptide derivative comprising the following basic structure:

H₂N-W-COOH

wherein W is an amino acid sequence selected from the group consisting of:

His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (sequence ID NO: 1) and

His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg (sequence ID NO: 6)

When processed in a mammal, the derivative forms a polypeptide derivative having insulinotropic activity;

(d) a polypeptide derivative comprising the following basic structure:

H₂N-R-COOH

wherein R is an amino acid sequence selected from the group consisting of:

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg (sequence ID NO: 3);

(1) a pharmaceutically acceptable acid addition salt of said peptide;

(2) a pharmaceutically acceptable carboxylate salt of said peptide;

(3) a pharmaceutically acceptable base addition salt of said peptide;

(4) a pharmaceutically acceptable lower alkyl ester of said peptide;

(ii) Pharmaceutically acceptable water-immiscible oily suspensions which enable prolonged administration of the compounds.

Particularly preferred compositions are those wherein the oil is selected from the group consisting of peanut oil, sesame oil, almond oil, castor oil, camellia oil, cottonseed oil, olive oil, corn oil, soybean oil, safflower oil, coconut oil, esters of fatty acids and esters of fatty alcohols.

Further particularly preferred compositions further comprise a wetting agent, especially a nonionic surfactant.

More particularly preferred compositions further comprise a suspending agent.

(i) a compound selected from the group consisting of:

(a) a peptide having the following amino acid sequence:

(b) a peptide having the following amino acid sequence:

Wherein X is selected from the following compounds:

(A)Lys，

(B)Lys-Gly，

(C)Lys-Gly-Arg；

(c) a polypeptide derivative comprising the following basic structure:

H₂N-W-COOH

wherein W is an amino acid sequence selected from the group consisting of:

(d) a polypeptide derivative comprising the following basic structure:

H₂N-R-COOH

wherein R is an amino acid sequence selected from the group consisting of:

(1) pharmaceutically suitable acid addition salts of said peptides;

(2) pharmaceutically suitable carboxylic acid salts of said peptides;

(3) pharmaceutically suitable base addition salts of said peptides;

(4) pharmaceutically suitable lower alkyl esters of said peptides;

(5) a pharmaceutically suitable amide of said peptide, wherein said pharmaceutically suitable amide is selected from the group consisting of an amide, a lower alkyl amide and a lower dialkyl amide.

(ii) Zinc (II) complexed with a peptide.

Preferred compositions are capable of sustaining glycaemic effects.

Particularly preferred are compositions wherein the zinc product is amorphous.

Also particularly preferred are compositions wherein the zinc product is crystalline.

(i) a compound selected from the group consisting of:

(a) a peptide having the following amino acid sequence:

(b) a peptide having the following amino acid sequence:

Wherein X is selected from the following compounds:

(A)Lys，

(B)Lys-Gly，

(C)Lys-Gly-Arg；

(c) polypeptide derivatives comprising the following basic structure

H₂N-W-COOH

Wherein W is an amino acid sequence selected from the group consisting of:

And which, when processed in a mammal, forms a polypeptide derivative having insulinotropic activity;

(d) a polypeptide derivative comprising the following basic structure:

H₂N-R-COOH

wherein R is an amino acid sequence selected from the group consisting of:

and derivatives of said peptides (a) to (d), wherein said derivatives are selected from the group consisting of:

(1) a pharmaceutically acceptable acid addition salt of said peptide;

(2) a pharmaceutically acceptable carboxylate salt of said peptide;

(3) a pharmaceutically acceptable base addition salt of said peptide;

(4) a pharmaceutically acceptable lower alkyl ester of said peptide;

(5) a pharmaceutically acceptable amide of said peptide, wherein said pharmaceutically acceptable amide is selected from the group consisting of an amide, a lower alkyl amide, and a lower dialkyl amide,

(ii) a metal selected from the group consisting of Ni (II), Co (II), Mg (II), Ca (II), K (I), Mn (II), Fe (II) and Cu (II).

In another embodiment, the invention is directed to a composition comprising:

(i) a compound selected from the group consisting of:

(a) a peptide having the amino acid sequence:

(b) a peptide having the amino acid sequence:

Wherein X is derived from the following compounds:

(A)Lys，

(B)Lys-Gly，

(C)Lys-Gly-Arg；

(c) a polypeptide derivative comprising the following basic structure:

H₂N-W-COOH

wherein W is an amino acid sequence selected from the group consisting of:

(d) polypeptide derivatives comprising the following basic structure,

H₂N-R-COOH

wherein R is an amino acid sequence selected from the group consisting of:

(1) a pharmaceutically acceptable acid addition salt of said peptide;

(2) a pharmaceutically acceptable carboxylate salt of said peptide;

(3) a pharmaceutically acceptable base addition salt of said peptide;

(4) a pharmaceutically acceptable lower alkyl ester of said peptide;

and (5) a pharmaceutically acceptable amide of said peptide, wherein said pharmaceutically acceptable amide is selected from the group consisting of an amide, a lower alkyl amide, and a lower dialkyl amide,

(ii) a basic polypeptide, wherein the composition is an aqueous suspension capable of sustained glycemic control.

Particularly preferred are compositions wherein the basic polypeptide is protamine.

In another embodiment, the invention is directed to a composition of matter comprising the following compounds:

(i) a compound selected from the group consisting of:

(a) a peptide having the amino acid sequence:

(b) a peptide having the amino acid sequence:

Wherein X is selected from the following compounds:

(A)Lys，

(B)Lys-Gly，

(C)Lys-Gly-Arg；

(c) polypeptide derivatives comprising the following basic structure

H₂N-W-COOH

Wherein W is an amino acid sequence selected from the group consisting of:

(d) a polypeptide derivative comprising the following basic structure:

H₂N-R-COOH

wherein R is an amino acid sequence selected from the group consisting of:

(1) a pharmaceutically acceptable acid addition salt of said peptide;

(2) a pharmaceutically acceptable carboxylate salt of said peptide;

(3) a pharmaceutically acceptable base addition salt of said peptide;

(4) a pharmaceutically acceptable lower alkyl ester of said peptide; the pharmaceutically acceptable amide is selected from the group consisting of amides, lower alkyl amides and lower dialkyl amides, and

(ii) a phenolic compound, wherein the composition is an aqueous suspension capable of sustained glycemic control.

Particularly preferred compositions are those wherein the phenolic compound is selected from the group consisting of phenol, cresol, resorcinol and methyl paraben.

(i) a compound selected from the group consisting of:

(a) a peptide having the amino acid sequence:

(b) a peptide having the amino acid sequence:

Wherein X is selected from the following compounds:

(A)Lys，

(B)Lys-Gly，

(C)Lys-Gly-Arg；

(c) a polypeptide derivative comprising the following basic structure:

H₂N-W-COOH

wherein W is an amino acid sequence selected from the group consisting of:

(d) polypeptide derivatives comprising the following basic structure

H₂N-R-COOH

Wherein R is an amino acid sequence selected from the group consisting of:

(1) a pharmaceutically acceptable acid addition salt of said peptide;

(2) a pharmaceutically acceptable carboxylate salt of said peptide;

(3) a pharmaceutically acceptable base addition salt of said peptide;

(4) a pharmaceutically acceptable lower alkyl ester of said peptide;

(5) a pharmaceutically acceptable amide of said peptide, wherein said pharmaceutically acceptable amide is selected from the group consisting of amides, lower alkyl amides, and lower dialkyl amides, and

(ii) a basic polypeptide and a phenolic compound, wherein the composition is an aqueous suspension capable of sustained glycemic control.

In another embodiment, the object of the invention is a composition of matter comprising:

(i) a compound selected from the group consisting of:

(a) a peptide having the amino acid sequence:

(b) A peptide having the amino acid sequence:

Wherein X is selected from the following compounds:

(A)Lys，

(B)Lys-Gly，

(C)Lys-Gly-Arg；

(c) polypeptide compound comprising the following basic structure

H₂N-W-COOH

Wherein W is an amino acid sequence selected from the group consisting of:

(d) polypeptide derivatives comprising the following basic structure,

H₂N-R-COOH

wherein R is an amino acid sequence selected from the group consisting of:

(1) a pharmaceutically acceptable acid addition salt of said peptide;

(2) a pharmaceutically acceptable carboxylate salt of said peptide;

(3) a pharmaceutically acceptable base addition salt of said peptide;

(4) a pharmaceutically acceptable lower alkyl ester of said peptide;

and (5) a pharmaceutically acceptable amide of said peptide, wherein said pharmaceutically acceptable amide is selected from the group consisting of amides, lower alkyl amides, and lower dialkyl amides, and

(ii) a basic polypeptide, a phenolic compound, and a metal ion, wherein said composition is an aqueous suspension capable of sustained glycemic control.

A preferred composition is wherein the basic polypeptide is protamine.

Also preferred is a composition wherein the metal ion is zinc.

In another embodiment, the object of the invention is a composition of matter comprising the following compounds:

(i) a compound selected from the group consisting of:

(a) a peptide having the following amino acid sequence:

(b) a peptide having the following amino acid sequence:

Wherein X is selected from the following compounds:

(A)Lys，

(B)Lys-Gly，

(C)Lys-Gly-Arg；

(c) polypeptide derivatives comprising the following basic structure

H₂N-W-COOH

Wherein W is an amino acid sequence selected from the group consisting of:

(d) polypeptide derivatives comprising the following basic structure

H₂N-R-COOH

Wherein R is an amino acid sequence selected from the group consisting of:

and

derivatives of said peptides from (a) to (d), wherein said derivatives are selected from the group consisting of:

(1) a pharmaceutically acceptable acid addition salt of said peptide;

(2) a pharmaceutically acceptable carboxylate salt of said peptide;

(3) a pharmaceutically acceptable base addition salt of said peptide;

(4) a pharmaceutically acceptable lower alkyl ester of said peptide;

(ii) the peptides and derivatives thereof are subjected to conditions that result in the formation of an amorphous or crystalline form.

Preferred compositions are those wherein the conditions are high shear, contact with salt, or a combination thereof.

Particularly preferred compositions are those wherein the salt is selected from the group consisting of ammonium sulfate, sodium sulfate, lithium chloride, sodium citrate, ammonium citrate, sodium phosphate, potassium phosphate, sodium chloride, potassium chloride, ammonium chloride, sodium acetate, ammonium acetate, magnesium sulfate, calcium chloride, ammonium nitrate, and sodium formate, and combinations thereof.

In yet another embodiment, the present invention is directed to a composition of matter comprising the following compounds:

(i) a compound selected from the group consisting of:

(a) a polypeptide having the amino acid sequence:

(b) a polypeptide having the amino acid sequence:

Wherein X is selected from the following compounds:

(A)Lys，

(B)Lys-Gly，

(C)Lys-Gly-Arg；

(c) a polypeptide derivative comprising the following basic structure:

H₂N-W-COOH

wherein W is an amino acid sequence selected from the group consisting of:

(d) a polypeptide derivative comprising the following basic structure:

H₂N-R-COOH

wherein R is an amino acid sequence selected from the group consisting of:

(1) a pharmaceutically acceptable acid addition salt of said peptide;

(2) a pharmaceutically acceptable carboxylate salt of said peptide;

(3) a pharmaceutically acceptable base addition salt of said peptide;

(4) a pharmaceutically acceptable lower alkyl ester of said peptide;

(ii) a liposome delivery system.

Particularly preferred compositions are those wherein the liposomes are phospholipid based.

Also particularly preferred are compositions wherein the liposomes are non-phospholipid based.

It is also an object of the present invention to treat non-insulin related diabetes mellitus in a mammal, which treatment comprises administering a composition of the present invention for an extended period of time when such treatment is desired.

Unless otherwise indicated, the term "derivative" as used in this specification and the appended claims includes, but is not limited to, polypeptides containing the indicated basic structure, including one or more L-amino acids at its C-terminus; wherein the C-terminal carboxyl group is substituted with (C)₁-C₆) Linear or branched alkyl forms esters; wherein the C-terminal carboxyl group forms a carboxy-amide (car-boxamide) or a substituted carboxy-amide; wherein the acidic amino acid residue (Asp and/or Glu) forms an ester or a carboxamide; and combinations thereof.

