U.S. patent application number 11/006990 was filed with the patent office on 2006-01-05 for prolonged delivery of peptides.
Invention is credited to Dennis E. Danley, Robert A. Gelfand, Kieran F. Geoghegan, Yesook Kim, William J. Lambert, Hong Qi.
Application Number | 20060003918 11/006990 |
Document ID | / |
Family ID | 26721207 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060003918 |
Kind Code |
A1 |
Kim; Yesook ; et
al. |
January 5, 2006 |
Prolonged delivery of peptides
Abstract
There are disclosed methods for the treatment of non-insulin
dependent diabetes mellitus in a mammal comprising the prolonged
administration of GLP-1 (7-37), and related peptides. Also
disclosed are compositions to prolong the administration of the
peptides.
Inventors: |
Kim; Yesook; (Branford,
CT) ; Lambert; William J.; (East Lyme, CT) ;
Qi; Hong; (East Lyme, CT) ; Gelfand; Robert A.;
(Madison, CT) ; Geoghegan; Kieran F.; (Mystic,
CT) ; Danley; Dennis E.; (Waterford, CT) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
12531 HIGH BLUFF DRIVE
SUITE 100
SAN DIEGO
CA
92130-2040
US
|
Family ID: |
26721207 |
Appl. No.: |
11/006990 |
Filed: |
December 7, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09943084 |
Aug 31, 2001 |
6828303 |
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11006990 |
Dec 7, 2004 |
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08472349 |
Jun 7, 1995 |
6284727 |
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09943084 |
Aug 31, 2001 |
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08181655 |
Jan 25, 1994 |
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08472349 |
Jun 7, 1995 |
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08044133 |
Apr 7, 1993 |
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08181655 |
Jan 25, 1994 |
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Current U.S.
Class: |
514/7.2 ;
514/340; 514/369; 514/6.9 |
Current CPC
Class: |
A61K 31/4439 20130101;
A61K 38/26 20130101; A61K 31/426 20130101; C07K 14/605
20130101 |
Class at
Publication: |
514/002 ;
514/340; 514/369 |
International
Class: |
A61K 38/22 20060101
A61K038/22; A61K 31/4439 20060101 A61K031/4439; A61K 31/426
20060101 A61K031/426 |
Claims
1. A method for the treatment of non-insulin dependent diabetes
mellitus in a mammal in need of such treatment comprising the
repeated administration over an extended period of time of a
compound with prolonged action after each administration, said
prolonged action necessary to achieve sustained glycemic control in
mammals.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/943,084, filed Aug. 31, 2001, now U.S. Pat.
No. 6,828,303, which is a continuation of U.S. patent application
Ser. No. 08/472,349, filed Jun. 7, 1995, now U.S. Pat. No.
6,284,727 B1, which is a continuation of U.S. patent application
Ser. No. 08/181,655, filed Jan. 25, 1994, now abandoned, which is a
continuation-in-part of U.S. patent application Ser. No.
08/044,133, filed Apr. 7, 1993, now abandoned. The contents of
these documents are incorporated herein by reference in their
entirety.
BACKGROUND ART
[0002] The present invention relates to compositions and methods
for the treatment of Diabetes Mellitus. More specifically, the
present invention relates to compositions to prolong the
administration of glucagon-like peptide 1 (GLP-1), and derivatives
thereof. These compositions are useful in treatment of Non-Insulin
Dependent Diabetes Mellitus (NIDDM).
[0003] The amino acid sequence of GLP-1 is known as: TABLE-US-00001
His-Asp-Glu-Phe-Glu-Arg-His-Ala- (SEQUENCE ID NO:1)
Glu-Gly-Thr-The-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
[0004] GLP-1 is disclosed by Lopez, L. C., et al., P.N.A.S., USA
80: 5485-5489 (1983); Bell, G. I., et al., Nature 302: 716-718
(1983); Heinrich, G. et al., Endocrinol. 115: 2176-2181 (1984) and
Ghiglione, M., et al., Diabetologia 27: 599-600 (1984).
[0005] During processing in the pancreas and intestine, GLP-1 is
converted to a 31 amino acid peptide having amino acids 7-37 of
GLP-1, hereinafter this peptide is referred to as GLP-1 (7-37).
[0006] This peptide has been shown to have insulinotropic activity,
that is, it is able to stimulate, or cause to be stimulated, the
synthesis or expression of the hormone insulin. Because of this
insulinotropic activity, GLP-1 (7-37) is alternatively referred to
as insulinotropin.
[0007] GLP-1 (7-37) has the following amino acid sequence:
TABLE-US-00002 His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:2)
Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-
Trp-Leu-Val-Lys-Gly-Arq-Gly.
[0008] GLP-1 (7-37), certain derivatives thereof and the use
thereof to treat Diabetes mellitus in a mammal are disclosed in
U.S. Pat. Nos. 5,118,666 ('666 patent) and 5,120,712 ('712 patent),
the disclosures of these patents being incorporated herein by
reference. The derivatives of GLP-1 (7-37) disclosed in the '666
and '712 patents include polypeptides which contain or lack one of
more amino acids that may not be present in the naturally occurring
sequence. Further derivatives of GLP-1 (7-37) disclosed in the '666
and '712 patents include certain C-terminal salts, esters and
amides where the salts and esters are defined as OM where M is a
pharmaceutically acceptable cation or a lower (C.sub.1-C.sub.6)
branched or unbranched alkyl group and the amides are defined as
--NR.sup.2, R.sup.3 where R.sup.2 and R.sup.3 are the same or
different and are selected from the group consisting of hydrogen
and a lower (C.sub.1-C.sub.6) branched or unbranched alkyl
group.
[0009] Certain other polypeptides, alternatively referred to as
truncated GLP-1 or truncated insulinotropin, having insulinotropic
activity and the derivatives thereof are disclosed in PCT/US
89/01121 (WO 90/11296). Those polypeptides, referred to therein as
GLP-1 (7-36), GLP-1 (7-35) and GLP-1 (7-34) have the following
amino acid sequences, respectively. TABLE-US-00003
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:3)
Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-
Trp-Leu-Val-Lys-Gly-Arg; His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE
ID NO:4) Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly; and
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:5)
Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-
Trp-Leu-Val-Lys;
[0010] Derivatives of the polypeptides disclosed in PCT/US89/01121
include polypeptides having inconsequential amino acid
substitutions, or additional amino acids to enhance coupling to
carrier protein or to enhance the insulinotropic effect thereof.
Further derivatives of insulinotropin disclosed in PCT/US89/01121
include certain C-terminal salts, esters and amides where the salts
and esters are defined as OM where M is a pharmaceutically
acceptable cation or a lower branched or unbranched alkyl group and
the amides are defined as --NR.sup.2, R.sup.3 where R.sup.2 and
R.sup.3 are the same or different and are selected from the group
consisting of hydrogen and a lower branched or unbranched alkyl
group.
DISCLOSURE OF THE INVENTION
[0011] In one embodiment, the present invention is directed to a
method for the treatment of non-insulin dependent diabetes mellitus
in a mammal in need of such treatment comprising the repeated
administration over an extended period of time of a compound with
prolonged action after each administration, said prolonged action
necessary to achieve sustained glycemic control in such mammals,
said compound selected from the group consisting of:
[0012] (a) a peptide having the amino acid sequence: TABLE-US-00004
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:2)
Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-
Trp-Leu-Val-Lys-Gly-Arg-Gly;
[0013] (b) a peptide having the amino acid sequence: TABLE-US-00005
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:7)
Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-
Trp-Leu-Val-X
[0014] wherein X is selected from the group consisting of:
TABLE-US-00006 (A) Lys, (B) Lys-Gly, and (C) Lys-Gly-Arg;
[0015] (c) a derivative of a polypeptide comprising the primary
structure H.sub.2N--W--COOH
[0016] wherein W is an amino acid sequence selected from the group
consisting of TABLE-US-00007 His-Asp-Glu-Phe-Glu-Arg-His-Ala-
(SEQUENCE ID NO:1) 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 and His-Asp-Glu-Phe-Glu-Arg-His-Ala- (SEQUENCE
ID NO:6) 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
[0017] which derivative when processed in a mammal results in a
polypeptide derivative having an insulinotropic activity; [0018]
(d) a derivative of a polypeptide comprising the primary structure
H.sub.2N--R--COOH
[0019] wherein R is an amino acid sequence selected from the group
consisting of TABLE-US-00008 His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-
(SEQUENCE ID NO:2) Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly-Arg-Gly;
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:3)
Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-
Trp-Leu-Val-Lys-Gly-Arg; His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE
ID NO:4) Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly; and
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:5)
Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-
Trp-Leu-Val-Lys;
[0020] and [0021] (e) a derivative of said peptides (a) through (d)
wherein said derivative is selected from the group consisting of:
[0022] (1) a pharmaceutically acceptable acid addition salt of said
peptides; [0023] (2) a pharmaceutically acceptable carboxylate salt
of said peptides; [0024] (3) a pharmaceutically acceptable alkali
addition salt of said peptides; [0025] (4) a pharmaceutically
acceptable lower alkyl ester of said peptides; and [0026] (5) a
pharmaceutically acceptable amide of said peptides wherein said
pharmaceutically acceptable amide is selected from the group
consisting of amide, lower alkyl amide and lower dialkyl amide.
[0027] Preferred is the method wherein said administration is
subcutaneous.
[0028] Also preferred is the method wherein said administration is
intramuscular.
[0029] Also preferred is the method wherein said administration is
transdermal.
[0030] Also especially preferred is the method wherein said
administration is by an infusion pump.
[0031] Also preferred is the method wherein said administration is
by oral inhalation.
[0032] Also preferred is the method wherein said administration is
by nasal inhalation.
[0033] Also preferred is the method wherein said administration is
gastrointestinal.
[0034] In another embodiment, the present invention is directed to
a composition of matter comprising; [0035] (i) a compound selected
from the group consisting of:
[0036] (a) a peptide having the amino acid sequence: TABLE-US-00009
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:2)
Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-
Trp-Leu-Val-Lys-Gly-Arg-Gly;
[0037] (b) a peptide having the amino acid sequence: TABLE-US-00010
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:7)
Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala
Trp-Leu-Val-X
[0038] wherein X is selected from the group consisting of:
TABLE-US-00011 (A) Lys, (B) Lys-Gly, and (C) Lys-Gly-Arg;
[0039] (c) a derivative of a polypeptide comprising the primary
structure H.sub.2N--W--COOH
[0040] wherein W is an amino acid sequence selected from the group
consisting of TABLE-US-00012 (SEQUENCE ID NO:1)
His-Asp-Glue-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 and (SEQUENCE
ID NO:6) 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
[0041] which derivative when processed in a mammal results in a
polypeptide derivative having an insulinotropic activity; [0042]
(d) a derivative of a polypeptide comprising the primary structure
H.sub.2N--W--COOH
[0043] wherein R is an amino acid sequence selected from the group
consisting of TABLE-US-00013 (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-Gly (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-Arg (SEQUENCE ID NO:4)
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 and (SEQUENCE ID NO:5)
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;
[0044] a derivative of said peptides (a) through (d) wherein said
derivative is selected from the group consisting of: [0045] (1) a
pharmaceutically acceptable acid addition salt of said peptides;
[0046] (2) a pharmaceutically acceptable carboxylate salt of said
peptides; [0047] (3) a pharmaceutically acceptable alkali addition
salt of said peptides; [0048] (4) a pharmaceutically acceptable
lower alkyl ester of said peptides; and [0049] (5) a
pharmaceutically acceptable amide of said peptides wherein said
pharmaceutically acceptable amide is selected from the group
consisting of amide, lower alkyl amide and lower dialkyl amide, and
[0050] (ii) a polymer capable of prolonging the action of said
compound to achieve sustained glycemic control.
[0051] Especially preferred is the composition wherein said polymer
is a low molecular weight polymer.
[0052] Further especially preferred is a composition wherein said
polymer is selected from the group consisting of polyethylene
glycol, polyvinylpyrrolidone, polyvinylalcohol,
polyoxyethylene-polyoxypropylene copolymers, polysaccharides
selected from the group consisting of cellulose, cellulose
derivatives, chitosan, acacia gum, karaya gum, guar gum, xanthan
gum, tragacanth, alginic acid, carrageenan, agarose, and
furcellarans, dextran, starch, starch derivatives, hyaluronic acid,
polyesters, polyamides, polyanhydrides, and polyortho esters, with
especially preferred polymers selected from the group consisting of
polyethylene glycol and polyvinylpyrrolidone.
[0053] In another embodiment, the present invention is directed to
a composition of matter comprising; [0054] (i) a compound selected
from the group consisting of:
[0055] (a) a peptide having the amino acid sequence: TABLE-US-00014
(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-Gly;
[0056] (b) a peptide having the amino acid sequence: TABLE-US-00015
(SEQUENCE ID NO:7) 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
[0057] wherein X is selected from the group consisting of:
TABLE-US-00016 (A) Lys, (B) Lys-Gly, and (C) Lys-Gly-Arg;
[0058] (c) a derivative of a polypeptide comprising the primary
structure H.sub.2N--W--COOH
[0059] wherein W is an amino acid sequence selected from the group
consisting of TABLE-US-00017 (SEQUENCE ID NO:1)
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 and (SEQUENCE
ID NO:6) 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
[0060] which derivative when processed in a mammal results in a
polypeptide derivative having an insulinotropic activity; [0061]
(d) a derivative of a polypeptide comprising the primary structure
H.sub.2N--W--COOH
[0062] wherein R is an amino acid sequence selected from the group
consisting of TABLE-US-00018 (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-Gly; (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-Arg; (SEQUENCE ID NO:4)
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; and (SEQUENCE ID NO:5)
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;
[0063] and [0064] a derivative of said peptides (a) through (d)
wherein said derivative is selected from the group consisting of:
[0065] (1) a pharmaceutically acceptable acid addition salt of said
peptides; [0066] (2) a pharmaceutically acceptable carboxylate salt
of said peptides; [0067] (3) a pharmaceutically acceptable alkali
addition salt of said peptides; [0068] (4) a pharmaceutically
acceptable lower alkyl ester of said peptides; and [0069] (5) a
pharmaceutically acceptable amide of said peptides wherein said
pharmaceutically acceptable amide is selected from the group
consisting of amide, lower alkyl amide and lower dialkyl amide, and
[0070] (ii) a pharmaceutically acceptable water-immiscible oil
suspension capable of prolonging administration of said
compound.
