U.S. patent application number 10/281379 was filed with the patent office on 2003-12-18 for use of creatine analogues and creatine kinase modulators for the prevention and treatment of glucose metabolic disorders.
This patent application is currently assigned to Avicena Group, Inc.. Invention is credited to Kaddurah-Daouk, Rima, Teicher, Beverly A..
Application Number | 20030232793 10/281379 |
Document ID | / |
Family ID | 24157358 |
Filed Date | 2003-12-18 |
United States Patent
Application |
20030232793 |
Kind Code |
A1 |
Kaddurah-Daouk, Rima ; et
al. |
December 18, 2003 |
Use of creatine analogues and creatine kinase modulators for the
prevention and treatment of glucose metabolic disorders
Abstract
The present invention relates to the use of creatine compounds
including cyclocreatine and creatine phosphate for treating or
preventing a metabolic disorder consisting of hyperglycemia,
insulin dependent diabetes mellitus, impaired glucose tolerance,
hyperinsulinemia, insulin insensitivity, diabetes related diseases
in a patient experiencing said disorder. The creatine compounds
which can be used in the present method include (1) analogues of
creatine which can act as substrates or substrate analogues for the
enzyme creatine kinase; (2) compounds which can act as activators
or inhibitors of creatine kinase; (3) compounds which can modulate
the creatine transporter (4) N-phosphocreatine analogues bearing
transferable or non-transferable moieties which mimic the
N-phosphoryl group. (5) compounds which modify the association of
creatine kinase with other cellular components.
Inventors: |
Kaddurah-Daouk, Rima;
(Belmont, MA) ; Teicher, Beverly A.; (Carmel,
IN) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Avicena Group, Inc.
|
Family ID: |
24157358 |
Appl. No.: |
10/281379 |
Filed: |
October 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10281379 |
Oct 25, 2002 |
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09539963 |
Mar 31, 2000 |
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09539963 |
Mar 31, 2000 |
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08914887 |
Aug 19, 1997 |
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6075031 |
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08914887 |
Aug 19, 1997 |
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08540894 |
Oct 11, 1995 |
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Current U.S.
Class: |
514/114 ;
514/553; 514/565; 514/575 |
Current CPC
Class: |
A61K 31/495 20130101;
A61P 3/08 20180101; A61K 31/197 20130101; A61K 31/397 20130101;
A61P 3/10 20180101; A61K 31/401 20130101; A61K 31/662 20130101;
A61K 31/675 20130101; A61K 31/663 20130101; A61P 3/00 20180101;
A61K 31/198 20130101; A61K 31/4168 20130101; A61K 31/661 20130101;
A61K 31/4172 20130101 |
Class at
Publication: |
514/114 ;
514/553; 514/575; 514/565 |
International
Class: |
A61K 031/66; A61K
031/198; A61K 031/185 |
Claims
What is claimed is:
1. A method of treating or preventing a glucose metabolic disorder
in a subject afflicted with said disorder, comprising administering
to the subject an amount of a creatine compound, or a
pharmaceutically acceptable salt thereof, effective to treat,
reduce, or prevent said disorder.
2. The method of claim 1 wherein said disorder is
hyperglycemia.
3. The method of claim 1 wherein said disorder is insulin dependent
diabetes mellitus.
4. The method of claim 1 wherein said disorder is impaired glucose
tolerance.
5. The method of claim 1 wherein said disorder is
hyperinsulinemia.
6. The method of claim 1 wherein said disorder is insulin
insensitivity.
7. The method of claim 1 wherein said disorder is diabetes related
diseases.
8. The method of claim 1 wherein the subject is a mammal.
9. The method of claim 8 wherein the subject is a human.
10. A method for treating a metabolic disorder selected from the
group consisting of hyperglycemia, insulin dependent diabetes
mellitus, impaired glucose tolerance, hyperinsulinemia, insulin
insensitivity, diabetes related diseases, in a subject afflicted
with said disorder, comprising administering to the subject a
therapeutic amount of a creatine analogue having the general
formula: 5and pharmaceutically acceptable salts thereof, wherein:
a) Y is selected from the group consisting of: --CO.sub.2H--NHOH,
--NO.sub.2, --SO.sub.3H, --C(.dbd.O)NHSO.sub.2J and
--P(.dbd.O)(OH)(OJ), wherein J is selected from the group
consisting of: hydrogen, C.sub.1-C.sub.6 straight chain alkyl,
C.sub.3-C.sub.6 branched alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.3-C.sub.6 branched alkenyl, and aryl; b) A is selected from
the group consisting of: C, CH, C.sub.1-C.sub.5alkyl,
C.sub.2-C.sub.5alkenyl, C.sub.2-C.sub.5alkynyl, and
C.sub.1-C.sub.5alkoyl chain, each having 0-2 substituents which are
selected independently from the group consisting of: 1) K, where K
is selected from the group consisting of: C.sub.1-C.sub.6 straight
alkyl, C.sub.2-C.sub.6 straight alkenyl, C.sub.1-C.sub.6 straight
alkoyl, C.sub.3-C.sub.6 branched alkyl, C.sub.3-C.sub.6 branched
alkenyl, and C.sub.4-C.sub.6 branched alkoyl, K having 0-2
substituents independently selected from the group consisting of:
rromo, chloro, epoxy and acetoxy; 2) an aryl group selected from
the group consisting of: a 1-2 ring carbocycle and a 1-2 ring
heterocycle, wherein the aryl group contains 0-2 substituents
independently selected from the group consisting of: --CH.sub.2L
and --COCH.sub.2L where L is independently selected from the group
consisting of: bromo, chloro, epoxy and acetoxy; and 3) --NH--M,
wherein M is selected from the group consisting of: hydrogen,
C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4 alkenyl, C.sub.1-C.sub.4
alkoyl, C.sub.3-C.sub.4 branched alkyl, C.sub.3-C.sub.4 branched
alkenyl, and C.sub.4 branched alkoyl; c) X is selected from the
group consisting of NR.sub.1, CHR.sub.1, CR.sub.1, O and S, wherein
R.sub.1 is selected from the group consisting of: 1) hydrogen; 2) K
where K is selected from the group consisting of: C.sub.1-C.sub.6
straight alkyl, C.sub.2-C.sub.6 straight alkenyl, C.sub.1-C.sub.6
straight alkoyl, C.sub.3-C.sub.6 branched alkyl, C.sub.3-C.sub.6
branched alkenyl, and C.sub.4-C.sub.6 branched alkoyl, K having O-2
substituents independently selected from the group consisting of:
bromo, chloro, epoxy and acetoxy; 3) an aryl group selected from
the group consisting of a 1-2 ring carbocycle and a 1-2 ring
heterocycle, wherein the aryl group contains 0-2 substituents
independently selected from the group consisting of: --CH.sub.2L
and --COCH.sub.2L where L is independently selected from the group
consisting of: bromo, chloro, epoxy and acetoxy; 4) a
C.sub.5-C.sub.9 a-amino-w-methyl-w-adenosylcarboxylic acid attached
via the w-methyl carbon; 5) 2 C.sub.5-C.sub.9
a-amino-w-aza-w-methyl-w-adenos- ylcarboxylic acid attached via the
w-methyl carbon; and 6) a C.sub.5-C.sub.9
a-amino-w-thia-w-methyl-w-adenosylcarboxylic acid attached via the
w-methyl carbon; d) Z.sub.1 and Z.sub.2 are chosen independently
from the group consisting of: .dbd.O, --NHR.sub.2,
--CH.sub.2R.sub.2, --NR.sub.2OH; wherein Z.sub.1 and Z.sub.2 may
not both be .dbd.O and wherein R.sub.2 is selected from the group
consisting of: 1) hydrogen; 2) K, where K is selected from the
group consisting of: C.sub.1-C.sub.6 straight alkyl;
C.sub.2-C.sub.6 straight alkenyl, C.sub.1-C.sub.6 straight alkoyl,
C.sub.3-C.sub.6 branched alkyl, C.sub.3-C.sub.6 branched alkenyl,
and C.sub.4-C.sub.6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of: bromo, chloro,
epoxy and acetoxy; 3) an aryl group selected from the group
consisting of a 1-2 ring carbocycle and a 1-2 ring heterocycle,
wherein the aryl group contains 0-2 substituents independently
selected from the group consisting of: --CH.sub.2L and
--COCH.sub.2L where L is independently selected from the group
consisting of: bromo, chloro, epoxy and acetoxy; 4) 2
C.sub.4-C.sub.8 a-amino-carboxylic acid attached via the w-carbon;