Polypeptides having homology to the above-described peptides sufficient for the insulinotropic activity of such polypeptides are included within the scope of the present invention. Variants of the above-described polypeptides, which comprise incoherent amino acid substitutions and have insulinotropic activity, are also included in the scope of the present invention.

Glucagon-like peptide-1 (7-37), its isolation, characterization, and use in the treatment of diabetes are disclosed in US 5118666 and 5120712. These patent documents are incorporated herein by reference in their entirety.

In the present invention, it has now been found that prolonged plasma elevation of GLP-1 and related polypeptides is essential for achieving sustained glycemic control during and after a meal in a patient with non-insulin related diabetes. It has surprisingly been found that just before and after a meal period, even up to more than 1 hour, the elevation of GLP-1 and related peptides does not adequately control its glucose content. Thus, administration of GLP-1 and related peptides requires an extended release system. This extended release system results in increased insulin action.

The phrase "increasing insulin action" as used herein and in the appended claims includes, but is not limited to, one or more of increasing insulin synthesis, increasing insulin secretion, increasing glucose uptake by muscle and fat, and decreasing hepatic glucose production.

The polypeptides of the invention can be prepared by various methods well known to those skilled in the art. For example, polypeptides can be synthesized using an automated peptide synthesizer, such as an Applied Biosystem (ABI)430A solid phase peptide synthesizer. Alternatively, the polypeptides of the invention may be prepared by recombinant DNA techniques in which the DNA sequence encoding the polypeptide is operably linked to an expression vector and used to transform a suitable host cell. The transformed host cell is then cultured under conditions in which the polypeptide will be expressed. And recovering the polypeptide from the culture. Synthesis may also be used in combination with recombinant DNA techniques to obtain the amide and ester derivatives of the invention and/or to obtain fragments of the desired polypeptide, which fragments are then ligated by methods well known to those skilled in the art.

The polypeptide derivatives of the present invention can be prepared by methods well known to those skilled in the art. For example, in the presence of a catalytic acid such as HCl, will be desired(C₁-C₆) An alkanol is reacted with the desired polypeptide to produce a C-terminal alkyl ester derivative of the polypeptide of the invention. Suitable reaction conditions for forming such alkyl esters include a reaction temperature of about 50 ℃ and a reaction time of about 1 hour to about 3 hours. Similarly, (C) containing Asp and/or Glu residues in the polypeptide₁-C₆) Alkyl ester polypeptide derivatives of the invention may also be so produced.

Carboxyamide derivatives of the polypeptides of the invention may also be prepared by solid phase peptide synthesis methods well known to those skilled in the art. For example, seeSolid Phase Pep- tide SynthesisStewart, JM et al, Pierce Chem, Co, Press, 1984.

Alternatively, or in combination with the above, the polypeptide derivative of the present invention may be prepared by altering the DNA coding sequence of such a polypeptide. Thus, a basic amino acid residue is replaced by a different basic amino acid residue or by an acid or acidic or neutral amino acid residue, or an acidic amino acid residue is replaced by a different acidic amino acid residue or by a basic or neutral amino acid residue, or a neutral amino acid residue is replaced by a different neutral amino acid residue or by an acidic or basic amino acid residue. Such changes in the basic sequence of the polypeptide may also be effected by direct synthesis of the derivative. Such methods are well known to those skilled in the art. Thus, these derivatives are useful in the practice of the present invention, and they must achieve a insulinotropic effect.

The insulinotropic activity of the polypeptide derivative of the present invention was determined as follows.

Using Lacy, PE et alDiabetes,16: 35-39(1967) islets were isolated from pancreatic tissue from normal mice by isolating collagenase digests of pancreatic tissue on Ficoll gradients (pH 7.427%, 23%, 20.5% and 11% in Hanks' balanced salt solution). Islets were collected from the 20.5%/11% interface, washed and hand-removed under a stereomicroscope for exocrine and other tissues. In the presence of 10% bovine fetal serum and RPMI16 containing 11mM glucose40 medium at 37 ℃ and 95% air/5% CO₂The islets were cultured under conditions overnight. The islets were then transferred to RPMI1640 medium supplemented with 10% fetal bovine serum and containing 5.6mM glucose. At 37 deg.C, 95% air/5% CO₂The islands were incubated under conditions for 60 minutes. The polypeptide derivatives to be investigated were prepared in RPMI medium containing 10% bovine fetal serum and 16.7mM glucose at 1nM and 10nM concentrations. Approximately 8-10 isolated islets were then transferred with a pipette to a total volume of 250. mu.l of the medium-containing polypeptide derivative in a 96-well microtiter plate. 95% air/5% CO at 37 ℃ in the presence of a polypeptide derivative₂The islets were incubated under conditions for 90 minutes. Aliquots of the islet-free medium were then collected and analyzed for the amount of insulin present in 100 μ l using the equal insulin RIA kit (Binax, inc., Portland, ME) in a radioimmunoassay.

When the polypeptide derivative of the present invention is administered, for example, intravenously, intramuscularly or subcutaneously, the dose effective for treating early adult diabetes is about 1Pg/kg to 1000 μ g/kg per day. The preferred dosage range for intravenous infusion during and between meals is about 4-10 ng/kg/min or about 0.6-1.4 μ g/day per 100kg patient. However, it is recognized that dosages outside this range are also possible and are within the scope of the invention. The physician of the prescribing will and will determine the appropriate dosage and will be the exact result of the conditions to be treated and the response to administration of the derivative and the patient's age, weight, sex and medical history.

Prolonged administration can be achieved by subcutaneous, intramuscular or transdermal means, oral inhalation, nasal inhalation, gastrointestinal tract or by infusion pump means.

Prolonged administration of GLP-1 and related peptides can also be achieved by formulating the solution in various water-soluble polymers. These polymers are generally low molecular weight (< 15kDa) polymers. Non-limiting examples of such low molecular weight polymers include polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, and polyoxyethylene-polyoxypropylene copolymer. Higher molecular weight polymers may also be used. Non-limiting examples of higher molecular weight polymers include polysaccharides such as cellulose and its derivatives, chitosan, acacia, karaya, guar, Xanthan, tragacanth, alginic acid, carrageenan, agarose, furcelleran. In the latter case, polymers which degrade in vivo enzymatically or hydrolytically are preferred, such as dextran, starch and its derivatives, hyaluronic acid, polyesters, polyamides, polyanhydrides and polyorthoesters. Tissue accumulation associated with high molecular weight non-biodegradable polymers can be avoided by using low molecular weight polymers or biodegradable polymers. The formulation typically contains GLP-1 or related peptide at a concentration of about 1mg/ml, depending on the polymer, but typically at a concentration as high as 50cp in viscosity, and may have suitable buffers, tonicity agents and preservatives. In vivo data in mice and humans demonstrate that the formulation achieves measurable amounts of blood insulinotropic hormone, for example, up to over 24 hours. In contrast, for example, insulinotropic hormone formulated in phosphate buffered saline solution resulted in rapid (-15 minutes) peak plasma levels that fell below the detection limit just beyond 4 hours. The plasma concentration versus time plot shows that the rate of adsorption of insulinotropic hormone in the presence of the polymer is greatly reduced, e.g., from the point of injection.

GLP-1 and related peptides can also be formulated as particles suspended in a pharmaceutically acceptable oil. The preferred oil is a triglyceride. Non-limiting examples of such oils include peanut oil, sesame oil, almond oil, castor oil, camellia oil, cottonseed oil, olive oil, corn oil, soybean oil, safflower oil, and coconut oil. Other types of oils are also useful, such as esters of fatty acids and esters of fatty alcohols, provided that the oil is immiscible with water and is a poor solvent for the peptide. The formulation may also contain suitable preservatives, wetting agents and suspending agents. The weight percentage of insulinotropic hormone in the formulation may be, for example, in the range of 0.01 to 10%. Data in rats demonstrate that these formulations reach measurable blood levels of insulinotropic hormone for up to 24 hours. In contrast, for example, insulinotropic hormone formulated in phosphate buffered saline results in rapid (-15 minutes) peak plasma levels, with plasma levels falling below the monitorable limit at just over 4 hours. The plasma concentration versus time plot shows that the rate of absorption of insulinotropic hormone from the point of injection is greatly reduced in oil suspensions.

GLP-1 and its related peptides can also be administered in a low solubility form by binding to metal ions, preferably in the form of a salt. Preferably the ion is zinc (II). This combination may cause the composition to be amorphous or crystalline. Other metal ions that may also be used include Ni (II), Co (II), Mg (II), Ca (II), K (I), Mn (II), Fe (II), and Cu (II).

Other forms of prolonged administration include liposomes, or multilamellar or unilamellar sheets, the preparation of which is well known to those skilled in the art. Liposomes, whether multilamellar or unilamellar, can be phospholipid-based or non-phospholipid-based.

Other extended release formulations are aqueous suspensions of insulinotropic hormone precipitates or aggregates formed by the use of precipitating agents such as phenolic compounds or basic polypeptides or metal ions or salts and/or the use of high shear forces. More than one precipitating agent may be used simultaneously. The precipitate may be amorphous in crystalline form.

The insulinotropic hormone crystals can be obtained from a solution of the drug in water by using a pH gradient method (either from high to low or from low to high) and/or a temperature gradient method and/or a solubility-reducing salt method. Salts include ammonium citrate, sodium or potassium phosphate, sodium or potassium chloride or ammonium, sodium or ammonium acetate, magnesium sulfate, calcium chloride, ammonium nitrate, sodium formate and any other salt that may reduce the solubility of the drug. If the salt used for crystallization is not pharmaceutically acceptable, its mother liquor may be replaced with a pharmaceutically acceptable medium after completion of crystallization. If further reduction of drug solubility is desired to achieve the desired pharmacokinetic profile, the crystals may be treated with metal ions such as zinc or calcium and/or a phenolic compound. The treatment can be carried out by simply adding these additives to the crystal suspension.

The solubility of insulinotropic hormone precipitates or aggregates can be from below 1. mu.g/ml to 500. mu.g/ml under physiological conditions. The data in mice demonstrate that the formulation is capable of reaching measurable blood levels of insulinotropic hormone for at least 30 hours.

The aqueous medium used in the above formulation may be any injectable buffer system, or even pure water. The pH of the final formulation may be any value as long as the formulation is injectable. Protamine can be added in any salt form (e.g., sulfate, chloride, etc.) or as protamine base. Example concentration ranges of components that can be used in the preparation of the formulation are as follows: phenol (0.5-5.0 mg/ml), m-cresol (0.5-5.5 mg/ml), protamine (0.02-1.0 mg/ml), zinc (0.1-6 molar ratio of zinc/insulinotropic hormone), sodium chloride (up to 100mg/ml) and phosphate buffer (5-500 mM).

Other phenolic or non-phenolic compounds may also be used. Non-limiting examples of such compounds include resorcinol, methyl paraben, propyl paraben, benzyl alcohol, chlorocresol, cresol, benzaldehyde, catechol, pyrogallol, hydroquinone, n-propyl gallate, butylated hydroxyanisole, butylated hydroxytoluene. Non-limiting examples of basic polypeptides are polylysine, polyarginine, and the like.

Having generally described the invention, reference will now be made to specific embodiments. It is to be understood that these examples are not intended to limit the invention, the scope of which is defined by the appended claims.

Example 1

Insulinotropic hormone (1mg/ml) suspension

Preparation of solution A1

10mg of insulinotropic hormone was weighed into a 5ml volumetric flask. Approximately 4ml of Phosphate Buffered Saline (PBS) was added to the volumetric flask in order to disperse and dissolve the drug. Sufficient PBS (appropriate) was added to fill the flask. 20mg of insulinotropic hormone are weighed into a10 ml volumetric flask. About 8ml of PBS was added to the vial to disperse and dissolve the drug. An appropriate amount of PBS was added to the flask. The solutions in the two vials were passed through a 0.22 μ filter using a glass syringe

(low bound protein) they were combined by filtration into 10ml glass vials. The PBS of solution A1 contained 2mg/ml of insulinotropic hormone.

