U.S. patent application number 11/693877 was filed with the patent office on 2007-07-26 for use of glp-1 analogs and derivatives administered peripherally in regulation of obesity.
Invention is credited to Richard DiMarchi, Suad Efendic.
Application Number | 20070173452 11/693877 |
Document ID | / |
Family ID | 26705788 |
Filed Date | 2007-07-26 |
United States Patent
Application |
20070173452 |
Kind Code |
A1 |
DiMarchi; Richard ; et
al. |
July 26, 2007 |
USE OF GLP-1 ANALOGS AND DERIVATIVES ADMINISTERED PERIPHERALLY IN
REGULATION OF OBESITY
Abstract
This invention relates the use of glucagon-like peptides such as
GLP-1, a GLP-1 analog, or a GLP-1 derivative in methods and
compositions for reducing body weight.
Inventors: |
DiMarchi; Richard; (Carmel,
IN) ; Efendic; Suad; (Lidingo, SE) |
Correspondence
Address: |
ELI LILLY & COMPANY
PATENT DIVISION
P.O. BOX 6288
INDIANAPOLIS
IN
46206-6288
US
|
Family ID: |
26705788 |
Appl. No.: |
11/693877 |
Filed: |
March 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10429522 |
May 5, 2003 |
7211557 |
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11693877 |
Mar 30, 2007 |
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09585186 |
Jun 1, 2000 |
6583111 |
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10429522 |
May 5, 2003 |
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08961405 |
Oct 30, 1997 |
6191102 |
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09585186 |
Jun 1, 2000 |
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60030213 |
Nov 5, 1996 |
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Current U.S.
Class: |
514/5.3 ;
514/11.7; 514/20.3 |
Current CPC
Class: |
Y10S 514/909 20130101;
A61K 38/26 20130101; C07K 14/605 20130101; Y10S 514/866 20130101;
A61P 3/04 20180101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 38/26 20060101
A61K038/26 |
Claims
1.-14. (canceled)
15. A method of reducing body weight in a subject in need of body
weight reduction by administering to the subject a composition
comprising an agonist of the GLP-1 receptor wherein the agonist is
a peptide that is resistant to cleavage by the enzyme
dipeptidyl-peptidase IV.
16. The method of claim 15 wherein the composition further
comprises a pharmaceutically acceptable buffer.
17. The method of claim 16 wherein the composition further
comprises an anti-microbial agent.
18. The method of claim 17 wherein the anti-microbial agent is
selected from phenol and meta-cresol.
19. The method of claim 17 further comprising an isotonicity
agent.
20. The method of claim 15 wherein the composition is administered
by a non-parenteral route selected from the group consisting of:
oral, rectal, nasal, and lower respiratory.
21. The method of claim 15 wherein the composition is a controlled
release preparation
22. The method of claim 21 wherein the controlled release
preparation comprises particles of a polymeric material.
Description
[0001] This application claims priority from co-pending U.S.
provisional application 60/030,213, filed Nov. 5, 1996.
BACKGROUND OF THE INVENTION
[0002] This invention relates to the use of glucagon-like peptide-1
(GLP-1), analogs and derivatives of GLP-1, in methods and
compositions, in particular pharmaceutical formulations, that
promote is weight-loss.
[0003] Obesity, and especially upper body obesity, is the most
common nutritional disorder in the over-nourished populations of
the world. Numerous studies indicate that lowering body weight
dramatically reduces risk for chronic diseases, such as diabetes,
hypertension, hyperlipidemia, coronary heart disease, and
musculoskeletal diseases. For example, various measures of obesity,
including, simple body weight, waist-to-hip ratios, and mesenteric
fat depot, are strongly correlated with risk for non-insulin
dependent diabetes (NIDDM), also known as type II diabetes.
According to the American Diabetes Association (1995) about 80% of
NIDDM patients are overweight. Weight-reduction is a specific goal
of medical treatment of many chronic diseases, including NIDDM.
[0004] Current methods for promoting weight loss are not completely
satisfactory. Some obese patients may lose weight through
deliberate modification of behavior, such as changing diet and
increased exercise. Failure to achieve weight loss by these methods
may be due to genetic factors that cause increased appetite, a
preference for high-fat foods, or a tendency for lipogenic
metabolism. Unfortunately, an estimated 33 billion dollars a year
are spent on weight-loss measures that are largely futile. Thus,
new methods and compositions such as pharmaceutical agents that
promote weight-loss are urgently needed to complement old
approaches.
[0005] Glucagon-like peptide 1 (GLP-1) is known to play a critical
role in the regulation of the physiological response to feeding.
GLP-1 is processed from proglucagon and is released into the blood
from the endocrine L-cells mainly located in the distal small
intestine and colon in response to ingestion of a meal (Nilsson et
al., 1991; Krcymann et al., 1987: Mojsov at al. 1986). GLP-1 acts
through a G protein-coupled cell surface receptor (GLP-1R) and
enhances nutrient-induced insulin synthesis (Fehmann et all 1992)
and release (Fehmann et al., 1995). GLP-1 stimulates insulin
secretion (insulinotropic action) and cAMP formation (Mojsov et
al., 1992). GLP-1(-7-36) amide stimulates insulin releaser lowers
glucagon secretion, and inhibits gastric secretion and emptying
(Nauck, 1993; Gutniak et al, 1992). These gastrointestinal effects
of GLP-1 are not found in vagotomized subjects, pointing to a
centrally-mediated effect (Orskov et al., 1995). GLP-1 binds with
high affinity to isolated rat adipocytes, activating cAMP
production (Valverde et al., 1993) and stimulating lipogenesis
(Oben, et al., 1991) or lipolysis (Ruiz-Grande et al., 1992). GLP-1
stimulates glycogen synthesis, glucose oxidation, and lactate
formation in rat skeletal muscle (Villanueva et al., 1994).
[0006] m-RNA encoding the pancreatic-type GLP-1 receptor is found
in relatively high quantities in rat pancreatic islets, lung,
hypothalamus, and stomach (Billock et al., 1996). Interestingly,
despite the knowledge that both GLP-1 and GLP-1 receptors are found
in the hypothalamus (Krcymann et al., 1989; Kanse et al., 1988), no
central role for GLP-1 was determined until a recent report that
GLP-1 administered by the intracerebroventricular route (ICV)
markedly inhibits feeding in fasted rats (Turton et al., 1996). The
same report indicates that after ICV administration of GLP-1,
c-fos, a marker of neuronal activation, appears exclusively in the
paraventricular nucleus of the hypothalamus and in the central
nucleus of the amygdala, two regions of the brain of primary
importance in the regulation of feeding (Morley, 1987) ICV GLP-1
also significantly reduces food intake following injection of the
powerful feeding stimulant, neuropeptide Y, in animals fed ad
libitum, (Turton et al., 1996). A subsequent report demonstrates
that GLP-1 administered centrally or peripherally is involved in
control of body temperature regulation, but does not affect food
intake after acute intraperitoneal administration in rats (O'Shea
et al., 1996). A recent article reports that lateral ventricular
injections of GLP-1 in sated rats induce extensive stimulation of
Fos-ir in the paraventricular nucleus and parvocellular central
nucleus of the amygdala, substantiating Turton, et al. (Rowland et
al., 1996). Additionally, these investigators described strong
activation of other centers involved in the regulation of feeding,
including the immediate early gene protein product in the nucleus
of the tractus solitarius, the pontine lateral parabrachial
nucleus, the basal nucleus of the stria terminals, and the area
postrema. GLP-1 receptors accessible to peripheral GLP-1 are found
in the rat subfornical organ and area postrema (Orskov et al.,
1996).