5) B, wherein B is selected from the group consisting of:
--CO.sub.2H--NHOH, 13 SO.sub.3H, --NO.sub.2, OP(.dbd.O)(OH)(OJ) and
--P(.dbd.O)(OH)(OJ), wherein J is selected from the group
consisting of: hydrogen, C.sub.1-C.sub.6 straight alkyl,
C.sub.3-C.sub.6 branched alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.3-C.sub.6 branched alkenyl, and aryl, wherein B is optionally
connected to the nitrogen via a linker selected from the group
consisting of: C.sub.1-C.sub.2 alkyl, C.sub.2 alkenyl, and
C.sub.1-C.sub.2 alkoyl; 6) --D--E, wherein D is selected from the
group consisting of: C.sub.1-C.sub.3 straight alkyl, C.sub.3
branched alkyl, C.sub.2-C.sub.3 straight alkenyl, C.sub.3 branched
alkenyl, C.sub.1-C.sub.3 straight alkoyl, aryl and aroyl; and E is
selected from the group consisting of: --(PO.sub.3).sub.nNMP, where
n is 0-2 and NMP is ribonucleotide monophosphate connected via the
5'-phosphate, 3'-phosphate or the aromatic ring of the base;
--[P(.dbd.O)(OCH.sub.3)(O)].sub.m--Q, where m is 0-3 and Q is a
ribonucleoside connected via the ribose or the aromatic ring of the
base; --[P(.dbd.O)(OH)(CH.sub.2)].sub.m--Q, where m is 0-3 and Q is
a ribonucleoside connected via the ribose or the aromatic ring of
the base; and an aryl group containing 0-3 substituents chosen
independently from the group consisting of: Cl, Br, epoxy, acetoxy,
--OG, --C(.dbd.O)G, and --CO.sub.2G, where G is independently
selected from the group consisting of: C.sub.1-C.sub.6 straight
alkyl, C.sub.2-C.sub.6 straight alkenyl, C.sub.1-C.sub.6 straight
alkoyl, C.sub.3-C.sub.6 branched alkyl, C.sub.3-C.sub.6 branched
alkenyl, C.sub.4-C.sub.6 branched alkoyl, wherein E may be attached
to any point to D, and if D is alkyl or alkenyl, D may be connected
at either or both ends by an amide linkage; and 7) --E, wherein E
is selected from the group consisting of --(PO.sub.3).sub.nNMP,
where n is 0-2 and NMP is a ribonucleotide monophosphate connected
via the 5'-phosphate, 3'-phosphate or the aromatic ring of the
base; --[P(.dbd.O)(OCH.sub.3)(O)].sub.m--Q, where m is 0-3 and Q is
a ribonucleoside connected via the ribose or the aromatic ring of
the base; --[P(.dbd.O)(OH)(CH.sub.2)].sub.m--Q, where m is 0-3 and
Q is a ribonucleoside connected via the ribose or the aromatic ring
of the base; and an aryl group containing 0-3 substituents chose
independently from the group consisting of: Cl, Br, epoxy, acetoxy,
--OG, --C(.dbd.O)G, and --CO.sub.2G, where G is independently
selected from the group consisting of: C.sub.1-C.sub.6 straight
alkyl, C.sub.2-C.sub.6 straight alkenyl, C.sub.1-C.sub.6 straight
alkoyl, C.sub.3-C.sub.6 branched alkyl, C.sub.3-C.sub.6 branched
alkenyl, C.sub.4-C.sub.6 branched alkoyl; and if E is aryl, E may
be connected by an amide linkage; e) if R.sub.1 and at least one
R.sub.2 group are present, R.sub.1 may be connected by a single or
double bond to an R.sub.2 group to form a cycle of 5 to 7 members;
f) if two R.sub.2 groups are present, they may be connected by a
single or a double bond to form a cycle of 4 to 7 members; and g)
if R.sub.1 is present and Z.sub.1 or Z.sub.2 is selected from the
group consisting of --NHR.sub.2, --CH.sub.2R.sub.2 and
--NR.sub.2OH, then R.sub.1 may be connected by a single or double
bond to the carbon or nitrogen of either Z.sub.1 or Z.sub.2 to form
a cycle of 4 to 7 members.
11. The method of claim 10 wherein the creatine compound is
administered in combination with insulin or a sulphonylurea
compound.
12. The method of claim 10 wherein the creatine compound is
cyclocreatine.
13. The method of claim 10 wherein the creatine compound is
creatine phosphate.
14. A method for treating a glucose metabolic disorder selected
from the group consisting of hyperglycemia, insulin dependent
diabetes mellitus, impaired glucose tolerance, hyperinsulinemia,
insulin insensitivity, diabetes related diseases in a subject
afflicted with said disorder comprising administering to the
subject an effective therapeutic amount of a substrate of creatine
kinase.
15. A method for treating a glucose metabolic disorder selected
from the group consisting of hyperglycemia, insulin dependent
diabetes mellitus, impaired glucose tolerance, hyperinsulinemia,
insulin insensitivity, diabetes related diseases in a subject
afflicted with said disorder comprising administering to the
subject an effective therapeutic amount of a creatine phosphate
analogue.
16. A process for designing analogues of cyclocreatine and creatine
phosphate effective for the treatment of diseases related to
glucose level regulation comprising utilizing creatine kinase
structural coordinates as a basis for said analogues and chemically
modifying said coordinates to achieve a pharmacologically active
analogue.
Description
RELATED APPLICATIONS
[0001] The present invention is a continuation-in-part of Ser. No.
08/540,894, filed Oct. 11, 1995, the entire disclosure of which is
incorporated herein by reference.
FIELD OF INVENTION
[0002] The present invention provides for new use for creatine
compounds (compounds which modulate one or more of the structural
or functional components of the creatine kinase/creatine phosphate
system) as therapeutic agents. More particularly, the present
invention provides a method of treating or preventing certain
metabolic disorders of human and animal metabolism, e.g.,
hyperglycemia, insulin dependent diabetes mellitus, impaired
glucose tolerance, insulin insensitivity, hyperinsulinemia and
related diseases secondary to diabetes.
BACKGROUND OF THE INVENTION
[0003] There are several metabolic diseases of human and animal
glucose metabolism, eg., hyperglycemia, insulin dependent diabetes
mellitus, impaired glucose tolerance, hyperinsulinemia, and insulin
insensitivity, such as in non-insulin dependent diabetes mellitus
(NIDDM). Hyperglycemia is a condition where the blood glucose level
is above the normal level in the fasting state, following ingestion
of a meal or during a glucose tolerance test. It can occur in NIDDM
as well as in obesity. Hyperglycemia can occur without a diagnosis
of NIDDM. This condition is called impaired glucose tolerance or
pre-diabetes. Impaired glucose tolerance occurs when the rate of
metabolic clearance of glucose from the blood is less than that
commonly occurring in the general population after a standard dose
of glucose has been orally or parenterally administered. It can
occur in NIDDM as well as obesity, pre-diabetes and gestational
diabetes. Hyperinsulinemia is defined as having a blood insulin
level that is above normal level in fasting state or following
ingestion of a meal. It can be associated with or causative of
hypertension or atherosclerosis. Insulin insensitivity, or insulin
resistance occurs when the insulin-dependent glucose clearance rate
is less than that commonly occurring in the general population
during diagnostic procedures.
[0004] A number of compounds have been tried to alleviate symptoms
associated with glucose metabolism disorders. For example,
guanidine, monoguanidine and biguanidine compounds have been shown
to produce hypoglycemia. Watanabe, C., J. Biol. Chem., 33: 253-265
(1918); Bischoff, F. et al., Guanidine Structures and Hypoglycemia,
81: 325-349 (1929). However these compounds were shown to be toxic.
Biguanide derivatives, e.g., phenformin and metformin, have been
used clinically as antidiabetic agents. Some members of this class
continue to be used today, while others have been withdrawn from
the market. Schafer, G., Diabetes Metabol. (Paris) 9:148-163
(1983). Gamma-guanidinobutyramide, also known as Tyformin, and its
salt derivative, Augmentin, were investigated as potential
anti-diabetic agents from the mid 1960's to mid 1970's. While
Augmentin produced hypoglycemia, it was reported to have major
undesirable side effects such as hypertension and circulatory
collapse. Malaisse, W. et al., Horm. Metab. Res., 1:258-265 (1969);
ibid, 3:76-81 (1971).