Preparation of solution B1

8mg of protamine sulfate and 44mg of phenol were weighed into a10 ml volumetric flask. An appropriate amount of PBS was added to dissolve protamine sulfate and phenol. This solution was filtered through a 0.22 μ filter (low in bound protein) into a10 ml glass vial. Solution B1 contained 0.6mg/ml protamine base and 4.4mg/ml phenol in PBS.

Aqueous suspension 1

1.5ml of solution A1 was pipetted into a 3.5ml glass vial type I. When 1.5ml of solution B1 was pipetted into the vial, the solution in the vial was magnetically stirred. The vial was covered and sealed with an aluminum shell. The solution in the vial was stirred slowly for 16 hours to form a suspension. Aqueous suspension 1 contained 1mg/ml insulinotropic hormone, 0.3mg/ml protamine base, 2.2mg/ml phenol in PBS. The suspension was used for pharmacokinetic studies in mice.

Example 2

Insulinotropic hormone (1mg/ml) suspension

Preparation of solution A2

10mg of insulinotropic hormone are weighed into a 5ml volumetric flask. About 4ml PBS was added to the vial to disperse and dissolve the drug. Add appropriate amount of PBS to the vial. 20mg of insulinotropic hormone was weighed into a10 ml volumetric flask, and about 8ml of PBS was added to the flask to disperse and dissolve the drug. An appropriate amount of PBS was added to the bottle. The two vial solutions were filtered through a 0.22 μ filter into a10 ml glass vial using a glass syringe. Solution A2 contained 2mg/ml insulinotropic hormone in PBS.

Preparation of solution B2

2mg of protamine sulfate and 44mg of phenol were weighed into a10 ml volumetric flask. An appropriate amount of PBS was added to the bottle to dissolve protamine sulfate and phenol. This solution was filtered through a 0.22 μ filter into a10 ml glass vial. The PBS of solution B2 contained 0.15mg/ml protamine base and 4.4mg/ml phenol.

Aqueous suspension 2

1.5ml of solution A2 was pipetted into a 3.5ml glass vial type I. When 1.5ml of solution B2 was pipetted into the bottle, the solution in the bottle was stirred magnetically. The bottle was covered with an aluminum shell and sealed. The solution in the bottle was stirred for 16 hours to enable the formation of a suspension. Aqueous suspension 2 in PBS contained 1mg/ml insulinotropic hormone, 0.075mg/ml protamine base and 2.2mg/ml phenol. This suspension was used for pharmacokinetic studies in mice.

Example 3

Insulinotropic hormone (1mg/ml) suspension

Preparation of solution A3

20mg of insulinotropic hormone are weighed into a10 ml volumetric flask. About 8ml PBS was added to the vial to disperse and dissolve the drug. An appropriate amount of PBS was added to the flask. Solution A3 was filtered through a 0.22 μ filter into a10 ml glass vial using a syringe. Solution A3 contained 2mg/ml insulinotropic hormone in PBS.

Preparation of solution B3

8mg of protamine sulphate, 44mg of phenol and 323mg of glycerol are weighed into a10 ml volumetric flask. An appropriate amount of PBS was added to the bottle to dissolve protamine sulfate, phenol and glycerol. This solution was filtered through a 0.22 μ filter into a10 ml glass vial using a syringe. The PBS of solution B3 contained 0.6mg/ml protamine base, 4.4mg/ml phenol and 32mg/ml glycerol.

Aqueous suspension 3

Pipette 1.5ml of solution A3 into a 3.5ml type I glass vial. When 1.5ml of solution B3 was pipetted into the bottle, the solution in the bottle was stirred magnetically. The vial was covered and sealed with an aluminum shell. The solution in the bottle was stirred for 16 hours to form a suspension. Aqueous suspension 3 in PBS contained 1mg/ml insulinotropic hormone, 0.3mg/ml protamine base, 2.2mg/ml phenol and 16mg/ml glycerol. The suspension was used for pharmacokinetic studies in mice.

Example 4

Insulinotropic hormone (1mg/ml) suspension

Preparation of solution A4

20mg of insulinotropic hormone are weighed into a10 ml volumetric flask. About 8ml of PBS was added to the vial to disperse and dissolve the drug. Add appropriate amount of PBS to the vial. Solution A4 was filtered into a10 ml glass vial using a glass syringe through a 0.22 μ filter (Millipore Millex-GV). Solution A4 contained 2mg/ml insulinotropic hormone in PBS.

Preparation of solution B4

8mg of protamine sulfate and 52mg of m-cresol are weighed into a10 ml measuring flask. An appropriate amount of PBS was added to the bottle to dissolve protamine sulfate and m-cresol. This solution was filtered through a 0.22 μ filter into a10 ml glass vial. The PBS of solution B4 contained 0.6mg/ml protamine base and 5mg/ml m-cresol.

Aqueous suspension 4

1.5ml of solution A4 was pipetted into a 3.5ml glass vial type I. When 1.5ml of solution B4 was pipetted into the bottle, the solution in the bottle was stirred magnetically. The vial was covered and sealed with an aluminum shell. The solution in the flask was stirred for 16 hours to form crystals. The PBS of aqueous suspension 4 contained 1mg/ml insulinotropic hormone, 0.3mg/ml protamine base and 2.5mg/ml m-cresol. This suspension was used for pharmacokinetic studies in mice.

Example 5

Insulinotropic hormone (1mg/ml) suspension

Preparation of solution A5

50mg of insulinotropic hormone are weighed into a25 ml volumetric flask. About 23ml of PBS was added to the vial to disperse and dissolve the drug. An appropriate amount of PBS was added to the flask. Solution a5 was filtered through a 0.22 μ filter into a 50ml glass vial using a syringe. The BPS of solution A5 contained 2mg/ml insulinotropic hormone.

Preparation of phenol feed solution

0.44g of phenol was weighed into a100 ml volumetric flask. About 95ml PBS was added to the flask to dissolve the phenol. An appropriate amount of PBS was added to the bottle to dissolve the phenol. The resulting solution (4.4mg/ml phenol) was used to prepare solution B5.

Preparation of solution B5

Solution B5 was prepared by filtering 25ml of the phenol feed solution through a 0.2 μ filter into a 50ml glass vial. Solution B5 contained 4.4mg/ml phenol in PBS.

Aqueous suspension 5

1.25ml of solution A5 was pipetted into a 3.5ml glass vial type I. When 1.25ml of solution B5 was pipetted into the bottle, the solution in the bottle was magnetically stirred. The bottle was covered with an aluminum shell and sealed. The solution in the bottle was stirred for 16 hours to form a suspension. Aqueous suspension 5 contained 1mg/ml insulinotropic hormone and 2.2mg/ml phenol in PBS. This suspension was used for pharmacokinetic studies in mice.

Example 6

Insulinotropic hormone (1mg/ml) suspension

Preparation of solution A6

50mg of insulinotropic hormone are weighed into a25 ml volumetric flask. About 23ml of PBS was added to the vial to disperse and dissolve the drug. An appropriate amount of PBS was added to the flask. Solution a6 was filtered through a 0.22 μ filter into a 50ml glass vial using a syringe. Solution A6 contained 2mg/ml insulinotropic hormone in PBS.

Preparation of phenol feed solution

0.44g of phenol was weighed into a100 ml volumetric flask. About 95ml PBS was added to the flask to dissolve the phenol. An appropriate amount of PBS was added to the bottle to dissolve the phenol. The resulting solution (4.4mg/ml phenol) was used to prepare solution B6.

Preparation of solution B6

Solution B6 was prepared by weighing 1.25mg of protamine sulfate in a25 ml volumetric flask. About 20ml of phenol feed solution was added to the bottle to dissolve protamine sulfate. Add an appropriate amount of phenol feed to the bottle. Solution B6 was filtered through a 0.22 μ filter into a 50ml glass vial. The PBS of solution B6 contained 4.4mg/ml phenol and 0.038mg/ml protamine base.

Aqueous suspension 6

1.25ml of solution A6 was pipetted into a 3.5ml glass vial type I. When 1.25ml of solution B6 was pipetted into the vial, the solution in the vial was magnetically stirred. The vial was covered and sealed with an aluminum shell. The solution in the bottle was stirred for 16 hours to form a suspension. The aqueous suspension 6 contained 1mg/ml insulinotropic hormone, 2.2mg/ml phenol, 0.019mg/ml protamine base in PBS. This suspension was used for pharmacokinetic studies in mice.

Example 7

Insulinotropic hormone (1mg/ml) suspension

Preparation of solution A7

50mg of insulinotropic hormone are weighed into a25 ml volumetric flask. About 23ml of PBS was added to the vial to disperse and dissolve the drug. An appropriate amount of PBS was added to the flask. Solution a7 was filtered through a 0.22 μ filter into a 50ml glass vial using a syringe. The BPS of solution A7 contained 2mg/ml insulinotropic hormone.

Preparation of phenol feed solution

0.44g of phenol was weighed into a100 ml volumetric flask. About 95ml of PBS was added to the flask to dissolve the phenol. An appropriate amount of PBS was added to the bottle to dissolve the phenol. The resulting solution (4.4mg/ml phenol) was used to prepare solution B7.

Preparation of solution B7

Solution B7 was prepared by weighing 2.5mg of protamine sulfate in a25 ml volumetric flask. To this bottle was added about 20ml of phenol feed solution to dissolve protamine sulfate. An appropriate amount of phenol feed solution was added to the flask. Solution B7 was filtered through a 0.22 μ filter into a 50ml glass vial. The PBS of solution B7 contained 4.4mg/ml phenol and 0.075mg/ml protamine base.

Aqueous suspension 7

1.25ml of solution A7 was removed in a 3.5ml glass vial type I. When 1.25ml of solution B7 was pipetted into the bottle, the solution in the bottle was magnetically stirred. The bottle was covered with an aluminum shell and sealed. The solution in the bottle was stirred for 16 hours to form a suspension. The aqueous suspension 7 in PBS contained 1mg/ml insulinotropic hormone, 2.2mg/ml phenol and 0.038mg/ml protamine base. This suspension was used for pharmacokinetic studies in mice.

Example 8

Insulinotropic hormone (1mg/ml) suspension

Preparation of solution A12

20mg of insulinotropic hormone are weighed into a10 ml volumetric flask. About 8ml PBS was added to the vial to disperse and dissolve the drug. The appropriate amount of PBS was added to the vial. Solution A12 was filtered through a 0.22 μ filter into a10 ml glass vial using a syringe. The BPS of solution A12 contained 2mg/ml insulinotropic hormone.

Preparation of solution B12

Solution B12 was prepared by weighing 20mg of phenol in a10 ml volumetric flask. About 8ml of PBS was added to the bottle to dissolve the phenol. The bottle was filled with an appropriate amount of PBS. Solution B12 was filtered through a 0.22 μ filter into a10 ml glass vial. The BPS of solution B12 contained 2mg/ml phenol.

Aqueous suspension 12

Pipette 4ml of solution A12 into a10 ml glass vial type I. The solution in the bottle was stirred while 4ml of solution B12 was pipetted into the vial. The bottle was covered with an aluminum shell and sealed. The solution in the bottle was stirred for 16 hours to form a suspension. The PBS of aqueous suspension 12 contained 1mg/ml insulinotropic hormone and 1mg/ml phenol. This suspension was used for pharmacokinetic studies in mice.

Example 9

Insulinotropic hormone (1mg/ml) suspension

Preparation of solution A15

20mg of insulinotropic hormone are weighed into a10 ml volumetric flask. About 8ml of Phosphate Buffer (PB) was added to the vial to dissolve the drug. The appropriate amount of PB was added to the bottle. Solution A15 was filtered through a 0.22 μ filter into a10 ml glass vial using a syringe. The PB of solution A15 contained 2mg/ml insulinotropic hormone.

Preparation of solution B15

Solution B15 was prepared by weighing 8mg of protamine sulfate in a10 ml volumetric flask. About 8ml of PB was added to the bottle to dissolve protamine sulfate. An appropriate amount of PB was added to the bottle. Solution B15 was filtered through a 0.22 μ filter into a10 ml glass vial. The PB of solution B15 contained 0.6mg/ml protamine base.