[0007] Turton et al. (1996) specifically state that the effects of
GLP-1 on body weight and food intake are caused only by
administration of GLP-1 directly in the cerebroventriculum, that
intraperitoneal administration of GLP-1, even at relatively high
does, does not affect early dark-phase feeding, and that GLP-1
fragments are inactive when administered peripherally, citing
(Suzuki et al., 1989). Such statements discourage the use of GLP-1
as a composition (pharmaceutical agent) for reducing body weight,
because central routes of administration, such as the ICV route,
are not feasible for treating obesity in humans. The physiological
effects of GLP-1 documented above have led to the suggestion of its
beneficial use for treating diabetes and obesity by transplanting
recombinant cell lines encoding GLP-1 or GLP or GLP-1 receptors,
for example (WO 96/25487).
[0008] Another publication discouraged the use of GLP-1 by
interpreting the art to show that "peripheral administration of
GLP-1 had no effect on feeding behavior." (WO 97/31943, page 3).
This publication also reported an effect of GLP-2 on food intake
when administered peripherally.)
SUMMARY OF THE INVENTION
[0009] Methods and compositions, in particular pharmaceutical
formulations, medicaments, using glucagon-like peptide-1 analogs,
derivatives, and active peptides thereof, are effective in reducing
body weight and in treating obesity. The definition of obesity
varies with geographical location, clinical focus, and social
preferences. The methods and compositions of the present invention
however, are suitable for any subject in which weight reduction is
desired. The invention is not limited for use in, e.g. diabetic
patients.
[0010] Peripheral administration of GLP-1 (7-36) amide to obese
patients quite unexpectedly, and contrary to the implications of
Turton et al. (1996), causes a significant reduction in body
weight. Thus, an aspect of the present invention is a method of
reducing body weight which includes preparing a composition having
a glucagon-like peptide-1 compound and administering it to a
subject. Suitable glucagon-like peptide-1 compounds include GLP-1
GLP-1 analogs, GLP-1 derivatives, agonists of the GLP-1 receptor,
agonists of the GLP-1 signal transduction cascade, compounds that
stimulate synthesis of endogenous GLP-1, compounds that stimulate
release of endogenous GLP-1, and pharmaceutically-acceptable salts
thereof. A pharmaceutically effective dose, that is, a dose
sufficient to cause reduction in body weight, is administered.
DETAILED DESCRIPTION OF THE INVENTION
[0011] Methods and compositions, in particular medicaments
(pharmaceutical compositions or formulations) using glucagon-like
peptide-1, analogs or derivatives thereof, are effective in
reducing body weight and in treating obesity. Analogs and
derivatives of GLP-1 that are useful for the practice of the
invention are those with an increased half life compared to GLP-1
and the ability to effect weight lose when administered to a
subject over a period of time. The definition of obesity varies
with geographical location, clinical focus, and social preferences.
The methods and compositions of the present invention, however, are
suitable for any subject in which weight reduction is desired. The
invention is not limited for use in, e.g. diabetic patients.
Compounds
[0012] GLP-1 analogs, derivatives, variants, precursors and
homologues are all suitable for the practice of the invention as
long as the active fragment that effects weight loss is
included.
[0013] "GLP-1" means GLP-1(7-37). By custom in the art, the
amino-terminus of GLP-1(7-37) has been assigned number 7 and the
carboxy-terminus, number 37. The amino acid sequence of GLP-1(7-37)
is well-known in the art, but is presented below for the reader's
convenience: TABLE-US-00001 (SEQ ID NO:1)
NH.sub.2-His.sup.7-Als-Glu-Gly.sup.10-
Thr-Phe-Thr-Ser-Asp.sup.15-Val-Ser-Ser-Tyr-Leu.sup.20-
Glu-Gly-Gln-Ala-Ala.sup.25-Lys-Glu-Phe-Ile-Ala.sup.30-
Trp-Leu-Val-Lys-Gly.sup.35-Arg-Gly.sup.37-COOH
[0014] A "GLP-1 analog" is defined as a molecule having a
modification including one or more amino acid substitutions,
deletions, inversions, or additions when compared with GLP-1. GLP-1
analogs known in the art include, for example, GLP-1(7-34) and
GLP-1(7-35), GLP-1(7-36), Val.sup.8-GLP-1(7-37),
Gln.sup.9-GLP-1(7-37), D-Gln.sup.9-GLP-1(7-37),
Thr.sup.16-Lys.sup.18-GLP-1(7-37), and Lys.sup.18-GLP-1(7-37).
Preferred GLP-1 analogs are GLP-1(7-34) and GLP-1(7-35) which are
disclosed in U.S. Pat. No. 5,118,666, and also GLP-1(7-36). These
compounds are the biologically processed forms of GLP-1 having
insulinotropic properties. Other GLP-1 analogs are disclosed in
U.S. Pat. No. 5,545,618.
[0015] A "GLP-1 derivative" is defined as a molecule having the
amino acid sequence of GLP-1 or of a GLP-1 analog, but additionally
having at least one chemical modification of one or more of its
amino acid side groups, a-carbon atoms, terminal amino group, or
terminal carboxylic acid group. A chemical modification includes
adding chemical moieties, creating new bonds, and removing chemical
moieties. Modifications at amino acid side groups include acylation
of lysine e-amino groups, N-alkylation of arginine, histidine, or
lysine, alkylation of glutamic or aspartic carboxylic acid groups,
and deamidation of glutamine or asparagine. Modifications of the
terminal amino include the des-amino, N-lower alkyl, N-di-lower
alkyl, and N-acyl modifications. Modifications of the terminal
carboxy group include the amide, lower alkyl amide, dialkyl amide,
and lower alkyl ester modifications. A lower alkyl is a
C.sub.1-C.sub.4 alkyl. Furthermore, one or more side groups, or
terminal groups, may be protected by protective groups known to the
ordinarily-skilled protein chemist. The .alpha.-carbon of an amino
acid may be mono- or di-methylated.
[0016] In the present invention a preferred group of GLP-1 analogs
and derivatives for use in the present invention is composed of the
various GLP-1 molecules claimed in U.S. Pat. No. 5,545,618 ('618).
Effective analogs of the active GLP-1 peptides, 7-34, 7-35, 7-36
and 7-37 have amino acid substitutions as positions 7-10 and/or are
truncated at the C-terminus and/or contain various other amino acid
substitutions in the basic peptide. Analogs having D-amino acid
substitutions in the 7 and 8 positions and/or N-alkylated or
N-acylated amino acids in the 7 position are particularly resistant
to degradation in vivo.