[0005] British patent 1,153,424 discloses the use of certain esters
and amides of guanidino-aliphatic acids in the treatment of
diabetes mellitus where hyperuremia is present. The patent does not
disclose that these compounds have an effect on hyperglycemia or
any other symptom or pathological state related to disease.
Canadian patent 891509 discloses the use of esters and amides of
guanidinoaliphatic acids were disclosed for treating hyperuremia
and hyperglycemia in diabetes mellitus.
[0006] British patents 1,195,199, and 1,195,200 disclose the use of
guanidino alkanoic acids or their amides or esters for the
treatment of hyperglycemia occurring in diabetes. A variety of
British patents (1,552,179/1,195,199/1,195,200/1,552,179) describe
the low potency of the guanidino alkanoic acid derivatives as
single agents but describe their use in combination with other
modalities.
[0007] Aynsley-Green and Alberti injected rats intravenously with
beta guanidino propionic acid, arginine, guanidine, 4
guanidinobutyramide and 4 guanidinobutyric acid. Arginine and beta
guanidino propionic acid stimulated insulin release but did not
affect glucose levels. Also the treatment of animals with large
amounts of beta gunidino propionic acid for several weeks was shown
not to affect glucose levels, Moerland, T. et al., Am. J. Physiol.,
257:C810-C816 (1989). Under different conditions beta gunidino
propionic acid was shown to have an effect as described later. The
two other compounds did stimulate insulin release but increased
glucose levels. Aynsley-Green, A. et al., Horm. Metab., 6:115-120
(1974).
[0008] It is an object of the present invention to provide methods
for treatment of metabolic diseases that relate to glucose level
regulation by administering to an afflicted individual an amount of
a compound or compounds which modulate one or more of the
structural or functional components of the creatine kinase/creatine
phosphate system sufficient to prevent, reduce or ameliorate the
symptoms of the disease. These compounds are collectively referred
to as "creatine compounds." The experiments described herein
demonstrate that the creatine kinase system is directly related to
control of blood glucose levels in animals. Creatine analogues are
shown herein to be effective hypoglycemic agents for treatment of
glucose metabolic diseases.
SUMMARY OF THE INVENTION
[0009] The present invention provides a method of treating or
preventing a glucose metabolic disorder using creatine, creatine
phosphate, or a compound or compounds which modulates one or more
of the structural or functional components of the creatine
kinase/creatine phosphate system. Disorders which may be treated
using the present invention include, for example, those selected
from the group consisting of hyperglycemia, insulin dependent
diabetes mellitus, impaired glucose tolerance, hyperinsulinemia and
diabetes related complications. The method of the invention
comprises administering to a subject afflicted with or susceptible
to said disorder an amount of a creatine compound (compounds which
modulate one or more of the structural or functional components of
the creatine kinase/creatine phosphate system) sufficient to
alleviate or prevent the symptoms of the disorder. The creatine
compound may be in the form of a pharmacologically acceptable salt
or combined with an adjuvant or other pharmaceutical agent
effective to treat or prevent the disease or condition.
[0010] Prior to the present invention, the creatine kinase system
had not been implicated in glucose metabolic disorders. The
substrates for the creatine kinase enzyme, i.e., creatine and
creatine phosphate, are both guanidino compounds. The present
inventors have discovered that the creatine kinase (CK) enzyme
modifies key events involved in glucose regulation by potentially
regulating energy (ATP) involved in the release of insulin or the
uptake of glucose in tissue. It is now possible to modify the CK
system and design compounds that can prevent or ameliorate these
diseases. The present invention demonstrates that at least two
creatine compounds, creatine phosphate and cyclocreatine, are
hypoglycemic agents. That is, these compounds cause glucose levels
to drop significantly in a subject.
[0011] As stated herein above, a variety of guanidino compounds
have been shown to act as hypoglycemic agents including the
compound beta guanidino propionic acid (see, for example, PCT
Publication Number WO 91/12799). The target for these compounds and
their mode of action is not fully understood. However, beta
guanidino propionic acid was shown not to affect glucose levels in
normal animals, but had an effect on glucose levels in a model for
non-insulin dependent diabetes mellitus. This compound has some
structural similarity to creatine, but does not form a part of this
invention. Compounds useful in the present invention are creatine
compounds which modulate the creatine kinase system.
[0012] The present invention also provides pharmaceutical
compositions containing creatine compounds in combination with a
pharmaceutically acceptable carrier. The present compositions may
be used in combination with effective amounts of standard
chemotherapeutic agents which act on regulating glucose levels,
such as insulin or sulphonlylureas, to prophylactically and/or
therapeutically treat a subject with a disease related to glucose
levels.
[0013] Packaged drugs for treating subjects having a disease
relating to glucose level regulation also are the subject of the
present invention. The packaged drugs include a container holding
the creatine compound, in combination with a pharmaceutically
acceptable carrier, along with instructions for administering the
same for the purpose of preventing, ameliorating, arresting or
eliminating a disease related to glucose level regulation.
[0014] By treatment is meant the amelioration of one or more
symptoms of, or total avoidance of, the metabolic disorder as
described herein. By prevention is meant the avoidance of a
currently recognized disease state, as described herein, in a
patient evidencing some or all of the glucose metabolic disorders
described above. The present compositions may be administered in a
sustained release formulation. By sustained release is meant a
formulation in which the drug becomes biologically available to the
patient at a measured rate over a prolonged period. Such
compositions are well known in the art.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 graphically illustrates the effect of selected
creatine compounds on glucose levels in rats: Panel (A): glucose
levels in control (unmanipulated animals); Panel(B): glucose levels
in cyclocreatine treated animals; Panel (C): glucose levels in
beta-guanidino propionic acid treated animals; and Panel (D):
glucose levels in creatine phosphate treated animals.
[0016] FIG. 2 graphically illustrates the effect of the selected
compounds on glucose levels in rats: Panel (A): control
(unmanipulated animals); Panel (B): cyclocreatine treated; Panel
(C): beta-guanidino propionic acid treated; Panel (D): creatine
phosphate treated animals.
[0017] FIG. 3 graphically illustrates the average effect of
selected creatine compounds on glucose levels in rats over time:
Panel (A): illustrates average glucose levels in cyclocreatine
treated animals as compared to the average of the control
(unmanipulated animals); Panel (B): illustrates average glucose
levels in beta-guanidino propionic acid treated animals as compared
to the average of the control (unmanipulated animals); Panel (C):
illustrates average glucose levels in creatine phosphate treated
animals as compared to the average of the control (unmanipulated
animals).
[0018] FIG. 4 graphically illustrates the effect of cyclocreatine
on glucose levels in rabbits infused with cyclocreatine. Rabbits or
HCMV infected rabbits were infused in a continuous intravenious
mode with cyclocreatine as outlined in Example 4. The cyclocreatine
solution was prepared in saline at 5 mg/ml, 10 mg/ml or 15 mg/ml
and was infused to deliver amounts of drug 375-1125 mg/Kg/day.
Cyclocreatine was infused daily for up to 7 days. Glucose levels
were determined using standard procedures (BioPure, Boston, Mass.)
on several days and up to the end of the cyclocreatine infusion.
Glucose levels were determined on several days and up to 7 days
post cyclocreatine infusion. Panel (A) illustrates the effect of
cyclocreatine infused at 15 mg/ml on blood glucose levels of normal
rabbits. Each bar represents glucose levels in separate animals.
Panel (B) illustrates the effect of cyclocreatine infused at 10
mg/ml on blood glucose levels in infected rabbits. Each bar
represents glucose levels in separate animals. Panel (C)
illustrates the effect of cyclocreatine infused at 5 mg/ml on blood
glucose levels of infected rabbits. Each line represents glucose
levels in separate animals.
[0019] FIG. 5 graphically illustrates the average effect of infused
cyclocreatine on glucose levels in rabbits. Animals infused with
saline or cyclocreatine at different concentrations were examined
for glucose levels for up to seven days post infusion. At each
concentration of cyclocreatine glucose levels were determined in
3-4 animals and average values were calculated and presented as
bars.
[0020] FIG. 6 graphically illustrate the average effect of
cyclocreatine over time on glucose levels in the Male Zuker lean
littermates (+/?) as compared to Day 0. Three animals per group
were treated with the compound in the feed as described in Example
5. Solid circles (.circle-solid.) are averages in untreated groups
while open circles (.largecircle.) are averages in treated
groups.