Aqueous suspension 15

3ml of solution A15 were pipetted into a10 ml glass vial type I. The solution in the bottle was stirred while 3ml of solution B15 was pipetted into the bottle. The bottle was covered with an aluminum shell and sealed. The solution in the bottle was stirred for 16 hours to form a suspension. The PB of the aqueous suspension 15 contained 1mg/ml insulinotropic hormone and 0.3mg/ml protamine base. This suspension was used for pharmacokinetic testing in rats.

Example 10

Insulinotropic hormone (1mg/ml) suspension

Preparation of solution A16

20mg of insulinotropic hormone are weighed into a10 ml volumetric flask. About 8ml of PB was added to the vial to dissolve the drug. An appropriate amount of PB was added to the bottle. Solution A16 was filtered through a 0.22 μ filter into a10 ml glass vial using a syringe. BP in solution A16 contained 2mg/ml insulinotropic hormone.

Preparation of solution B16

Solution B16 was prepared by weighing 44g of phenol in a10 ml volumetric flask. About 8ml of PB was added to the volumetric flask to dissolve the phenol. The appropriate amount of PB was added to the bottle. Solution B16 was filtered through a 0.22 μ filter into a10 ml glass vial. The PB of solution B16 contained 4.4mg/ml phenol.

Aqueous suspension 16

3ml of solution A16 were pipetted into a10 ml glass vial type I. The solution in the bottle was magnetically stirred while 3ml of solution B16 was pipetted into the bottle. The bottle was covered with an aluminum shell and sealed. The solution in the bottle was stirred for 16 hours to form a suspension. The aqueous suspension 16 contained 1mg/ml insulinotropic hormone and 2.2mg/ml phenol in PB. This suspension was used for pharmacokinetic studies in mice.

Example 11

Insulinotropic hormone (1mg/ml) suspension

Aqueous suspension 17

10mg of insulinotropic hormone was weighed into a10 ml volumetric flask. About 8ml of PB was added to the vial to dissolve the drug. The bottle is added with a proper amount of PB. The solution in the vial was filtered through a 0.22 μ filter into a10 ml type I glass vial using a syringe. The bottle was covered with an aluminum shell and sealed. The solution in the bottle was stirred for 16 hours to form a suspension. The aqueous suspension 17 contained 1mg/ml of insulinotropic hormone in PB. This suspension was used for pharmacokinetic studies in mice.

Example 12

Insulinotropic hormone (1mg/ml) suspension

Aqueous suspension 18

10mg of insulinotropic hormone was weighed into a10 ml volumetric flask. About 8ml PBS was added to the vial to dissolve the drug. The bottle was filled with an appropriate amount of PBS. The solution in the vial was filtered through a 0.22 μ filter into a10 ml type I glass vial using a syringe. The bottle was covered with an aluminum shell and the bottle was sealed. The solution in the flask was stirred slowly for 16 hours (to ensure that no foam or bubbles formed) to form a suspension. Aqueous suspension 18 contained 1mg/ml insulinotropic hormone in PBS. This suspension was used for pharmacokinetic studies in mice.

Example 13

Insulinotropic hormone (0.2mg/ml) suspension

Preparation of solution A22

Solution A22 was prepared by weighing 2mg of insulinotropic hormone into a 5ml volumetric flask. About 3ml of PBS was added to the vial to dissolve the drug. The appropriate amount of PBS was added to the vial. Solution A22 was filtered through a 0.22 μ filter into a10 ml glass vial using a syringe. Solution A22 contained 0.4mg/ml insulinotropic hormone in PBS.

Preparation of solution B22

Solution B22 was prepared by weighing 44mg of phenol in a10 ml volumetric flask. About 8ml of PBS was added to the flask to dissolve the phenol. The bottle was then filled with PBS. Solution B22 was filtered through a 0.22 μ filter into a10 ml glass vial. Solution B22 contained 4.4mg/ml phenol in PBS.

Aqueous suspension 22

Pipette 1.5ml of solution A22 into a 3.5ml type I glass vial. The solution in the flask was magnetically stirred while 1.5ml of solution B22 was pipetted into the flask. The bottle was covered with an aluminum shell and sealed. The solution in the flask was stirred for 16 hours to form a suspension. The aqueous suspension 22 contained 0.2mg/ml insulinotropic hormone and 2.2mg/ml phenol in PBS. This suspension was used for pharmacokinetic studies in mice.

Example 14

Insulinotropic hormone (0.2mg/ml) suspension

Preparation of solution A23

Solution A23 was prepared by weighing 2mg of insulinotropic hormone into a 5ml volumetric flask. About 3ml of PBS was added to the vial to dissolve the drug. The bottle was filled with an appropriate amount of PBS. Solution A23 was filtered through a 0.22 μ filter into a10 ml glass vial using a syringe. Solution A23 contained 0.4mg/ml insulinotropic hormone in PBS.

Preparation of solution B23

Solution B23 was prepared by weighing 8.8mg of phenol in a10 ml volumetric flask. About 8ml of PBS was added to the bottle to dissolve the phenol. The bottle was filled with an appropriate amount of PBS. Solution B23 was filtered through a 0.22 μ filter into a10 ml glass vial. Solution B23 contained 0.88mg/ml phenol in PBS.

Aqueous suspension 23

1.5ml of solution A23 was pipetted into a 3.5ml glass vial type I. The solution in the flask was magnetically stirred while 1.5ml of solution B23 was pipetted into the flask. The bottle was covered and sealed with an aluminum shell. The solution in the flask was stirred for 16 hours to form a suspension. The aqueous suspension 23 contained 0.2mg/ml insulinotropic hormone and 0.44mg/ml phenol in PBS. This suspension was used for pharmacokinetic studies in mice.

Example 15

Insulinotropic hormone (1mg/ml) suspension

Preparation of solution A24

Solution A24 was prepared by weighing 10mg of insulinotropic hormone into a 5ml volumetric flask. About 3ml of PBS was added to the vial to dissolve the drug. The bottle was filled with an appropriate amount of PBS. Solution a24 was filtered through a 0.22 μ filter into a10 ml glass vial using a syringe. Solution A24 contained 2mg/ml insulinotropic hormone in PBS.

Preparation of solution B24

Solution B24 was prepared by weighing 8mg of protamine sulfate in a10 ml volumetric flask. About 8ml of PBS was added to the flask to dissolve protamine sulfate. The bottle was filled with an appropriate amount of PBS. Solution B24 was filtered through a 0.22 μ filter into a10 ml glass vial. Solution B24 contained 0.6mg/ml protamine base in PBS.

Aqueous suspension 24

1.5ml of solution A24 was pipetted into a 3.5ml glass vial type I. The solution in the flask was magnetically stirred while 1.5ml of solution B24 was pipetted into the flask. The bottle was covered with an aluminum shell and sealed. The solution in the flask was stirred for 16 hours to form a suspension. The aqueous suspension 24 contained 1mg/ml insulinotropic hormone and 0.3mg/ml protamine base in PBS. This suspension was used for pharmacokinetic studies in mice.

Example 16

Insulinotropic hormone (1mg/ml) suspension

Preparation of solution A25

Solution A25 was prepared by weighing 10mg of insulinotropic hormone into a 5ml volumetric flask. About 3ml of PBS was added to the vial to dissolve the drug. The appropriate amount of PBS was added to the vial. Solution a25 was filtered through a 0.22 μ filter into a10 ml glass vial using a syringe. Solution A25 contained 2mg/ml insulinotropic hormone in PBS.

Preparation of solution B25

Solution B25 was prepared by weighing 53mg of m-cresol in a10 ml volumetric flask. About 8ml of PBS was added to the bottle to dissolve m-cresol. An appropriate amount of PBS was added to the flask. Solution B25 was filtered through a 0.22 μ filter into a10 ml glass vial. The PBS of solution B25 contained 5.3mg/ml m-cresol.

Aqueous suspension 25

1.5ml of solution A25 was pipetted into a 3.5ml glass vial type I. The solution in the flask was magnetically stirred while 1.5ml of solution B25 was pipetted into the flask. The bottle was covered with an aluminum shell and sealed. The solution in the bottle was stirred for 16 hours to form a suspension. The aqueous suspension 25 contained 1mg/ml insulinotropic hormone and 2.5mg/ml m-cresol in PBS. This suspension was used for pharmacokinetic studies in mice.

Example 17

Insulinotropic hormone (0.5mg/ml) suspension

Preparation of solution A29

Solution A29 was prepared by weighing 25mg of insulinotropic hormone into a25 ml volumetric flask. About 20ml of PBS was added to the vial to dissolve the drug. The appropriate amount of PBS was added to the vial. Solution A29 was filtered through a 0.22 μ filter into a 50ml glass vial using a syringe. Solution A29 contained 1mg/ml insulinotropic hormone in PBS.

Preparation of solution B29

Solution B29 was prepared by weighing 50mg of phenol in a 50ml volumetric flask. About 40ml of PBS was added to the bottle to dissolve the phenol. The bottle was filled with an appropriate amount of PBS. Solution B29 was filtered through a 0.22 μ filter into a 50ml glass vial. Solution B29 contained 1.0mg/ml phenol in PBS.

Aqueous suspension 29

1.5ml of solution A29 was pipetted into a 3.5ml glass vial type I. The solution in the bottle was magnetically stirred while 1.5ml of solution B29 was pipetted into the bottle. The bottle was covered with an aluminum shell and sealed. The solution in the flask was stirred for 16 hours to form a suspension. Aqueous suspension 29 contained 0.5mg/ml insulinotropic hormone and 0.5mg/ml phenol in PBS. This suspension was used for pharmacokinetic studies in mice.

Example 18

Insulinotropic hormone (1mg/ml) suspension

Preparation of solution A31

10mg of insulinotropic hormone are weighed into a 5ml volumetric flask. About 4ml of PBS was added to the vial to disperse and dissolve the drug. The appropriate amount of PBS was added to the vial. Solution A31 was filtered through a 0.22 μ filter into a10 ml glass vial using a syringe. Solution A31 contained 2mg/ml insulinotropic hormone in PBS.

Preparation of solution B31

Solution B31 was prepared by weighing 50mg of phenol in a 50ml volumetric flask. About 40ml of PBS was added to the bottle to dissolve the phenol. The appropriate amount of PBS was added to the vial. Solution B31 was filtered through a 0.22 μ filter into a 50ml glass vial. Solution B31 contained 1mg/ml phenol in PBS.

Aqueous suspension 31

1.5ml of solution A31 was pipetted into a 3.5ml glass vial type I. The solution in the bottle was magnetically stirred while 1.5ml of solution B31 was pipetted into the bottle. The bottle was covered with an aluminum shell and sealed. The solution in the bottle was stirred for 16 hours to form a suspension. The aqueous suspension 31 contained 1mg/ml insulinotropic hormone and 0.5mg/l phenol in PBS. This suspension was used for pharmacokinetic studies in mice.

Example 19

Insulinotropic hormone (4mg/ml) suspension

Preparation of solution A51

22.2mg of insulinotropic hormone was weighed into a10 ml glass vial. Pipette 5ml of PBS into the vial to dissolve the drug. This solution was filtered through a 0.22 μ filter (low in bound protein) into a10 ml glass vial. Solution A51 contained 4.44mg/ml insulinotropic hormone in PBS.

Preparation of solution B51

110mg phenol and 30mg protamine sulfate were weighed into a 5ml volumetric flask. About 4ml of PBS was added to the bottle to dissolve phenol and protamine sulfate. The volumetric flask was filled to the mark with PBS. The solution was filtered through a 0.22 μ filter (low bound protein) into a10 ml glass vial. The PBS of solution B51 contained 22mg/ml phenol and 4.5mg/ml protamine base.