[0017] The analogs of the invention in '618 which show enhanced
insulin stimulating properties have the sequence, of native GLP-1,
7-34, 7-35, 7-36, or 7-37, or the C-terminal amide thereof, with at
least one modification selected from the group consisting of:
[0018] (a) substitution of a neutral amino acid, arginine, or a D
form of lysine for lysine at position 26 and/or 34 and/or a neutral
amino acid, lysine, or a D form of arginine for arginine at
position 36;
[0019] (b) substitution of an oxidation-resistant amino acid for
tryptophan at position 31;
[0020] (c) substitution according to at least one of:
[0021] Y for V at position 16;
[0022] K for S at position 18;
[0023] D for E at position 21;
[0024] S for G at position 22;
[0025] R for Q at position 23;
[0026] R for A at position 24; end
[0027] Q for K at position 26;
(Using the Single Letter Codes for Amino Acids)
[0028] (d) a substitution comprising at least one of:
[0029] an alternative small neutral amino acid for A at position
8;
[0030] an alternative acidic amino acid or neutral amino acid for E
at position 9.
[0031] an alternative neutral amino acid for G at position 10;
and
[0032] an alternative acidic amino acid for D at position 15;
and
[0033] (e) substitution of an alternative neutral amino acid or the
D or N-acylated or alkylated form of histidine for histidine at
position 7.
[0034] With respect to modifications (a), (b), (d) and (e), the
substituted amino acids may be in the D form. The amion acids
substituted at position 7 can also be in the N-acylated or
N-alkylated forms.
[0035] In another aspect, the invention of '618 is directed to
peptides which show enhanced degradation resistance in plasma as
compared to GLP-1 (7-37) wherein this enhanced resistance to
degradation. In these analogs, any of the abovementioned truncated
forms of GLP-1(7-34) to GLP-1(7-37) or their C-terminal amidated
forms is modified by
[0036] (a) substitution of a D-neutral or D-acidic amino acid for H
at position 7, or
[0037] (b) substitution of a D-amino acid for A at position 8,
or
[0038] (c) both, or
[0039] (d) substitution of an N-acylated or N-alkylated form of any
naturally occurring amino acid for H at position 7.
[0040] Thus analogs which are resistant to degradation include
(N-acyl (1-6C) AA).sup.7 GLP-1(7-37) and (N-alkyl (1-6C AA).sup.7
GLP-1 (7-37) wherein when AA is a lysyl residue, one or both
nitrogens may be alkylated or acylated, AA symbolizes any amino
acid consistent with retention of insulin stimulating activity.
[0041] For substitutions of D-amino acids in the 7 and 8 positions,
the D residue of any acidic or neutral amino acid can be used at
position 7 and of any amino acid at position 8, again consistent
with insulin stimulating activity. Either or both of position 7 and
8 can be substituted by a D-amino acid; the D-amino acid at
position 7 can also be acylated or alkylated. These modified forms
are applicable not only to GLP-1(7-37) but also to shorter
truncated analogs.
[0042] Thus, among the preferred analogs of the '618 invention are
those wherein the (7-34), (7-35), or (7-37) form of GLP-1 has been
modified only by substitution of a neutral amino acid, arginine, or
a D form of lysine for lysine at position 26 and/or 34 and/or a
neutral amino acid, lysine, or a D form of arginine for arginine at
position 36 (section (a)). Particularly preferred are those wherein
the amino acid substituted for lysine at position 26 and 34 is
selected from the croup consisting of K.sup.+,G, S, A, L, I, Q, R,
R.sup.+ and M, and for arginine at position 36 is selected from the
group of K, K.sup.+, G, S, A, L, I, Q, M, and R.sup.+. (where
.sup.+ indicates a D form).
[0043] Also preferred are analogs wherein the sole modification is
the substitution of an oxidation-resistant amino acid for
tryptophan at position 31 (section (b)). Particularly favored
substitutions are selected from the group consisting of F, V, L, I,
A, and Y.
[0044] Also preferred are those analogs wherein the only
modification is at least one of those specific substitutions set
forth in section (c). Particularly preferred are those analogs
wherein combined substitutions of S for G at position 22, R at
positions 23 and 24 for Q and A respectively, and Q for K at
position 26 have been made, ox substitutions of Y for V at position
26 and K for S at position 18 have been made, or these
substitutions plus D for E at positions 21 have been made.
[0045] Also preferred are analogs wherein the sole modifications
are those set forth in section (d). Particularly preferred among
these are those wherein the small neutral amino acid substituted
for alanine at position 8 is selected from the group consisting of
S, S.sup.+, GC, C.sup.+, Sar, A.sup.+, beta-ala and Aib; and/or the
acidic or neutral amino acid substituted for glutamic acid at
position 9 is selected from the group consisting of E.sup.+, D,
D.sup.+, Cya T, T.sup.+, N, N.sup.+, Q, Q.sup.+, Cit, MSO, and
acetyl-K; and/or the alternative neutral amino acid substituted for
glycine at position 10 is selected from the group consisting of S,
S.sup.+, Y, Y.sup.+, T, T.sup.+, N, N.sup.+, Q, Q.sup.+, Cit, MSO,
acetyl-K, F, and F+; and/or wherein D is substituted for E at
position 15.
[0046] Also preferred are analogs wherein position 7 alone has been
altered (section (e)). Preferred substitutions are those wherein
the amino acid substituted for histidine at position 7 is selected
from the group consisting of H.sup.+, Y, Y.sup.+, F, F.sup.+, R,
R.sup.+, Orn, Orn.sup.+, M, M.sup.+, N-formyl-H, N-formyl-H.sup.+,
N-acetyl-H, N-acetyl-H.sup.+, N-isopropyl-H, N-isopropyl-H.sup.+,
N-acetyl-K; N-acetyl-K.sup.+, P and P.sup.+.
[0047] Also preferred are embodiments with a combination of only
two of the above-referenced classes of modified forms, in addition
to the following specific embodiments.
[0048] The following specific analogs are preferred:
[0049] (H.sup.+).sup.7-GLP-1(7-37);
[0050] (Y).sup.7-GLP-1(7-37);
[0051] (N-acetyl-H).sup.7-GLP-1(7-37);
[0052] (N-isopropyl-H).sup.7-GLP-1(7-37);
[0053] (A.sup.+).sup.8-GLP-1(7-37);
[0054] (E.sup.+).sup.9-GLP-1(7-37);
[0055] (D).sup.9-GLP-1(7-37);
[0056] (D.sup.+).sup.9-GLP-1(7-37);
[0057] (F.sup.+).sup.10-GLP-1(7-37);
[0058] (S).sup.22(R).sup.23(R).sup.24(Q).sup.26-GLP-1(7-37);
[0059] (S).sup.22(R).sup.23(R).sup.24(Q).sup.26-GLP-1(7-37);
[0060] Preferred forms of analogs with enhanced stability also have
only one, or at most two, amino acid modifications.
[0061] Preferred substitutions for the histidine at position 7
include the D-forms of acidic or neutral amino acids or the D-forms
of histidines. Preferred are P.sup.+, D.sup.+, E.sup.+, N.sup.+,
Q.sup.+, L.sup.+, V.sup.+, I.sup.+ and H.sup.+.