[0021] FIG. 7 graphically illustrates the average effect of
cyclocreatine over time on glucose levels in the Male Zuker
diabetic fatty (ZDF-fa/fa) rats as compared to Day 0. Three animals
per group were treated with the compound in the feed as described
in Example 5. Solid squares (.box-solid.) are averages in untreated
groups while open squares (.quadrature.) are averages in treated
groups.
[0022] FIG. 8 graphically illustrate the average effect of creatine
over time on glucose level in the Male Zuker lean littermates (+/?)
as compared to Day 0. Three animals per group were treated with the
compound in the feed as described in Example Five. Solid circles
(.circle-solid.) are averages in untreated groups while open
circles (.largecircle.) are averages in treated groups.
[0023] FIG. 9 graphically illustrates the average effect of
creatine over time on glucose levels in the Male Zuker diabetic
fatty (ZDF-fa/fa) rats as compared to Day 0. Three animals per
group were treated with the compound in the feed as described in
Example 5. Solid squares (.box-solid.) are averages in untreated
groups while open squares (.quadrature.) are averages in treated
groups.
[0024] FIG. 10 graphically illustrate the average effect of
cyclocreatine over time on insulin level in the Male Zuker lean
littermates (+/?) as compared to Day 0. Three animals per group
were treated with the compound in the feed as described in Example
5. Solid circles (.circle-solid.) are averages in untreated groups
while open circles (.largecircle.) are averages in treated
groups.
[0025] FIG. 11 graphically illustrates the average effect of
cyclocreatine over time on insulin levels in the Male Zuker
diabetic fatty (ZDF-fa/fa) rats as compared to Day 0. Three animals
per group were treated with the compound in the feed as described
in Example 5. Solid squares (.box-solid.) are averages in untreated
groups while open squares (.quadrature.) are averages in treated
groups.
[0026] FIG. 12 graphically illustrate the average effect of
creatine over time on insulin level in the Male Zuker lean
littermates (+/?) as compared to Day 0. Three animals per group
were treated with the compound in the feed as described in Example
5. Solid circles (.circle-solid.) are averages in untreated groups
while open circles (.largecircle.) are averages in treated
groups.
[0027] FIG. 13 graphically illustrates the average effect of
creatine over time on insulin levels in the Male Zuker diabetic
fatty (ZDF-fa/fa) rats as compared to Day 0. Three animals per
group were treated with the compound in the feed as described in
Example 5. Solid squares (.box-solid.) are averages in untreated
groups while open squares (.quadrature.) are averages in treated
groups.
[0028] FIG. 14 graphically illustrates three individual patients'
glucose levels upon treatment with cyclocreatine at a dose of 60
mg/Kg. Patients were treated with cyclocreatine via a 3 hour
continuous infusion in a one liter volume of saline using the
schedule of administration as described in Example 6.
[0029] FIG. 15 graphically illustrates three individual patients'
glucose levels upon treatment with cyclocreatine at a dose of 80
mg/Kg. Patients were treated with cyclocreatine via a 3 hour
continuous infusion in a one liter volume of saline using the
schedule of administration as described in Example 6.
[0030] FIG. 16 graphically illustrates glucose levels in a diabetic
cancer patient upon treatment with cyclocreatine at a dose of 10
mg/Kg. The patient was treated with cyclocreatine via a 3 hour
continuous infusion in a one liter volume of saline using the
schedule of administration as described in Example 6.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The method of the present invention generally comprises
administering to an individual afflicted with a disease or
susceptible to a disease involving glucose level regulation, an
amount of a compound or compounds which modulate one or more of the
structural or functional components of the creatine
kinase/phosphocreatine (CK/CrP) system sufficient to prevent,
reduce or ameliorate symptoms of the disease. Components of the
CK/CrP system which can be modulated include the enzyme creatine
kinase (CK), the substrates creatine, creatine phosphate, ADP, ATP,
and the transporter of creatine. As used herein, the term
"modulate" means to change, affect or interfere with the
functioning of the component in the CK/CrP enzyme system.
[0032] The CK/CrP is an energy generating system operative
predominantly in the brain, muscle, heart, retina, and the
pancreas. Wallimann et. al., Biochem. J., 281, 21-401 (1992). The
components of the system include the enzyme creatine kinase (CK),
the substrates creatine (Cr), creatine phosphate (CrP), ATP, ADP,
and the creatine trasporter. The enzyme reversibly catalyzes the
transfer of a phosphoryl group from CrP to ADP to generate ATP. It
is found to be localized at sites where rapid rate of ATP
replenishment is needed. Some of the functions associated with this
system include efficient regeneration of energy in the form of ATP
in cells with fluctuating and high energy demand, energy transport
to different parts of the cell, phosphoryl transfer activity, ion
transport regulation, and involvement in signal transduction
pathways.
[0033] The substrate creatine is a compound which is naturally
occurring and is found in mammalian brain, skeletal muscle, retina
and the heart. It's phosphorylated form CrP is also found in the
same organs and is the product of the CK reaction. Both compounds
can be easily synthesized and are believed to be non-toxic to man.
A series of creatine analogues have also been synthesized and used
as probes to study the active site of the enzyme. Kaddurah-Daouk et
al. (WO 92/08456 published May 29, 1992 and WO 90/09192, published
Aug. 23, 1990; U.S. Pat. No. 5,321,030; and U.S. Pat. No.
5,324,731, the entire disclosures of which are hereby incorporated
herein by reference) described methods for inhibiting growth,
transformation, or metastasis of mammalian cells using related
compounds. Examples of such compounds include cyclocreatine,
homocyclocreatine and beta guanidino propionic acid. These same
inventors have also demonstrated the efficacy of such compounds for
combating viral infections (U.S. Pat. No. 5,321,030). Elgebaly in
U.S. Pat. No. 5,091,404 discloses the use of cyclocreatine for
restoring functionality in muscle tissue. Cohn in PCT publication
No. W094/16687 describes a method for inhibiting the growth of
several tumors using creatine and related compounds. No prior work
has established a direct link between the creatine kinase system
and diseases related to glucose level regulation such as
hyperglycemia, insulin dependent or independent diabetes and
related diseases secondary to diabetes.
[0034] Compounds which are particularly effective for use in the
present invention include cyclocreatine, creatine phosphate and
analogues thereof which are described below. The term "creatine
compound" will be used herein to include Cr, CrP, cyclocreatine,
compounds which are structurally similar to Cr, CrP, and
cyclocreatine, and analogues of Cr, CrP, and cyclocreatine. The
term "creatine compound" also includes compounds which "mimic" the
activity of cyclocreatine and creatine phosphate or creatine
analogues i.e., compounds which modulate the creatine kinase
system. The term "mimics" is intended to include compounds which
may not be structurally similar to creatine but mimic the
therapeutic activity of the creatine analogues cyclocreatine and
creatine phosphate or structurally similar compounds. The term
creatine compounds will also include inhibitors of creatine kinase,
ie. compounds which inhibit the activity of the enzyme creatine
kinase, molecules that inhibit the creatine transporter or
molecules that inhibit the binding of the enzyme to other
structural proteins or enzymes or lipids. The term "modulators" of
the creatine kinase system" are compounds which modulate the
activity of the enzyme, or the activity of the transporter of
creatine, or the ability of the enzyme to associate with other
cellular components. These could be substrates for the enzyme and
they would have the ability to build in their phosphorylated state
intracellularly. These types of molecules are also included in our
term creatine compounds. The term creatine "analogue" is intended
to include compounds which are structurally similar to creatine
such as cyclocreatine and creatine phosphate, compounds which are
art-recognized as being analogues of creatine, and/or compounds
which share the same function as cyclocreatine and creatine
phosphate.
[0035] Creatine (also known as N-(aminoiminomethyl)-N-methyl
glycine; methylglycosamine or N-methyl-guanidino acetic acid) is a
well-known substance. See, The Merck Index, Eleventh Edition No.
2570 (1989). Creatine is phosphorylated chemically or enzymatically
to creatine kinase to generate creatine phosphate, which is also
well known (see, The Merck Index, No.7315). Both creatine and
creatine phosphate (phosphocreatine) can be extracted from animals
or tissue or synthesized chemically. Both are commercially
available.
[0036] Cyclocreatine is an essentially planar cyclic analogue of
creatine. Although cyclocreatine is structurally similar to
creatine, the two compounds are distinguishable both kinetically
and thermodynamically. Cyclocreatine is phosphlorylated efficiently
by the enzyme creatine kinase in the forward reaction, both in
vitro and in vivo. Rowley, G. L., J.AM. Chem.Soc., 93:5542-5551
(1971); McLaughlin, A. C. et. al., J. Biol. Chem., 247, 4382-4388
(1972). It represents a class of substrate analogues of creatine
kinase and which are believed to be active.