Aqueous suspension 51

3ml of solution A51 and 0.33ml of solution B51 were pipetted into a 3.5ml type I glass vial. The solution in the flask was shaken slowly to ensure uniform mixing. The vial was left at room temperature for 16 hours. The aqueous suspension 51 contained 4mg/ml insulinotropic hormone, 0.44mg/ml protamine base and 2.2mg/ml phenol in PBS. This suspension was used for pharmacokinetic studies in mice.

Example 20

Insulinotropic hormone (4mg/ml) suspension

Preparation of solution A52

22.2mg of insulinotropic hormone were weighed into a10 ml glass vial. 5ml of PBS was pipetted into the vial to dissolve the drug. This solution was filtered through a 0.22 μ filter (low protein binding) into a10 ml glass vial. Solution A52 contained 4.44mg/ml insulinotropic hormone in PBS.

Preparation of solution B52

110mg of phenol and 15.6mg of zinc acetate dihydrate are weighed into a 5ml volumetric flask. About 4ml of water for injection was added to the bottle to dissolve phenol and zinc acetate dihydrate. Fill the flask with water for injection to the mark. The solution was filtered through a 0.22 μ filter (low protein binding) into a10 ml glass vial. Solution B52 contained 22mg/ml phenol and 7.8mg/ml zinc acetate dihydrate in the water for injection.

Aqueous suspension 52

3ml of solution A52 and 0.33ml of solution B52 were pipetted into a 3.5ml type I glass vial. The solution in the bottle was shaken slowly to ensure uniform mixing. The vial was left at room temperature for 16 hours. The aqueous suspension 52 contained 4mg/ml insulinotropic hormone, 0.78mg/ml zinc acetate dihydrate and 2.2mg/ml phenol in PBS. This suspension was used for pharmacokinetic studies in mice.

Example 21

Insulinotropic hormone (4mg/ml) suspension

Preparation of phenol solution

244mg of phenol were weighed into a100 ml volumetric flask. About 90ml of water for injection was added to the bottle to dissolve the phenol. Fill the flask with water for injection to the mark. The pH of this solution was adjusted to pH9.0 with 5% NaOH solution. The phenol solution contained 2.44mg/ml phenol in water for injection at pH 9.0.

Preparation of solution A71

22.2mg of insulinotropic hormone was weighed into a10 ml glass vial. 5ml of phenol solution was pipetted into the vial to dissolve the drug. This solution was filtered through a 0.22 μ filter (low in bound protein) into a10 ml glass vial. Solution A71 contained 4.44mg/ml insulinotropic hormone and 2.44mg/ml phenol in water for injection.

Preparation of solution B71

116mg of protamine sulfate was weighed into a10 ml volumetric flask. About 8ml of water for injection was added to the bottle to dissolve protamine sulfate. The volumetric flask is filled with water for injection to the scale. The solution was filtered through a 0.22 μ filter (low bound protein) into a10 ml glass vial. Solution B71 contained 8.7mg/ml protamine base in the water for injection.

Preparation of solution C71

156mg of zinc acetate dihydrate and 1.632g of NaCl were weighed into a10 ml volumetric flask. About 8ml of water for injection was added to the bottle to dissolve the zinc acetate dihydrate and NaCl. The volumetric flask is filled with water for injection to the scale. The solution was filtered through a 0.22 μ filter (low bound protein) into a10 ml glass vial. Solution C71 contained 15.6mg/ml zinc acetate dihydrate and 163.2mg/ml NaCl in the water for injection.

Aqueous suspension 71

3ml of solution A71, 0.165ml of solution B71 and 0.165ml of solution C71 were pipetted into a 3.5ml glass vial type I. The solution in the bottle was shaken slowly to ensure uniform mixing. The vial was left at room temperature for 16 hours. Aqueous suspension 71 contained 4mg/ml insulinotropic hormone, 0.435mg/ml protamine base, 0.78mg/ml zinc acetate dihydrate, 8.16mg/ml NaCl and 2.2mg/ml phenol in water for injection. This suspension was used for pharmacokinetic studies in mice.

Example 22

Insulinotropic hormone (4mg/ml) suspension

Preparation of m-cresol solution

244mg of m-cresol are weighed into a100 ml measuring flask. About 90ml of water for injection was added to the bottle to dissolve m-cresol. The volumetric flask is filled with water for injection to the scale. The pH of this solution was adjusted to pH9.0 with 5% NaOH solution. The m-cresol solution contained 2.44mg/ml m-cresol in water for injection at pH 9.0.

Preparation of solution A100

22.2mg of insulinotropic hormone was weighed into a10 ml glass vial. 5ml of the m-cresol solution was pipetted into a bottle to dissolve the drug. This solution was filtered through a 0.22 μ filter (low in bound protein) into a10 ml glass vial. Solution A100 contained 4.44mg/ml insulinotropic hormone and 2.44mg/ml m-cresol in water for injection.

Preparation of solution B100

116mg of protamine sulfate was weighed into a10 ml volumetric flask. About 8ml of water for injection was added to the bottle to dissolve protamine sulfate. The volumetric flask is filled with water for injection to the scale. This solution was filtered through a 0.22 μ filter (low in bound protein) into a10 ml glass vial. Solution B100 contained 8.7mg/ml protamine base in the water for injection.

Preparation of solution C100

156mg of zinc acetate dihydrate and 1.632g of NaCl were weighed into a10 ml volumetric flask. About 8ml of water for injection was added to the flask to dissolve the zinc acetate dihydrate and NaCl. The volumetric flask is filled with water for injection to the scale. This solution was filtered through a 0.22 μ filter (low in bound protein) into a10 ml glass vial. Solution C100 contained 15.6mg/ml zinc acetate dihydrate and 163.2mg/ml NaCl in water for injection.

Aqueous suspension 100

3ml of solution A100, 0.165ml of solution B100 and 0.165ml of solution C100 were pipetted into a 3.5ml glass vial type I. The solution in the bottle was shaken slowly to ensure uniform mixing. The vial was left at room temperature for 16 hours. The aqueous suspension 100 contained 4mg/ml insulinotropic hormone, 0.435mg/ml protamine base, 0.78mg/ml zinc acetate dihydrate, 8.16ml/ml NaCl and 2.2mg/ml m-cresol in water for injection. This suspension was used for pharmacokinetic studies in mice.

Example 23

Insulinotropic hormone (4mg/ml) suspension

Preparation of solution A68

22.2mg of insulinotropic hormone was weighed into a10 ml glass vial. 5ml of PBS was pipetted into the vial to dissolve the drug. This solution was filtered through a 0.22 μ filter (low in bound protein) into a10 ml glass vial. Solution A68 contained 4.44mg/ml insulinotropic hormone in PBS.

Preparation of solution B68

116mg of protamine sulfate was weighed into a10 ml volumetric flask. About 8ml of water for injection was added to the bottle to dissolve protamine sulfate. The volumetric flask is filled with water for injection to the scale. The solution was filtered through a 0.22 μ filter (low bound protein) into a10 ml glass vial. Solution B68 contained 8.7mg/ml protamine base in the water for injection.

Preparation of solution C68

156mg of zinc acetate dihydrate and 440mg of phenol were weighed into a10 ml volumetric flask. About 8ml of water for injection was added to the bottle to dissolve the zinc acetate dihydrate and phenol. The bottle is filled to the scale with water for injection. The solution was filtered through a 0.22 μ filter (low in bound protein) into a10 ml glass vial. Solution C68 contained 15.6mg/ml zinc acetate dihydrate and 44mg/ml phenol in water for injection.

Aqueous suspension 68

3ml of solution A68, 0.165ml of solution B68 and 0.165ml of solution C68 were pipetted into a 3.5ml glass vial type I. The solution in the bottle was shaken slowly to ensure uniform mixing. The vial was left at room temperature for 16 hours. The aqueous suspension 68 in PBS contained 4mg/ml insulinotropic hormone, 0.435mg/ml protamine base, 0.78mg/ml zinc acetate dihydrate and 2.2mg/ml phenol. This suspension was used for pharmacokinetic studies in mice.

Example 24

Insulinotropic hormone (4mg/ml) suspension

Preparation of solution A67

22.2mg of insulinotropic hormone was weighed into a10 ml glass vial. 5ml of PBS was pipetted into the vial to dissolve the drug. This solution was filtered through a 0.22 μ filter (low in bound protein) into a10 ml glass vial. Solution A67 contained 4.44mg/ml insulinotropic hormone in PBS.

Preparation of solution B67

116mg of protamine sulfate was weighed into a10 ml volumetric flask. About 8ml of water for injection was added to the bottle to dissolve protamine sulfate. The volumetric flask was filled to the mark with water for injection. The solution was filtered through a 0.22 μ filter (low in bound protein) into a10 ml glass vial. Solution B67 contained 8.7mg/ml protamine base in the water for injection.

Preparation of solution C67

156mg of zinc acetate dihydrate and 440mg of m-cresol were weighed into a volumetric flask. About 8ml of water for injection was added to the bottle to dissolve the zinc acetate dihydrate and m-cresol. The volumetric flask was filled to the mark with water for injection. The solution was filtered through a 0.22 μ filter (low in bound protein) into a10 ml glass vial. Solution C67 contained 15.6mg/ml zinc acetate dihydrate and 44mg/ml m-cresol in water for injection.

Aqueous suspension 67

3ml of solution A67, 0.165ml of solution B67 and 0.165ml of solution C67 were pipetted into a 3.5ml glass vial type I. The solution in the bottle was shaken slowly to ensure uniform mixing. The vial was left at room temperature for 16 hours. The PBS of aqueous suspension 67 contained 4mg/ml insulinotropic hormone, 0.435mg/ml protamine base, 0.78mg/ml zinc acetate dihydrate and 2.2mg/ml m-cresol. This suspension was used for pharmacokinetic studies in mice.

Example 25

Preparation of solution A39

67.6mg of insulinotropic hormone was weighed into a glass vial. About 22ml of water for injection is added to the bottle to dissolve the insulinotropic hormone. The pH of the solution in the vial was adjusted to 9.6 with NaOH to make a clear solution. Water for injection was added to the vial to achieve a final drug concentration of 2.5 mg/ml.

Preparation of solution B39

386.8mg of zinc acetate dihydrate were weighed into a100 ml volumetric flask. About 80ml of water for injection was added to the bottle to dissolve the zinc acetate dihydrate. The volumetric flask was filled to the mark with water for injection. Solution B39 contained 3.9mg/ml zinc acetate dihydrate in the water for injection.

Preparation of solution C39

1.095g of phenol were weighed into a 50ml volumetric flask. About 40ml of water for injection was added to the bottle to dissolve the phenol. The volumetric flask was filled to the mark with water for injection. Solution C39 contained 21.9mg/ml phenol in the water for injection.

Preparation of solution D39

Weigh 2.25g NaCl in a25 ml volumetric flask. About 20ml of solution C39 was added to the bottle to dissolve NaCl. The volumetric flask was filled to the mark with solution C39. The solution D39 contained 9% (w/v) NaCl and 21.9mg/ml phenol in water for injection.

Aqueous suspension 39

All solutions were filtered through a 0.22 μ filter (low bound protein). 9ml of solution A39 were transferred to a10 ml sample vial. To the bottle was added 1ml of solution B39 while stirring slowly. A precipitate formed immediately. The pH was determined to be 7.0. The vial was left at room temperature for about 18 hours. 4ml of the sample was transferred to another 10ml vial and 0.44ml of solution D39 was added to the vial. The sample was stirred slowly for 5 minutes and then left at room temperature overnight.

The aqueous suspension 39 contained 2mg/ml insulinotropic hormone, 2.2mg/ml phenol, 0.9% NaCl and 0.39mg/ml zinc acetate. This suspension was used for pharmacokinetic studies in mice.

Example 26

Preparation of solution A53

32.5mg of insulinotropic hormone was weighed into a10 ml glass vial. 6ml of water for injection is added to the bottle. The pH of the solution in the vial was adjusted to 9.6 with 1% (w/v) NaOH to make it a clear solution. Adding appropriate amount of injectable water to make the concentration of the medicine 5.0 mg/ml.