[0062] The histidine at position 7, or a replacement (D or L), can
also be N-alkylated (1-6C) or N-acylated (1-6C). Alkyl groups are
straight or branched chain (including cyclic) hydrocarbyl residues
of the indicated member of C. Acyl groups are of the formula
RCO-wherein R is alkyl. Preferred alkyl groups are t-propyl,
.alpha.-propyl and ethyl; preferred acyl are acetyl and propionyl.
Preferred residues which may be alkylated or acylated include P, D,
E, N, Q, V, L, I, K and H in either the D or L form.
[0063] Preferred substitutions for alanine at position 8 are the
D-forms of P, V, L, I and A; also preferred are the D-forms of D,
E, N, Q, K, T, S and H.
[0064] Some specific analogs show both enhanced insulin release
stimulating activity and enhanced stability.
[0065] A preferred group of GLP-1 analogs and derivatives for use
in the present invention is composed of molecules of the formula:
TABLE-US-00002 (SEQ ID NO:2) R.sub.1-X-Glu-Gly.sup.10-
Thr-Phe-Thr-Ser-Asp.sup.15-Val-Ser-Ser-Tyr-Leu.sup.20- Y
-Gly-Gln-Ala-Ala.sup.25-Lys- Z -Phe-Ile-Ala.sup.30-
Trp-Leu-Val-Lys-Gly.sup.35-Arg-R.sub.2
[0066] and the pharmaceutically-acceptable salts thereof, wherein:
R.sub.1 is selected from the group consisting of L-histidine,
D-histidine, desamino-histidine, 2-amino-histidine,
b-hydroxy-histidine, homohistidine, alpha-fluoromethyl-histidine,
and alpha-methyl-histidine; X is selected from the group consisting
of Ala, Gly, Val, Thr, Ile, and alpha-methyl-Ala; Y is selected
from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly; Z is
selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and
Gly, and R.sub.2 is selected from the group consisting of NH.sub.2,
and Gly-OH; provided that the compound has an isoelectric point in
the range from about 6.0 to about 9.0 and further providing that
when R.sub.1 is His, X is Ala, Y is Glu, and Z is Glu, R.sub.2 must
be NH.sub.2.
[0067] Numerous GLP-1 analogs and derivatives having an isoelectric
point in the range from about 6.0 to about 9.0 have been disclosed
and include, for example;
[0068] GLP-1(7-36)NH.sub.2
[0069] Gly.sup.8-GLP-1(7-36)NH.sub.2
[0070] Gln.sup.9-GLP-1(7-37)
[0071] D-Gln.sup.9-GLP-1(7-37)
[0072] acetyl-Lys.sup.9-GLP-1(7-37)
[0073] Thr.sup.9-GLP-1(7-37)
[0074] D-Thr.sup.9-GLP-1(7-37)
[0075] Asn.sup.9-GLP-1(7-37)
[0076] D-Asn.sup.9-GLP-1(7-37)
[0077] Ser.sup.22-Arg.sup.23-Arg.sup.24-Gln.sup.26-GLP-1(7-37)
[0078] Thr.sup.16-Lys.sup.18-GLP-1(7-37)
[0079] Lys.sup.18-GLP-1(7-37)
[0080] Arg.sup.23-GLP-1(7-37)
[0081] Arg.sup.24-GLP-1(7-37)
[0082] Another preferred group of active compounds for use in the
present invention is disclosed in WO 91/11457, (related to U.S.
Pat. No. 5,545,618) and includes GLP-1(7-34), GLP-1(7-35),
GLP-1(7-36), or GLP-1(7-37), or the amide form thereof, and
pharmaceutically-acceptable salts thereof, having at least one
modification including those shown below:
[0083] (a) substitution of glycine, serine, cysteine, threonine,
asparagine, glutamine, tyrosine, alanine, valine, isoleucine,
leucine, methionine, phenylalanine, arginine, or D-lysine for
lysine at position 26 and/or position 34; or substitution of
glycine, serine, cysteine, threonine, asparagine, glutamine,
tyrosine, alanine, valine, isoleucine, leucine, methionine,
phenylalanine, lysine, or a D-arginine for arginine at position
36;
[0084] (b) substitution of an oxidation-resistant amino acid for
tryptophan at position 31;
[0085] (c) substitution of at least one of: tyrosine for valine at
position 16; lysine for serine at position 18; aspartic acid for
glutamic acid at position 21; serine for glycine at position 22;
arginine for glutamine at position 23; arginine for alanine at
position 24; and glutamine for lysine at position 26; and
[0086] (d) substitution of at least one of: glycine, serine, or
cysteine for alanine at position 8; aspartic acid, glycine, serine,
cysteine, threonine, asparagine, glutamine, tyrosine, alanine,
valine, isoleucine, leucine, methionine, or phenylalanine for
glutamic acid at position 9; serine, cysteine, threonine,
asparagine, glutamine, tyrosine, alanine, valine, isoleucine,
leucine, methionine, or phenylalanine for glycine at position 10;
and glutamic acid for aspartic acid at position 15; and
[0087] (e) substitution of glycine, serine, cysteine, threonine,
asparagine, glutamine, tyrosine, alanine, valine, isoleucine,
leucine, methionine, or phenylalanine, or the D- or N-acylated or
alkylated form of histidine for histidine at position 7; wherein,
in the substitutions is (a), (b), (d), and (e), the substituted
amino acids can optionally be in the D-form and the amino acids
substituted at position 7 can optionally be in the N-acylated or
N-alkylated form.
[0088] Because the enzyme, dipeptidyl-peptidase IV (DPP IV), may be
responsible for the observed rapid in vivo inactivation of
administered GLP-1, (Mentlein et al., 1993), administration of
GLP-1 analogs and derivatives that are protected from the activity
of DPP IV is preferred, and the administration of
Gly.sup.8-GLP-1(7-36)NH.sub.2, Val.sup.8-GLP-1(7-37)OH,
a-methyl-Ala.sup.8-GLP-1(7-36)NH.sub.2, and
Gly.sup.8-Gln.sup.21-GLP-1(7-37)OH, or pharmaceutically-acceptable
salts thereof, is more preferred.
[0089] The use in the present invention of a molecule claimed in
U.S. Pat. No. 5,188,666 ('666) is also preferred. Such a molecule
includes a peptide having one of the following amino acid
sequences. TABLE-US-00003 (SEQ ID NO:3)
NH.sub.2-His.sup.7-Ala-Glu-Gly.sup.10-
Thr-Phe-Thr-Ser-Asp.sup.15-Val-Ser-Ser-Tyr-Leu.sup.20-
Glu-Gly-Gln-Ala-Ala.sup.25-Lys-Glu-Phe-Ile-Ala.sup.30-
Trp-Leu-Val-X
[0090] wherein X may be Lys and Lys-Gly; or a derivative of said
peptide, and wherein said peptide may be a
pharmaceutically-acceptable acid addition salt of said peptide; a
pharmaceutically-acceptable carboxylate salt of said peptide; a
pharmaceutically-acceptable lower alkylester of said peptide; or a
pharmaceutically-acceptable amide of said peptide selected from the
group consisting of amide, lower alkyl amide, and lower dialkyl
amide.
[0091] The invention in '666 pertains to a peptide fragment which
is insulinotropic and is derivable from a naturally occurring amino
acid sequence.
[0092] The invention comprises a compound selected from the group
consisting of:
[0093] (A) a peptide comprising the sequence:
[0094]
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gl-
n-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-X wherein X is selected
form the group consisting of:
[0095] (a) Lys,
[0096] (b) Lys-Gly,
[0097] (c) Lys-Gly-Arg,
[0098] and (B) a derivative of the peptide; wherein the compound is
substantially free of natural contaminants, and has an
insulinotropic activity which exceeds the insulinotropic activity
of GLP-1 (1-36) or GLP-1 (1-37).
[0099] The invention also includes a compound selected from the
group consisting of:
[0100] (A) a peptide comprising the sequence: TABLE-US-00004
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
wherein X is selected form the group consisting of:
[0101] (a) Lys,
[0102] (b) Lys-Gly,
[0103] (c) Lys-Gly-Arg; [0104] and (B) a derivative of the peptide;
wherein the compound is substantially free of natural contaminants,
and has an insulinotropic activity at a concentration of at least
10.sup.-10 M,
[0105] Of particular interest are peptides of the following
formula: H.sub.2N--X--CO--R.sup.1 (1) wherein R.sup.1 is OH, OM, or
--NR.sup.2R.sup.3; [0106] M is a pharmaceutically acceptable cation
or a lower branched or unbranched alkyl group; [0107] R.sup.2 and
R.sup.3 are the same or different and selected from the croup
consisting of hydrogen and a lower branched or unbranched alkyl
group;
[0108] X is a peptide comprising the sequence: TABLE-US-00005
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
[0109] NH.sup.2 is the amine group of the amino terminus of X; and
CO is the carbonyl group of the carboxy terminus of X; [0110] (2)
the aced addition salts thereof; and [0111] (3) the protected or
partially protected derivatives thereof; wherein said compound has
an insulinotropic activity which exceeds the insulinotropic
activity of GLP-1 (1-36) or GLP-1 (1-37).
[0112] Another preferred group of molecules for use in the present
invention consists of compounds claimed in U.S. Pat. No. 5,512,549
having the general formula: TABLE-US-00006 (SEQ ID NO:4)
R.sup.1-Ala-Glu-Gly.sup.10-
Thr-Phe-Thr-Ser-Asp.sup.15-Val-Ser-Ser-Tyr-Leu.sup.20-
Glu-Gly-Gln-Ala-Ala.sup.25-Xaa-Glu-Phe-Ile-Ala.sup.30-
Trp-Leu-Val-Lys-Gly.sup.35-Arg-R.sup.3 | R.sup.2
[0113] and pharmaceutically-acceptable salts thereof, wherein
R.sup.1 may be 4-imidazopropionyl, 4-imidazoacetyl, or 4-imidazo-a,
a dimethyl-acetyl; R.sup.2 may be C.sub.6-C.sub.10 unbranched acyl,
or absent; R.sup.3 may be Gly-OH or NH.sub.2; and, Xaa is Lys or
Arg.
[0114] More preferred compounds of SEQ ID NO:4 for use in the
present invention are those in which Xaa is Arg and R.sup.2 is a
C.sub.6-C.sub.10 unbranched acyl.
[0115] Highly preferred compounds of SEQ ID NO:4 for use in the
present invention are those in which Xaa is Arg, R.sup.2 is
C.sub.6-C.sub.10 unbranched acyl, and R.sup.3 is Gly-OH.
[0116] More highly preferred compounds of SEQ ID NO:4 for use in
the present invention are those in which Xaa is Arg, R.sup.2 is a
C.sub.6-C.sub.10 unbranched acyl, R.sup.3 is Gly-OH, and R.sup.1 is
4-imidazopropionyl.
[0117] The most preferred compound of SEQ ID NO:4 for use in the
present invention is that in which Xaa is Arg, R.sup.2 is C.sub.8
unbranched acyl, R.sup.3 is Gly-OH and R.sup.1 is
4-imidazopropionyl.
[0118] The use in the present invention of a molecule claimed in
U.S. Pat. No. 5,120,712 is highly preferred. Such a molecule
includes a peptide having the amino acid sequence: TABLE-US-00007
(SEQ ID NO:1) NH.sub.2-His.sup.7-Ala-Glu-Gly.sup.10-
Thr-Phe-Thr-Ser-Asp.sup.15-Val-Ser-Ser-Tyr-Leu.sup.20-
Glu-Gly-Gln-Ala-Ala.sup.25-Lys-Glu-Phe-Ile-Ala.sup.30-
Trp-Leu-Val-Lys-Gly.sup.35-Arg-Gly.sup.37-OH
[0119] and a derivative of said peptide, wherein said peptide may
be a pharmaceutically-acceptable acid addition salt of said
peptide; a pharmaceutically-acceptable carboxylate salt of said
peptide; a pharmaceutically-acceptable lower alkylester of said
peptide; or a pharmaceutically-acceptable amide of said peptide
wherein the amide may be an amide, lower alkyl amide, or lower
dialkyl amide.
[0120] The use of GLP-1(7-36) amide, or a
pharmaceutically-acceptable salt thereof, in the present invention
is most highly preferred. The amino acid sequence of GLP-1 (7-36)
amide is; TABLE-US-00008 (SEQ ID NO:5)
NH.sub.2-His.sup.7-Ala-Glu-Gly.sup.10-
Thr-Phe-Thr-Ser-Asp.sup.15-Val-Ser-Ser-Tyr-Leu.sup.20-
Glu-Gly-Gln-Ala-Ala.sup.25-Lys-Glu-Phe-Ile-Ala.sup.30-
Trp-Leu-Val-Lys-Gly.sup.35-Arg-NH.sub.2
[0121] The use of Val.sup.8-GLP-1(7-37)OH, or a
pharmaceutically-acceptable salt thereof, in the present invention
is most highly preferred. The amino acid sequence of
Val.sup.8-GLP-1(7-37)OH is: TABLE-US-00009 (SEQ ID NO:6)
NH.sub.2-His.sup.7-Ala-Glu-Gly.sup.10-
Thr-Phe-Thr-Ser-Asp.sup.15-Val-Ser-Ser-Tyr-Leu.sup.20-
Glu-Gly-Gln-Ala-Ala.sup.25-Lys-Glu-Phe-Ile-Ala.sup.30-
Trp-Leu-Val-Lys-Gly.sup.35-Arg-Gly.sup.37-OH
Preparation of the Compounds
[0122] Methods for preparing the active compounds used in the
present invention, namely, GLP-1, an GLP-1 analog, or a GLP-1
derivative, or any related compound including an active fragment
effecting weight loss when administered peripherally, are
well-known, and are described in U.S. Pat. Nos. 5,118,666,
5,120,712, and 5,523,549.
[0123] The amino acid portion of the active compound used in the
present invention, or a precursor thereto, is made by 1)
solid-phase synthetic chemistry; 2) purification of GLP molecules
from natural sources; 3) recombinant DNA technology or 4) a
combination of these methods.
[0124] Solid phase chemical synthesis of polypeptides is well known
in the art and may be found in general texts in the area such as
Dugas and Penney 1981; Merrifield 1962; Stewart and Young 1969.
[0125] For example, the amino acid portion may be synthesized by
solid-phase methodology utilizing a 430A peptide synthesizer
(PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster
City, Calif. 94404) and synthesis cycles supplied by PE-Applied
Biosystems. BOC-amino acids and other reagents are commercially
available from PE-Applied Biosystems and other chemical supply
houses. Sequential BOC chemistry using double couple protocols are
applied to the starting p-methyl benzhydryl amine resins for the
production of C-terminal carboxamides. For the production of
C-terminal acids, the corresponding PAM resin is used. Asn, Gln,
and Arg are coupled using preformed hydroxy benzotriazole esters.
The following side chain protecting groups may be used:
[0126] Arg, Tosyl
[0127] Asp, cyclohexyl
[0128] Glu, cyclohexyl
[0129] Ser, Benzyl
[0130] Thr, Benzyl
[0131] Tyr, 4-bromo carbobenzoxy
[0132] BOC deprotection may be accomplished with trifluoroacetic
acid in methylene chloride. Following completion of the synthesis
the peptides may be deprotected and cleaved from the resin with
anhydrous hydrogen fluoride (HF) containing 10%: meta-cresol.
Cleavage of the side chain protecting group(s) and of the peptide
from the resin as carried out at -5.degree. C. to 5.degree. C.,
preferably on ice for 60 minutes. After removal of the HF, the
peptide/resin is washed with ether, and the peptide extracted with
glacial acetic acid and lyophilized.
[0133] Techniques well-known to the ordinarily-skilled artisan in
recombinant DNA technology may be used to prepare the active
compound used in present invention. In fact, recombinant DNA
methods may be preferable because of higher yield. The basic steps
in recombinant production are:
[0134] a) isolating a natural DNA sequence encoding a GLP-1
molecule of the present invention or constructing a synthetic or
semi-synthetic DNA coding sequence for a GLP-1 molecule,
[0135] b) placing the coding sequence into an expression vector in
a manner suitable for expressing proteins either alone or as a
fusion proteins,
[0136] c) transforming an appropriate eukaryotic or prokaryotic
host cell with the expression vector,
[0137] d) culturing the transformed host cell under conditions that
will permit expression of a GLP-1 molecule, and
[0138] e) recovering and purifying the recombinantly produced GLP-1
molecule.
[0139] As previously stated, the coding sequences may be wholly
synthetic or the result of modifications to the larger, native
glucagon-encoding DNA. A DNA sequence that encodes preproglucagon
is presented in Lund et al. 1982 and may be used as starting
material in the semisynthetic production of the compounds of the
present invention by altering the native sequence to achieve the
desired results.
[0140] Synthetic genes, the in vitro or in vivo transcription and
translation of which results in the production of a GLP-1 molecule,
may be constructed by techniques well known in the art. Owing to
the natural degeneracy of the genetic code, the skilled artisan
will recognize that a sizable yet definite number of DNA sequences
may be constructed, all of which encode GLP-1 molecules of the
present invention.
[0141] The methodology of synthetic gene construction is well-known
in the art (Brown et al. 1979.) The DNA sequence is designed from
the desired amino acid sequence using the genetic code, which is
easily ascertained by the ordinarily-skilled biologist. Once
designed, the sequence itself may be generated using conventional
DNA synthesizing apparatus such as the Model 380A or 380B DNA
synthesizers (PE-Applied Biosystems, Inc., 850 Lincoln Center
Drive, Foster City, Calif. 94404).
[0142] To express the amino acid portion of a compound used in the
present invention, an engineered synthetic DNA sequence is inserted
in any one of many appropriate recombinant DNA expression vectors
through the use of appropriate restriction endonucleases (Maniatis
et al., 1989). Restriction endonuclease cleavage sites are
engineered into either end of the GLP-1 molecule-encoding DNA to
facilitate isolation from, and integration into, amplification and
expression vectors well-known in the art. The particular
endonucleases employed will be dictated by the restriction
endonuclease cleavage pattern of the parent expression vector
employed. Restriction sites are chosen to properly orient the
coding sequence with control sequences, thereby achieving proper
in-frame reading and expression of the protein of interest. The
coding sequence must be positioned to be in proper reading frame
with the promoter and ribosome binding site of the expression
vector, both of which are functional in the host cell in which the
protein is to be expressed.
[0143] To achieve efficient transcription of the synthetic gene, it
must be operably associated with a promoter-operator region.
Therefore, the promoter-operator region of the synthetic gene is
placed in the same sequential orientation with respect to the ATG
start codon of the synthetic gene.
[0144] A variety of expression vectors useful for transforming
prokaryotic and eukaryotic cells are well known in the art (Promega
Catalogue, 1992; Stratagene Catalogue, 1992). Also, U.S. Pat. No.
4,710,473 describes circular DNA plasmid transformation vectors
useful for expression of exogenous genes in E. coli at high levels.
These plasmids are useful as transformation vectors in recombinant
DNA procedures and
[0145] (a) confer on the plasmid the capacity for autonomous
replication in a host cell;
[0146] (b) control autonomous plasmid replication in relation to
the temperature at which host cell cultures are maintained;
[0147] (c) stabilize maintenance of the plasmid in host cell
populations;
[0148] (d) direct synthesis of a protein product indicative of
plasmid maintenance in a host cell population;
[0149] (e) provide in-series restriction endonuclease recognition
sites unique to the plasmid; and
[0150] (f) terminate mRNA transcription. These circular DNA
plasmids are useful as vectors in recombinant DNA procedures for
securing high levels of expression of exogenous genes.
[0151] Having constructed an expression vector for the amino acid
portion of a compound used in the present invention, the next step
is to place the vector into a suitable cell and thereby construct a
recombinant host cell useful for expressing the polypeptide.
Techniques for transforming cells with recombinant DNA vectors are
well known in the art and may be found in such general references
as Maniatis, et al. supra. Host cells made be constructed from
either eukaryotic or prokaryotic cells.
[0152] Prokaryotic host cells generally produce the protein at
higher rates and are easier to culture. Proteins expressed in
high-level bacterial expression systems characteristically
aggregate in granules or inclusion bodies, which contain high
levels of the overexpressed protein. Such protein aggregates
typically must be recovered, solubilized, denatured and refolded
using techniques well known in the art (Kreuger et al., 1990; U.S.
Pat. No. 4,923,967).
Preparation of GLP-1 Analogs and Derivatives
[0153] Alterations to a precursor GLP-1 or GLP-1 amino acid
sequence to produce a desired GLP-1 analog or GLP-1 derivative, or
active fragment thereof, are made by well-known methods: chemical
modification, enzymatic modification, or a combination of chemical
and enzymatic modifications. The techniques of classical solution
phase methods and semi-synthetic methods may also be useful for
preparing the GLP-1 molecules used in the present invention.
Methods for preparing the GLP-1 molecules of the present invention
are well known to an ordinarily skilled peptide chemist.
[0154] Addition of an acyl group to the epsilon amino group of
Lys.sup.34 may be accomplished using any one of a variety of
methods known in the art (Bioconjugate Chem. 1990: Hashimoto et
al., 1989),
[0155] For example, an N-hydroxy-succinimide ester of octanoic acid
can be added to the lysyl-epsilon amine using 50% acetonitrile in
borate buffer. The peptide can be acylated either before or after
the imidazolic group is added. Moreover, if the peptide is prepared
recombinantly, acylation prior to enzymatic cleavage is possible.
Also, the lysine in the GLP-1 derivative can be acylated as taught
in WO 96/29342.
[0156] The existence and preparation of a multitude of protected,
unprotected, and partially-protected, natural and unnatural,
functional analogs and derivatives of GLP-1 (7-36)amide and GLP-1
(7-37) molecules have been described (U.S. Pat. Nos. 5,120,712;
5,545,618 and 5,118,666; Orskov et al., 1989; WO 91/11457).
[0157] Optionally, the amino and carboxy terminal amino acid
residues of GLP-1 derivatives may be protected, or, optionally,
only one of the termini is protected. Reactions for the formation
and removal of such protecting groups are described in works known
to those of skill in the art including, for example, Protective
Groups in Organic Chemistry 1973; Green, 1981; Schroder and Lubke,
1965. Representative amino-protecting groups include, for example,
formyl, acetyl, isopropyl, butoxycarbonyl,
fluorenylmethoxycarbonyl, carbobenzyloxy, and the like.
Representative carboxy-protecting groups include, fox example,
benzyl ester, methyl ester, ethyl ester, t-butyl ester, p-nitro
phenyl ester, and the like.
[0158] Carboxy-terminal, lower-alkyl-ester, GLP-1 derivatives used
in the present invention are prepared by reacting the desired
(C.sub.1-C.sub.4) alkanol with the desired polypeptide in the
presence of a catalytic acid such as hydrochloric acid. Appropriate
conditions for such alkyl ester formation include a reaction
temperature of about 50.degree. C. and reaction time of about 1
hour to about 3 hours. Similarly, alkyl ester derivatives of the
Asp and/or Glu residues can be formed.
[0159] Preparation of a carboxamide derivative of a compound used
in the present invention is formed, for example, as described in
Stewart et al., 1984.
[0160] A pharmaceutically-acceptable salt form of GLP-1, of a GLP-1
analog, or of a GLP-1 derivative may be used in the present
invention. Acids commonly employed to form acid addition salts are
inorganic acids such as hydrochloric acid, hydrobromic acid,
hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and
organic acids such, as p-toluenesulfonic acid, methanesulfonic
acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid,
succinic acid, citric acid, benzoic acid, acetic acid, and the
like. Examples of such salts include the sulfate, pyrosulfate,
bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,
dihydrogenphosphate, metaphosphate, pyrophosphate, chloride,
bromide, iodide, acetate, propionate, decanoate, caprylate,
acrylate, formate, isobutyrate, caproate, heptanoate, propiolate,
oxalate, malonate, succinate, suberate, sebacate, fumarate,
maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate,
chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate,
methoxybenzoate, phthalate, sulfonate, xylenesulfonate,
phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,
gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,
propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,
mandelate, and the like. Preferred acid addition salts are those
formed with mineral acids such as hydrochloric acid and hydrobromic
acid, and, especially, hydrochloric acid.
[0161] Base addition salts include those derived from inorganic
bases, such as ammonium or alkali or alkaline earth metal
hydroxides, carbonates, bicarbonates, and the like. Such bases
useful in preparing the salts of this invention thus include sodium
hydroxide, potassium hydroxide, ammonium hydroxide, potassium
carbonate, and the like. The salt forms are particularly
preferred,
[0162] A GLP-1, GLP-1 analog, or GLP-1 derivative used in the
present invention may be formulated with one or more excipients
before use in the present invention. For example, the active
compound used in the present invention may be complexed with a
divalent metal cation by well-known methods. Such metal cations
include, for example, Zn.sup.++, Mn.sup.++, Fe.sup.++, Co.sup.++,
Cd.sup.++, Ni.sup.++, and the like.
COMPOSITIONS OF THE INVENTION
[0163] Optionally, the active compound used in the present
invention may be combined with a pharmaceutically-acceptable
buffer, and the pH adjusted to provide acceptable stability, and a
pH acceptable for parenteral administration.
[0164] Optionally, one or more pharmaceutically-acceptable
anti-microbial agents may be added. Meta-cresol and phenol are
preferred pharmaceutically-acceptable anti-microbial agents. One or
more pharmaceutically-acceptable salts may be added to adjust the
ionic strength or tonicity. One or more excipients may be added to
further adjust the isotonicity of the formulation. Glycerin is an
example of an isotonicity-adjusting excipient.
[0165] GLP-1 receptors and the signal transduction cascade
initiated by ligand binding to the GLP-1 receptor are described in
WO 96/25487; Thorens, 1992; Thorens et al., 1993; Widmann et al.,
1994. The GLP-1 receptor is a membrane protein with seven
transmembrane domains, coupled to heterotrimeric G-proteins that
link activation of the receptor by ligand binding to production of
intracellular secondary messengers, especially, cyclic adenosine
monophosphate (cAMP). cAMP, in turn, activates a specific protein
kinase, cAMP-dependent protein kinase (protein kinase A, PKA). This
enzyme phosphorylates a number of key response elements present in
the promoter region of certain genes. In pancreatic b-cells and
other neuroendocrine cells, phosphorylation of some specific
proteins of the regulated secretary pathway stimulates peptide
secretion by stimulating exocytosis of secretory granules.
[0166] Various compounds are known to stimulate secretion of
endogenous GLP-1. For example, exposure of STC-1 cells to certain
secretagogues, such as, the adenylate cyclase activator, forskolin,
or the protein kinase-C-stimulating agent,
12-O-tetradecanoylphobol-13-acetate (TPA), caused significant
increases in release of GLP-1 (Abello et al., 1994). The STC-1 cell
line originated from an intestinal tumor in transgenic mice
carrying insulin-promoting oncogenes, and STC-1 cells are known to
contain m-RNA transcripts of pro-glucagon, from which GLP-1 is
generated. Other compounds, such as, somatostatin, gastric
inhibitory polypeptide, glucose-dependent insulinotropic peptide,
bombesin, calcitonin gene-related peptide, gastrin-releasing
peptide, cholinergic agonists, the b-adrenergic agonist,
isoproterenol, and the muscarinic cholinergic agonist, bethanechol,
are similarly known to cause release of endogenous GLP-1
(Plaisancie et al., 1994; Orskov et al., 1986; Brubaker, 1991;
Buchan,
Administration of Compositions
[0167] Administration may be via any route known to be effective by
the physician of ordinary skill, except that parenteral
administration directly into the central nervous system is not a
route taught or claimed in this invention. Peripheral, parenteral
administration is preferred. Parenteral administration is commonly
understood in the medical literature as the injection of a dosage
form into the body by a sterile syringe or some other mechanical
device such as an infusion pump. For the purpose of this invention,
peripheral parenteral routes include intravenous, intramuscular,
subcutaneous, and intraperitoneal routes of administration.