[0037] Examples of creatine analogues known or believed to modify
the creatine kinase/creatine phosphate system are listed in Tables
1 and 2. 123
[0038] Most of these compounds have been previously synthesized for
other purposes. Rowley et. al., J.Am.Chem.Soc., 93:5542-5551
(1971); Mclaughlin et. al., J.Biol.Chem., 247:4382-4388 (1972);
Nguyen, A. C. K., "Synthesis and enzyme studies using creatine
analogues", Thesis, Dept of Pharmaceutical Chemistry, Univ. Calif.,
San Francisco, (1983); Lowe et al., J. Biol. Chem., 225:3944-3951
(1980); Roberts et. al., J. Biol. Chem, 260:13502-13508 (1995);
Roberts et. al., Arch. biochem. Biophy., 220:563-571 (1983), and
Griffiths et. al., J.Biol. Chem., 251:2049-2054 (1976). The
contents of all of the aforementioned references are expressly
incorporated herein by reference. Further to the aforementioned
references, Kaddurah-Daouk et. al., (WO 92/08456; WO 90/09192; U.S.
Pat. No. 5,324,731; U.S. Pat. No. 5,321,030) also provide citations
for the synthesis of a plurality of creatine analogues. The
contents of all the aforementioned references and patents are
hereby incorporated herein by reference.
[0039] It is possible to modify the substances described below to
produce analogues which have enhanced characteristics, such as
greater specificity for the enzyme, enhanced solubility or
stability, enhanced cellular uptake, or better binding activity.
Salts of products may be exchanged to other salts using standard
protocols.
[0040] Bisubstrate analogues of creatine kinase and non
hydrolyizable substrate analogues of creatine phosphate (non
transferable moieties which mimic the N phosphoryl group of
creatine phosphate) can be designed readily and would be examples
of creatine kinase modulators. Creatine phosphate compounds can be
synthesized chemically or enzymatically. The chemical synthesis is
well known. Annesley, T. M., Walker, J. B., Biochem.Biophys.Res.
Commun., 74:185-190 (1977); Cramer, F., Scheiffele, E., Vollmar,
A., Chem.Ber., 95:1670-1682 (1962).
[0041] Creatine compounds which are particularly useful in this
invention include those encompassed by the following general
formula: 4
[0042] and pharmaceutically acceptable salts thereof, wherein:
[0043] a) Y is selected from the group consisting of:
--CO.sub.2H--NHOH, --NO.sub.2, --SO.sub.3H, --C(.dbd.O)NHSO.sub.2J
and --P(.dbd.O)(OH)(OJ), wherein J is selected from the group
consisting of: hydrogen, C.sub.1-C.sub.6 straight chain alkyl,
C.sub.3-C.sub.6 branched alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.3-C.sub.6 branched alkenyl, and aryl;
[0044] b) A is selected from the group consisting of: C, CH,
C.sub.1-C.sub.5alkyl, C.sub.2-C.sub.5alkenyl,
C.sub.2-C.sub.5alkynyl, and C.sub.1-C.sub.5alkoyl chain, each
having 0-2 substituents which are selected independently from the
group consisting of:
[0045] 1) K, where K is selected from the group consisting of:
C.sub.1-C.sub.6 straight alkyl, C.sub.2-C.sub.6 straight alkenyl,
C.sub.1-C.sub.6 straight alkoyl, C.sub.3-C.sub.6 branched alkyl,
C.sub.3-C.sub.6 branched alkenyl, and C.sub.4-C.sub.6 branched
alkoyl, K having 0-2 substituents independently selected from the
group consisting of: rromo, chloro, epoxy and acetoxy;
[0046] 2) an aryl group selected from the group consisting of: a
1-2 ring carbocycle and a 1-2 ring heterocycle, wherein the aryl
group contains 0-2 substituents independently selected from the
group consisting of: --CH.sub.2L and --COCH.sub.2L where L is
independently selected from the group consisting of: bromo, chloro,
epoxy and acetoxy; and
[0047] 3) --NH--M, wherein M is selected from the group consisting
of: hydrogen, C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4 alkenyl,
C.sub.1-C.sub.4 alkoyl, C.sub.3-C.sub.4 branched alkyl,
C.sub.3-C.sub.4 branched alkenyl, and C.sub.4 branched alkoyl;
[0048] c) X is selected from the group consisting of NR.sub.1,
CHR.sub.1, CR.sub.1, O and S, wherein R.sub.1 is selected from the
group consisting of:
[0049] 1) hydrogen;
[0050] 2) K where K is selected from the group consisting of:
C.sub.1-C.sub.6 straight alkyl, C.sub.2-C.sub.6 straight alkenyl,
C.sub.1-C.sub.6 straight alkoyl, C.sub.3-C.sub.6 branched alkyl,
C.sub.3-C.sub.6 branched alkenyl, and C.sub.4-C.sub.6 branched
alkoyl, K having O-2 substituents independently selected from the
group consisting of: bromo, chloro, epoxy and acetoxy;
[0051] 3) an aryl group selected from the group consisting of a 1-2
ring carbocycle and a 1-2 ring heterocycle, wherein the aryl group
contains 0-2 substituents independently selected from the group
consisting of: --CH.sub.2L and --COCH.sub.2L where L is
independently selected from the group consisting of: bromo, chloro,
epoxy and acetoxy;
[0052] 4) a C.sub.5-C.sub.9 a-amino-w-methyl-w-adenosylcarboxylic
acid attached via the w-methyl carbon;
[0053] 5) 2 C.sub.5-C.sub.9
a-amino-w-aza-w-methyl-w-adenosylcarboxylic acid attached via the
w-methyl carbon; and
[0054] 6) a C.sub.5-C.sub.9
a-amino-w-thia-w-methyl-w-adenosylcarboxylic acid attached via the
w-methyl carbon;
[0055] d) Z.sub.1 and Z.sub.2 are chosen independently from the
group consisting of: .dbd.O, --NHR.sub.2, --CH.sub.2R.sub.2,
--NR.sub.2OH; wherein Z.sub.1 and Z.sub.2 may not both be .dbd.O
and wherein R.sub.2 is selected from the group consisting of:
[0056] 1) hydrogen;
[0057] 2) K, where K is selected from the group consisting of:
C.sub.1-C.sub.6 straight alkyl; C.sub.2-C.sub.6 straight alkenyl,
C.sub.1-C.sub.6 straight alkoyl, C.sub.3-C.sub.6 branched alkyl,
C.sub.3-C.sub.6 branched alkenyl, and C.sub.4-C.sub.6 branched
alkoyl, K having 0-2 substituents independently selected from the
group consisting of: bromo, chloro, epoxy and acetoxy;
[0058] 3) an aryl group selected from the group consisting of a 1-2
ring carbocycle and a 1-2 ring heterocycle, wherein the aryl group
contains 0-2 substituents independently selected from the group
consisting of: --CH.sub.2L and --COCH.sub.2L where L is
independently selected from the group consisting of: bromo, chloro,
epoxy and acetoxy;
[0059] 4) 2 C.sub.4-C.sub.8 a-amino-carboxylic acid attached via
the w-carbon;
[0060] 5) B, wherein B is selected from the group consisting of:
--CO.sub.2H--NHOH, --SO.sub.3H, --NO.sub.2, OP(.dbd.O)(OH)(OJ) and
--P(.dbd.O)(OH)(OJ), wherein J is selected from the group
consisting of: hydrogen, C.sub.1-C.sub.6 straight alkyl,
C.sub.3-C.sub.6 branched alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.3-C.sub.6 branched alkenyl, and aryl, wherein B is optionally
connected to the nitrogen via a linker selected from the group
consisting of: C.sub.1-C.sub.2 alkyl, C.sub.2 alkenyl, and
C.sub.1-C.sub.2 alkoyl;
[0061] 6) --D--E, wherein D is selected from the group consisting
of: C.sub.1-C.sub.3 straight alkyl, C.sub.3 branched alkyl,
C.sub.2-C.sub.3 straight alkenyl, C.sub.3 branched alkenyl,
C.sub.1-C.sub.3 straight alkoyl, aryl and aroyl; and E is selected
from the group consisting of: --(PO.sub.3).sub.nNMP, where n is 0-2
and NMP is ribonucleotide monophosphate connected via the
5'-phosphate, 3'-phosphate or the aromatic ring of the base;
--[P(.dbd.O)(OCH.sub.3)(O)].sub.m--Q, where m is 0-3 and Q is a
ribonucleoside connected via the ribose or the aromatic ring of the
base; --[P(.dbd.O)(OH)(CH.sub.2)].sub.m--Q, where m is 0-3 and Q is
a ribonucleoside connected via the ribose or the aromatic ring of
the base; and an aryl group containing 0-3 substituents chosen
independently from the group consisting of: Cl, Br, epoxy, acetoxy,
--OG, --C(.dbd.O)G, and --CO.sub.2G, where G is independently
selected from the group consisting of: C.sub.1-C.sub.6 straight
alkyl, C.sub.2-C.sub.6 straight alkenyl, C.sub.1-C.sub.6 straight
alkoyl, C.sub.3-C.sub.6 branched alkyl, C.sub.3-C.sub.6 branched
alkenyl, C.sub.4-C.sub.6 branched alkoyl, wherein E may be attached
to any point to D, and if D is alkyl or alkenyl, D may be connected
at either or both ends by an amide linkage; and
[0062] 7) --E, wherein E is selected from the group consisting of
--(PO.sub.3).sub.nNMP, where n is 0-2 and NMP is a ribonucleotide