Preparation of solution B53

390mg of zinc acetate dihydrate were weighed into a 50ml volumetric flask. About 40ml of water for injection was added to the bottle to dissolve the zinc acetate dihydrate. The bottle is filled to the scale with water for injection. Solution B53 contained 7.8mg/ml zinc acetate dihydrate in the water for injection.

Aqueous suspension 53

All solutions were filtered through a 0.22 μ filter (low bound protein). 2.4ml of solution A53 were transferred into a 3.5ml vial. To the flask was added 300. mu.l of solution B53 while stirring slowly. Immediately after the addition, a birefringent precipitate formed and was determined to have a pH of 6.8. After the vial had been left at room temperature for 20 hours, 7.5. mu. l m-cresol was added directly to the supernatant of the settled suspension. The suspension was then stirred slowly to dissolve the m-cresol. 300 μ l of 9% NaCl solution was added to the suspension with stirring. The aqueous suspension 53 contained 4mg/ml of insulinotropic hormone, 0.9% NaCl, 0.78mg/ml of zinc acetate and 2.5mg/ml of m-cresol in water for injection. This suspension was used for pharmacokinetic studies in mice.

Example 27

Preparation of solution A54

32.5mg of insulinotropic hormone was weighed into a10 ml glass vial. 6ml of water for injection is added to the bottle. The pH of the solution in the vial was adjusted to 9.6 with 1% (w/v) NaOH to make it a clear solution. Adding appropriate amount of injectable water to make the concentration of the medicine reach 5.0 mg/ml.

Preparation of solution B54

390mg of zinc acetate dihydrate were weighed into a 50ml volumetric flask. About 40ml of water for injection was added to the bottle to dissolve the zinc acetate dihydrate. The volumetric flask is filled to the scale with water for injection. Solution B54 contained 7.8mg/ml zinc acetate dihydrate in the water for injection.

Preparation of solution C54

1.1g phenol and 4.5g NaCl were weighed into a 50ml volumetric flask. About 40ml of water for injection was added to the bottle to dissolve phenol and NaCl. The volumetric flask is filled to the scale with water for injection. Solution C54 contained 22mg/ml phenol and 90mg/ml NaCl.

Aqueous suspension 54

All solutions were filtered through a 0.22 μ filter (low bound protein). 2.4ml of solution A54 were transferred into a 3.5ml vial. 300 μ l of solution B54 was added to the flask with stirring. Upon completion of the addition, a birefringent precipitate formed. The pH was determined to be 6.8. The sample was left at room temperature for 20 hours. 300 μ l of solution C54 was added with slow stirring. Aqueous suspension 54 contained 4mg/ml insulinotropic hormone, 0.78mg/ml zinc acetate dihydrate, 2.2mg/ml phenol, and 9mg/ml NaCl in water for injection. This suspension was used for pharmacokinetic studies in mice.

Example 28

Preparation of solution A57

15mg of insulinotropic hormone was weighed into a10 ml glass vial. 3ml of water for injection was added to the bottle. The pH of the solution in the vial was adjusted to 9.9 with 5% NaOH to completely dissolve the drug. Solution A57 contained 5.0mg/ml of water for injection.

Preparation of solution B57

780mg of zinc acetate dihydrate were weighed into a100 ml volumetric flask. About 80ml of water for injection was added to the flask to dissolve the zinc acetate dihydrate. The volumetric flask is filled to the scale with water for injection. Solution B57 contained 7.8mg/ml zinc acetate dihydrate in the water for injection.

Preparation of solution C57

2.2g phenol and 9g NaCl were weighed into a100 ml volumetric flask. To the bottle was added 80ml of water for injection to dissolve phenol and NaCl. The volumetric flask is filled to the scale with water for injection. Solution C57 contained 22mg/ml phenol and 90mg/ml NaCl in the water for injection.

Aqueous suspension 57

2.4ml of solution A57 were transferred into a 3.5ml vial. The solution was stirred slowly while 300. mu.l of solution B57 was added. A precipitate formed immediately after the addition of solution B57. The pH was determined to be 7.1. The sample was left at room temperature for 24 hours. 300. mu.l of solution C57 were added with slow stirring. Aqueous suspension 57 contains 4mg/ml insulinotropic hormone, 0.78mg/ml zinc acetate dihydrate, 2.2mg/ml phenol, and 9mg/ml NaCl in water for injection. This suspension was used for pharmacokinetic studies in mice.

Example 29

Preparation of solution A64

53.3mg of insulinotropic hormone was weighed into a 30ml glass vial. After addition of 11ml of water for injection, the pH of the vial solution was adjusted to 8.3 with 5% NaOH (w/v) to dissolve the insulinotropic hormone. The pH was adjusted down to 6.0 with dilute HCl to ensure that the solution was still clear. Adding appropriate amount of injectable water to make the concentration of the medicine reach 4.4 mg/ml. Solution a64 was filtered through a 0.22 μ filter (low bound protein) into a 3.5ml sample vial. 1.8ml of the filtered solution was transferred to an additionally sterilized 3.5ml vial and the vial was left to crystallize at room temperature for 3 days.

Preparation of solution B64

780mg of zinc acetate dihydrate were weighed into a 50ml volumetric flask. About 40ml of water for injection was added to the bottle to dissolve the zinc acetate dihydrate. The volumetric flask is filled to the scale with water for injection. Solution B64 contained 15.6mg/ml zinc acetate dihydrate in the water for injection.

Preparation of solution C64

18g NaCl was weighed into a100 ml volumetric flask. About 80ml of water for injection was added to the bottle to dissolve NaCl. The volumetric flask is filled to the scale with water for injection. Solution C64 contained 180mg/ml NaCl in the water for injection.

Aqueous suspension 64

After crystallization of solution A64 was complete, 100. mu.l of solution B64 were added to 1.8ml of the slowly stirred crystal suspension. The samples were then left at room temperature for 3 days. 100. mu.l of solution C64 were added to the crystal suspension with slow stirring. The pH of the suspension was adjusted to pH7.3 with dilute NaOH. 5.0 μm-cresol was added directly to the pH-adjusted crystal suspension. The aqueous suspension 64 contained 4mg/ml of insulinotropic hormone, 0.78mg/ml of zinc acetate dihydrate, 9mg/ml of NaCl and 2.5mg/ml of m-cresol in water for injection. This suspension was used for pharmacokinetic studies in mice.

Example 30

Preparation of solution A69

Weigh 1g NaCl in a100 ml volumetric flask. About 80ml of water for injection was added to the bottle to dissolve NaCl. The bottle is filled to the scale with water for injection. Solution A69 contained 1% (w/v) NaCl in the water for injection.

Preparation of solution B69

390mg of zinc acetate dihydrate were weighed into a100 ml volumetric flask. About 80ml of water for injection was added to the bottle to dissolve the zinc acetate dihydrate. The volumetric flask is filled to the scale with water for injection. Solution B69 contained 3.9mg/ml zinc acetate dihydrate in the water for injection.

Preparation of emulsion C69

2.5ml of sterile filtered (0.22. mu. low protein bound) m-cresol was transferred to a100 ml volumetric flask. The volumetric flask was filled to the mark with water for injection and sonicated to obtain a homogeneous suspension. Emulsion C69 contained 25mg/ml m-cresol in water for injection.

Aqueous suspension 69

35.74mg of insulinotropic hormone was weighed into a10 ml glass vial. 7ml of solution A69 were added. The pH of the solution in the vial was adjusted to 9.2 to dissolve the drug. The pH of the solution was then adjusted to 6.5 with dilute HCl. Adding appropriate amount of injectable water to make the concentration of the medicine to be 4.4 mg/ml. The solution was filtered through a 0.22 μ filter (low protein binding). The solution was left at room temperature for 6 days during which the insulinotropic hormone crystallized out. 1.5ml of the crystal suspension was transferred to another vial. 167. mu.l of solution B69 were added with slow stirring. The sample was left at room temperature for 1 day. 167. mu.l of emulsion C69 were added to the supernatant of the clear suspension. The sample was stirred to dissolve m-cresol. The aqueous suspension 69 contains, in water for injection, 3.6mg/ml insulinotropic hormone, 0.36mg/ml zinc acetate, 8.17mg/ml NaCl and 2.28mg/ml m-cresol. This suspension was used for pharmacokinetic studies in mice.

Example 31

Preparation of solution A101

10g of sodium acetate are weighed into a100 ml volumetric flask. About 80ml of water for injection was added to the bottle to dissolve the sodium acetate. The volumetric flask is filled to the scale with water for injection. Solution A101 contained 100mg/ml sodium acetate in the water for injection.

Aqueous suspension 101

44.4mg of insulinotropic hormone were weighed into a10 ml glass vial. 8ml of water for injection was added to the bottle. The pH of the solution in the bottle was adjusted to 9.3 to give a clear solution. 1ml of solution A101 was added to the insulinotropic hormone solution. The pH was then adjusted to 6.5 and the solution was filtered through a 0.22 μ filter (low bound protein). The filtrate was left at room temperature for 3 days to allow crystallization to occur. Aqueous suspension 101 contains 4.9mg/ml insulinotropic hormone and 11.1mg/ml sodium acetate in water for injection. This suspension was used for pharmacokinetic studies in mice.

Example 32

Preparation of solution A82

Weigh 9g NaCl in 100ml volumetric flask. About 80ml of water for injection was added to the bottle to dissolve NaCl. The bottle is filled to the scale with water for injection. Solution A82 contained 9% (w/v) NaCl in the water for injection.

Preparation of solution B82

789mg of zinc acetate dihydrate were weighed into a100 ml volumetric flask. About 80ml of water for injection was added to the bottle to dissolve the zinc acetate dihydrate. The volumetric flask is filled to the scale with water for injection. The water for injection of solution B82 contained 7.89mg/ml zinc acetate dihydrate.

Preparation of emulsion C82

2.5ml of sterile filtered (0.22. mu. low protein binding) m-cresol was transferred into a100 ml volumetric flask. The bottle was filled to scale with water for injection and sonicated to give a homogeneous suspension. Emulsion C82 contained 25mg/ml m-cresol in water for injection.

Aqueous suspension 82

All solutions were filtered through a 0.22 μ filter (low protein binding). To a10 ml vial (8 ml water added) was added 45.34mg of insulinotropic hormone. The pH was adjusted to 9.3 with 5% NaOH. After 1ml of solution A82 was added to the vial, the pH of the solution was adjusted to 6.55 with dilute HCl and the solution (5mg/ml insulinotropic hormone) was filtered through a 0.22 μ filter (low bound protein). To the sterile filtered insulinotropic hormone solution was added 81. mu.l of the aqueous suspension 101 (see example 31) and the sample was shaken to disperse. The sample was then left at room temperature for 72 hours to generate a crystal suspension. 2.4ml of the suspension were transferred to a 3.5ml vial. Add 300. mu.l of solution B82 to the bottle while stirring slowly. The pH of the solution in the bottle was adjusted to 7.3 with dilute NaOH. To the supernatant of the settled suspension was added 300. mu.l of emulsion C82. The aqueous suspension 82 contained 4mg/ml of insulinotropic hormone, 0.79mg/ml of zinc acetate dihydrate, 2.5mg/ml of lmm-cresol and 0.9% of NaCl in water for injection. This suspension was used for pharmacokinetic studies in mice.

Example 33

GLP-1(7-36) amide (1mg/ml) suspension

Preparation of solution A26

Solution A26 was prepared by weighing 10mg of GLP-1(7-36) amide in a 5ml volumetric flask. About 3ml of PBS was added to the vial to dissolve the drug. The appropriate amount of PBS was added to the vial. Solution A26 was filtered through a 0.22 μ filter into a10 ml glass vial. Solution A26 contained 2mg/ml GLP-1(7-36) in PBS.

Preparation of solution B26

Solution B26 was prepared by weighing 44mg of phenol in a10 ml volumetric flask. About 8ml of PBS was added to the bottle to dissolve the phenol. An appropriate amount of PBS was added to the flask. Solution B26 was filtered through a 0.22 μ filter into a10 ml glass vial. Solution B26 contained 4.4mg/ml phenol in PBS.

Aqueous suspension 26

Pipette 1.5ml of solution A26 into a 3.5ml type I glass vial. The solution in the vial was magnetically stirred while 1.5ml of solution B26 was pipetted into the vial. The bottle was covered with an aluminum shell and sealed. The solution in the flask was stirred slowly (to ensure no foam or no foam formation) for 18 hours to form a suspension. Aqueous suspension 26 contained 1mg/ml GLP-1(7-36) amide and 2.2mg/ml phenol in PBS. This suspension was used for pharmacokinetic studies in mice.

Example 34

According to one form of the invention, GLP-1(7-37) in low solubility form is prepared by combining 2-15mg/ml GLP-1(7-37) in a buffer having a pH of 7-8.5 with a solution of a metal ion salt to give a 1-8mg/ml GLP-1(7-37) solution wherein the molar ratio of zinc to GLP-1(7-37) is from 1: 1 to 270: 1. A reprecipitate was formed and left overnight at room temperature. The solubility of GLP-1(7-37) in metal ion solutions varies depending on the metal used. Subsequent determination of the solubility of the GLP-1(7-37) precipitate in a metal-free solvent such as PBS or water indicates that the zinc, cobalt and nickel ions produce the low-solubility form of GLP-1 (7-37).

TABLE 1

Ability of various metal ion salts to form GLP-1(7-37) of low solubility

Metal ion salts	Solubility in Metal solutions	Solubility in PBS
Metal ion salts	Solubility in Metal solutions	Solubility in PBS	Zinc acetate, cobalt chloride, nickel sulfate, manganese chloride, magnesium chloride and calcium chloride	0.04μg/ml0.04μg/ml0.11μg/ml0.14μg/ml0.23μg/ml1.75μg/ml1.98μg/ml	0.04 mu g/ml0.03 mu g/ml0.04 mu g/ml0.07 mu g/ml1.64 mu g/ml without precipitation and without precipitation

Note: in each case, 100. mu.l of 5mM metal ion solution was added to 100. mu.l of 5mg/ml GLP-1(7-37), mixed and left overnight. The insoluble precipitate was removed by centrifugation. The concentration of GLP-1(7-37) in the metal ion solution was determined. The pellet was then suspended in Phosphate Buffered Saline (PBS), sonicated and left overnight. The insoluble material was then agglomerated and the GLP-1(7-37) concentration was measured.

Example 35

Microcrystalline forms of GLP-1(7-37) can be obtained by mixing solutions of GLP-1(7-37) in buffer solution at pH7-8.5 with a combination of certain salts with low molecular weight polyethylene glycol (PEG). Table 2 describes the conditions for obtaining six specific groups of GLP-1(7-37) in microcrystalline form.

TABLE 2

Obtaining selected agents of microcrystals

Reagent	Salt (salt)	Buffer solution	Precipitating agent
Reagent	Salt (salt)	Buffer solution	Precipitating agent	123456	0.2M MgCl without 0.2M sodium citrate₂0.2M MgCl₂0.5M K₂HPO₄Is free of	No 0.1M Tris pH8.50.1M HEPES pH7.50.1M HEPES pH7.5 none	0.4M Potassium tartrate, sodium 30% PEG 40028% PEG 40030% PEG 40020% PEG 800030% PEG 1500

Note: 5mg/ml GLP-1(7-37) feed solution in 50mM Tris pH8.1 was added with the reagents in a ratio of 1: 1. The drop was observed and the presence or absence of crystalline or amorphous insoluble GLP-1(7-37) was noted. In general, low mW PEG appears to be prone to crystal formation. Tris is Tris (hydroxymethyl) aminomethane and HEPES is N-2- (hydroxyethyl) piperazine-N-2-ethanesulfonic acid.

Example 36

Specific combinations of GLP-1(7-37) and PEG concentrations are required to give microcrystalline forms and high yields. Table 3 shows that a particular combination of PEG 600 and GLP-1(7-37) concentrations, as opposed to the amorphous form of the drug, results in a microcrystalline form. The yield of the insoluble form of GLP-1(7-37) is also shown.

TABLE 3

Form/yield of crystalline form GLP-1(7-37)

GLP-1(7-37)	15％PEG 600	22.5％PEG 600	30％PEG 600
GLP-1(7-37)	15％PEG 600	22.5％PEG 600	30％PEG 600	2.0mg/ml (form/yield)	Amorphous form/8%	Amorphous/10%	Amorphous form/8%
3.5mg/ml (form/yield)	Crystal form/62%	Crystal form/26%	Crystal form/59%	2.0mg/ml (form/yield)	Amorphous form/8%	Amorphous/10%	Amorphous form/8%
3.5mg/ml (form/yield)	Crystal form/62%	Crystal form/26%	Crystal form/59%	5.0mg/ml (form/yield)	Amorphous/34%	Crystal form/63%	Crystal form/72%
6.5mg/ml (form/yield)	Amorphous/52%	Crystal form/76%	Crystal form/82%	5.0mg/ml (form/yield)	Amorphous/34%	Crystal form/63%	Crystal form/72%
6.5mg/ml (form/yield)	Amorphous/52%	Crystal form/76%	Crystal form/82%	8.0mg/ml (form/yield)	Amorphous/55%	Crystal form/82%	Amorphous/66%
9.5mg/ml (form/yield)	Amorphous/69%	Crystal form/85%	Amorphous/83%	8.0mg/ml (form/yield)	Amorphous/55%	Crystal form/82%	Amorphous/66%

Note: a20 mg/ml GLP-1(7-37) solution in pH8 Tris buffer was mixed with a solution in H₂And mixing O and 60% polyethylene glycol 600(PEG 600) of a Tris buffer solution with the pH of 8 to obtain 15-30% PEG and 3-10 mg/ml GLP-1, thereby preparing the GLP-1(7-37) microcrystal. After standing overnight, the yield of GLP-1(7-37) microcrystals in solution was 50-85%.

Example 37

This example illustrates another mode of the present invention which comprises treating pre-formed GLP-1(7-37) microcrystals with various metal ions to obtain a low-solubility microcrystalline form. GLP-1(7-37) microcrystals prepared with 8mg/ml GLP-1(7-37) and 22.5% PEG have the same solubility as pure lyophilized GLP-1(7-37) as described in example 22. In order to have the low solubility properties required for long acting drug release, the solubility can be measured as metal: GLP-1(7-37) these pre-formed microcrystals are treated overnight at room temperature with a metal salt solution in a ratio of 1: 1 to 260: 1. Excess metal salts can be removed by centrifugation/washing. Table 4 lists the results of treatment with several divalent cationic metal salts.

TABLE 4

Solubility of GLP-1(7-37) crystals at different treatments

Additive agent	GLP-1(7-37) (mg/ml) (in treatment solution)	GLP-1(7-37) (mg/ml) (in PBS)	GLP-1(7-37) (mg/ml) (in PBS/EDTA)
Additive agent	GLP-1(7-37) (mg/ml) (in treatment solution)	GLP-1(7-37) (mg/ml) (in PBS)	GLP-1(7-37) (mg/ml) (in PBS/EDTA)	Citrate No (PBS) pH5.2ZnCl₂ pH5.2ZnAc pH5.2ZnAc pH5.2MgSO₄ pH5.2NiSO₄ pH5.2MnCl₂ pH5.2CaCl₂ pH5.2	1.20.150.030.010.060.500.100.100.40	1.2 untested 0.030.020.020.550.040.100.27	Untested 1.11.10.92 untested 0.45 untested

Note: GLP-1(7-37) crystals were grown from a solution of 8mg/ml IST in 50mM Tris pH8 with 22.5% PEG 600 added to water. All additional treatment solutions were 100mM divalent ion salt in 10mM sodium citrate pH5.2 or Na MES pH 6.5.

Example 38

Both amorphous and microcrystalline low solubility formulations are prepared using zinc acetate using the methods described herein. Mice were injected subcutaneously (three animals per formulation) and plasma levels of GLP-1(7-37) were determined by radioimmunoassay at 24 hours. FIG. 8 shows that the time of the drug in plasma is extended compared to the soluble GLP-1(7-37) subcutaneous control injection.

Example 39

45% (w/v) polyethylene glycol 3350(PEG)

1mg/ml insulinotropic hormone

20mM phosphate buffer

Sterilized water for injection (SWFI)

A50% (w/w) PEG solution was prepared with SWFI. A200 mM phosphate buffer was also prepared using anhydrous sodium dihydrogen phosphate (26.85mg/ml) and sodium monohydrogen phosphate monohydrate (1.41 mg/ml). The pH of the buffer is adjusted to pH8 with sodium hydroxide or hydrochloric acid, if necessary. The appropriate amount of insulinotropic hormone is dissolved in sufficient buffer to achieve a10 mg/ml solution of insulinotropic hormone. To the insulinotropic hormone solution is added an appropriate amount of PEG solution and the solution is adjusted to the desired volume with sufficient SWFI. The final solution was sterile filtered through a 0.2 μ filter and aseptically filled into vials. The solution (0.5ml) was injected subcutaneously into mice and plasma insulinotropic hormone content was measured by RIA.

Example 40

1.32% (w/v) hydroxyethyl cellulose (HEC)

1mg/ml insulinotropic hormone

20mM phosphate buffer

100mM sodium chloride

Proper amount of sterile water for injection (SWFI)

A2% (w/w) solution of hydroxyethylcellulose was prepared with SWFI. A200 mM phosphate buffer was also prepared from anhydrous sodium dihydrogenphosphate (26.85mg/ml) and disodium hydrogenphosphate monohydrate (1.41mg/ml), and the pH of the buffer was adjusted to pH8 with sodium hydroxide or hydrochloric acid, if necessary. Appropriate amounts of insulinotropic hormone and sodium chloride are dissolved in sufficient buffer solution to obtain a10 mg/ml insulinotropic hormone solution. Add the appropriate amount of HEC solution to the insulinotropic hormone solution and adjust the solution to the desired volume with a sufficient amount of SWFI. The final solution was sterile filtered with a 0.2 μ filter and aseptically filled into vials. This solution (0.5ml) was injected subcutaneously into mice and plasma insulinotropic hormone levels were measured using RIA.

EXAMPLE 41

18％(w/v)Pluronic F127

1mg/ml insulinotropic hormone

20mM phosphate buffer

Proper amount of sterile water for injection (SWFI)

A20% (w/w) solution of Pluronic F127 was prepared with SWFI. The polymer was dissolved using a Polytron (probe homogenizer) with an ice bath. 200mM phosphate buffer was also prepared using anhydrous sodium dihydrogen phosphate (26.85mg/ml) and disodium hydrogen phosphate monohydrate (1.41 mg/ml). If necessary, the pH of the buffer was adjusted to pH8 with sodium hydroxide or hydrochloric acid. An appropriate amount of insulinotropic hormone is dissolved in a sufficient amount of buffer to obtain a10 mg/ml insulinotropic hormone solution. The appropriate amount of Pluronic solution was added to the insulinotropic hormone solution and the solution was adjusted to the desired volume with a sufficient amount of SWFI. The final solution was sterile filtered using a 0.2 μm filter and aseptically filled into vials. This solution (0.5ml) was injected subcutaneously into mice, and then plasma insulinotropic hormone content was measured by RIA.

Example 42

Peanut oil suspension (ball mill)

1mg/ml insulinotropic hormone

1％Tween80

Tween80 was added at 1% to the peanut oil. This solution was sterile filtered through a 0.2 μm filter. Solid insulinotropic hormone is then suspended in the oil. The particle size was reduced by milling with a szesvariAttritor ball mill at 40RPM for 18 hours (cold water jacket). This suspension was then filled into vials. The suspension (0.5ml) was injected subcutaneously into mice, and then plasma insulinotropic hormone content was measured by RIA.

Example 43

22.6% (w/v) Glucan

1mg/ml insulinotropic hormone

20mM phosphate buffer

Proper amount of sterile water for injection

A50% (w/w) Dextran (Dextran) solution was prepared with SWFI. 200mM phosphate buffer was also prepared using anhydrous sodium dihydrogen phosphate (26.85mg/ml) and disodium hydrogen phosphate monohydrate (1.41 mg/ml). The pH of the buffer is adjusted to pH8 with sodium hydroxide or hydrochloric acid, if necessary. An appropriate amount of insulinotropic hormone is dissolved in a sufficient amount of buffer solution to obtain a 5.0mg/ml insulinotropic hormone solution. The appropriate weight of dextran solution was added to the insulinotropic hormone solution and the solution was adjusted to the desired volume with a sufficient amount of SWFI. The final solution was sterile filtered through a 0.2 μm filter and filled into vials. This solution (0.5ml) was injected subcutaneously into mice, and then plasma insulinotropic hormone content was measured with RIA.

Example 44

Insulinotropic hormone was crystallized from a mixture of Phosphate Buffered Saline (PBS), ethanol and m-cresol. A homogeneous slurry of insulinotropic hormone (10mg/ml) was prepared in a glass vial with PBS and a large volume of ethanol (up to 9 times the slurry) was added to the vial while the solution in the vial was magnetically stirred. Very fine amorphous insulinotropic hormone particles are produced. M-cresol was added to the bottle so as to obtain a m-cresol concentration of 1% (v/v). The bottle was capped to prevent evaporation of the solution. The crystalline mixture was stored at room temperature for two days and needle-shaped crystalline platelets were grown from the amorphous particles. The length of the crystal is 50 to 200 μm, and the width is 2 to 4 μm.

Example 45

Insulinotropic hormone (1-4 mg/ml) is dissolved in 1% sodium sulfate (or sodium acetate, sodium chloride or ammonium sulfate) solution with pH value higher than 8, and dilute HCl is used to reduce the pH value of the solution to 6.0-7.5. And (3) placing the clear solution at room temperature for two days, and obtaining needle-shaped or sheet-shaped crystals according to crystallization conditions.

Example 46

GLP-1(7-37) is dissolved in 50mM glycerol buffer solution containing 0.1-0.2M NaCl and having a pH of 8.5-9.5, and the amount of the buffer solution is 1-5 mg/ml. The zinc salt (acetate or chloride) solution is added to obtain a zinc salt solution having a molar ratio of zinc to GLP-1(7-37) of 0.5: 1 to 1.5: 1. GLP-1(7-37) crystals grow overnight at room temperature, and the yield is 70-97%.

Example 47

GLP-1(7-37) crystals can be grown by a steam diffusion method using 10-20 mg/ml of peptide dissolved in 100mM Tris at a pH of 8-9.5. The peptide solution was mixed with the same buffer solution containing 0.5-2.5M NaCl at a ratio of 1: 1 and then equilibrated in a sealed system for very high concentration buffer solutions (i.e., TRis with 0.5-2.5M NaCl).

Sequence table

(1) Data of seq. ID No.1

(i) Sequence characterization

(A) Length: 37 amino acid

(B) Type (2): amino acids

(C) And (3) stranding: is made of

(D) Topological structure: straight line

(ii) Molecular type: peptides

(iii) Suppose that: is free of

(iv) Antisense: is free of

(v) Fragment type: n-terminal

(vi) The source is as follows:

(A) an organism: N/A

(B) The strain is as follows: N/A

(C) Individual isolates: N/A

(E) Single mode specimen: N/A

(H) Cell lines: N/A

(vii) The direct source is as follows:

(A) library: N/A

(B) Cloning: N/A

(viii) Position in the genome:

(A) chromosome/segment: N/A

(B) Position of the figure: N/A

(C) Unit: N/A

(xi) Description of the sequence: seq. ID. No.1

His Asp Glu Phe Glu Arg His Ala Glu Gly Thr phe Thr Ser Asp Val

1 5 10 15

Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu

20 25 30

Val Lys Gly Arg Gly

35

(2) Data of seq. ID No.2

(i) Sequence characteristics:

(A) length: 31 amino acid

(B) Type (2): amino acids

(C) And (3) stranding: is made of

(D) Topological structure: straight line

(ii) Molecular type: peptides

(iii) The assumption is that: is free of

(iv) Antisense: is free of

(v) Fragment type: n-terminal

(vi) The source is as follows:

(A) an organism: N/A

(B) The strain is as follows: N/A

(C) Individual isolates: N/A

(E) Single mode specimen: N/A

(H) Cell lines: N/A

(vii) The direct source is as follows:

(A) library: N/A

(B) Cloning: N/A

(viii) Position in the genome:

(A) chromosome/segment: N/A

(B) The figure position is as follows: N/A

(C) Unit: N/A

(xi) Description of the sequence: SEQ. ID. No.2

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly

20 25 30

(3) Data of SEQ. ID No.3

(i) Sequence characterization

(A) Length: 30 amino acids

(B) Type (2): amino acids

(C) And (3) stranding: is made of

(D) Topological structure: straight line

(ii) Molecular type: peptides

(iii) Suppose that: is free of

(iv) Antisense: is free of

(v) Fragment type: n-terminal

(vi) The source is as follows:

(A) an organism: N/A

(B) The strain is as follows: N/A

(C) Individual isolates: N/A

(E) Single mode specimen: N/A

(H) Cell lines: N/A

(vii) The direct source is as follows:

(A) library: N/A

(B) Cloning: N/A

(viii) Position in the genome:

(A) chromosome/segment: N/A

(B) The figure position is as follows: N/A

(C) Unit: N/A

(xi) Description of the sequence: SEQ. ID. No.3

His Ala Glu Gly Thr Phe Thr Ser Asp Val ser ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg

20 25 30

(4) Data of SEQ. ID No.4

(i) Sequence characterization

(A) Length: 29 amino acids

(B) Type (2): amino acids

(C) And (3) stranding: is made of

(D) Topological structure: straight line

(ii) Molecular type: peptides

(iii) Suppose that: is free of

(iv) Antisense: is free of

(v) Fragment type: n-terminal

(vi) The source is as follows:

(A) an organism: N/A

(B) The strain is as follows: N/A

(C) Individual isolates: N/A

(E) Single mode specimen: N/A

(H) Cell lines: N/A

(vii) The direct source is as follows:

(A) library: N/A

(B) Cloning: N/A

(viii) Position in the genome:

(A) chromosome/segment: N/A

(B) The figure position is as follows: N/A

(xi) Description of the sequence: SEQ. ID. No.4

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly

20 25

(5) Data of SEQ. ID No.5

(i) Sequence characterization

(A) Length: 28 amino acids

(B) Type (2): amino acids

(C) And (3) stranding: is made of

(D) Topological structure: straight line

(ii) Molecular type: peptides

(iii) Suppose that: is free of

(iv) Antisense: is free of

(v) Fragment type: n-terminal

(vi) The source is as follows:

(A) an organism: N/A

(B) The strain is as follows: N/A

(C) Individual isolates: N/A

(E) Single mode specimen: N/A

(H) Cell lines: N/A

(vii) The direct source is as follows:

(A) library: N/A

(B) Cloning: N/A

(viii) Position in the genome:

(A) chromosome/segment: N/A

(B) The figure position is as follows: N/A

(xi) Description of the sequence: SEQ. ID. No.5

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys

20 25

(6) Data of SEQ. ID No.6

(i) Sequence characterization

(A) Length: 36 amino acids

(B) Type (2): amino acids

(C) And (3) stranding: is made of

(D) Topological structure: straight line

(ii) Molecular type: peptides

(iii) Suppose that: is free of

(iv) Antisense: is free of

(v) Fragment type: n-terminal

(vi) The source is as follows:

(A) an organism: N/A

(B) The strain is as follows: N/A

(C) Individual isolates: N/A

(E) Single mode specimen: N/A

(H) Cell lines: N/A

(vii) The direct source is as follows:

(A) library: N/A

(B) Cloning: N/A

(viii) Position in the genome:

(A) chromosome/segment: N/A

(B) The figure position is as follows: N/A

(xi) Description of the sequence: SEQ. ID. No.6

His Asp Glu Phe Glu Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val

1 5 10 15

Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu

20 25 30

Val Lys Gly Arg

35

(7) Data of SEQ. ID No.7

(i) Sequence characterization

(A) Length: 27 amino acids

(B) Type (2): amino acids

(C) And (3) stranding: is made of

(D) Topological structure: straight line

(ii) Molecular type: peptides

(iii) Suppose that: is free of

(iv) Antisense: is free of

(v) Fragment type: n-terminal

(vi) The source is as follows:

(A) an organism: N/A

(B) The strain is as follows: N/A

(C) Individual isolates: N/A

(E) Single mode specimen: N/A

(H) Cell lines: N/A

(vii) The direct source is as follows:

(A) library: N/A

(B) Cloning: N/A

(viii) Position in the genome:

(A) chromosome/segment: N/A

(B) The figure position is as follows: N/A

(xi) Description of the sequence: SEQ. ID. No.7

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val

20 25

Claims

1. Use of a polypeptide in the manufacture of a medicament for treating non-insulin dependent diabetes in a patient in need thereof, the polypeptide comprising a polypeptide of the formula:

Z-H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W-L-V-X

wherein Z represents no residue, or H-D-E-F-E-R-;

x is selected from K, K-G, K-G-R or K-G-R-G;

and when Z is H-D-E-F-E-R-, X is K-G-R or K-G-R-G;

the polypeptide has a solubility equal to or lower than 500 μ g/ml under physiological conditions; and

the medicament provides to a patient receiving the medicament a plasma concentration of the polypeptide sufficient to enhance insulin action for a time required to achieve continuous glycemic control in the treatment of non-insulin dependent diabetes mellitus.

2. The use of claim 1, wherein the polypeptide is in crystalline or amorphous form.

3. The use of claim 1, wherein said polypeptide is a pharmaceutically acceptable acid addition salt of said polypeptide.

4. The use of claim 1, wherein said polypeptide is a pharmaceutically acceptable carboxylate salt of said polypeptide.

5. The use of claim 1, wherein said polypeptide is a pharmaceutically acceptable base addition salt of said polypeptide.

6. The use of claim 1, wherein said polypeptide is a pharmaceutically acceptable lower alkyl ester of said polypeptide.

7. The use of claim 1, wherein said polypeptide is a pharmaceutically acceptable amide of said polypeptide, wherein said pharmaceutically acceptable amide is selected from the group consisting of an amide, a lower alkyl amide, and a lower dialkyl amide.

8. The use of claim 1, wherein the medicament is a medicament for subcutaneous, intramuscular, transdermal, oral, nasal or gastrointestinal administration.

9. The use of claim 1, wherein the medicament further comprises a polymer selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyoxyethylene-polyoxypropylene copolymer, polysaccharide selected from the group consisting of cellulose, cellulose derivatives, chitosan, gum arabic, karaya resin, guar gum, Xanthan resin, tragacanth gum, alginic acid, carrageenan, agarose and furcellarans, dextran, starch derivatives, hyaluronic acid, polyesters, polyamides, polyanhydrides or polyorthoesters.

10. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable water-immiscible oil suspension selected from peanut oil, sesame oil, almond oil, castor oil, camellia oil, cottonseed oil, olive oil, corn oil, soybean oil, safflower oil, coconut oil, esters of fatty acids or esters of fatty alcohols.

11. The use of claim 1, wherein the medicament further comprises a wetting agent, the wetting agent being a non-ionic surfactant.

12. The use of claim 1, wherein the medicament further comprises a suspending agent.

13. The use of claim 1, wherein the medicament further comprises a metal selected from Zn (II), Ni (II), Co (II), Mg (II), Ca (II), K (I), Mn (II), Fe (II), or Cu (II).

14. The use of claim 1, wherein the medicament is an aqueous suspension capable of sustained glycemic control.

15. The use of claim 1, wherein the medicament further comprises a phenolic compound selected from the group consisting of phenol, cresol, resorcinol, or methyl paraben.

16. The use of any one of claims 3, 4 or 5, wherein the salt is selected from ammonium sulfate, sodium sulfate, lithium chloride, sodium citrate, ammonium citrate, sodium phosphate, potassium phosphate, sodium chloride, potassium chloride, ammonium chloride, sodium acetate, ammonium acetate, magnesium sulfate, calcium chloride, ammonium nitrate or sodium formate, or a combination thereof.