[0071] Especially preferred is the composition wherein said oil is
selected from the group consisting of peanut oil, sesame oil,
almond oil, castor oil, camellia oil, cotton seed oil, olive oil,
corn oil, soy oil, safflower oil, coconut oil, esters of fatty
acids, and esters of fatty alcohols. Further especially preferred
is the composition further comprising a wetting agent, especially a
nonionic surfactant.
[0072] More further especially preferred is the composition further
comprising a suspending agent.
[0073] In another embodiment, the present invention is directed to
a composition of matter comprising; [0074] (i) a compound selected
from the group consisting of:
[0075] (a) a peptide having the amino acid sequence: TABLE-US-00019
(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-Gly;
[0076] (b) a peptide having the amino acid sequence: TABLE-US-00020
(SEQUENCE ID NO:7) 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
[0077] wherein X is selected from the group consisting of:
TABLE-US-00021 (A) Lys, (B) Lys-Gly, and (C) Lys-Gly-Arg;
[0078] (c) a derivative of a polypeptide comprising the primary
structure H.sub.2N--W--COOH
[0079] wherein W is an amino acid sequence selected from the group
consisting of TABLE-US-00022 (SEQUENCE ID NO:1)
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 and (SEQUENCE
ID NO:6) 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
[0080] which derivative when processed in a mammal results in a
polypeptide derivative having an insulinotropic activity; [0081]
(d) a derivative of a polypeptide comprising the primary structure
H.sub.2N--W--COOH
[0082] wherein R is an amino acid sequence selected from the group
consisting of TABLE-US-00023 (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-Gly (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-Arg (SEQUENCE ID NO:4)
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 and (SEQUENCE ID NO:5);
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;
[0083] and [0084] a derivative of said peptides (a) through (d)
wherein said derivative is selected from the group consisting of:
[0085] (1) a pharmaceutically acceptable acid addition salt of said
peptides; [0086] (2) a pharmaceutically acceptable carboxylate salt
of said peptides; [0087] (3) a pharmaceutically acceptable alkali
addition salt of said peptides; [0088] (4) a pharmaceutically
acceptable lower alkyl ester of said peptides; and [0089] (5) a
pharmaceutically acceptable amide of said peptides wherein said
pharmaceutically acceptable amide is selected from the group
consisting of amide, lower alkyl amide and lower dialkyl amide, and
[0090] (ii) zinc (II), which is complexed with the peptide.
[0091] Preferred is the composition capable of sustained glycemic
action.
[0092] Especially preferred is the composition wherein the zinc
product is amorphous.
[0093] Also especially preferred is the composition wherein the
zinc product is crystalline.
[0094] In yet another embodiment, the present invention is directed
to a composition of matter comprising; [0095] (i) a compound
selected from the group consisting of:
[0096] (a) a peptide having the amino acid sequence: TABLE-US-00024
(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-Gly;
[0097] (b) a peptide having the amino acid sequence: TABLE-US-00025
(SEQUENCE ID NO:7) 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
[0098] wherein X is selected from the group consisting of:
TABLE-US-00026 (A) Lys, (B) Lys-Gly, and (C) Lys-Gly-Arg;
[0099] (c) a derivative of a polypeptide comprising the primary
structure H.sub.2N--W--COOH
[0100] wherein W is an amino acid sequence selected from the group
consisting of TABLE-US-00027 (SEQUENCE ID NO:1)
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 and (SEQUENCE
ID NO:6) 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
[0101] and which derivative when processed in a mammal results in a
polypeptide derivative having an insulinotropic activity; [0102]
(d) a derivative of a polypeptide comprising the primary structure
H.sub.2N--W--COOH
[0103] wherein R is an amino acid sequence selected from the group
consisting of TABLE-US-00028 (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-Gly (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-Arg (SEQUENCE ID NO:4)
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 and (SEQUENCE ID NO:5)
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;
[0104] and [0105] a derivative of said peptides (a) through (d)
wherein said derivative is selected from the group consisting of:
[0106] (1) a pharmaceutically acceptable acid addition salt of said
peptides; [0107] (2) a pharmaceutically acceptable carboxylate salt
of said peptides; [0108] (3) a pharmaceutically acceptable alkali
addition salt of said peptides; [0109] (4) a pharmaceutically
acceptable lower alkyl ester of said peptides; and [0110] (5) a
pharmaceutically acceptable amide of said peptides wherein said
pharmaceutically acceptable amide is selected from the group
consisting of amide, lower alkyl amide and lower dialkyl amide, and
[0111] (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).
[0112] In yet another embodiment, the present invention is directed
to a composition of matter comprising; [0113] (i) a compound
selected from the group consisting of:
[0114] (a) a peptide having the amino acid sequence: TABLE-US-00029
(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-Gly;
[0115] (b) a peptide having the amino acid sequence: TABLE-US-00030
(SEQUENCE ID NO:7) 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
[0116] wherein X is selected from the group consisting of:
TABLE-US-00031 (A) Lys, (B) Lys-Gly, (C) Lys-Gly-Arg;
[0117] (c) a derivative of a polypeptide comprising the primary
structure H.sub.2N--W--COOH
[0118] wherein W is an amino acid sequence selected from the group
consisting of TABLE-US-00032 (SEQUENCE ID NO:1)
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 and (SEQUENCE
ID NO:6) 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
[0119] which derivative when processed in a mammal results in a
polypeptide derivative having an insulinotropic activity; [0120]
(d) a derivative of a polypeptide comprising the primary structure
H.sub.2N--W--COOH
[0121] wherein R is an amino acid sequence selected from the group
consisting of TABLE-US-00033 (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-Gly; (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-Arg; (SEQUENCE ID NO:4)
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; and (SEQUENCE ID NO:5)
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-
Tyr-Leu-Gly-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-
Trp-Leu-Val-Lys;
[0122] and [0123] a derivative of said peptides (a) through (d)
wherein said derivative is selected from the group consisting of:
[0124] (1) a pharmaceutically acceptable acid addition salt of said
peptides; [0125] (2) a pharmaceutically acceptable carboxylate salt
of said peptides; [0126] (3) a pharmaceutically acceptable alkali
addition salt of said peptides; [0127] (4) a pharmaceutically
acceptable lower alkyl ester of said peptides; and [0128] (5) a
pharmaceutically acceptable amide of said peptides wherein said
pharmaceutically acceptable amide is selected from the group
consisting of amide, lower alkyl amide and lower dialkyl amide, and
[0129] (ii) a basic polypeptide, wherein such composition is an
aqueous suspension capable of sustained glycemic control.
[0130] Especially preferred is the composition wherein the basic
polypeptide is protamine.
[0131] In yet another embodiment, the present invention is directed
to a composition of matter comprising; [0132] (i) a compound
selected from the group consisting of:
[0133] (a) a peptide having the amino acid sequence: TABLE-US-00034
(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-Gly;
[0134] (b) a peptide having the amino acid sequence: TABLE-US-00035
(SEQUENCE ID NO:7) 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
[0135] wherein X is selected from the group consisting of:
TABLE-US-00036 (A) Lys, (B) Lys-Gly, (C) Lys-Gly-Arg;
[0136] (c) a derivative of a polypeptide comprising the primary
structure H.sub.2N--W--COOH
[0137] wherein W is an amino acid sequence selected from the group
consisting of TABLE-US-00037 (SEQUENCE ID NO:1)
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 and (SEQUENCE
ID NO:7) 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
[0138] which derivative when processed in a mammal results in a
polypeptide derivative having an insulinotropic activity; [0139]
(d) a derivative of a polypeptide comprising the primary structure
H.sub.2N--W--COOH
[0140] wherein R is an amino acid sequence selected from the group
consisting of TABLE-US-00038 (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-Gly; (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-Arg; (SEQUENCE ID NO:4)
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; and (SEQUENCE ID NO:5)
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;
[0141] and [0142] a derivative of said peptides (a) through (d)
wherein said derivative is selected from the group consisting of:
[0143] (1) a pharmaceutically acceptable acid addition salt of said
peptides; [0144] (2) a pharmaceutically acceptable carboxylate salt
of said peptides; [0145] (3) a pharmaceutically acceptable alkali
addition salt of said peptides; [0146] (4) a pharmaceutically
acceptable lower alkyl ester of said peptides; and [0147] (5) a
pharmaceutically acceptable amide of said peptides wherein said
pharmaceutically acceptable amide is selected from the group
consisting of amide, lower alkyl amide and lower dialkyl amide, and
[0148] (ii) a phenolic compound, wherein such composition is an
aqueous suspension capable of sustained glycemic control.
[0149] Especially preferred is the composition wherein said
phenolic compound is selected from the group consisting of phenol,
cresol, resorcinol, and methyl/araben.
[0150] In yet another embodiment, the present invention is directed
to a composition of matter comprising; [0151] (i) a compound
selected from the group consisting of:
[0152] (a) a peptide having the amino acid sequence: TABLE-US-00039
(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-Gly;
[0153] (b) a peptide having the amino acid sequence: TABLE-US-00040
(SEQUENCE ID NO:7) 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
[0154] wherein X is selected from the group consisting of:
TABLE-US-00041 (A) Lys, (B) Lys-Gly, (C) Lys-Gly-Arg;
[0155] (c) a derivative of a polypeptide comprising the primary
structure H.sub.2N--W--COOH
[0156] wherein W is an amino acid sequence selected from the group
consisting of TABLE-US-00042 (SEQUENCE ID NO:1)
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 and (SEQUENCE
ID NO:6) 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
[0157] which derivative when processed in a mammal results in a
polypeptide derivative having an insulinotropic activity; [0158]
(d) a derivative of a polypeptide comprising the primary structure
H.sub.2N--W--COOH
[0159] wherein R is an amino acid sequence selected from the group
consisting of TABLE-US-00043 (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-Gly; (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-Arg; (SEQUENCE ID NO:4)
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; and (SEQUENCE ID NO:5)
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;
[0160] and [0161] a derivative of said peptides (a) through (d)
wherein said derivative is selected from the group consisting of:
[0162] (1) a pharmaceutically acceptable acid addition salt of said
peptides; [0163] (2) a pharmaceutically acceptable carboxylate salt
of said peptides; [0164] (3) a pharmaceutically acceptable alkali
addition salt of said peptides; [0165] (4) a pharmaceutically
acceptable lower alkyl ester of said peptides; and [0166] (5) a
pharmaceutically acceptable amide of said peptides wherein said
pharmaceutically acceptable amide is selected from the group
consisting of amide, lower alkyl amide and lower dialkyl amide, and
[0167] (ii) a basic polypeptide and a phenolic compound, wherein
such composition is an aqueous suspension capable of sustained
glycemic control.
[0168] In another embodiment, the present invention is directed to
a composition of matter comprising; [0169] (i) a compound selected
from the group consisting of:
[0170] (a) a peptide having the amino acid sequence: TABLE-US-00044
(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-Gly;
[0171] (b) a peptide having the amino acid sequence: TABLE-US-00045
(SEQUENCE ID NO:7) 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
[0172] wherein X is selected from the group consisting of:
TABLE-US-00046 (A) Lys, (B) Lys-Gly, and (C) Lys-Gly-Arg;
[0173] (c) a derivative of a polypeptide comprising the primary
structure H.sub.2N--W--COOH
[0174] wherein W is an amino acid sequence selected from the group
consisting of TABLE-US-00047 (SEQUENCE ID NO:1)
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 and (SEQUENCE
ID NO:6) 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
[0175] which derivative when processed in a mammal results in a
polypeptide derivative having an insulinotropic activity; [0176]
(d) a derivative of a polypeptide comprising the primary structure
H.sub.2N--W--COOH
[0177] wherein R is an amino acid sequence selected from the group
consisting of TABLE-US-00048 (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-Gly; (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-Arg; (SEQUENCE ID NO:4)
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; and (SEQUENCE ID NO:5)
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;
[0178] and [0179] a derivative of said peptides (a) through (d)
wherein said derivative is selected from the group consisting of:
[0180] (1) a pharmaceutically acceptable acid addition salt of said
peptides; [0181] (2) a pharmaceutically acceptable carboxylate salt
of said peptides; [0182] (3) a pharmaceutically acceptable alkali
addition salt of said peptides; [0183] (4) a pharmaceutically
acceptable lower alkyl ester of said peptides; and [0184] (5) a
pharmaceutically acceptable amide of said peptides wherein said
pharmaceutically acceptable amide is selected from the group
consisting of amide, lower alkyl amide and lower dialkyl amide, and
[0185] (ii) a basic polypeptide, a phenolic compound, and a metal
ion wherein said composition is an aqueous suspension capable of
sustained glycemic control.
[0186] Preferred is the composition wherein said basic polypeptide
is protamine.
[0187] Also preferred is the composition wherein said metal ion is
zinc.
[0188] In another embodiment, the present invention is directed to
a composition of matter comprising; [0189] (i) a compound selected
from the group consisting of:
[0190] (a) a peptide having the amino acid sequence: TABLE-US-00049
(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-Gly;
[0191] (b) a peptide having the amino acid sequence: TABLE-US-00050
(SEQUENCE ID NO:7) 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
[0192] wherein X is selected from the group consisting of:
TABLE-US-00051 (A) Lys, (B) Lys-Gly, (C) Lys-Gly-Arg;
[0193] (c) a derivative of a polypeptide comprising the primary
structure H.sub.2N--W--COOH
[0194] wherein W is an amino acid sequence selected from the group
consisting of TABLE-US-00052 (SEQUENCE ID NO:1)
His-Asp-Glu-Phe-Glu-Arq-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 and (SEQUENCE
ID NO:6) 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
[0195] which derivative when processed in a mammal results in a
polypeptide derivative having an insulinotropic activity; [0196]
(d) a derivative of a polypeptide comprising the primary structure
H.sub.2N--W--COOH
[0197] wherein R is an amino acid sequence selected from the group
consisting of TABLE-US-00053 (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-Gly; (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-Arg; (SEQUENCE ID NO:4)
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; and (SEQUENCE ID NO:5)
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;
[0198] and [0199] a derivative of said peptides (a) through (d)
wherein said derivative is selected from the group consisting of:
[0200] (1) a pharmaceutically acceptable acid addition salt of said
peptides; [0201] (2) a pharmaceutically acceptable carboxylate salt
of said peptides; [0202] (3) a pharmaceutically acceptable alkali
addition salt of said peptides; [0203] (4) a pharmaceutically
acceptable lower alkyl ester of said peptides; and [0204] (5) a
pharmaceutically acceptable amide of said peptides wherein said
pharmaceutically acceptable amide is selected from the group
consisting of amide, lower alkyl amide and lower dialkyl amide, and
[0205] (ii) said peptides and derivatives thereof having been
subjected to conditions resulting in amorphous crystalline
formation.
[0206] Preferred is the composition wherein said conditions are
high shear, exposure to salts; or combinations thereof.
[0207] Especially preferred is the composition wherein said salt is
selected from the group consisting of ammonium sulfate, sodium
sulfate, lithium 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.
[0208] In still another embodiment, the present invention is
directed to a composition of matter comprising; [0209] (i) a
compound selected from the group consisting of:
[0210] (a) a peptide having the amino acid sequence: TABLE-US-00054
(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-Gly;
[0211] (b) a peptide having the amino acid sequence: TABLE-US-00055
(SEQUENCE ID NO:7) 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
[0212] wherein X is selected from the group consisting of:
TABLE-US-00056 (A) Lys, (B) Lys-Gly, (C) Lys-Gly-Arg;
[0213] (c) a derivative of a polypeptide comprising the primary
structure H.sub.2N--W--COOH
[0214] wherein W is an amino acid sequence selected from the group
consisting of TABLE-US-00057 (SEQUENCE ID NO:1)
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 and (SEQUENCE
ID NO:6) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-
Thr-Ser-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys Gly-Arg
[0215] which derivative when processed in a mammal results in a
polypeptide derivative having an insulinotropic activity; [0216]
(d) a derivative of a polypeptide comprising the primary structure
H.sub.2N--W--COOH
[0217] wherein R is an amino acid sequence selected from the group
consisting of TABLE-US-00058 (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-Gly; (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-Arg; (SEQUENCE ID NO:4)
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; and (SEQUENCE ID NO:5)
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;
[0218] and [0219] a derivative of said peptides (a) through (d)
wherein said derivative is selected from the group consisting of:
[0220] (1) a pharmaceutically acceptable acid addition salt of said
peptides; [0221] (2) a pharmaceutically acceptable carboxylate salt
of said peptides; [0222] (3) a pharmaceutically acceptable alkali
addition salt of said peptides; [0223] (4) a pharmaceutically
acceptable lower alkyl ester of said peptides; and [0224] (5) a
pharmaceutically acceptable amide of said peptides wherein said
pharmaceutically acceptable amide is selected from the group
consisting of amide, lower alkyl amide and lower dialkyl amide, and
[0225] (ii) a liposome delivery system.
[0226] Especially preferred is the composition wherein said
liposome is phospholipid based.
[0227] Also especially preferred is the composition wherein said
liposome is non-phospholipid based.
[0228] The present invention is also directed to the treatment of
non-insulin dependent diabetes mellitus in a mammal in need of such
treatment comprising the prolonged administration of the
compositions of the present invention. *
BRIEF DESCRIPTION OF THE DRAWINGS
[0229] FIG. 1 shows the effect of a prolonged infusion (7 hours) of
4 ng/kg/min insulinotropin on plasma glucose levels in patients
with NIDDM.
[0230] FIG. 2 shows the effect of a short infusion (60 minutes) of
10 ng/kg/min insulinotropin on plasma glucose levels in patients
with NIDDM.
[0231] FIG. 3 shows the effect of a prolonged infusion (7 hours) of
2 ng/kg/min and 4 ng/kg/min of insulinotropin on plasma glucose
levels in patients with NIDDM.
[0232] FIG. 4. Mean (n=3) Plasma Concentration of Insulinotropin in
Rats After Subcutaneous Administration of Single 0.5 mg/0.5 ml
Doses in Different Aqueous Suspensions (AS).
[0233] FIG. 5. Mean (n=3) Plasma Concentration of Insulinotropin in
Rats After Subcutaneous Administration of Single 0.5 mg/0.5 ml
Doses in Different Aqueous Suspensions (AS).
[0234] FIG. 6. Mean (n=3) Plasma Concentration of Insulinotropin in
Rats After Subcutaneous Administration of Single 0.5 mg/0.5 ml
Doses in Different Aqueous Suspensions (AS).
[0235] FIG. 7. Mean (n=3) Plasma Concentration of Insulinotropin in
Rats After Subcutaneous Administration of Single 0.5 mg/0.5 ml
Doses in Different Aqueous Suspensions (AS).
[0236] FIG. 8. Mean (n=3) Plasma Concentration of Insulinotropin in
Rats After Subcutaneous Administration of Single 0.5 mg/0.13 ml
Doses in Different Aqueous Suspensions (AS).
[0237] FIG. 9. Mean (n=3) Plasma Concentration of Insulinotropin in
Rats After Subcutaneous Administration of Single 0.5 mg/0.13 ml
Doses in Different Aqueous Suspensions (AS).
[0238] FIG. 10 shows pharmacokinetic studies of an insulinotropin
zinc precipitate.
MODES OF CARRYING OUT THE INVENTION
[0239] Unless otherwise indicated, the term "derivative", as used
throughout this Specification and the appendant claims, includes,
but is not limited to, polypeptides comprising the primary
structure shown, wherein one or more L-amino acids are included at
the C-terminus thereof; wherein the C-terminal carboxyl group forms
an ester with a (C.sub.1-C.sub.6) straight or branched chain alkyl
group; wherein the C-terminal carboxyl group forms a carboxamide or
substituted carboxamide; wherein the acidic amino acid residues
(Asp and/or Glu) form an ester or carboxamide; and combinations
thereof.
[0240] Included within the scope of this invention are polypeptides
having homology to the peptides described above, which homology is
sufficient to impart insulinotropic activity to such polypeptides.
Also included within the scope of this invention are variants of
the polypeptides described above, which variants comprise
inconsequential amino acid substitutions and have insulinotropic
activity.
[0241] Glucagon-like Peptide-1 (7-37), its isolation,
characterization, and use to treat Diabetes mellitus are disclosed
in U.S. Pat. Nos. 5,118,666 and 5,120,712, the disclosures of these
patents in their entirety being incorporated herein by
reference.
[0242] In the present invention, it has now been discovered that
prolonged plasma elevations of GLP-1, and related polypeptides, are
necessary during the meal arid beyond to achieve sustained glycemic
control in patients with Non Insulin Dependent Diabetes Mellitus.
It has surprisingly been found that raising GLP-1, and related
peptides, around meal time alone, even for periods of up to one
hour, will not adequately control the glucose levels. Thus,
administration of GLP-1, and related peptides, requires a prolonged
delivery system. This prolonged delivery system leads to an
enhancing of insulin action.
[0243] The phrase "enhancing insulin action", as used throughout
this Specification and the appendant 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 glucose production by the liver.
[0244] The polypeptides of this invention are prepared by various
methods well known to those skilled in the art. For example, the
polypeptides can be synthesized using automated peptide
synthesizers such as an Applied Biosystems (ABI) 430A solid phase
peptide synthesizer. Alternatively, the polypeptides of this
invention can be prepared using recombinant DNA technology wherein
a DNA sequence coding for the polypeptide is operably linked to an
expression vector and used to transform an appropriate host cell.
The transformed host cell is then cultured under conditions whereby
the polypeptide will be expressed. The polypeptide is then
recovered from the culture. Further still, a combination of
synthesis and recombinant DNA techniques can be employed to produce
the amide and ester derivatives of this invention and/or to produce
fragments of the desired polypeptide which are then joined by
methods well known to those skilled in the art.
[0245] Derivatives of the polypeptides according to this invention
are prepared by methods well known to those skilled in the art. For
example, C-terminal alkyl ester derivatives of the polypeptides of
this invention are prepared by reacting the desired
(C.sub.1-C.sub.6) alkanol with the desired polypeptide in the
presence of a catalytic acid such as HCl. Appropriate reaction
conditions for such alkyl ester formation include a reaction
temperature of about 50.degree. C. and reaction times of about 1
hour to about 3 hours. Similarly, derivatives of the polypeptides
of this invention comprising (C.sub.1-C.sub.6) alkyl esters of the
Asp and/or Glu residues within the polypeptide can be so formed.
.degree.
[0246] Carboxamide derivatives of the polypeptides of this
invention are also prepared by solid phase peptide synthesis
methods well known to those skilled in the art. For example, see,
Solid Phase Peptide Synthesis, Stewart, J. M. et al., Pierce Chem.
Co. Press, 1984.
[0247] Alternatively, or in combination with the above, derivatives
of the polypeptides of this invention can be prepared by modifying
the DNA coding sequence for such polypeptide so that a basic amino
acid residue is replaced with a different basic amino acid residue
or with an acid acidic or neutral amino acid residue, or an acidic
amino acid residue is replaced with a different acidic amino acid
residue or with a basic or neutral amino acid residue, or a neutral
amino acid residue is replaced with a different neutral amino acid
residue or with an acidic or basic amino acid residue. Such changes
in polypeptide primary sequence can also be accomplished by direct
synthesis of the derivative. Such methods are well known to those
skilled in the art. Of course, such derivatives, to be useful in
the practice of this invention, must achieve an insulinotropic
effect.
[0248] The insulinotropic activity of a polypeptide derivative
according to this invention is determined as follows.
[0249] Pancreatic islets are isolated from pancreatic tissue from
normal rats by a modification of the method of Lacy, P. E., et al.,
Diabetes, 16:35-39 (1967) in which the collagenase digest of
pancreatic tissue is separated on a Ficoll gradient (27%, 23%,
20.5% and 11% in Hanks' balanced salt solution, pH 7.4). The islets
are collected from the 20.5%/11% interface, washed and handpicked
free of exocrine and other tissue under a stereomicroscope. The
islets are incubated overnight in RPMI 1640 medium supplemented
with 10% fetal bovine serum and containing 11 mM glucose at
37.degree. C. and 95% air/5% CO.sub.2. The islets are then
transferred to RPMI 1640 medium supplemented with 10% fetal bovine
serum and containing 5.6 mM glucose. The islets are incubated for
60 minutes at 37.degree. C., 95% air/5% CO.sub.2. The polypeptide
derivative to be studied is prepared at 1 nM and 10 nM
concentrations in RPMI medium containing 10% fetal bovine serum and
16.7 mM glucose. About 8 to 10 isolated islets are then transferred
by pipette to a total volume of 250 .mu.l of the polypeptide
derivative containing medium in 96 well microtiter dishes. The
islets are incubated in the presence of the polypeptide derivative
at 37.degree. C., 95% air/5% CO.sub.2 for 90 minutes. Then,
aliquots of islet-free medium are collected and 100 .mu.l thereof
are assayed for the amount of insulin present by radioimmunoassay
using an Equate Insulin RIA Kit (Binax, Inc., Portland, Me.).
[0250] Dosages effective in treatment of adult onset diabetes will
range from about 1 pg/kg to 1,000 .mu.g/kg per day when a
polypeptide derivative of this invention is administered, for
example, intravenously, intramuscularly or subcutaneously. A
preferred dosage range for intravenous infusion during and between
meals is about 4 to 10 ng/kg/min or about 0.6 to 1.4 .mu.g/day
based on a 100 kg patient. It is to be appreciated, however, that
dosages outside of that range are possible and are also within the
scope of this invention. The appropriate dosage can and will be
determined by the prescribing physician and will be a result of the
severity of the condition being treated as well as the response
achieved with the derivative being administered and the age,
weight, sex and medical history of the patient.
[0251] The prolonged administration may be achieved by
subcutaneous, intramuscular, or transdermal means, oral inhalation,
nasal inhalation, gastrointestinal, or by means of an infusion
pump.
[0252] Prolonged administration of GLP-1, and related peptides, may
also be achieved by formulation as a solution in various
water-soluble polymers. These polymers are generally low molecular
weight (<15 kDa) polymers. Non-limiting examples of such low
molecular weight polymers include polyethylene glycol,
polyvinylpyrrolidone, polyvinylalcohol and
polyoxyethylene-polyoxypropylene copolymers. Higher molecular
weight polymers may be used. Non-limiting examples of higher
molecular weight polymers include polysaccharides such as cellulose
and its derivatives, chitosan, acacia gum, karaya gum, guar gum,
xanthan gum, tragacanth, alginic acid, carrageenan, agarose,
furcelleran. In the later case, polymers which are degraded in vivo
either enzymatically or by hydrolysis are preferred, for example,
dextran; starch and its derivatives, hyaluronic acid, polyesters,
polyamides, polyanhydrides and polyortho esters. The tissue
accumulation associated with high molecular weight,
non-biodegradable polymers is avoided by using low molecular weight
polymers or biodegradable polymers. The formulations typically
contain GLP-1, or related peptides, at approximately 1 mg/ml, with
concentration dependent on the polymer, but typically at
concentrations up to that which will attain a 50 cps viscosity, and
possibly a suitable buffer, tonicity agent, and preservative. In
vivo data in rats and man demonstrate that the formulations are
capable of achieving measurable blood insulinotropin, for example,
levels for up to 24 hours. In contrast, insulinotropin, for
example, formulated in phosphate-buffered saline results in rapid
(.about.15 minutes) peak plasma levels, with plasma level dropping
below detection limits in just over 4 hours. Plasma concentration
versus time plots suggest that insulinotropin absorption rate, for
example, from the injection site has been significantly reduced in
the presence of the polymers.
[0253] GLP-1, and related peptides, may also be formulated as
particles suspended in a pharmaceutically acceptable oil. The
preferred oils are triglycerides. Non-limiting examples of such
oils include peanut oil, sesame oil, almond oil, castor oil,
camellia oil, cotton seed oil, olive oil, corn oil, soy oil,
safflower oil, and coconut oil. Oils of other classes are
acceptable, for example, esters of fatty acids and esters of fatty
alcohols, as long as the oil is immiscible with water and is a poor
solvent for the peptide. The formulation may also contain
appropriate preservatives, wetting agents, and suspending agents.
The weight percent of insulinotropin, for example, in the
formulation may vary from 0.01 to 10%. In vivo data in rats
demonstrate that these formulations are capable of achieving
measurable insulinotropin blood levels, for example, for up to 24
hours. In contrast, insulinotropin, for example, formulated in
phosphate-buffered saline results in rapid (.about. 15 minutes)
peak plasma levels, with plasma level dropping below detection
limits in just over 4 hours. Plasma concentration versus time plots
suggest that insulinotropin absorption rate from the injection site
have been significantly reduced in the oil suspensions.
[0254] GLP-1, and related peptides, may also be formulated as a low
solubility form for administration by combination with a metal ion,
preferably in the form of a salt. A preferred ion is zinc (II). The
combination may result in a composition which is amorphous or
crystalline. Other metal ions may also be used including Ni(II),
Co(II), Mg(II), Ca(II), K(I), Mn(II), Fe(II) and Cu(II).
[0255] Other forms of prolonged administration include liposomes,
either multilamellar or unilamellar, the preparation of which is
well known to those skilled in the art. The liposomes, whether
multilamellar or unilamellar, may be phospholipid or
non-phospholipid based.
[0256] Another type of prolonged delivery formulation is an aqueous
suspension of insulinotropin precipitates or aggregates formed by
using precipitants for example, phenolic compounds or basic
polypeptides or metal ions or salts, and/or by using high shear.
More than one precipitant can be used at one time. The precipitates
can be either crystalline or amorphous.
[0257] Insulinotropin crystals can be obtained from a solution of
the drug in water by using pH gradient (either high to low or low
to high) and/or temperature gradient and/or salts to reduce
solubility. The salts include ammonium citrate, sodium or potassium
phosphate, sodium or potassium or ammonium chloride, sodium or
ammonium acetate, magnesium sulfate, calcium chloride, ammonium
nitrate, sodium formate, and any other salts which can reduce the
solubility of the drug. If the salt used for crystallization is not
pharmaceutically acceptable, the mother liquor can be substituted
by pharmaceutically acceptable medium after crystallization is
completed. If further reduction of drug solubility is necessary to
achieve a desirable pharmacokinetic profile, the crystals can be
treated by metal ions such as zinc or calcium and/or phenolic
compounds. The treatment can be done by simply incorporating those
additives to the crystal suspension.
[0258] The solubility of the insulinotropin precipitates or
aggregates can range from less than 1 .mu.g/mL to 500 .mu.g/mL
under physiological conditions. In vivo data in rats demonstrate
that the formulations are capable of achieving measurable
insulinotropin blood levels, for example, for at least 30
hours.
[0259] Aqueous media used for the above formulations can be any
kind of buffer system which can be used for injection or even with
pure water. The pH of the final formulation can be any value as
long as the formulation is injectable. Protamine can be added as
any kind of salt form (e.g. sulfate, chloride, etc.) or protamine
base. Exemplary concentration ranges of the components which can be
used for the formulation preparation are as follows: phenol (0.5 to
5.0 mg/ml), m-cresol (0.5 to 5.5 mg/ml), protamine (0.02 to 1.0
mg/ml), zinc (0.10 to 6 zinc/insulinotropin molar ratio), sodium
chloride (up to 100 mg/ml), and phosphate buffer (5-500 mM).
[0260] Other phenolic on non phenolic compounds may also be used.
Non-limiting examples of such compounds include resorcinol,
methylparaben, propylparaben, benzyl alcohol, chlorocresol, cresol,
benzaldehyde, catecol, pyrogallol, hydroquinone, n-propyl gallate,
butylated hydroxyanisole, butylated hydroxytoluene. Non-limiting
examples of basic polypeptides are polylysine, polyarginine,
etc.
[0261] Having described the invention in general terms, reference
is now made to specific examples. It is to be understood that these
examples are not meant to limit the present invention, the scope of
which is determined by the appended claims.
EXAMPLE 1
Insulinotropin (1 mg/ml) Suspension Solution A1 Preparation
[0262] 10 mg of insulinotropin was weighed into a 5 ml volumetric
flask. Approximately 4 ml of phosphate buffered saline (PBS) was
added to the flask to disperse and dissolve the drug. Sufficient
PBS (q.s. amount) was added to fill the flask. 20 mg of
insulinotropin was weighed into a 10 ml volumetric flask.
Approximately 8 ml of PBS was added to the flask to disperse and
dissolve the drug. The q.s. amount of PBS was added to the flask.
The volumes in both flasks were combined by filtering them by a
glass syringe through a 0.22.mu. filter (low protein binding) into
a 10 ml glass vial. Solution A1 contained insulinotropin 2 mg/ml in
PBS.
[0263] Solution B1 preparation 8 mg of protamine sulfate and 44 mg
of phenol were weighed into a 10 ml volumetric flask. The q.s.
amount of PBS was added to dissolve the protamine sulfate and the
phenol. This solution was filtered through a 0.22.mu. filter (low
protein binding) into a 10 ml glass vial. Solution B1 contained
protamine base 0.6 mg/ml and phenol 4.4 mg/ml in PBS.
[0264] Aqueous Suspension 1
[0265] 1.5 ml of solution A1 was pipetted into a 3.5 ml type I
glass vial. The contents of the vial were stirred magnetically
while 1.5 ml of solution B1 was pipetted into the vial. The vial
was stoppered and sealed with an aluminum shell. The vial contents
were stirred gently for 16 hours to allow suspension formation.
Aqueous Suspension 1 contained insulinotropin 1 mg/ml, protamine
base 0.3 mg/ml and phenol 2.2 mg/ml in PBS. This suspension was
used for in vivo pharmacokinetic studies in rats.
EXAMPLE 2
Insulinotropin (1 mg/ml) Suspension Solution A2 Preparation
[0266] 10 mg of insulinotropin was weighed into a 5 ml volumetric
flask. Approximately 4 ml of PBS was added to the flask to disperse
and dissolve the drug. The q.s. amount of PBS was added to the
flask. 20 mg of insulinotropin was weighed into a 10 ml volumetric
flask. Approximately 8 ml of PBS was added to the flask to disperse
and dissolve the drug. The q.s. amount of the PBS was added to the
flask. The volumes in both flasks were combined by filtering them
by a glass syringe through a 0.22.mu. filter into a 10 ml glass
vial. Solution A2 contained insulinotropin 2 mg/ml in PBS.
[0267] Solution B2 Preparation
[0268] 2 mg of protamine sulfate and 44 mg of phenol were weighed
into a 10 ml volumetric flask. The q.s. amount of PBS was added to
the flask to dissolve the protamine sulfate and phenol. This
solution was filtered through a 0.22.mu. filter into a 10 ml glass
vial. Solution B2 contained protamine base 0.15 mg/ml and phenol
4.4 mg/ml in PBS.
[0269] Aqueous Suspension 2
[0270] 1.5 ml of solution A2 was pipetted into a 3.5 ml type I
glass vial. The contents of the vial were stirred magnetically
while 1.5 ml of solution B2 was pipetted into the vial. The vial
was stoppered and sealed with an aluminum shell. The vial contents
were stirred for 16 hours to allow suspension formation. Aqueous
Suspension 2 contained insulinotropin 1 mg/ml, protamine base 0.075
mg/ml, and phenol 2.2 mg/ml in PBS. This suspension was used for in
vivo pharmacokinetic studies in rats.
EXAMPLE 3
Insulinotropin (1 mg/ml) Suspension
[0271] Solution A3 Preparation
[0272] 20 mg of insulinotropin was weighed into a 10 ml volumetric
flask. Approximately 8 ml of PBS was added to the flask to disperse
and dissolve the drug. The q.s. amount of PBS was added to the
flask. Solution A3 was filtered by a syringe through a 0.22.mu.
filter into a 10 ml glass vial. Solution A3 contained
insulinotropin 2 mg/ml in PBS.
[0273] Solution B3 Preparation
[0274] 8 mg of protamine sulfate, 44 mg of phenol, and 323 mg of
glycerin were weighed into a 10 ml volumetric flask. The q.s.
amount of PBS was added to the flask to dissolve the protamine
sulfate, the phenol, and the glycerin. This solution was filtered
by a syringe through a 0.22.mu. filter into a 10 ml glass vial.
Solution B3 contained protamine base 0.6 mg/ml, phenol 4.4 mg/ml,
and glycerin 32 mg/ml in PBS.
[0275] Aqueous Suspension 3
[0276] 1.5 ml of Solution A3 was pipetted into a 3.5 ml type I
glass vial. The contents of the vial were stirred magnetically
while 1.5 ml of Solution B3 was pipetted into the vial. The vial
was stoppered and sealed with an aluminum shell. The vial contents
were stirred for 16 hours to allow suspension formation. Aqueous
Suspension 3 contained insulinotropin 1 mg/ml, protamine base 0.3
mg/ml, phenol 2.2 mg/ml, and glycerin 16 mg/ml in PBS. This
suspension was used for in vivo pharmacokinetic studies in
rats.
EXAMPLE 4
Insulinotropin (1 mg/ml) Suspension
[0277] Solution A4 Preparation
[0278] 20 mg of insulinotropin was weighed into a 10 ml volumetric
flask. Approximately 8 ml of PBS was added to the flask to disperse
and dissolve the drug. The q.s. amount of PBS was added to the
flask. Solution A4 was filtered by a syringe through a 0.22.mu.
filter (Millipore Millex-GV) into a 10 ml glass vial. Solution A4
contained insulinotropin 2 mg/ml in PBS.
[0279] Solution B4 Preparation
[0280] 8 mg of protamine sulfate and 52 mg of m-cresol were weighed
into a 10 ml volumetric flask. The q.s. amount of PBS was added to
the flask to dissolve the protamine sulfate and the m-cresol. This
solution was filtered through a 0.22.mu. filter into a 10 ml glass
vial. Solution B4 contained protamine base 0.6 mg/ml and m-cresol 5
mg/ml in PBS.
[0281] Aqueous Suspension 4
[0282] 1.5 ml of solution A4 was pipetted into a 3.5 ml type I
glass vial. The contents of the vial were stirred magnetically
while 1.5 ml of solution B4 was pipetted into the vial. The vial
was stoppered and sealed with an aluminum shell. The vial contents
were stirred for 16 hours to allow crystal formation. Aqueous
Suspension 4 contained insulinotropin 1 mg/ml, protamine base 0.3
mg/ml and m-cresol 2.5 mg/ml in PBS. This suspension was used for
in vivo pharmacokinetic studies in rats.
EXAMPLE 5
Insulinotropin (1 mg/ml) Suspension
[0283] Solution A5 Preparation
[0284] 50 mg of insulinotropin was weighed into a 25 ml volumetric
flask. Approximately 23 ml of PBS was added to the flask to
disperse and dissolve the drug. The q.s. amount of PBS was added to
the flask. Solution A5 was filtered by a syringe through a 0.22.mu.
filter into a 50 ml glass vial. Solution A5 contained
insulinotropin 2 mg/ml in PBS.
[0285] Phenol Stock Solution Preparation
[0286] 0.44 g of phenol was weighed into a 100 ml volumetric flask.
Approximately 95 ml of PBS was added to the flask to dissolve the
phenol. The q.s. amount of PBS was added to the flask to dissolve
the phenol. The resulting solution (4.4 mg/ml phenol) was used to
prepare Solution B5.
[0287] Solution B5 Preparation
[0288] Solution B5 was prepared by filtering 25 ml of the phenol
stock solution through a 0.2.mu. filter into a 50 ml glass vial.
Solution B5 contained phenol 4.4 mg/ml in PBS.
[0289] Aqueous Suspension 5
[0290] 1.25 ml of solution A5 was pipetted into a 3.5 ml type I
glass vial. The contents of the vial were stirred magnetically
while 1.25 ml of solution B5 was pipetted into the vial. The vial
was stoppered and sealed with an aluminum shell. The vial contents
were stirred for 16 hours to allow suspension formation. Aqueous
Suspension 5 contained insulinotropin 1 mg/ml and phenol 2.2 mg/ml
in PBS. This suspension was used for in vivo pharmacokinetic
studies in rats.
EXAMPLE 6
Insulinotropin (1 mg/ml) Suspension
[0291] Solution A6 Preparation
[0292] 50 mg of insulinotropin was weighed into a 25 ml volumetric
flask. Approximately 23 ml of PBS was added to the flask to
disperse and dissolve the drug. The q.s. amount of PBS was added to
the flask. Solution A6 was filtered by a syringe through a 0.22.mu.
filter into a 50 ml glass vial. Solution A6 contained
insulinotropin 2 mg/ml in PBS.
[0293] Phenol Stock Solution Preparation
[0294] 0.44 g of phenol was weighed into a 100 ml volumetric flask.
Approximately 95 ml of PBS was added to the flask to dissolve the
phenol. The q.s. amount of PBS was added to the flask to dissolve
the phenol. The resulting solution (4.4 mg/ml phenol) was used to
prepare Solution B6.
[0295] Solution B6 Preparation
[0296] Solution B6 was prepared by weighing 1.25 mg of protamine
sulfate into a 25 ml volumetric flask. Approximately 20 ml of
phenol stock solution was added to the flask to dissolve the
protamine sulfate. The q.s. amount of phenol stock solution was
added to the flask. Solution B6 was filtered through a 0.22.mu.
filter into a 50 ml glass vial. Solution B6 contained phenol 4.4
mg/ml and protamine base 0.038 mg/ml in PBS.
[0297] Aqueous Suspension 6
[0298] 1.25 ml of solution A6 was pipetted into a 3.5 ml type I
glass vial. The contents of the vial were stirred magnetically
while 1.25 ml of solution B6 was pipetted into the vial. The vial
was stoppered and sealed with an aluminum shell. The vial contents
were stirred for 16 hours to allow suspension formation. Aqueous
Suspension 6 contained insulinotropin 1 mg/ml, phenol 2.2 mg/ml,
and protamine base 0.019 mg/ml in PBS. This suspension was used for
in vivo pharmacokinetic studies in rats.
EXAMPLE 7
Insulinotropin (1 mg/ml) Suspension
[0299] Solution A7 Preparation
[0300] 50 mg of insulinotropin was weighed into a 25 ml volumetric
flask. Approximately 23 ml of PBS was added to the flask to
disperse and dissolve the drug. The q.s. amount of PBS was added to
the flask. Solution A7 was filtered by a syringe through a 0.22.mu.
filter into a 50 ml glass vial. Solution A7 contained
insulinotropin 2 mg/ml in PBS.
[0301] Phenol Stock Solution Preparation
[0302] 0.44 g of phenol was weighed into a 100 ml volumetric flask.
Approximately 95 ml of PBS was added to the flask to dissolve the
phenol. The q.s. amount of PBS was added to the flask to dissolve
the phenol. The resulting solution (4.4 mg/ml phenol) was used to
prepare Solution B7.
[0303] Solution B7 Preparation
[0304] Solution B7 was prepared by weighing 2.5 mg of protamine
sulfate into a 25 ml volumetric flask. Approximately 20 ml of
phenol stock solution was added to the flask to dissolve the
protamine sulfate. The q.s. amount of phenol stock solution was
added to the flask. Solution B7 was filtered through a 0.22.mu.
filter into a 50 ml glass vial. Solution B7 contained phenol 4.4
mg/ml and protamine base 0.075 mg/ml in PBS.
[0305] Aqueous Suspension 7
[0306] 1.25 ml of solution A7 was pipetted into a 3.5 ml type I
glass vial. The contents of the vial were stirred magnetically
while 1.25 ml of solution B7 was pipetted into the vial. The vial
was stoppered and sealed with an aluminum shell. The vial contents
were stirred for 16 hours to allow suspension formation. Aqueous
Suspension 7 contained insulinotropin 1 mg/ml, phenol 2.2 mg/ml,
and protamine base 0.038 mg/ml in PBS. This suspension was used for
in vivo pharmacokinetic studies in rats.
EXAMPLE 8
Insulinotropin (1 mg/ml) Suspension
[0307] Solution A12 Preparation
[0308] 20 mg of insulinotropin was weighed into a 10 ml volumetric
flask. Approximately 8 ml of PBS was added to the flask to disperse
and dissolve the drug. The q.s. amount of PBS was added to the
flask. Solution A12 was filtered by a syringe through a 0.22.mu.
filter into a 10 ml glass vial. Solution A12 contained
insulinotropin 2 mg/ml in PBS.
[0309] Solution B12
[0310] Solution B12 was prepared by weighing 20 mg of phenol into a
10 ml volumetric flask. Approximately 8 ml of PBS was added to the
flask to dissolve the phenol. The q.s. amount of PBS was added to
the flask. Solution B12 was filtered through a 0.22.mu. filter into
a 10 ml glass vial. Solution B12 contained phenol 2 mg/ml in
PBS.
[0311] Aqueous Suspension 12
[0312] 4 ml of solution A12 was pipetted into a 10 ml type I glass
vial. The contents of the vial were stirred while 4 ml of solution
B12 was pipetted into the vial. The vial was stoppered and sealed
with an aluminum shell. The vial contents were stirred for 16 hours
to allow suspension formation. Aqueous Suspension 12 contained
insulinotropin 1 mg/ml and phenol 1 mg/ml in PBS. This suspension
was used for in vivo pharmacokinetic studies in rats.
EXAMPLE 9
Insulinotropin (1 mg/ml) Suspension
[0313] Solution A15 Preparation
[0314] 20 mg of insulinotropin was weighed into a 10 ml volumetric
flask. Approximately 8 ml of phosphate buffer (PB) was added to the
flask to dissolve the drug. The q.s. amount of PB was added to the
flask. Solution A15 was filtered by a syringe through a 0.22.mu.
filter into a 10 ml glass vial. Solution A15 contained
insulinotropin 2 mg/ml in PB.
[0315] Solution B15 Preparation
[0316] Solution B15 was prepared by weighing 8 mg of protamine
sulfate into a 10 ml volumetric flask. Approximately 8 ml of PB was
added to the flask to dissolve the protamine sulfate. The q.s.
amount of PB was added to the flask. Solution B15 was filtered
through a 0.22.mu. filter into a 10 ml glass vial. Solution B15
contained protamine base 0.6 mg/ml in PBS.
[0317] Aqueous Suspension 15
[0318] 3 ml of solution A15 was pipetted into a 10 ml type I glass
vial. The contents of the vial were stirred while 3 ml of solution
B15 was pipetted into the vial. The vial was stoppered and sealed
with an aluminum shell. The vial contents were stirred for 16 hours
to allow suspension formation. Aqueous Suspension 15 contained
insulinotropin 1 mg/ml and protamine base 0.3 mg/ml in PB. This
suspension was used for In vivo pharmacokinetic studies in
rats.
EXAMPLE 10
Insulinotropin (1 mg/ml) Suspension
[0319] Solution A16 Preparation
[0320] 20 mg of insulinotropin was weighed into a 10 ml volumetric
flask. Approximately 8 ml of PB was added to the flask to dissolve
the drug. The q.s. amount of PB was added to the flask. Solution
A16 was filtered by a syringe through a 0.22.mu. filter into a 10
ml glass vial. Solution A16 contained insulinotropin 2 mg/ml in
PB.
[0321] Solution B16 Preparation
[0322] Solution B16 was prepared by weighing 44 mg of phenol into a
10 ml volumetric flask. Approximately 8 ml of PB was added to the
flask to dissolve the phenol. The q.s. amount of PB was added to
the flask. Solution B16 was filtered through a 0.22.mu. filter into
a 10 ml glass vial. Solution B16 contained phenol 4.4 mg/ml in
PB.
[0323] Aqueous Suspension 16
[0324] 3 ml of Solution A16 was pipetted into a 10 ml type I glass
vial. The contents of the vial were stirred magnetically while 3 ml
of Solution B16 was pipetted into the vial. The vial was stoppered
and sealed with an aluminum shell. The vial contents were stirred
for 16 hours to allow suspension formation. Aqueous Suspension 16
contained insulinotropin 1 mg/ml and phenol 2.2 mg in PB. This
suspension was used for in vivo pharmacokinetic studies in
rats.
EXAMPLE 11
Insulinotropin (1 mg/ml) Suspension
[0325] Aqueous Suspension 17
[0326] 10 mg of insulinotropin was weighed into a 10 ml volumetric
flask. Approximately 8 ml of PB was added to the flask to dissolve
the drug. The q.s. amount of PB was added to the flask. The
contents of the flask was filtered by syringe through a 0.22.mu.
filter into a 10 ml type I glass vial. The vial was stoppered and
sealed with an aluminum shell. The vial contents were stirred for
16 hours to allow suspension formation. Aqueous Suspension 17
contained insulinotropin 1 mg/ml in PB. This suspension was used
for in vivo pharmacokinetic studies in rats.
EXAMPLE 12
Insulinotropin (1 mg/ml) Suspension
[0327] Aqueous Suspension 18
[0328] 10 mg of insulinotropin was weighed into a 10 ml volumetric
flask. Approximately 8 ml of PBS was added to the flask to dissolve
the drug. The q.s. amount of PBS was added to the flask. The
contents of the flask were filtered by a syringe through a 0.22.mu.
filter into a 10 ml type I glass vial. The vial was stoppered and
sealed with an aluminum shell. The vial contents were stirred
gently (making sure no foam or bubble formed) for 16 hours to allow
suspension formation. Aqueous Suspension 18 contained
insulinotropin 1 mg/ml in PBS. This suspension was used for in vivo
pharmacokinetic studies in rats.
EXAMPLE 13
Insulinotropin (0.2 mg/ml) Suspension
[0329] Solution A22 Preparation
[0330] Solution A22 was prepared by weighing 2 mg of insulinotropin
into a 5 ml volumetric flask. Approximately 3 ml of PBS was added
to the flask to dissolve lhe drug. The q.s. amount of PBS was added
to the flask. Solution A22 was filtered by a syringe through a
0.22.mu. filter into a 10 ml glass vial. Solution A22 contained
insulinotropin 0.4 mg/ml in PBS.
[0331] Solution B22 Preparation
[0332] Solution B22 was prepared by weighing 44 mg of phenol into a
10 ml volumetric flask. Approximately 8 ml of PBS was added to the
flask to dissolve the phenol. The q.s. amount of PBS was added to
the flask. Solution B22 was filtered through a 0.22.mu. filter into
a 10 ml glass vial. Solution B22 contained phenol 4.4 mg/ml in
PBS.
[0333] Aqueous Suspension 22
[0334] 1.5 ml of solution A22 was pipetted into a 3.5 ml type I
glass vial. The contents of the vial were stirred magnetically
while 1.5 ml of solution B22 was pipetted into the vial. The vial
was stoppered and sealed with an aluminum shell. The vial contents
were stirred for 16 hours to allow suspension formation. Aqueous
Suspension 22 contained insulinotropin 0.2 mg/ml and phenol 2.2
mg/ml in PBS. This suspension was used for in vivo pharmacokinetic
studies in rats.
EXAMPLE 14
Insulinotropin (0.2 mg/ml) Suspension
[0335] Solution A23 Preparation
[0336] Solution A23 was prepared by weighing 2 mg of insulinotropin
into a 5 ml volumetric flask. Approximately 3 ml of PBS was added
to the flask to dissolve the drug. The q.s. amount of PBS was added
to the flask. Solution A23 was filtered by a syringe through a
0.22.mu. filter into a 10 ml glass vial. Solution A23 contained
insulinotropin 0.4 mg/ml in PBS.
[0337] Solution B23 Preparation
[0338] Solution B23 was prepared by weighing 8.8 mg of phenol into
a 10 ml volumetric flask. Approximately 8 ml of PBS was added to
the flask to dissolve the phenol. The q.s. amount of PBS was added
to the flask. Solution B23 was filtered through a 0.22.mu. filter
into a 10 ml glass vial. Solution B23 contained phenol 0.88 mg/ml
in PBS.
[0339] Aqueous Suspension 23
[0340] 1.5 ml of solution A23 was pipetted into a 3.5 ml type I
glass vial. The contents of the vial were stirred magnetically
while 1.5 ml of solution B23 was pipetted into tie vial. The vial
was stoppered and sealed with an aluminum shell. The vial contents
were stirred for 16 hours to allow suspension formation. Aqueous
Suspension 23 contained insulinotropin 0.2 mg/ml and phenol 0.44
mg/ml in PBS. This suspension was used for in vivo pharmacokinetic
studies in rats.
EXAMPLE 15
Insulinotropin (1 mg/ml) Suspension
[0341] Solution A24 Preparation
[0342] Solution A24 was prepared by weighing 10 mg of
insulinotropin into a 5 ml volumetric flask. Approximately 3 ml of
PBS was added to the flask to dissolve the drug. The q.s. amount of
PBS was added to the flask. Solution A24 was filtered by a syringe
through a 0.22.mu. filter into a 10 ml glass vial. Solution A24
contained insulinotropin 2 mg/ml in PBS.
[0343] Solution B24 Preparation
[0344] Solution B24 was prepared by weighing 8 mg of protamine
sulfate into a 10 ml volumetric flask. Approximately 8 ml of PBS
was added to the flask to dissolve the protamine sulfate. The q.s.
amount of PBS was added to the flask. Solution B24 was filtered
through a 0.22.mu. filter into a 10 ml glass vial. Solution B24
contained protamine base 0.6 mg/ml in PBS.
[0345] Aqueous Suspension 24
[0346] 1.5 ml of solution A24 was pipetted into a 3.5 ml type I
glass vial. The contents of the vial were stirred magnetically
while 1.5 ml of solution B24 was pipetted into the vial. The vial
was stoppered and sealed with an aluminum shell. The vial contents
were stirred for 16 hours to allow suspension formation. Aqueous
Suspension 24 contained insulinotropin 1 mg/ml and protamine base
0.3 mg/ml in PBS. This suspension was used for in vivo
pharmacokinetic studies in rats.
EXAMPLE 16
Insulinotropin (1 mg/ml) Suspension
[0347] Solution A25 Preparation
[0348] Solution A25 was prepared by weighing 10 mg of
insulinotropin into a 5 ml volumetric flask. Approximately 3 ml of
PBS was added to the flask to dissolve the drug. The q.s. amount of
PBS was added to the flask. Solution A25 was filtered by a syringe
through a 0.22.mu. filter into a 10 ml glass vial. Solution A25
contained insulinotropin 2 mg/ml in PBS.
[0349] Solution B25 Preparation
[0350] Solution B25 was prepared by weighing 53 mg of m-cresol into
a 10 ml volumetric flask. Approximately 8 ml of PBS was added to
the flask to dissolve the m-cresol. The q.s. amount of PBS was
added to the flask. Solution B25 was filtered through a 0.22.mu.
filter into a 10 ml glass vial. Solution B25 contained m-cresol 5.3
mg/ml in PBS.
[0351] Aqueous Suspension 25
[0352] 1.5 ml of solution A25 was pipetted into a 3.5 ml type I
glass vial. The contents of the vial were stirred magnetically
while 1.5 ml of solution B25 was pipetted into the vial. The vial
was stoppered and sealed with an aluminum shell. The vial contents
were stirred for 16 hours to allow suspension formation. Aqueous
Suspension 25 contained insulinotropin 1 mg/ml and m-cresol 2.5
mg/ml in PBS. This suspension was used for in vivo pharmacokinetic
studies in rats.
EXAMPLE 17
Insulinotropin (0.5 mg/ml) Suspension
[0353] Solution A29 Preparation
[0354] Solution A29 was prepared by weighing 25 mg of
insulinotropin into a 25 ml volumetric flask. Approximately 20 ml
of PBS was added to the flask to dissolve the drug. The q.s. amount
of PBS was added to the flask. Solution A29 was filtered by a
syringe through a 0.22.mu. filter into a 50 ml glass vial. Solution
A29 contained insulinotropin 1 mg/ml in PBS.
[0355] Solution B29 Preparation
[0356] Solution B29 was prepared by weighing 50 mg of phenol into a
50 ml volumetric flask. Approximately 40 ml of PBS was added to the
flask to dissolve the phenol. The q.s. amount of PBS was added to
the flask. Solution B29 was filtered through a 0.22.mu. filter into
a 50 ml glass vial. Solution B29 contained phenol 1.0 mg/ml in
PBS.
[0357] Aqueous Suspension 29
[0358] 1.5 ml of solution A29 was pipetted into a 3.5 ml type I
glass vial. The contents of the vial were stirred magnetically
while 1.5 ml of solution B29 was pipetted into the vial. The vial
was stoppered and sealed with an aluminum shell. The vial contents
were stirred for 16 hours to allow suspension formation. Aqueous
Suspension 29 contained insulinotropin 0.5 mg/ml and phenol 0,5
mg/ml in PBS. This suspension was used for in vivo pharmacokinetic
studies in rats.
EXAMPLE 18
Insulinotropin (1 mg/ml) Suspension
[0359] Solution A31 Preparation
[0360] 10 mg of insulinotropin was weighed into a 5 ml volumetric
flask. Approximately 4 ml of PBS was added to the flask to disperse
and dissolve the drug. The q.s. amount of PBS was added to the
flask. Solution A31 was filtered by a syringe through a 0.22.mu.
filter into a 10 ml glass vial. Solution A31 contained
insulinotropin 2 mg/ml in PBS.
[0361] Solution B31 Preparation
[0362] Solution B31 was prepared by weighing 50 mg of phenol into a
50 ml volumetric flask. Approximately 40 ml of PBS was added to the
flask to dissolve the phenol. The q.s. amount of PBS was added to
the flask. Solution B31 was filtered through a 0.22.mu. filter into
a 50 ml glass vial. Solution B31 contained phenol 1 mg/ml in
PBS.
[0363] Aqueous Suspension 31
[0364] 1.5 ml of solution A31 was pipetted into a 3.5 ml type I
glass vial. The contents of the vial were stirred magnetically
while 1.5 ml of solution B31 was pipetted into the vial. The vial
was stoppered and sealed with an aluminum shell. The vial contents
were stirred for 16 hours to allow suspension formation. Aqueous
Suspension 31 contained insulinotropin 1 mg/ml and phenol 0.5 mg/ml
in PBS. This suspension was used for in vivo pharmacokinetic
studies in rats.
EXAMPLE 19
Insulinotropin (4 mg/mL) Suspension
[0365] Solution A51 Preparation
[0366] 22.2 mg of insulinotropin was weighed into a 10 mL glass
vial. 5 mL of PBS was pipetted into the vial to dissolve the drug.
This solution was filtered through a 0.22.mu. filter (low protein
binding) into a 10 mL glass vial. Solution A51 contained
insulinotropin 4.44 mg/mL in PBS.
[0367] Solution B51 Preparation
[0368] 110 mg of phenol and 30 mg of protamine sulfate were weighed
into a 5 mL volumetric flask. Approximately 4 mL of PBS was added
to the flask to dissolve the phenol and protamine sulfate. The
flask was filled to the mark with PBS. The solution was filtered
through a 0.22.mu. filter (low protein binding) into a 10 mL glass
vial. Solution B51 contained phenol 22 mg/mL and protamine base 4.5
mg/mL in PBS.
[0369] Aqueous Suspension 51
[0370] 3 mL of Solution A51 and 0.33 mL of Solution B51 were
pipetted into a 3.5 mL type I glass vial. The contents of the vial
were shaken gently to ensure a homogeneous mix. The vial was
allowed to sit at ambient temperature for 16 hours. Aqueous
Suspension 51 contained insulinotropin 4 mg/mL, protamine base 0.44
mg/mL, and phenol 2.2 mg/mL in PBS. This suspension was used for in
vivo pharmacokinetic studies in rats
EXAMPLE 20
Insulinotropin (4 mg/mL) Suspension
[0371] Solution A52 Preparation
[0372] 22.2 mg of insulinotropin was weighed into a 10 mL glass
vial. 5 mL of PBS was pipetted into the vial to dissolve the drug.
This solution was filtered through a 0.22.mu. filter (low protein
binding) into a 10 mL glass vial. Solution A52 contained
insulinotropin 4.44 mg/mL in PBS.
[0373] Solution B52 Preparation
[0374] 110 mg of phenol and 15.6 mg of zinc acetate dihydrate were
weighed into a 5 mL volumetric flask. Approximately 4 mL of water
for injection was added to the flask to dissolve the phenol and
zinc acetate dihydrate. The flask was filled to the mark with water
for injection. The solution was filtered through a 0.22.mu. filter
(low protein binding) into a 10 mL glass vial. Solution B52
contained phenol 22 mg/mL and zinc acetate dihydrate 7.8 mg/mL in
water for injection.
[0375] Aqueous Suspension 52
[0376] 3 mL of Solution A52 and 0.33 mL of Solution 652 were
pipetted into a 3.5 mL type I glass vial. The contents of the vial
were shaken gently to ensure a homogeneous mix. The vial was
allowed to sit at ambient temperature for 16 hours. Aqueous
Suspension 52 contained insulinotropin 4 mg/mL, zinc acetate
dihydrate 0.78 mg/mL, and phenol 2.2 mg/mL in PBS. This suspension
was used for in vivo pharmacokinetic studies in rats.
EXAMPLE 21
Insulinotropin (4 mg/mL) Suspension
[0377] Phenol Solution Preparation
[0378] 244 mg of phenol was weighed into a 100 mL volumetric flask.
Approximately 90 mL of water for injection was added to the flask
to dissolve the phenol. The flask was filled to the mark with water
for injection. The pH of this solution was adjusted to pH 9.0 with
5% NaOH solution. The Phenol Solution contained phenol 2.44 mg/mL
in water for injection pH 9.0.
[0379] Solution A71 Preparation
[0380] 22.2 mg of insulinotropin was weighed into a 10 mL glass
vial. 5 mL of the Phenol Solution was pipetted into the vial to
dissolve the drug. This solution was filtered through a 0.22.mu.
filter (low protein binding) into a 10 mL glass vial. Solution A71
contained insulinotropin 4.44 mg/mL and phenol 2.44 mg/mL in water
for injection.
[0381] Solution B71 Preparation
[0382] 116 mg of protamine sulfate was weighed into a 10 mL
volumetric flask. Approximately 8 mL of water for injection was
added to the flask to dissolve the protamine sulfate. The flask was
filled to the mark with water for injection. The solution was
filtered through a 0.22.mu. filter (low protein binding) into a 10
mL glass vial. Solution B71 contained protamine base 8.7 mg/mL in
water for injection.
[0383] Solution C71 Preparation
[0384] 156 mg of zinc acetate dihydrate and 1.632 g of NaCl were
weighed into a 10 mL volumetric flask. Approximately 8 mL of water
for injection was added to the flask to dissolve the zinc acetate
dihydrate and NaCl. The flask was filled to the mark with water for
injection. The solution was filtered through a 0.22.mu. filter (low
protein binding) into a 10 mL glass vial. Solution C71 contained
zinc acetate dihydrate 15.6 mg/mL and NaCl 163.2 mg/mL in water for
injection.
[0385] Aqueous Suspension 71
[0386] 3 mL of Solution A71, 0.165 mL of Solution B71, and 0.165 mL
of Solution C71 were pipetted into a 3.5 mL type I glass vial. The
contents of the vial were shaken gently to ensure a homogeneous
mix. The vial was allowed to sit at ambient temperature for 16
hours. Aqueous Suspension 71 contained insulinotropin 4 mg/mL,
protamine base 0.435 mg/mL, zinc acetate dihydrate 0.78 mg/mL, NaCl
8.16 mg/mL, and phenol 2.2 mg/mL in water for injection. This
suspension was used for in vivo pharmacokinetic studies in
rats.
EXAMPLE 22
Insulinotropin (4 mg/mL) Suspension
[0387] m-Cresol Solution Preparation
[0388] 244 mg of m-cresol was weighed into a 100 mL volumetric
flask. Approximately 90 mL of water for injection was added to the
flask to dissolve the m-cresol. The flask was filled to the mark
with water for injection. The pH of this solution was adjusted to
pH 9.0 with 5% NaOH solution. The m-cresol Solution contained
m-cresol 2.44 mg/mL in water for injection pH 9.0.
[0389] Solution A100 Preparation
[0390] 22.2 mg of insulinotropin was weighed into a 10 mL glass
vial. 5 mL of the m-cresol Solution was pipetted into the vial to
dissolve the drug. This solution was filtered through a 0.22 .mu.
filter (low protein binding) into a 10 mL glass vial. Solution A100
contained insulinotropin 4.44 mg/mL and m-cresol 2.44 mg/mL in
water for injection.
[0391] Solution B100 Preparation
[0392] 116 mg of protamine sulfate was weighed into a 10 mL
volumetric flask. Approximately 8 mL of water for injection was
added to the flask to dissolve the protamine sulfate. The flask was
filled to the mark with water for injection. The solution was
filtered through a 0.22.mu. filter (low protein binding) into a 10
mL glass vial. Solution B100 contained protamine base 8.7 mg/mL in
water for injection.
[0393] Solution C100 Preparation
[0394] 156 mg of zinc acetate dihydrate and 1.632 g of NaCl were
weighed into a 10 mL volumetric flask. Approximately 8 mL of water
for injection was added to the flask to dissolve the zinc acetate
dihydrate and NaCl. The flask was filled to the mark with water for
injection. The solution was filtered through a 0.22.mu. filter (low
protein binding) into a 10 mL glass vial. Solution C100 contained
zinc acetate dihydrate 15.6 mg/mL and NaCl 163.2 mg/mL in water for
injection.
[0395] Aqueous Suspension 100
[0396] 3 mL of Solution A100, 0.165 mL of Solution B100, and 0.165
mL of Solution C100 were pipetted into a 3.5 mL type I glass vial.
The contents of the vial were shaken gently to ensure a homogeneous
mix. The vial was allowed to sit at ambient temperature for 16
hours. Aqueous Suspension 100 contained insulinotropin 4 mg/mL,
protamine base 0.435 mg/mL, zinc acetate dihydrate 0.78 mg/mL, NaCl
8.16 mg/mL, and m-cresol 2.2 mg/mL in water for injection. This
suspension was used for in vivo pharmacokinetic studies in
rats.
EXAMPLE 23
Insulinotropin (4 mg/mL) Suspension
[0397] Solution A68 Preparation
[0398] 22.2 mg of insulinotropin was weighed into a 10 mL glass
vial. 5 mL of the PBS was pipetted into the vial to dissolve the
drug. This solution was filtered through a 0.22.mu. filter (low
protein binding) into a 10 mL glass vial. Solution A68 contained
insulinotropin 4.44 mg/mL in PBS.
[0399] Solution B68 Preparation
[0400] 116 mg of protamine sulfate was weighed into a 10 mL
volumetric flask. Approximately 8 mL of water for injection was
added to the flask to dissolve the protamine sulfate. The flask was
filled to the mark with water for injection. The solution was
filtered through a 0.22.mu. filter (low protein binding) into a 10
mL glass vial. Solution B68 contained protamine base 8.7 mg/mL in
water for injection.
[0401] Solution C68 Preparation
[0402] 156 mg of zinc acetate dihydrate and 440 mg of phenol was
weighed into a 10 mL volumetric flask. Approximately 8 mL of water
for injection was added to the flask to dissolve the zinc acetate
dihydrate and phenol. The flask was filled to the mark with water
for injection. The solution was filtered through a 0.22.mu. filter
(low protein binding) into a 10 mL glass vial. Solution C68
contained zinc acetate dihydrate 15.6 mg/mL and phenol 44 mg/mL in
water for injection.
[0403] Aqueous Suspension 68
[0404] 3 mL of Solution A68, 0.165 mL of Solution B68, and 0.165 mL
of Solution C68 were pipetted into a 3.5 mL type I glass vial. The
contents of the vial were shaken gently to ensure a homogeneous
mix. The vial was allowed to sit at ambient temperature for 16
hours. Aqueous Suspension 68 contained insulinotropin 4 mg/mL,
protamine base 0.435 mg/mL, zinc acetate dihydrate 0.78 mg/mL, and
phenol 2.2 mg/mL in PBS. This suspension was used for in vivo
pharmacokinetic studies in rats.
EXAMPLE 24
Insulinotropin (4 mg/mL) Suspension
[0405] Solution A67 Preparation
[0406] 22.2 mg of insulinotropin was weighed into a 10 mL glass
vial. 5 mL of the PBS was pipetted into the vial to dissolve the
drug. This solution was filtered through a 0.22.mu. filter (low
protein binding) into a 10 mL glass vial. Solution A67 contained
insulinotropin 4.44 mg/mL in PBS.
[0407] Solution B67 Preparation
[0408] 116 mg of protamine sulfate was weighed into a 10 mL
volumetric flask. Approximately 8 mL of water for injection was
added to the flask to dissolve the protamine sulfate. The flask was
filled to the mark with water for injection. The solution was
filtered through a 0.22.mu. filter (low protein binding) into a 10
mL glass vial. Solution B67 contained protamine base 8.7 mg/mL in
water for injection.
[0409] Solution C67 Preparation
[0410] 156 mg of zinc acetate dihydrate and 440 mg of m-cresol were
weighed into a 10 mL volumetric flask. Approximately 8 mL of water
for injection was added to the flask to dissolve the zinc acetate
dihydrate and m-cresol. The flask was filled to the mark with water
for injection. The solution was filtered through a 0.22.mu. filter
(low protein binding) into a 10 mL glass vial. Solution C67
contained zinc acetate dihydrate 15.6 mg/mL and m-cresol 44 mg/mL
in water for injection.
[0411] Aqueous Suspension 67
[0412] 3 mL of Solution A67, 0.165 mL of Solution B67, and 0.165 mL
of Solution C67 were pipetted into a 3.5 mL type I glass vial. The
contents of the vial were shaken gently to ensure a homogeneous
mix. The vial was allowed to sit at ambient temperature for 16
hours. Aqueous Suspension 67 contained insulinotropin 4 mg/mL,
protamine base 0.435 mg/mL, zinc acetate dihydrate 0.78 mg/mL, and
m-cresol 2.2 mg/mL in PBS. This suspension was used for in vivo
pharmacokinetic studies in rats.
EXAMPLE 25
[0413] Solution A39 Preparation
[0414] 67.6 mg of insulinotropin was weighed into a glass vial.
Approximately 22 mL of water for injection was added to the vial to
dissolve the insulinotropin. The pH of the vial content was
adjusted to 9.6 using NaOH to make a clear solution. Water for
injection was added to the vial to make the final drug
concentration to be 2.5 mg/ml.
[0415] Solution B39 Preparation
[0416] 386.8 mg of zinc acetate dihydrate was weighed into a 100 ml
volumetric flask. Approximately 80 mL of water for injection was
added to the flask to dissolve the zinc acetate dihydrate. The
flask was filled to the mark with water for injection. Solution B39
contained zinc acetate dihydrate 3.9 mg/mL in water for
injection.
[0417] Solution C39 Preparation
[0418] 1.095 g of phenol was weighed into a 50 ml volumetric flask.
Approximately 40 mL of water for injection was added to the flask
to dissolve the phenol. The flask was filled to the mark with water
for injection. Solution C39 contained phenol 21.9 mg/mL in water
for injection.
[0419] Solution D39 Preparation
[0420] 2.25 g of NaCl was weighed into a 25 mL volumetric flask.
Approximately 20 mL of Solution C39 was added to the flask to
dissolve the NaCl. The flask was filled to the mark with Solution
C39. Solution D39 contained NaCl 9% (w/v) and phenol 21.9 mg/mL in
water for injection.
[0421] Aqueous Suspension 39
[0422] All solutions were filtered through 0.22 .mu.l filters (low
protein binding). 9 ml of Solution A39 was transferred to a 10 ml
sample vial. 1 ml of Solution B39 was added to the vial while
stirring gently. Precipitates were formed immediately. The pH was
measured to be 7.0. The vial was allowed to sit at ambient
temperature for about 18 hours. 4 ml of the sample was transferred
to a separate 10 ml vial, and 0.44 ml of Solution D39 was added to
the vial. The sample was stirred gently for 5 minutes and was then
allowed to sit at ambient temperature overnight.
[0423] Aqueous Suspension 39 contained insulinotropin 2 mg/ml,
phenol 2.2 mg/ml, NaCl 0.9%, and zinc acetate 0.39 mg/ml. This
suspension was used for in vivo pharmacokinetic studies in
rats.
EXAMPLE 26
[0424] Solution A53 Preparation
[0425] 32.5 mg of insulinotropin was weighed into a 10 ml glass
vial. 6 ml of water for injection was added to the vial. The pH of
the vial content was adjusted to 9.6 using 1% (w/v) NaOH to make a
clear solution. Appropriate amount of water for injection was added
to make the drug concentration to be 5.0 mg/ml.
[0426] Solution B53 Preparation
[0427] 390 mg of zinc acetate dihydrate was weighed into a 50 ml
volumetric flask. Approximately 40 mL of water for injection was
added to the flask to dissolve the zinc acetate dihydrate. The
flask was filled to the mark with water for injection. Solution B53
contained zinc acetate dihydrate 7.8 mg/mL in water for
injection.
[0428] Aqueous Suspension 53
[0429] All solutions were filtered through 0.22.mu. filters (low
protein binding). 2.4 mL of Solution A53 was transferred to a 3.5
ml vial. 300 .mu.l of Solution B53 was added to the vial while
stirring gently. Birefringent precipitates were formed immediately
after the addition. The pH was measured to be 6.8. After the vial
was allowed to sit at ambient temperature for 20 hours, 7.5 .mu.l
of m-cresol was added directly to the supernatant of the settled
suspension. The suspension was then stirred gently to dissolve the
m-cresol. 300 .mu.l of 9% NaCl solution was added to the suspension
with stirring. Aqueous Suspension 53 contained insulinotropin 4
mg/mL, 0.9% NaCl, 0.78 mg/mL zinc acetate, and 2.5 mg/mL m-cresol
in water for injection. This suspension was used for in vivo
pharmacokinetic studies in rats.
EXAMPLE 27
[0430] Solution A54 Preparation
[0431] 32.5 mg of insulinotropin was weighed into a 10 ml glass
vial. 6 ml of water for injection was added to the vial. The pH of
the vial content was adjusted to 9.6 using 1% (w/v) NaOH to make a
clear solution. Appropriate amount of water for injection was added
to make the drug concentration to be 5.0 mg/ml.
[0432] Solution B54 Preparation
[0433] 390 mg of zinc acetate dihydrate was weighed into a 50 ml
volumetric flask. Approximately 40 mL of water for injection was
added to the flask to dissolve the zinc acetate dihydrate. The
flask was filled to the mark with water for injection. Solution B54
contained zinc acetate dihydrate 7.8 mg/mL in water for
injection.
[0434] Solution C54 Preparation
[0435] 1.1 g of phenol and 4.5 g of NaCl were weighed into a 50 ml
volumetric flask. Approximately 40 mL of water for injection. The
flask was filled to the mark with water for injection. Solution C54
contained phenol 22 mg/mL and NaCl 90 mg/mL.
[0436] Aqueous Suspension 54
[0437] All solutions were filtered through 0.22.mu. filters (low
protein binding). 2.4 ml of Solution A54 was transferred to a 3.5
ml vial. 300 .mu.l of Solution B54 was added to the vial with
stirring. Birefringent precipitates were formed immediately after
the addition. The pH was measured to be 6.8. The sample was allowed
to sit for 20 hours at ambient temperature. 300 .mu.l of Solution
C54 was added with gentle stirring. Aqueous Suspension 54 contained
insulinotropin 4 mg/mL, zinc acetate dihydrate 0.78 mg/mL, phenol
2.2 mg/mL, and NaCl 9 mg/mL in water for injection. This suspension
was used for in vivo pharmacokinetic studies in rats.
EXAMPLE 28
[0438] Solution A57 Preparation
[0439] 15 mg of insulinotropin was weighed into a 10 mL glass vial.
3 mL of water for injection was added to the vial. The pH of the
vial content was adjusted to 9.9 using 5% NaOH to dissolve the drug
completely. Solution A57 contained insulinotropin 5.0 mg/mL in
water for injection.
[0440] Solution B57 Preparation
[0441] 780 mg of zinc acetate dihydrate was weighed into a 100 mL
volumetric flask. Approximately 80 mL of water for injection was
added to the flask to dissolve the zinc acetate dihydrate. The
flask was filled to the mark with water for injection. Solution B57
contained zinc acetate dihydrate 7.8 mg/mL in water for
injection.
[0442] Solution C57 Preparation
[0443] 2.2 g of phenol and 9 g of NaCl were weighed into a 100 mL
volumetric flask. Approximately 80 mL of water for injection was
added to the flask to dissolve the phenol and the NaCl. The flask
was filled to the mark with water for injection. Solution C57
contained phenol 22 mg/ml and NaCl 90 mg/mL in water for
injection.
[0444] Aqueous Suspension 57
[0445] 2.4 mL of Solution A57 was transferred to a 3.5 mL vial. The
solution was stirred gently during addition of 300 .mu.L of
Solution B57. Precipitates were formed immediately after the
addition of the Solution B57. The pH was measured and found to be
7.1. The sample was allowed to sit under ambient conditions for 24
hours. 300 .mu.L of Solution C57 was added with gentle stirring.
Aqueous Suspension 57 contained insulinotropin 4 mg/mL, zinc
acetate dihydrate 0.78 mg/mL, phenol 2.2 mg/mL, and NaCl 9 mg/mL in
water for injection. This suspension was used for in vivo
pharmacokinetic studies in rats.
EXAMPLE 29
[0446] Solution A64 Preparation
[0447] 53.3 mg of insulinotropin was weighed into a 30 mL glass
vial. After adding 11 mL of water for injection, the pH of the vial
contents was adjusted to 8.3 using 5% NaOH (w/v) to dissolve the
insulinotropin. The pH was adjusted down to 6.0 using dilute HCl
making sure that the solution still remained clear. Appropriate
amount of water for injection was added to make the drug
concentration to be 4.4 mg/ml. Solution A64 was filtered through a
0.22.mu. filter (low protein binding) into a 3.5 mL sample vial.
1.8 mL of the filtered solution was transferred to a separate
sterile 3.5 mL vial, and the vial was allowed to sit at ambient
temperature to crystallize for 3 days.
[0448] Solution B64 Preparation
[0449] 780 mg of zinc acetate dihydrate was weighed into a 50 mL
volumetric flask. Approximately 40 mL of water for injection was
added to the flask to dissolve the zinc acetate dihydrate. The
flask was filled to the mark with water for injection. Solution B64
contained zinc acetate dihydrate 15.6 mg/mL in water for
injection.
[0450] Solution C64 Preparation
[0451] 18 g of NaCl was weighed into a 100 mL volumetric flask.
Approximately 80 mL of water for injection was added to the flask
to dissolve the NaCl. The flask was filled to the mark with water
for injection. Solution C64 contained NaCl 180 mg/mL in water for
injection.
[0452] Aqueous Suspension 64
[0453] After crystallization was completed in Solution A64, 100
.mu.L of Solution B64 was added to 1.8 mL of the crystal suspension
was slow stirring. The sample was then allowed to sit at ambient
temperature for 3 days. 100 .mu.L of Solution C64 was added to the
crystal suspension with gentle stirring. The pH of the suspension
was adjusted to pH 7.3 using dilute NaOH. 5.0.mu. of m-cresol was
added directly to the pH adjusted crystal suspension. Aqueous
Suspension 64 contained insulinotropin 4 mg/mL, zinc acetate
dihydrate 0.78 mg/mL, NaCl 9 mg/mL, and m-cresol 2.5 mg/mL in water
for injection. This suspension was used for in vivo pharmacokinetic
studies in rats.
EXAMPLE 30
[0454] Solution A69 Preparation
[0455] 1 g of NaCl was weighed into a 100 mL volumetric flask.
Approximately 80 mL of water for injection was added to the flask
to dissolve the NaCl. The flask was filled to the mark with water
for injection. Solution A69 contained NaCl 1% (w/v) in water for
injection.
[0456] Solution B69 Preparation
[0457] 390 mg of zinc acetate dihydrate was weighed into a 100 mL
volumetric flask. Approximately 80 mL of water for injection was
added to the flask to dissolve the zinc acetate dihydrate. The
flask was filled to the mark with water for injection. Solution 669
contained zinc acetate dihydrate 3.9 mg/mL in water for
injection.
[0458] Emulsion C69 Preparation
[0459] 2.5 mL of sterile filtered (0.22.mu. low protein binding)
m-cresol was transferred to a 100 mL volumetric flask. The flask
was filled with water for injection to the mark and sonicated to
produce a homogenous suspension. Emulsion C69 contained m-cresol 25
mg/mL in water for injection.
[0460] Aqueous Suspension 69
[0461] 35.74 mg of insulinotropin was weighed into a 10 mL glass
vial. 7 mL of Solution A69 was added. The pH of the vial contents
was adjusted to 9.2 to dissolve the drug. The pH of the solution
was re-adjusted to 6.5 using dilute HCl. Appropriate amount of
water for injection was added to make the drug concentration to be
4.4 mg/ml. The solution was filtered through a 0.22.mu. filter (low
protein binding). The solution was allowed to sit at ambient
temperature for 6 days, during which insulinotropin was
crystallized. 1.5 mL of the crystal suspension was transferred to a
separate vial. 167 .mu.L of Solution B69 was added with gentle
stirring. The sample was allowed to sit at ambient temperature for
1 day. 167 .mu.L of emulsion C69 was added to the supernatant of
the settled suspension. The sample was stirred to dissolve the
m-cresol. Aqueous Suspension 69 contained insulinotropin 3.6 mg/ml,
zinc acetate 0.36 mg/ml, NaCl 8.17 mg/ml and m-cresol 2.28 mg/ml in
water for injection. This suspension was used for in vivo
pharmacokinetic studies in rats.
EXAMPLE 31
[0462] Solution A101 Preparation
[0463] 10 g of sodium acetate was weighed into a 100 ml volumetric
flask. Approximately 80 mL of water for injection was added to the
flask to dissolve the sodium acetate. The flask was filled to the
mark with water for injection. Solution A200 contained 100 mg/ml
sodium acetate in water for injection.
[0464] Aqueous Suspension 101
[0465] 44.4 mg of insulinotropin was weighed into a 10 ml glass
vial. 8 ml of water for injection was added to the flask. The pH of
the vial contents was adjusted to 9.3 to obtain a clear solution. 1
mL of Solution A200 was added to the insulinotropin solution. The
pH was then adjusted down to 6.5. The solution was filtered through
a 0.22.mu. filter (low protein binding). The filtered solution was
allowed to sit at ambient temperature for 3 days so that
crystallization could occur. Aqueous Suspension 101 contained
insulinotropin 4.9 mg/mL sodium acetate 11.1 mg/mL in water for
injection. This suspension was used for in vivo pharmacokinetic
study in rats.
EXAMPLE 32
[0466] Solution A82 Preparation
[0467] 9 g of NaCl was weighed into a 100 mL volumetric flask.
Approximately 80 mL of water for injection was added to the vial to
dissolve the NaCl. The flask was filled to the mark with water for
injection. Solution A82 contained NaCl 9% (w/v) in water for
injection.
[0468] Solution B82 Preparation
[0469] 789 mg of zinc acetate dihydrate was weighed into a 100 mL
volumetric flask. Approximately 80 mL of water for injection was
added to the vial to dissolve the zinc acetate dihydrate. The flask
was filled to the mark with water for injection. Solution B82
contained zinc acetate dihydrate 7.89 mg/mL in water for
injection.
[0470] Emulsion C82 Preparation
[0471] 2.5 mL of sterile filtered (0.22.mu. low protein binding)
m-cresol was transferred to a 100 mL volumetric flask. The flask
was filled with water for injection to the mark and sonicated to
produce a homogenous suspension. Emulsion C82 contained m-cresol 25
mg/mL in water for injection.
[0472] Aqueous Suspension 82
[0473] All solutions were filtered through 0.22.mu. filters (low
protein binding). 45.34 mg of insulinotropin was added to a 10 ml
vial to which 8 ml of water was added. The pH was adjusted to 9.3
using 5% NaOH. After 1 ml of Solution A82 was added to the vial,
the pH of the solution was adjusted down to 6.55 using dilute HCl.
The solution (5 mg/mL insulinotropin) was filtered through a
0.22.mu. filter (low protein binding). 81 .mu.l of Aqueous
Suspension 101 (see example 31) was added to the sterile filtered
insulinotropin solution and dispersed by shaking the sample. The
sample was then allowed to sit for 72 hours at ambient temperature
to form a crystal suspension. 2.4 ml of the suspension was
transferred to a 3.5 ml vial. 300 .mu.l of Solution B82 was added
to the vial with gentle stirring. The pH of the vial content was
adjusted to 7.3 using dilute NaOH. 300 .mu.l of Emulsion C82 was
added to the supernatant of the settled suspension. Aqueous
Suspension 82 contained insulinotropin 4 mg/ml, zinc acetate
dihydrate 0.79 mg/mL, m-cresol 2.5 mg/mL and 0.9% NaCl in water for
injection. This suspension was used for in vivo pharmacokinetic
studies in rats.
EXAMPLE 33
[0474] GLP-1 (7-36) Amide (1 mg/ml) Suspension
[0475] Solution A26 Preparation
[0476] Solution A26 was prepared by weighing 10 mg of GLP-1 (7-36)
Amide into a 5 ml volumetric flask. Approximately 3 ml of PBS was
added to the flask to dissolve the drug. The q.s. amount of PBS was
added to the flask. Solution A26 was filtered through a 0.22.mu.
filter into a 10 ml glass vial. Solution A26 contained GLP-1 (7-36)
2 mg/ml in PBS.
[0477] Solution B26 Preparation
[0478] Solution B26 was prepared by weighing 44 mg of phenol into a
10 ml volumetric flask. Approximately 8 ml of PBS was added to the
flask to dissolve the phenol. The q.s. amount of PBS was added to
the flask. Solution B26 was filtered through a 0.22.mu. filter into
a 10 ml glass vial. Solution B26 contained phenol 4.4 mg/ml in
PBS.
[0479] Aqueous Suspension 26
[0480] 1.5 ml of solution A26 was pipetted into a 3.5 ml type I
glass vial. The contents of the vial were stirred magnetically
while 1.5 ml of solution B26 was pipetted into the vial. The vial
was stoppered and sealed with an aluminum shell. The vial contents
were stirred gently (making sure no foam or bubble formed) for 18
hours to allow suspension formation. Aqueous Suspension 26
contained GLP-1 (7-36) Amide 1 mg/ml and phenol 2.2 mg/ml in PBS.
This suspension was used for in vivo pharmacokinetic studies in
rats.
EXAMPLE 34
[0481] In one form of the invention, a low solubility form of GLP-1
(7-37) is prepared by combining GLP-1 (7-37) at from 2-15 mg/ml in
buffer at pH 7-8.5 with a solution of a metal ion salt to obtain
solutions with from 1-8 mg/ml GLP-1 (7-37) at molar ratios of about
1:1 to 270:1 zinc to GLP-1 (7-37). A heavy precipitate forms and is
let stand overnight at room temperature. The solubility of GLP-1
(7-37) in the metal ion solution varies with the metal employed.
Subsequent measurement of the solubility of the GLP-1 (7-37) pellet
in a non metal-containing solvent such as PBS or water shows that
zinc, cobalt and nickel ions produce low solubility forms of GLP-1
(7-37). TABLE-US-00059 TABLE 1 Ability of Various metal ion salts
to produce low solubility GLP-1(7-37) Metal ion salt Solubility in
metal sol'n Solubility in PBS Zn Acetate 0.04 .mu.g/ml 0.04
.mu.g/ml Zn Chloride 0.04 .mu.g/ml 0.03 .mu.g/ml Co Chloride 0.11
.mu.g/ml 0.04 .mu.g/ml Ni Sulfate 0.14 .mu.g/ml 0.07 .mu.g/ml Mn
Chloride 0.23 .mu.g/ml 1.64 .mu.g/ml Mg Chloride 1.75 .mu.g/ml no
ppt. Ca Chloride 1.98 .mu.g/ml no ppt. Note: In each case, 100
.mu.l of metal ion solution at 5 mM was added to 100 .mu.l
GLP-1(7-37) at 5 mg/ml, mixed and allowed to stand overnight. The
insoluble pellet was removed by centrifugation. The concentration
of GLP-1(7-37) remaining in the metal ion solution was measured.
The pellet was resuspended in phosphate buffered saline (PBS),
sonicated and allowed to stand overnight. Again insoluble material
was pelleted and GLP-1(7-37) concentration measured.
EXAMPLE 35
[0482] Microcrystalline forms of GLP-1 (7-37) can be obtained by
mixing solutions of GLP-1 (7-37) in buffer pH 7-8.5 with certain
combinations of salts and low molecular weight polyethylene glycols
(PEG). Table 2 describes six specific sets of conditions to produce
microcrystalline forms of GLP-1 (7-37). TABLE-US-00060 TABLE 2
Selected Reagents Yielding Microcrystals Reagent # Salt Buffer
Precipitant 1 none none 0.4 M K, Na tartrate 2 0.2 M Na citrate 0.1
M Tris pH 8.5 30% PEG 400 3 0.2 M MgCl.sub.2 0.1 M HEPES pH 7.5 28%
PEG 400 4 0.2 M MgCl.sub.2 0.1 M HEPES pH 7.5 30% PEG 400 5 0.5 M
K.sub.2HPO.sub.4 none 20% PEG 8000 6 none none 30% PEG 1500 Note:
GLP-1(7-37) stock at 5 mg/ml in 50 mM Tris pH 8.1 was added 1:1
with reagent. Drops were viewed and scored for absence or presence
of insoluble GLP-1(7-37) in crystalline or amorphous form. In
general low mw PEG's appear to favor crystalline forms. Tris is
tris(hydroxymethyl)aminomethane and HEPES is
N-2-Hydroxyethyl)piperazine-N-2-ethanesulfonic acid.
EXAMPLE 36
[0483] Specific combinations of GLP-1 (7-37) and PEG concentrations
are required to obtain microcrystalline forms and high yields.
Table 3 shows specific combinations of PEG 600 and GLP-1 (7-37)
concentrations which produce microcrystalline as opposed to
amorphous forms of the drug. The yield of GLP-1 (7-37) in the
insoluble form is shown also. TABLE-US-00061 TABLE 3
Formation/yield of crystalline GLP-1(7-37) 15 22.5% 30% GLP-1(7-37)
PEG 600 PEG 600 PEG 600 2.0 mg/ml amorphous/8% amorphous/10%
amorphous/8% (Form/yield) 3.5 mg/ml crystalline/62% crystalline/26%
crystalline/59% (Form/yield) 5.0 mg/ml amorphous/34%
crystalline/63% crystalline/72% (Form/yield) 6.5 mg/ml
amorphous/52% crystalline/76% crystalline/82% (Form/yield) 8.0
mg/ml amorphous/55 crystalline/82% amorphous/66% (Form/yield) 9.5
mg/ml amorphous/69% crystalline/85% amorphous/83% (Form/yield)
Note: Microcrystals of GLP-1(7-37) are prepared by combining
solutions of GLP-1(7-37) at 20 mg/ml in tris buffer at pH 8, 60%
polyethylene glycol 600 (PEG 600) in H.sub.2O and tris buffer pH 8
to obtain a final concentrations of from 15-30% PEG and from 3-10
mg/ml GLP-1. After standing overnight, microcrystals of GLP-1(7-37)
form in the solution with yields from 50-85%.
EXAMPLE 37
[0484] This experiment exemplifies another form of the invention
which involves treating preformed microcrystals of GLP-1 (7-37)
with various metal ions to produce low solubility microcrystalline
forms. Microcrystals of GLP-1 (7-37) prepared at 8 mg/ml GLP-1
(7-37) and 22.5% PEG as described in Example 22 have a solubility
equivalent to pure lyophilized GLP-1 (7-37). In order to impart the
desired property of low solubility for long-acting drug delivery,
these preformed microcrystals can be treated with solutions of
metal salts at ratios of metal:GLP-1 (7-37) of from 1:1 to 260:1
overnight at room temp. The excess metal salt was removed by a
centrifugation/washing process. Table 4 shows the results with
several divalent cation metal salts as treatment. TABLE-US-00062
TABLE 4 Solubility of GLP-1(7-37) Crystals with Various Treatments
GLP-1(7-37) GLP-1(7-37) (mg/ml) in GLP-1(7-37) (mg/ml) in Additive
treatment sol'n (mg/ml) in PBS PBS/EDTA None (PBS) 1.2 1.2 ND
Citrate pH 5.2 0.15 ND ND ZnCl.sub.2 pH 5.2 0.03 0.03 1.1 ZnAc pH
5.2 0.01 0.02 1.1 ZnAc pH 6.5 0.06 0.02 0.92 MgSO.sub.4 pH 5.2 0.50
0.55 ND NiSO.sub.4 pH 5.2 0.10 0.04 0.45 MnCl.sub.2 pH 5.2 0.10
0.10 ND CaCl.sub.2 pH 5.2 0.40 0.27 ND Note: GLP-1(7-37) crystals
are grown from a solution of 8 mg/ml IST in 50 mM Tris pH 8 with
22.5% PEG 600 added in H.sub.2O. All additive treatment solutions
are 100 mM divalent ion salt in 10 mM Na citrate pH 5.2 or Na MES
pH 6.5.
EXAMPLE 38
[0485] Using the methods described herein, both amorphous and
microcrystalline low solubility formulations were prepared using
zinc acetate. Subcutaneous injections were made in rats (three
animals per formulation) and plasma levels of GLP-1 (7-37) were
measured by radioimmune assay over 24 hours. FIG. 8 shows the
extended duration of the drug in plasma compared to a subcutaneous
control injection of soluble GLP-1 (7-37).
EXAMPLE 39
[0486] 45% w/v Polyethylene Glycol 3350 (PEG) [0487] 1 mg/ml
Insulinotropin [0488] 20 mM Phosphate Buffer [0489] qs Sterile
Water for Injection (SWFI)
[0490] A 50% w/w PEG solution was prepared using SWFI. A 200 mM
phosphate buffer was separately prepared with anhydrous sodium
phosphate dibasic (26.85 mg/ml) and sodium phosphate monobasic
monohydrate (1.41 mg/ml). If necessary, the pH of the buffer
solution was brought to pH 8 with either sodium hydroxide or
hydrochloric acid. The appropriate amount of insulinotropin was
dissolved in enough of the buffer solution to make a 10 mg/ml
solution of insulinotropin. The appropriate weight of the PEG
solution was added to the insulinotropin solution, and a sufficient
quantity of SWFI was used to bring the solution to the desired
volume. The final solution was then sterile filtered with 0.2%
filter and aseptically filled into vials. The solution (0.5 ml) was
injected subcutaneously in rats, and plasma insulinotropin levels
followed by RIA assay.
EXAMPLE 40
[0491] 1.32% w/v Hydroxyethyl Cellulose (HEC) [0492] 1 mg/ml
Insulinotropin [0493] 20 mM Phosphate Buffer [0494] 100 mM Sodium
Chloride [0495] qs Sterile Water For Injection (SWFI)
[0496] A 2% w/w hydroxethyl cellulose solution was prepared using
SWFI. A 200 mM phosphate buffer was separately prepared with
anhydrous sodium phosphate dibasic (26.85 mg/ml) and sodium
phosphate monobasic monohydrate (1.41 mg/ml). If necessary, the pH
of the buffer solution was brought to pH 8 with either sodium
hydroxide or hydrochloric acid. The appropriate amount of
insulinotropin and sodium chloride were dissolved in enough of the
buffer solution to make a 10 mg/ml solution of insulinotropin. The
appropriate weight of the HEC solution was added to the
insulinotropin solution, and a sufficient quantity of SWFI was used
to bring the solution to the desired volume. The final solution was
then sterile filtered with a 0.2.mu. filter and aseptically filled
into vials. The solution (0.5 ml) was injected subcutaneously in
rats, and plasma insulinotropin followed by RIA assay.
EXAMPLE 41
[0497] 18% w/v Pluronic F127 [0498] 1 mg/ml Insulinotropin [0499]
20 mM Phosphate Buffer [0500] qs Sterile Water For Injection
(SWFI)
[0501] A 20% W/W Pluronic F 127 solution was prepared using SWFI. A
Polytron (probe homogenizer) with an ice bath was used to dissolve
the polymer. A 200 mM phosphate buffer was separately prepared with
anhydrous sodium phosphate dibasic (26.85 mg/ml) and sodium
phosphate monobasic monohydrate (1.41 mg/ml). If necessary, the pH
of the buffer solution was brought to pH 8 with either sodium
hydroxide or hydrochloric acid. The appropriate amount of
insulinotropin was dissolve in enough of the buffer solution to
make a 10 mg/ml solution of insulinotropin. The appropriate weight
of the Pluronic solution was added to the insulinotropin solution,
and a sufficient quantity of SWFI was used to bring the solution to
the desired volume. The final solution was then sterile filtered
with a 0.2.mu. filter and aseptically filled into vials. The
solution (0.5 ml) was injected subcutaneously in rats, and plasma
insulinotropin levels followed by RIA assay.
EXAMPLE 42
[0502] Peanut Oil Suspension (Ball Milled) [0503] 1 mg/ml
Insulinotropin [0504] 1% Tween 80
[0505] Tween 80 was added at 1% level to peanut oil. This solution
was sterile filtered with a 0.2 .mu.m filter. Solid insulinotropin
was then suspended in the oil. The particle size was reduced by
ball milling with a Szesvari Attritor at 40 RPM for 18 hours (cold
water jacket). This suspension was then filled into vials. The
suspension (0.5 ml) was injected subcutaneously in rats, and plasma
insulinotropin levels followed by RIA assay.
EXAMPLE 43
[0506] 22.6% w/v Dextran [0507] 1 mg/ml Insulinotropin [0508] 20 mM
Phosphate Buffer [0509] qs Sterile Water for Injection
[0510] A 50% w/w Dextran solution was prepared using SWFI. A 200 mM
phosphate buffer was separately prepared with anhydrous sodium
phosphate dibasic (26.85 mg/ml) and sodium phosphate monobasic
monohydrate (1.41 mg/ml). If necessary, the pH of the buffer
solution was brought to pH 8 with either sodium hydroxide or
hydrochloric acid. The appropriate amount of insulinotropin was
dissolved in enough of the buffer solution to make 5.0 mg/ml
solution of insulinotropin. The appropriate weight of the dextran
solution was added to the insulinotropin solution, and a sufficient
quantity of SWFI was used to bring the solution to the desired
volume. The final solution was then sterile filtered with 0.2 .mu.m
filter and aseptically filled into vials. The solution (0.5 ml) was
injected subcutaneously into rats, and plasma insulinotropin levels
were followed by RIA assay.
EXAMPLE 44
[0511] Insulinotropin was crystallized from the mixture of
phosphate buffered saline (PBS), ethanol, and m-cresol. A
homogeneous insulinotropin slurry (10 mg/ml) was made with PBS in a
glass vial, and a large volume of ethanol (9 times as much as the
slurry) was added to the vial while the vial content was stirred
magnetically. Very fine amorphous particles of insulinotropin
formed. m-Cresol was added to the vial so that the resulting
m-cresol concentration was 1% (v/v). The vial was capped to prevent
solvent from evaporating. The crystallization mixture was stored at
room temperature for a couple of days. Needle shape crystalline
plates grew from the amorphous particles. The lengths of the
crystals are between 50 and 200 .mu.m, and widths between 2 and 4
.mu.m.
EXAMPLE 45
[0512] Insulinotropin (1 to 4 mg/mL) was dissolved in 1% sodium
sulfate (or sodium acetate, or sodium chloride, or ammonium
sulfate) solution at higher pH values than 8, and the pH of the
solution was lowered down to 6.0 to 7.5 with d-HCl. The clear
solution was allowed to sit at ambient temperature. After a couple
of days, needle or plate shape crystals were obtained depending on
the crystallization conditions.
EXAMPLE 46
[0513] GLP-1 (7-37) was dissolved in 50 mM glycine buffer
containing 0.1 to 0.2 M NaCl at pH 8.5-9.5 at from 1 to 5 mg/ml. A
solution of zinc salt (acetate or chloride) was added to obtain a
molar ratio of from 0.5:1 to 1.5:1 zinc:GLP-1 (7-37). Crystals of
GLP-1 (7-37) grew overnight at room temperature with yields from 70
to 97%.
EXAMPLE 47
[0514] GLP-1 (7-37) crystals can be grown by vapor diffusion using
the peptide dissolved in 100 mM Tris at pH 8-9.5 at from 10-20
mg/ml. The peptide solution is mixed 1:1 with the same buffer
containing from 0.5 to 2.5 M NaCl then equilibrated in a sealed
system against the full strength buffer (i.e. Tris with 0.5-2.5 M
NaCl).
Sequence CWU 1
1
9 1 37 PRT Homo sapiens 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
31 PRT Homo sapiens 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 30 PRT Homo sapiens 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
29 PRT Homo sapiens 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 28 PRT Homo sapiens 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 36 PRT Homo sapiens 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 28 PRT Homo sapiens MUTAGEN 28 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 Lys 20 25 8 29
PRT Homo sapiens MUTAGEN 29 8 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 9 30 PRT Homo sapiens MUTAGEN 30 9
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
* * * * *