monophosphate connected via the 5'-phosphate, 3'-phosphate or the
aromatic ring of the base; --[P(.dbd.O)(OCH.sub.3)(O)].sub.m--Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose
or the aromatic ring of the base;
--[P(.dbd.O)(OH)(CH.sub.2)].sub.m--Q, where m is 0-3 and Q is a
ribonucleoside connected via the ribose or the aromatic ring of the
base; and an aryl group containing 0-3 substituents chose
independently from the group consisting of: Cl, Br, epoxy, acetoxy,
--OG, --C(.dbd.O)G, and --CO.sub.2G, where G is independently
selected from the group consisting of: C.sub.1-C.sub.6 straight
alkyl, C.sub.2-C.sub.6 straight alkenyl, C.sub.1-C.sub.6 straight
alkoyl, C.sub.3-C.sub.6 branched alkyl, C.sub.3-C.sub.6 branched
alkenyl, C.sub.4-C.sub.6 branched alkoyl; and if E is aryl, E may
be connected by an amide linkage;
[0063] e) if R.sub.1 and at least one R.sub.2 group are present,
R.sub.1 may be connected by a single or double bond to an R.sub.2
group to form a cycle of 5 to 7 members;
[0064] f) if two R.sub.2 groups are present, they may be connected
by a single or a double bond to form a cycle of 4 to 7 members;
and
[0065] g) if R.sub.1 is present and Z.sub.1 or Z.sub.2 is selected
from the group consisting of --NHR.sub.2, --CH.sub.2R.sub.2 and
--NR.sub.2OH, then R.sub.1 may be connected by a single or double
bond to the carbon or nitrogen of either Z.sub.1 or Z.sub.2 to form
a cycle of 4 to 7 members.
[0066] Currently preferred compounds include cyclocreatine,
creatine phosphate and those included in Tables 1 and 2
hereinabove.
[0067] The modes of administration for these compounds include, but
are not limited to, oral, transdermal, or parenteral (e.g.,
subcutaneous, intramuscular, intravenous, bolus or continuous
infusion). The actual amount of drug needed will depend on factors
such as the size, age and severity of disease in the afflicted
individual. Creatine has been administered to athletes in the range
of 2-8 gms/day to improve muscle function. Creatine phosphate was
administered to patients with congestive heart failure also in the
range of several gm/day, and was very well tolerated. In
experimental animal models of cancer or viral infections, where
creatine compounds have been shown to be active, amounts of 1
gm/kg/day were administered intravenously or intraperitoneially.
For this invention the creatine compound will be administered at
dosages and for periods of time effective to reduce, ameliorate or
eliminate the symptoms of the disease. Dose regimens may be
adjusted for purposes of improving the therapeutic or prophylactic
response of the compound. For example, several divided doses may be
administered daily, one dose, or cyclic administration of the
compounds to achieve the desired therapeutic result. Agents that
improve the solubility of these compounds could also be added.
[0068] The creatine compounds can be formulated with one or more
adjuvants and/or pharmaceutically acceptable carriers according to
the selected route of administration. The addition of gelatin,
flavoring agents, or coating material can be used for oral
applications. For solutions or emulsions in general, carriers may
include aqueous or alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles can include sodium chloride, potassium chloride among
others. In addition, intravenous vehicles can include fluid and
nutrient replenishers, electrolyte replenishers among others.
[0069] Preservatives and other additives can also be present. For
example, antimicrobial, antioxidant, chelating agents, and inert
gases can be added (see, generally, Remington's Pharmaceutical
Sciences, 16th Edition, Mack, (1980)).
[0070] The present invention is demonstrated more fully by the
following examples, which are not intended to be limiting in any
way:
EXAMPLE 1
Effect of Creatine Compounds on Glucose Levels in Rats Bearing
Tumors
[0071] Two creatine compounds, creatine phosphate and
cyclocreatine, were injected intravenously into tumor bearing rats,
and the level of glucose in the rats was monitored. Beta guanidino
propionic acid, also was administered. This compound was previously
shown to have no effect on glucose levels in normal animals but was
shown to modify glucose levels in NIDDM models. There was no
specific reason for using tumor bearing rats, except convenience
because the antitumor activity of these compounds also was being
studied. The presence of the tumors should not have any effect on
the ability of these compounds to regulate glucose levels.
[0072] The rats carrying the tumors were described by us previously
(see, Teisher et al., Cancer Chemother. Pharmacol, 35: 411-416,
1995). The schedule and dose selected in these experiments was
based on prior experience working with this class of compounds as
anticancer or antiviral chemotherapeutic agents. The rat mammary
adenocarcinoma 13762 was implanted in the female Fisher 344 rats on
day zero. The creatine compounds were administered intravenously on
days 4-8 and days 14-18. The amounts used were 1 gm/kg of
cyclocreatine, 0.93 gm/kg for beta guanidino propionic acid, and
2.32 gm/kg for creatine phosphate. We were targeting a 1 gm/kg
molar equivalent of creatine to achieve mM levels known typically
to be needed with creatine analogues to modulate the creatine
kinase system intracellularly. Plasma glucose levels were measured
at around 11a.m., by taking a drop of blood from the animals and
testing glucose levels using a commercial kit (CHEMSTRIP bG,
Boehringer Mannheim). For animals that were treated with drugs, the
treatment was around 9a.m., and bleeding was also at around
11a.m.
[0073] FIG. 1 shows the results of our first experiment
graphically. Panel (A): glucose levels in control (unmanipulated
animals); Panel(B): glucose levels in cyclocreatine treated
animals; Panel (C): glucose levels in beta-guanidino propionic acid
treated animals; and Panel (D): glucose levels in creatine
phosphate treated animals. The controls showed an average glucose
level in rats of 62 mg/dl. The treatment with cyclocreatine showed
two drops in glucose levels at the time of drug administration,
i.e., between days 4-8 and days 14-18. The drop in glucose level at
the second cycle of drug administration was more dramatic than the
first cycle, consistent with what is known about the continuous
build up of these compounds in organs high in creatine kinase
activity. Minimal changes in glucose levels were seen with beta
guanidino propionic acid treatment consistent with previous
published data. The compound creatine phosphate induced similar
pattern of drops in glucose levels as that seen with cyclocreatine,
although cyclocreatine seemed to be more potent.
EXAMPLE 2
Effect of Creatine Compounds on Glucose Levels in Rats Bearing
Tumors
[0074] The same experiment described above was repeated. FIG. 2
shows the effect of the selected compounds on glucose levels. Panel
(A): control (unmanipulated animals); Panel (B): cyclocreatine
treated; Panel (C): beta-guanidino propionic acid treated; Panel
(D): creatine phosphate treated animals. The same pattern seen in
Example 1 is also seen here. Cyclocreatine induced a drop in the
level of glucose after each administration. The drop in the second
cycle was more dramatic than the first. Beta-guanidino propionic
acid had minimal effect, and creatine phosphate seemed to mirror
the effect of cyclocreatine.
EXAMPLE 3
Effect of Creatine Compounds on Glucose Levels in Rats Bearing
Tumors
[0075] To examine more closely what occurred in the above two
experiments, the average readings of glucose levels from
experiments one and two were taken in the following time intervals
post drug treatment: Days 2-3, Days 4-8, Days 8-12, Days 14-18, Day
15 and Days 19-22. Day 15 demonstrates the largest effect on
glucose levels by this class of compounds. FIG. 3 outlines these
results. Cyclocreatine, Panel (A), shows a drop in glucose level
that could be as high as 50% on day 15. Beta-guanidino propionic
acid, Panel (B), shows minimal effects <15%, and creatine
phosphate, Panel (C), seems to drop glucose levels by 35% on day
15.