Intravenous, intramuscular, and subcutaneous routes of
administration of the compounds used in the present invention are
more preferred. Intravenous and subcutaneous routes of
administration of the compounds used in the present invention are
yet more highly preferred. For parenteral administration, an active
compound used in the present invention preferably is combined with
distilled water at an appropriate pH.
[0168] Certain compounds used in the present invention to effect
weight-loss may also be amenable to administration by the oral,
rectal, nasal, or lower respiratory routes, which are
non-parenteral routes. Of the said non-parenteral routes, the lower
respiratory route is preferred for administration of peptides used
in the instant invention. Various formulations of peptide compounds
for administration by the lower respiratory tract are disclosed in
U.S. Pat. Nos. 5,284,656 and 5,364,838. Publication WO 96/19197
discloses aerosol formulations of various peptides suitable for
enhancing lower respiratory tract absorption of the compounds used
in the instant invention. The oral route of administration is
preferred for compounds used in the instant invention.
[0169] Additional pharmaceutical methods may be employed to control
the duration of action. Controlled release preparations may be
achieved by the use of polymers to complex or absorb the active
compound used in the present invention. Extended duration may be
obtained by selecting appropriate macromolecules, for example,
polyesters, polyamino acids, polyvinylpyrrolidone, ethylenevinyl
acetate, methylcellulose, carboxymethylcellulose, or protamine
sulfate, and by selecting the concentration of macromolecules, as
well as the methods of incorporation, in order to prolong release.
Another possible method to extend the duration of action by
controlled release preparations is to incorporate an active
compound used in the present invention into particles of a
polymeric material such as polyesters, polyamino acids, hydrogels,
poly (lactic acid) or ethylene vinylacetate copolymers.
Alternatively, instead of incorporating a compound into these
polymeric particles, it is possible to entrap a compound used in
the present invention in microcapsules prepared, for example, by
coacervation techniques or by interfacial polymerization, for
example, hydroxymethylcellulose or gelatin-microcapsules,
respectively, or in colloidal drug delivery systems, for example,
liposomes, albumin microspheres, microemulsions, nanoparticles, and
nanocapsules, or in macroemulsions. Such teachings are known to
those of skill in the art and disclosed, e.g. in Remington's
Pharmaceutical Sciences, 1980.
Dose
[0170] The dose of GLP-1, GLP-1 analog, or GLP-1 derivatives, or
active fragments effective in a particular subject to cause
weight-loss will depend on a number of factors, among which are
included the subject's sex, weight and age, the underlying causes
of obesity, the route of administration and bioavailability, the
persistence of the administered compound in the body, the
formulation, and the potency. Where administration is intermittent,
the dose per administration should also take into account the
interval between doses, and the bioavailability of the administered
compound. Where administration is continuous, a suitable dosage
rate is between 0.25 and 6 pmol/kg body weight/min, preferably from
about 0.5 to about 1.2 pmol/kg/min. It is within the skill of the
ordinary physician to titrate the dose and rate of administration
of compositions containing GLP-1, GLP-1 analogs, or GLP-1
derivatives, or active fragments thereof to achieve the desired
clinical result, that is weight loss.
[0171] "Pharmaceutically acceptable" means suitable for
administration to a human, that is, does not contain toxic
elements, undesirable contaminants or the like, and does not
interfere with the activity of the active compounds therein.
[0172] The present invention will be more readily understood by
reference to a specific example, which is provided to illustrate,
not to limit, the present invention.
EXAMPLE 1
[0173] Four patients having non-insulin dependent diabetes mellitus
(NIDDM) (3 male, 1 female; age: 60.2.+-.1, 8 years; starting BMI:
33.5.+-.1.4 kg/m.sup.2; starting body weight: 97.5.+-.6.5 kg;
starting waist/hip: 0.946.+-.0.036; starting HbA.sub.1c:
7.1.+-.0.3%; fasting blood glucose:. 7.2.+-.1.1 mM) received
continuous, subcutaneous infusions of GLP-1(7-36) amide for four
weeks. Solutions of GLP-1 were prepared by combining 100 nmol of
GLP-1(7-36) amide and 0.025 mL human albumin solution (20%), then
adjusting the pH to 4 using 5 molar acetic acid, and finally
bringing the volume to 1 mL using normal saline. The solution was
administered at a GLP-1 dose rate of 1.2 pmol/kg/minute. The
volumetric delivery rate of the Minimed pump (Minimed Europe,
Paris) used to administer the GLP-1 solution was 0.05-0.07 mL/h.
The subcutaneous site of administration was the abdomen.
[0174] This treatment with GLP-1 was compared with two weeks of
intensive insulin therapy prior to and after the GLP-1 infusion.
During the insulin treatment periods, insulin was administered
subcutaneously before each meal (see Table 1). During the GLP-1
infusion, no insulin was administered. During both the insulin
treatment periods, and the GLP-1 treatment period, the patients
adhered to a standard diabetic diet consisting of, on a caloric
basis, about 55% carbohydrate, 30% fat, and 15% protein. No
exercise regimen was followed. The patients were not hospitalized,
and remained out-patients throughout the entire trial period.
[0175] During GLP-1 treatment, the four patients lost an average of
3.5.+-.1.2 kg body weight, while they lost only 1.3.+-.0.6 kg
during the first two weeks of intensive insulin treatment, and
actually gained weight, on average, during the second two weeks of
intensive insulin treatment. All values are individual values, or
mean.+-.SEM (standard error of the mean). No data are available for
patient MP for the second insulin treatment period. TABLE-US-00010
TABLE 1 Insulin Treatment Regimes. The four values represent the
amount of insulin administered subcutaneously (IU) to each patient
just prior to four daily meals. The first insulin treatment
preceded, and the second insulin treatment followed 4 weeks of
GLP-1 treatment First Insulin Second Insulin Treatment Treatment
Patient (2 weeks) (2 weeks) VN 47; 39; 35; 53 21; 20; 28; 26 NW 12;
13; 11; 12 11; 10; 12; 12 HF 11; 10; 12; 56 11; 10; 12; 12 MP 20;
14; 34; 30 --
[0176] TABLE-US-00011 TABLE 2 Patient Weight and Weight Change.
GLP-1 (7-36) amide was administered by continuous subcutaneous
infusion for four weeks, immediately preceded and followed by two
weeks of intensive insulin therapy. Patient Weight (kg) Weight
Change (kg) First Second First Second Insulin GLP-1 Insulin Insulin
GLP-1 Insulin Patient Initial 2 weeks 4 weeks 2 weeks 2 weeks 4
weeks 2 weeks VN 101.5 99.0 92.0 95.0 -2.5 -7.0 3.0 NW 113.0 111.0
108.0 108.0 -2.0 -3.0 0.0 HF 94.0 93.5 91.5 91.5 -0.5 -2.0 0.0 MP
82.0 81.9 80.0 -- -0.1 -1.9 -- 97.5 .+-. 6.5 96.4 .+-. 6.0 92.9
.+-. 5.8 98.2 .+-. 5.0 -1.3 .+-. 0.6 -3.5 .+-. 1.2 +1.0 .+-.
1.0
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