[0076] The experiments described above demonstrate that creatine
analogues which modulate the creatine kinase system, and that are
represented by cyclocreatine and creatine phosphate, can regulate
glucose levels. The creatine kinase enzyme system creatine kinase
emerges as a novel target for drug design for diseases related to
the control of glucose levels.
EXAMPLE 4
Effect of Cyclocreatine on Glucose Levels in Rabbits
[0077] The creatine compound cyclocreatine, was given as a
continuous intravenous infusion (IV) to normal rabbits or rabbits
infected with the human cytomegalovirus (HCMV) in their eyes
(Rabbit Chorioretinal model). Glucose levels were recorded over a
period of seven days. This compound was tested in infected as well
as in normal animals due to the fact that these compounds were also
being evaluated as anti viral agents, a biological activity that
were reported in the U.S. Pat. No. 5,321,030. As will become clear
in the data presented here the eye infection had no effect on the
levels of glucose recorded. The schedule and dose selected in these
experiments was based on prior experience working with this class
of compounds as antiviral agents.
[0078] A total of 11 NZW rabbits weighing 1.75-2.0 Kg were used in
the experiments. All animals were infused with various doses of
cyclocreatine over a period of 1-7 days in a continuous infusion
mode. Continuous infusions were achieved by surgical implantation
of an indwelling catheter implant into the jugular vein by standard
surgical procedures. The catheter was threaded through a steel
sleeve and swivel apparatus attached to the back of the animal's
neck which was anchored to a specially fitted vest. A Harvard
Apparatus 2200 unifusion pump maintained drug delivery at a
constant rate through out the experiment. This arrangement allowed
the animal unimpaired movement within its cage. Animals received a
bolus injection of antibiotics immediately after surgery and daily
if needed. After animals recovered from the anesthesia, some
animals were inoculated by intravitreal injection of AD169 HCMV
(10.sup.5 pfu). The remaining animals were left uninfected. Both
infected and uninfected animals received a continuous infusion of
cyclocreatine or saline for up to seven days. Concentrations of
cyclocreatine were 5, 10, or 15 mg cyclocreatine/ml saline and
infusion rates and volumes were adjusted to achieve the desired
dose of 375-1125 mg/Kg/day. These concentrations were based on
amounts required to achieve other biological activities such as
antiviral or anticancer. Volumes did not exceed the animal's normal
daily intake of fluids (based upon the assumed water consumption of
roughly 100-150 ml/Kg/day; Harknes and Wagner, 1985). The rest of
the animals received a similar volume of sterile saline. On days
0,1,3,5 and 7 blood was withdrawn from the ear veins and glucose
levels were determined.
[0079] Blood glucose levels in these rabbits that were allowed to
freely feed ranged from 169-201 mg/dl. The average level determined
in this assay was around 177 mg/dl which is slightly higher than
that reported for rabbits in the fasting state. Table 3 summarizes
levels of glucose in treated and untreated animals over a period of
up to seven days.
1TABLE 3 Glucose Levels In Cyclocreatine Infused Rabbits Drug Conc.
(mg/ml) 0 5 5 5 ave 5 10 10 10 10 ave 10 15 15 15 ave 15 Infected
(HCMV) yes yes yes yes yes yes yes no no no 0 177 177 177 177 177
169 194 201 184 187 177 177 177 177 1 152 104 94 93 97 118 89 76 81
91 3 179 70 49 47 55 103 98 74 85 90 48 59 54 4 59 69 87 65 70 5 29
41 33 34 45 62 55 53 54 7 22 15 6 22 22 (10 ml/rabbit 20%
Dextrose)* 7 73 73
[0080] FIG. 4 illustrates graphically the effect of cyclocreatine
glucose levels on each treated animal, and FIG. 5 illustrates the
average effects on glucose levels seen in these animals. As shown
in Table 3 and FIGS. (4 and 5), animals that were uninfected and
treated with cyclocreatine at a dose of 15 mg/ml (1125 mg/Kg/day)
experienced a significant drop in their glucose levels. By day
three glucose levels were in the range of 48-59 mg/dl; by day five
they were 7-22 mg/dl and the animals became very lethargic. The
administration of 10 mls of a 20% solution of dextrose on day 7
brought back the level of glucose to 70 mg/dl and the animals
seemed to quickly recover and resumed normal activity and eating.
These data clearly suggest that cyclocreatine is a potent regulator
of blood glucose levels and that the creatine kinase system must be
involved in glucose metabolism and homeostasis. Lower doses of
cyclocreatine were tested in infected animals. At doses of 10 mg/ml
(750 mg/Kg/day) and 5 mg/ml (375 mg/Kg/day) the same observation
was noted, ie a significant drop in blood glucose levels (Table 3
and FIGS. 4 and 5). As early as day one drops in glucose levels
were noted with averages going down to the 90 mg/dl range and by
day five the range was in the 30-50 mg/dl. Some glucose levels in
the animals treated with 5 mg/ml cyclocreatine seem to have a lower
level than those treated with 10 mg/ml. We believe this is
experimental variation due to the complexity of the setting
requiring experiments to be done on separate days. What is very
clear from all of these experiments is that cyclocreatine has
definite and very reproducible effects on lowering blood glucose
levels in rabbits. Infections in the eye do not seem to have an
impact on blood glucose levels, as animals infected and infused
with saline experienced no drop in blood glucose Table 3. These
saline infused animals also illustrate that saline alone has no
effect on blood glucose levels.
EXAMPLE 5
The Effect of Cyclocreatine on Glucose Levels and Insulin in a
Diabetic Animal
[0081] This preliminary study was initiated to gain insight into
the potential regulation of glucose levels by creatine compounds in
ZDF rats, a widely studied rodent model of NIDDM (Peterson, Lessons
from Animal Diabetes, 1994, Pages 225-230). Male Zuker diabetic
fatty (ZDF-fa/fa) rats and their lean Zuker littermates (ZDF +/?)
were from Genetic Models, Inc., Indianapolis, Ind. This model shows
diabetic characteristics which appear to mimic human adult onset
diabetes. Hyperglycemia is initially manifested at about 7 weeks of
age and all obese rats are fully diabetic by 12 weeks of age (fed
blood glucose of greater than 500 mg %). This level of
hyperglycemia increases slightly for several weeks thereafter.
Between 7 and 10 weeks, blood insulin levels are high but these
subsequently drop as the pancreatic beta cells cease to respond to
the glucose stimulus. The lean (ZDF/Gmi) rats are the control
counterparts of the diabetic animals. These rats have the same
genetics as the obese animals except for the obesity trait. No
phenotypic differences have been observed between these rats and
other typical lean control rats. Hence these animals represent an
excellent control for the obese diabetic animals.
[0082] Male Zuker diabetic fatty (ZDF-fa/fa) rats and Zuker lean
littermates (ZDF +/?) were 12 weeks old when dosing with creatine
compounds was initiated. The ZDF-fa/fa rats were completely
diabetic. The littermates were the same age. The average weight and
food intake was 360 gm and 28 gm/day for the ZDF fatty rats and 300
gm and 20gm/day for their lean littermates. Animals were housed and
dosed 3 per cage. Untreated animals were fed Purina modified lab
chow 5001. The creatine compounds cyclocreatine and creatine were
given in the feed as 1% of the diet. The Purina rodent chow (5001)
was formulated to contain 1% creatine or 1% cyclocreatine.
Formulations were prepared by Purina Test Diets, Richmond, Ind.
Both treated and untreated animals feed ad libitum and had free
access to water. Animals were bled regularly throughout the
experiment and glucose and insulin levels were determined using
standard procedures (Linco RI-13K). FIGS. 6 and 7 illustrate the
average (n=3) effect of cyclocreatine over time on glucose levels
in the lean and fatty diabetic animals respectively. FIGS. 8 and 9
illustrate the average (n=3) effect of creatine over time on
glucose levels in the lean and fatty diabetic animals respectively.
FIGS. 10 and 11 illustrate the average (n=3) effect of
cyclocreatine over time on insulin levels in the lean and fatty
diabetic animals respectively. FIGS. 12 and 13 illustrate the
average (n=3) effect of creatine over time on insulin levels in the
lean and fatty diabetic animals respectively. Cyclocreatine as 1%
of the diet dropped the level of glucose in the lean rats by about
15% (FIG. 6). In the obese diabetic animals, glucose levels in the
untreated groups continued to rise by up to 40% (FIG. 7) while
those on cyclocreatine experienced a drop of close to 20%. This
illustrates that cyclocreatine is capable of regulating glucose
levels in the diabetic state. Creatine had minimal effect on
glucose levels in both the lean and the diabetic animals (FIGS.
8,9).
[0083] FIG. 10 illustrates the average effect of cyclocreatine on
insulin levels in lean animals which seem to drop significantly
over 50%. FIG. 11 illustrates the average effect on insulin levels
in obese fatty animals which seem to be minimally affected. FIG. 12
illustrates the average effect of creatine on insulin levels in
lean animals which seems to show a modest up regulation, and FIG.
13 illustrates the average effect of creatine on insulin levels in
obese fatty animals which also seem to be slightly elevated.
EXAMPLE 6
The Effect of Cyclocreatine on Glucose Levels in Cancer
Patients
[0084] Cyclocreatine was tested in humans in a phase I/II open
label dose escalation study. The patient population was terminal
cancer patients because cyclocreatine has demonstrated antitumor
activity when used as a single agent or in combination therapy.
Cyclocreatine was administered at doses that ranged from 10 mg/Kg
to 100 mg/Kg. The schedule of administration of cyclocreatine is
described in Table 4.
2TABLE 4 Table II: Clinical Schedule of Cyclocreatine Dose
Administration in Cancer Patients WEEK .vertline. DAY 1 2 3 4 5 6 7
.vertline. 8 9 10 11 12 13 14 DOSING X .vertline. X X WEEK
.vertline. DAY 15 16 17 18 19 20 21 .vertline. 22 23 24 25 26 27 28
DOSING X X X .vertline. X X X X WEEK .vertline. DAY 29 30 31 32 33
34 35 .vertline. 36 37 38 39 40 41 42 DOSING X X X X X .vertline.
WEEK .vertline. DAY 43 44 45 46 47 48 49 .vertline. 50 51 52 53 54
55 56 DOSING .vertline. X X X X X WEEK .vertline. DAY 57 58 59 60
61 62 63 .vertline. 64 65 66 67 68 69 70 DOSING .vertline.
[0085] Cohorts of 3 patients were administered drug at each dose
level, via a 3 hour continuous infusion in one liter volume of
saline. The first week patients received cyclocreatine once, the
second week patients received cyclocreatine twice, the third week
three times, the fourth week four times, the fifth week five times.
On weeks six and seven, no drug was administered to allow the drug
to wash out. On week eight, cyclocreatine was given five times.
Tile study included a total of 23 patients (18 male, 5 female) with
a median age of 71 years (range 54-85). The patients had different
types of malignancies. Eligibility requirements included patients
who have failed standard therapy or for whom no therapy was
available, normal organ function, have recovered from prior
therapies, probability of survival of greater than three months.
Reasons for exclusion included: major surgery, life threatening
concurrent illness and CNS metastasis. Blood samples were collected
at baseline and 1 day before and after the last weekly drug
administration on days 1, 7, 9, 14, 17, 21, 25, 28, 33, 40, 47, 49,
54, 61, and 69. Glucose levels were determined for these collected
blood samples. Significant hypoglycemia was noted at the highest
tested drug concentrations (2 out of 3 patients treated at the 80
mg/Kg level and 2 out of 7 at the 100 mg/Kg dose). These patients
became lethargic and hypoglycemic and required immediate
intervention to revert glucose levels. At the lower tested drug
concentrations there seemed to be a trend towards a drop in glucose
levels shortly after drug administration. Not all patients
experienced a significant drop in glucose although the trend was
there. FIGS. 14 and 15 illustrate graphically individual patients'
glucose levels upon treatment with 60 mg/Kg or 80 mg/Kg
cyclocreatine. Patient (A) at the 80 mg/Kg dose was diabetic and
had many serious complications due to his disease. Insulin was
withdrawn in the middle of the study due to these complications and
that resulted in marked increase in his glucose level. His glucose
did not seem to respond well to cyclocreatine. Tables 5-10 give the
raw data for glucose levels in individual patients. It should be
noted that an insufficient number of readings was made shortly
after drug administration. It is interesting to note that several
patients who were diabetic or had higher glucose levels than normal
did respond to cyclocreatine, onen example being illustrated in
FIG. 16.
3TABLE 5 Glucose (mg/dl) (Normal 70-150) Patient Dose (mg/Kg) 11 12
13 Ave Study Day 60 60 60 60 0 110 83 147 116 1 78 89 100 89 7 141
72 48 37 9 89 92 91 14 108 105 93 102 21 90 136 2 10 25 90 68 75 78
26 74 56 65 33 113 5 99 40 3 67 75 47 95 142 119 49 98 106 102 54
72 72 61 106 64 85 69 112 141 127
[0086]
4TABLE 6 Glucose (mg/dl) (Normal 70-150) Patient Dose (mg/Kg) 14 15
16 Ave Study Day 80 80 80 80 0 172 100 182 151 1 33 85 102 73 7 243
93 102 146 9 376 65 68 170 14 198 88 94 127 17 291 63 47 134 21 271
72 30 172 25 352 62 207 28 364 89 227 33 345 67 206 40 175 106 141
47 77 77 49 280 89 185 54 416 87 252 61 81 81 69 81 81
[0087]
5TABLE 7 Glucose (mg/dl) (Normal 70-150) Patient Dose (mg/Kg) 17 18
19 20 21 22 23 Ave Study Day 100 100 100 100 100 100 100 100 0 121
122 115 104 158 137 168 132 1 122 98 101 104 178 170 216 141 7 127
104 87 160 99 108 228 130 9 145 84 92 77 246 91 173 130 14 71 126
90 100 87 95 17 150 97 151 84 184 133 21 104 5 50 76 77 163 101 25
102 80 111 77 257 125 28 125 71 137 109 325 153 33 136 119 165 143
26 170 40 162 9 141 107 130 47 108 14 101 121 111 49 80 95 99 119
98 54 114 49 91 71 81 61 122 0 109 104 69 86 4 107 92
[0088]
6TABLE 8 Glucose (mg/dl) (Normal 70-150) Patient Dose (mg/Kg) 1 2 3
2,1 2,2 2,3 Ave Study Day 10 10 10 10 10 10 10 0 180 122 121 99 108
109 123 7 229 119 77 85 83 144 123 9 170 115 116 83 118 109 119 14
126 81 126 105 110 17 67 72 108 84 83 21 82 223 108 101 89 225 18
25 82 141 96 100 93 102 28 59 70 103 90 97 84 33 84 97 94 76 110 54
86 40 136 89 98 80 109 214 121 47 58 117 95 86 128 97 49 147 93 191
223 164 54 180 103 85 107 56 102 61 125 128 100 171 131 69 104 124
104 90 335 151
[0089]
7TABLE 9 Glucose (mg/dl) (Normal 70-150) Patient Dose (mg/Kg) 4 5 6
7 2,4 2,5 2,6 Ave Study Day 20 20 20 20 20 20 20 20 0 84 117 85 240
151 77 84 120 1 77 91 83 201 232 96 121 129 7 85 114 141 120 80 96
137 110 9 79 84 161 104 131 112 14 93 207 181 99 138 144 17 60 89
135 68 93 89 21 73 136 154 180 84 139 128 23 98 100 89 102 97 28 88
180 172 67 128 127 33 70 89 174 77 94 101 40 74 121 159 106 99 112
47 202 86 99 129 49 88 90 201 126 54 69 60 120 91 90 61 74 102 128
98 101 69 83 114 156 77 108
[0090]
8TABLE 10 Glucose (mg/dl) (Normal 70-150) Patient Dose (mg/Kg) 8 9
10 2,7 2,8 2,9 Ave Study Day 40 40 40 40 40 40 40 0 94 118 100 115
222 98 125 1 114 114 95 116 87 105 7 96 73 156 153 117 119 9 115
130 84 123 148 148 125 14 87 151 123 102 184 126 129 17 91 145 93
117 251 139 139 21 103 156 106 104 90 112 25 96 210 143 122 278 114
161 28 125 151 91 154 87 122 33 122 109 244 121 149 40 67 128 94
139 92 104 47 116 75 100 105 99 49 122 129 132 128 54 136 145 79
195 105 132 61 102 121 92 104 105 69 135 89 74 205 92 119
[0091] Equivalents
[0092] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *