U.S. patent application number 09/824217 was filed with the patent office on 2002-03-07 for guanylhydrazones and their use to treat inflammatory conditions.
Invention is credited to Bianchi, Marina, Cerami, Anthony, Tracey, Kevin J., Ulrich, Peter.
Application Number | 20020028851 09/824217 |
Document ID | / |
Family ID | 26880231 |
Filed Date | 2002-03-07 |
United States Patent
Application |
20020028851 |
Kind Code |
A1 |
Bianchi, Marina ; et
al. |
March 7, 2002 |
Guanylhydrazones and their use to treat inflammatory conditions
Abstract
This invention concerns new methods and compositions that are
useful in preventing and ameliorating cachexia, the clinical
syndrome of poor nutritional status and bodily wasting associated
with cancer and other chronic diseases. More particularly, the
invention relates to aromatic guanylhydrazone (more properly termed
amidinohydrazone) compositions and their use to inhibit the uptake
of arginine by macrophages and/or its conversion to urea. These
compositions and methods are also useful in preventing the
generation of nitric oxide (NO) by cells, and so to prevent
NO-mediated inflammation and other responses in persons in need of
same. In another embodiment, the compounds can be used to inhibit
arginine uptake in arginine-dependent tumors and infections.
Inventors: |
Bianchi, Marina; (Milan,
IT) ; Cerami, Anthony; (Shelter Island, NY) ;
Tracey, Kevin J.; (Old Greenwich, CT) ; Ulrich,
Peter; (Old Tappan, NJ) |
Correspondence
Address: |
Supervisor, Patent Prosecution Services
PIPER MARBURY RUDNICK & WOLFE LLP
1200 Nineteenth Street, N.W.
Washington
DC
20036-2412
US
|
Family ID: |
26880231 |
Appl. No.: |
09/824217 |
Filed: |
April 3, 2001 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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09824217 |
Apr 3, 2001 |
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08479050 |
Jun 6, 1995 |
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6248787 |
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08479050 |
Jun 6, 1995 |
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08463568 |
Jun 5, 1995 |
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5750573 |
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08463568 |
Jun 5, 1995 |
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08315170 |
Sep 29, 1994 |
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5599984 |
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08315170 |
Sep 29, 1994 |
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08184540 |
Jan 21, 1994 |
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Current U.S.
Class: |
514/632 ;
514/597; 564/226; 564/50 |
Current CPC
Class: |
A61P 9/00 20180101; A61K
31/60 20130101; A61K 31/167 20130101; A61K 31/621 20130101; A61K
31/175 20130101; A61P 7/00 20180101; Y10S 514/886 20130101; Y10S
514/903 20130101; C07C 275/40 20130101; A61K 31/155 20130101; A61K
31/17 20130101; Y10S 514/825 20130101; C07C 275/38 20130101; A61P
3/00 20180101; A61K 31/00 20130101; A61P 29/00 20180101; A61P 35/00
20180101; C07C 281/18 20130101; A61P 43/00 20180101; A61P 33/00
20180101; A61P 31/00 20180101; A61K 31/195 20130101; A61K 31/44
20130101; A61K 31/4164 20130101 |
Class at
Publication: |
514/632 ;
514/597; 564/50; 564/226 |
International
Class: |
A61K 031/17; A61K
031/155; C07C 275/40 |
Claims
What is claimed is:
1. A compound having the formula: 5wherein: X.sub.2=GhyCH--,
GhyCCH.sub.3-- or H--; X.sub.1, X'.sub.1 and X'.sub.2,
independently=GhyCH-- or GhyCCH.sub.3--; Z=--NH(CO)NH--,
--(C.sub.6H.sub.4)--, --(C.sub.5NH.sub.3)-- or
--A--(CH.sub.2).sub.n--A--- , n=2-10, which is unsubstituted, mono-
or di-C-methyl substituted, or a mono- or di- unsaturated
derivative thereof; and A=--NH(CO)--, --NH(CO)NH--, --NH-- or --O--
and salts thereof.
2. The compound of claim 1 wherein, when X.sub.2 is GhyCH-- or
GhyCCH.sub.3--, X.sub.2 is meta or para to X.sub.1 and X'.sub.2 is
meta or para to X'.sub.1.
3. The compound of claim 2 which is
N,N'-bis(3,5-diacetylphenyl)decanediam- ide
tetrakis(amidinohydrazone) tetrahydrochloride.
4. A compound having the formula: 6wherein: X.sub.1, X.sub.2 and
X.sub.3, independently=GhyCH-- or GhyCCH.sub.3--; X'.sub.1,
X'.sub.2 and X'.sub.3, independently=H, GhyCH-- or GhyCCH.sub.3--;
Z=(C.sub.6H.sub.3), when m.sub.1, m.sub.2, m.sub.3=0, or Z=N, when,
independently, m.sub.1, m.sub.2, m.sub.3=2-6; and A=--NH(CO)--,
--NH(CO)NH--, --NH-- or --O-- and salts thereof.
5. The compound of claim 4 wherein, when any of X'.sub.1, X'.sub.2
and X'.sub.3 are other than H, then the corresponding substituent
of the group consisting of X.sub.1, X.sub.2 and X.sub.3 is meta or
para to X'.sub.1, X'.sub.2 and X'.sub.3, respectively.
6. The compound of claim 4 wherein, m.sub.1, m.sub.2, m.sub.3=0 and
A=--NH(CO)--.
7. The compound of claim 4 wherein, m.sub.1, m.sub.2, m.sub.3=2-6
and A=--NH(CO)NH--.
8. A pharmaceutical composition comprising the compound of claim 1,
3 or 4 and a pharmaceutically acceptable carrier.
9. A method for inhibiting synthesis of urea from plasma arginine
in a subject which comprises administering an effective amount of
the compound of claim 8 to the subject in need of said
inhibition.
10. A method for ameliorating cachexia in a subject which comprises
administering an effective amount of the compound of claim 8 to the
subject in need of said amelioration.
11. A method for ameliorating the deleterious effects of nitric
oxide synthesis in a subject which comprises administering an
effective amount of the compound of claim 8 to the subject in need
of said amelioration.
12. A method for ameliorating the deleterious effects of cytokine
secretion in a subject which comprises administering an effective
amount of the compound of claim 8 to the subject in need of said
amelioration.
13. The method of claim 12 wherein the subject is in need of
amelioration of the deliterious effects of Tumor Necrosis Factor
secretion.
14. A method for treatment of endotoxic shock in a subject which
comprises administering an effective amount of the compound of
claim 8 to a subject in need of said treatment.
15. A method for treatment of neoplastic disease in a subject which
comprises administering an effective amount of the compound of
claim 8 to a subject having a neoplasm responsive to said
administration.
16. A method for inhibiting synthesis of nitric oxide from plasma
arginine in a subject which comprises administering an effective
amount of the compound of claim 8 to the subject in need of said
inhibition.
17. A method for ameliorating cachexia in a subject in need of said
amelioration which comprises blocking the uptake of arginine by
macrophages in the subject.
18. A method for ameliorating cachexia in a subject in need of said
amelioration which comprises blocking the activity of argininase in
macrophages in the subject.
19. A method of ameliorating cachexia in a subject which comprises
administering to the subject in need of said amelioration a
compound effective to inhibit synthesis of urea by macrophages.
20. A method for ameliorating the deleterious effects of nitric
oxide synthesis in a subject which comprises blocking the uptake of
arginine by blood and blood vessel cells of the subject.
Description
1. INTRODUCTION
[0001] This invention concerns new methods and compositions that
are useful in treating inflammatory conditions, e.g., preventing
and ameliorating cachexia, the clinical syndrome of poor
nutritional status and bodily wasting associated with cancer and
other chronic diseases. More particularly, the invention relates to
aromatic guanylhydrazone ("Ghy", more properly termed
amidinohydrazone, i.e., NH.sub.2 (CNH)--NH--N.dbd.) compositions
and their use to inhibit the uptake of arginine by macrophages
and/or its conversion to urea. These compositions and methods are
also useful in preventing the generation of nitric oxide (NO) and
the secretion of cytokines by macrophages and other cell types, and
so to prevent NO-mediated inflammation and other responses in
persons in need of same. In another embodiment, the compounds can
be used to inhibit arginine uptake in arginine-dependent tumors and
infections.
2. BACKGROUND OF THE INVENTION
[0002] Cachexia is a syndrome characterized by the wasting of
tissue mass in diseased animals, and is grossly reflected as a loss
of host weight. Cachexia is a progressive and often fatal
complication found in many different chronic disease states and its
consequences require that the goals of therapy should not be solely
to redress the underlying disease. The loss of protein stores, loss
of body weight and generally poor nutritional status of cachectic
patients can be independent sources of morbidity and mortality.
Also, the debilitation associated with cachexia is a significant
limitation on the patient's ability to tolerate aggressive medical
and surgical therapies which are directed to the primary
etiology.
2.1. The Signs of Cachexia Distinct from those of Starvation
[0003] Cachexia is a severe, often life-threatening complication
commonly encountered in association with a variety of insults:
cancer, chemotherapy, radiation injury, chronic infection, trauma
and surgical stress. Food intake insufficient to meet the total
energy needs of the host is a constant element of the cachectic
syndrome. In addition to this relative hypophagia which is a
defining feature of cachexia, anorexia is also frequently
encountered.
[0004] However, studies of the syndrome indicate that cachexia is
not simply due to a dietary intake of protein and carbohydrate
below the needs of the host. Cachexia differs from unstressed
caloric deprivation in that the pattern of wasting seen during
partial or complete starvation is associated with an initial whole
body lipid loss concurrent with a relative conservation of tissue
protein. By contrast, cachexia is characterized by the significant
loss of both lipid and protein from tissue reservoirs 2.2. The
Implications of the Differences between Cachexia and Starvation
[0005] The most commonly accepted general explanation for cachexia
is that the host's proteins are broken down in the tissues to
provide a source of amino acids. These amino acids in turn are
thought to be needed for the synthesis of glucose, albumin and host
defense proteins in the liver.
[0006] This well-accepted theory suggests that therapies directed
toward increasing the total intake of calories and proteins should
substantially ameliorate the cachectic syndrome. However, even as
drastic an intervention as total parenteral nutrition is not able
to effectively treat cachexia. (Brennan, 1986 NEJM 305:375, Detsky,
et al., 1987, ANN INTERN MED 107:195, McGeer, et al., 1989, ANN
INTERN MED 110: 734, Koretz, 1984, J CLIN ONCOL 2:534).
[0007] In addition to the failure of supplementary nutrition as a
therapeutic modality, the fundamental difference in the pattern of
the losses of lipid and protein between cachexia and starvation
also indicates that cachexia is neither the result solely of the
abnormally increased nutritional needs due to the underlying
disease nor of anorexia due to the disease's disruption of the
physiologic regulation of appetite. Rather the differences suggest
the presence of some fundamental changes in the host's metabolism
due, directly or indirectly, to the underlying disease.
[0008] Further supporting this conclusion has been an accumulation
of evidence implicating soluble host-produced regulatory and
effector proteins, known as cytokines, in the chain of events which
leads to cachexia. Experiments have been conducted in which the
blood circulation of a normal and a cachectic animal were joined.
In these experiments with so-called "parabiosed" animals, it was
observed that the otherwise normal animal rapidly developed
cachexia although the underlying disease remains entirely with the
original host. These and other observations strongly implicate
circulating mediators as the proximal cause of cachexia, i.e., this
catabolic condition is not the passive result of the excessive
metabolic demands imposed by the growth of the invading cells or
organisms, nor the simple result of a lesser food intake than that
required to meet metabolic demands.
[0009] One possibility as to the identity of these humoral factors
was that the soluble mediators produced in cachexia were the same
molecules as had been already identified as host
immune/inflammatory-related molecules (cytokines), and shown to be
secreted by lymphocytes and macrophages. The theory that these
cytokines were involved in cachexia was confirmed by the
observation that the administration of exogenous Tumor Necrosis
Factor (also known as cachectin, herein abbreviated "TNF") to test
animals mimicked many features of cachexia. (Darling et al., 1990,
CANCER RES 50:4008; Beutler & Cerami, 1988, ADV IMMUNOL
42:213). Further, anti-TNF antisera are able to ameliorate many,
but not all, the signs of cachexia in experimental tumor systems.
(Sherry, et al., 1989, FASEB J 3:1956; Langstein, et al., 1989,
SURG FORUM 15: 408).
2.3. The Impact of the Role of Cytokines in Cachexia on the Search
for Therapies
[0010] It should be clear that even if all aspects of the cachectic
syndrome were attributable to some single cytokine or,
alternatively, to the activity of some combination of several
cytokines, hormones and other humoral factors, such knowledge would
not in and of itself provide a cellular or biochemical mechanism to
explain the metabolic changes that underlie cachexia, nor lead
directly to an effective therapy. Ultimately, cytokines must
interact with target cells and induce metabolic or phenotypic
changes in their targets to be of physiological and
pathophysiological significance. Thus the general identification of
cytokine mediation and the specific implications of particular
cytokine mediators are only intermediate objectives in the
determination of precisely what cellular and/or systemic metabolic
changes occur to bring about the full cachectic picture.
[0011] The findings that implicate cytokines as mediators of the
various cachectic syndromes, combined with the widespread focus on
liver and peripheral muscle as major contributors to the metabolic
changes of cachexia prompted many to look at the effects of known
cytokines on liver and muscle cells and to search for new cytokines
having an effect on these tissues. To date, however, no known
cytokines, individually or in combination, have been shown to
directly mobilize amino acids from protein stores in in vitro
systems using cells typical of these presumed sites of protein
breakdown in vivo.
2.4. Nitric Oxide as a Mediator of Endotoxic Shock
[0012] Nitric oxide (NO), a molecule produced enzymatically from
L-arginine by nitric oxide synthase (NOS), is a mediator of both
physiological homeostasis and inflammatory cytotoxicity. Moncada,
S. & Higgs, A., 1993, THE NEW ENGLAND JC-JRNAL OF MEDICINE 329,
2001-2012; Nathan, C., 1992, FASEB J 6, 3051-3064. NO production
via the constitutive and inducible isoforms of NOS in endothelial
cells for instance, causes vasodilation and governs blood pressure
and tissue perfusion. Kilbourn, R. G., Jubran, A., Gross, S. S., et
al., 1990, BIOCHEM. BIOPHYS. RES. COMMUN. 172, 1132-1138. NO
production by an inducible NOS in activated macrophages, on the
other hand, confers cytotoxic, increases vascular permeability, and
enhances the release of TNF.alpha. and IL-1. Ding, A. H., Nathan,
C. F. & Stuehr, D. J. J Immunol. 141, 2407-2412 (1988); Kubes,
P. & Granger, D. N. Am. J. Physiol. 262, H611-H615 (1992); Van
Dervort, A. L., Yan, L., Madara, P. J., et al. J Immunol. 152,
4102-4109 (1994); Bouskela, E. & Rubanyi, G. M. SHOCK 1,
347-353 (1994); Hibbs, J. B., Taintor, R. R. & Vavrin, Z.
Science 235, 473-476 (1987); Granger, D. L., Hibbs, J. B., Perfect,
J. R. & Durack, D. T. J. Clin. Invest. 85, 264-273 (1990).
Insight into the diverse biological actions of NO has been
facilitated by compounds that interfere with NOS to inhibit
production of NO. Because the NOS isoforms are highly conserved,
however, the available NOS inhibitors have not been found to
discriminate significantly between the activities of constitutive
versus inducible NOS.
[0013] Previously available NOS inhibitors have had limited success
in improving survival from endotoxemia, in part because they
indiscriminately suppress endothelial-derived relaxing factor
(EDRF), Cobb, J. P., et al., 1992, J. EXP. MED. 176: 1175-1182;
Minnard, E. A., et al., 1994, ARCH SURG. 129:142-148; Billiar, T.
R., 1990, et al., J LEUKOCYTE BIOL. 48:565-569. Suppression of EDRF
during endotoxemia may impair survival by causing vasoconstriction
and a diminution of blood flow to critical vascular beds. Hertofore
there have been reported no compounds that inhibit
cytokine-inducible macrophage NO without also inhibiting
endothelial-derived NO.
3. SUMMARY OF THE INVENTION
[0014] The present invention relates to methods and compounds for
treating cachexia by inhibiting the production of urea, more
particularly the production of urea by macrophages and the
inhibition of the transport processes which mediate arginine
uptake, particularly by macrophages. The method of the invention
may also be used to limit or prevent the damage induced by
NO-mediated responses associated with stroke, shock, inflammation
and other NO-related conditions. Another object of the invention
relates to inhibiting arginine uptake in the treatment of tumors or
infections, where the tumor cells, the infected cells or the
infectious agent requires arginine. A further object of the
invention is the inhibition of the deleterious secretion of
cytokines, such as Tumor Necrosis Factor, by activated
macrophages.
[0015] The class of compounds useful for the purposes of the
invention includes but is not limited to aromatics substituted with
multiple guanylhydrazone (Ghy) moieties, more properly termed
amidinohydrazones. The synthesis and use of such compounds is
described. The invention further encompasses screening assays to
test additional compounds for the above-noted activities, and
pharmaceutical compositions useful in the practice of this method
of therapy.
[0016] Because of the close relationship between urea production
and the physiological synthesis of nitric oxide, the compounds of
this invention can also be effective in limiting the cellular
production of NO, particularly by macrophages.
[0017] While not limited to any theory of how or why the therapies
described and claimed herein operate, the invention is based, in
part, on the Applicants' development of the following model for
cachexia: in cachexia, activated macrophages deplete the host's
nitrogen pool by converting circulating arginine to nitrogenous end
products that are eliminated from the body, requiring protein
catabolism by the muscle and/or liver and other organs in order to
replace the lost serum arginine. Thus, activated macrophages create
a "nitrogen sink" that persistently drains nitrogen from the
systemic pool, forcing the body to compensate by catabolizing
tissue proteins to liberate amino acids as new sources of nitrogen.
The model is based on the Applicants' experiments, that, while
seeking to identify a factor released by activated macrophages
which caused other tissues to make urea, found that activated
macrophages themselves directly synthesize urea by breaking down
arginine.
[0018] Activated macrophages break down arginine in two ways: (a)
into urea and ornithine, or (b) into citrulline and nitric oxide
(FIG. 1). The urea and nitric oxide so generated remove nitrogen
from the whole body nitrogen pool since these metabolites cannot be
efficiently recycled for reuse. Therefore, in cachexia, activated
macrophages deplete the plasma of arginine. The body's mechanisms
to maintain arginine homeostasis in the plasma will then require
catabolism of protein from the muscle, liver and other organs to
liberate sources of nitrogen and arginine. The discovery that
arginine breakdown in the activated macrophage is a proximal cause
of the inappropriate mobilization of tissue protein stores allowed
for the development of the therapies described herein--i.e.,
interfering with arginine uptake by activated macrophages, and/or
the production of urea from arginine to stem the inexorable loss of
urea down the metabolic "nitrogen sink" and thus inhibit the
further catabolism of protein from the tissues.
[0019] It is a further object of invention to provide compounds and
methods of their use to inhibit the production of nitric oxide by
macrophages to overcome the limitation imposed by the absence of a
macrophage-specific inhibitor of nitric oxide synthetase. Without
limitation as to theory, we reasoned that advantage could be made
of the requirement for arginine uptake by macrophages but not
endothelial cells induced to produce NO. The compounds of the
present invention were found to inhibit the production of NO by
macrophages while having no inhibition on the production of
vaso-active (endothelial derived) NO. Thus, the compounds of the
invention may advantageously be used to counteract
macrophage-induced, NO-mediated effects which accompany, for
example, endotoxic and septic shock.
[0020] The foregoing is presented by way of illustration and not
limitation. While the invention was developed with the background
knowledge of the model for the physiology of cachexia, the
invention itself involves the use of arginine analogs,
arginomimetics, and other compounds having a non-metabolizable
guanylhydrazone group(s) to inhibit the macrophage production of
urea. A general class of such compounds are aromatics containing
guanylhydrazones. These compounds can be synthesized by the
reaction of acetylbenzenes and benzaldehydes with aminoguanidine
and acid at high temperature in aqueous ethanol. (Ulrich, et al.,
1982, DRUG DEVELOPMENT RESEARCH 2:219; and Ulrich & Cerami,
1984 MEDICINAL CHEMISTRY 27:35, which are hereby incorporated by
reference.) The invention is demonstrated by working examples
describing the synthesis of compounds used in accordance with the
invention (Section 6, infra); a whole cell assay to identify
compounds that inhibit urea output (Section 7, infra); a whole cell
assay to identify compounds that inhibit arginine uptake (Section
8, infra); an arginase inhibition assay (Section 9, infra); the
demonstration of efficacy of the invention in an animal model
system for cachexia (Section 10, infra); the demonstration of
efficacy of the invention to reduce inflammation in the animal
model using carrageenan-induced paw swelling (Section 11, infra);
the demonstration of in vivo efficacy of the invention in
preventing lipopolysaccharide-induced fatality (Section 13, infra)
without blocking the effects of endothelial-derived relaxing factor
(Section 12, infra) and of in vitro efficacy of the invention in
preventing the secretion of Tumor Necrosis Factor by RAW 264.7
cells stimulated by LPS and .gamma.-interferon (Section 14, infra).
The results of Sections 12-14 are indicative of efficacy in
preventing the morbidity and mortality associated with toxic shock
or sytemic inflammatory response syndrome. Further examples show
the efficacy of Compound No. 14 in reducing the severity of
infarction induced by occlusion of the middle cerebral artery of a
rat (Section 15, infra) and in reducing the growth of an
experimental neoplasm in a nude mouse model (Section 16,
infra).
4. BRIEF DESCRIPTION OF FIGURES
[0021] FIG. 1. Biochemistry of arginine degradation by inflammatory
cells and inter-organ substrate cycling.
[0022] FIG. 2. Urea production by resident macrophages.
[0023] FIG. 3. Urea production by macrophages is stimulated by LPS
and .gamma.-interferon. Control -.smallcircle.-; IFN.gamma. (25
U/ml) -.circle-solid.-; LPS (100 ng/ml) -.diamond.-; IFN.gamma. (25
U/ml)+LPS (100 ng/ml) -.diamond-solid.-.
[0024] FIG. 4. Dose-response relationship of the effect of
.gamma.-interferon and LPS on RAW 264.7 cell production of urea in
the presence and absence of complementary drugs. FIG. 4A. Various
doses of .gamma.-interferon with and without 100 ng/ml LPS; FIG.
4B. Various doses of LPS with and without 25 U/ml
.gamma.-interferon.
[0025] FIG. 5. Dependence of RAW 264.7 urea production on
extracellular arginine. Control -.circle-solid.-; IFN.gamma. (25
U/ml)+LPS (100 ng/ml) -.tangle-solidup.-.
[0026] FIG. 6. Arginine transport. Uptake of tetra-.sup.3H-arginine
by RAW 264.7 cells a various times after stimulation.
[0027] FIG. 7. Chemical structures of exemplary compounds of the
invention. FIG. 7A. Nos. 1-7; FIG. 7B. Nos. 8-13; FIG. 7C. Nos.
14-20; FIG. 7D. Nos. 21-24; FIG. 7E. Nos. 25-27; FIG. 7F. Nos.
28-30; FIG. 7G. Nos. 31-33; FIG. 7H. Nos. 34-36; FIG. 7I Nos.
37-40; FIG. 7J. Nos. 41-43.
[0028] FIG. 8. The dose dependency of the protective effects
Compound No. 14 on .lambda.-carrageenan induced mouse paw
swelling.
[0029] FIG. 9. Comparison of the effect on acetylcholine-induced
hypotension of Compound No. 14 and of the known nitric oxide
synthase inhibitor, L-N.sup.G-methyl-arginine.
[0030] FIG. 10. Effects of Compound No. 14 on LPS-induced
mortality.
[0031] FIG. 11. Effects of Compound No. 14 on the secretion of TNF
by LPS/.gamma.-interferon-stimulated RAW 264.7 cells.
[0032] FIG. 12. Western blot of medium from
LPS/.gamma.-IFN-stimulated RAW 264.7 cells showing the production
of TNF by cells treated with 0, 1, 5 and 25 .mu.M Compound No.
14.
[0033] FIG. 13. Comparison of the combined effects of Compound No.
14 and extracellular arginine on the production of NO and TNF by
LPS/.gamma.-IFN-stimulated RAW 264.7 cells. FIG. 13A. NO
production: Control -.circle-solid.-; 1 .mu.M No. 14-.gradient.-; 5
.mu.M No. 14-.tangle-soliddn.-; 25 .mu.M No. 14 -.quadrature.-.
FIG. 13B. TNF production: Control -.circle-solid.-; 5 .mu.M No.
14-.tangle-soliddn.- ; 25 .mu.M No. 14-.gradient.-;
[0034] FIG. 14. Effects of Compound No. 14 on the production of
cytokines by LPS/.gamma.-IFN-stimulated PBMC. FIG. 14A. Tumor
Necrosis Factor; FIG. 14B. IL-6; FIG. 14C. Macrophage Inflammatory
Protein-1.alpha. FIG. 14D. Macrophage Inflammatory
Protein-1.beta..
[0035] FIG. 15. Effects of Compound No. 14 on the transport of
arginine in resting, -.circle-solid.-, and stimulated RAW 264.7
cells, -.gradient.-.
[0036] FIG. 16. Effects of Compound No. 14 on NO output of RAW
264.7 cells stimulated by .gamma.-IFN/LPS for 8 hours and exposed
to Compound No. 14 for further 4 hours.
[0037] FIG. 17. The plasma concentrations of Compound No. 14 in
rats following a single intravenous injection. The graph represents
the time course of Compound No. 14's disappearance from the blood
(solid line), and the extrapolated distribution and elimination
phases (dashed lines), as determined by the method of residuals.
Each square point represents the average.+-.standard deviation for
three rats, and each circular point the corresponding calculated
distribution phase residual point.
5. DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention relates to methods and compounds for
treating cachexia by inhibiting the production of urea, more
particularly the production of urea by macrophages. The method of
the invention may also be used to limit or prevent the damage
induced by NO-mediated responses associated with stroke, shock,
inflammation and other NO-related conditions. To this end, the
present invention relates to the inhibition of macrophage
production of urea and NO, and more particularly, inhibition of
induced excessive production of urea and the inhibition of the
transport processes which mediate arginine uptake by macrophages.
The invention further includes the inhibition of the deleterious
secretion of cytokines, e.g. Tumor Necrosis Factor (TNF). An
alternate embodiment of the invention relates to the inhibition of
arginine uptake in the treatment of tumors or infections, where the
tumor cells or the infectious agent requires arginine.
[0039] The class of compounds useful for the invention includes
aromatics substituted with multiple guanylhydrazone moieties, more
properly termed amidinohydrazones. The synthesis and use of such
compounds is described. The invention further encompasses screening
assays to test additional compounds for such activity and
pharmaceutical compositions useful in the practice of this method
of therapy.
[0040] Historically, our initial studies were directed to the
discovery of an hypothesized macrophage-produced soluble mediator
which caused cultured hepatocytes to produce more urea. More
particularly, we undertook to find a mediator produced by
stimulated or activated macrophages such as are present in many
chronic disease states associated with cachexia. Control studies
performed during the initial attempts to isolate a cytokine which
caused increased hepatocyte urea production in vitro led to the
serendipitous discovery that the activated macrophages themselves
produce urea in substantial amounts. That this was relevant to in
vivo conditions was confirmed by experiments demonstrating enhanced
urea production by macrophages isolated from mice made cachectic by
a transplanted tumor (FIG. 2). These studies were further confirmed
by the demonstration that the murine macrophage line RAW 264.7
responds to the macrophage activators LPS and .gamma.IFN with
increased urea production which depends on the extracellular
availability of arginine (FIGS. 3-5). These studies led to the
assay for pharmacologic activity, which is described in detail
herein below. The compounds used in the instant invention were
identified based on this assay and used in animal model systems
which demonstrate the efficacy of the invention (Sections 10, 11,
12, 13, 15 and 16, infra).
[0041] The foregoing results indicate that the secretion of
cytokines by activated macrophages is but one aspect of the
cachectic process. We developed the following model and hypothesis
which is presented for explanatory purposes only and without
limitation of the invention to any particular mechanism or
scientific model. Equally important, in this model, are the direct
metabolic processes of activated macrophages. The data indicate
that in cachexia, activated macrophages make and secrete urea
abundantly. The magnitude of the nitrogen loss which can be
directly attributed to macrophages can be estimated from
experimental culture data. Such studies show a single activated
macrophage produces about 50 pg of urea/day. If one estimates that
the fraction of activated macrophages is about 10% of the whole
body immune cell population in a human being, then there is a total
of about 10.sup.11 activated macrophages. This population could
correspondingly account for the loss of about 5 grams of nitrogen
per day which translates roughly to 30 grams of protein per day.
This magnitude of loss represents a significant fraction of the
weight loss that is observed clinically over time in chronic
cachexia.
[0042] In accordance with the invention, inhibition of urea
production, particularly that induced in macrophages, will reverse
the process. Macrophage cellular metabolism differs from hepatic
metabolism regarding the enzymes available to complete the
so-called "urea cycle." In both tissues the ultimate step in urea
production is a hydrolytic cleavage by the enzyme arginase of the
amidino moiety of arginine to yield ornithine and urea (FIG. 1).
One salient difference between macrophage and hepatic urea
production is that macrophages lack substantial quantities of the
enzyme ornithine transcarbamoylase and hence they cannot
efficiently salvage the ornithine produced by arginase nor can they
directly use ammonium (NH.sub.4) to form urea but rather must rely
on an exogenous supply of arginine. Secondly, it appears that there
are two arginase enzymes, both of which catalyze the hydrolytic
release of urea, one found especially in macrophages and a second
found typically in hepatocytes of the liver.
[0043] These differences between macrophage and hepatic urea
production have two implications: first, macrophages will
selectively deplete arginine from the plasma. This circulating
arginine must ultimately be replaced by protein breakdown in other
tissues because the conversion of nitrogen to urea is essentially
irreversible, i.e., urea cannot be further metabolized for re-use.
Arginine itself is synthesized from .alpha.-keto glutarate and
ammonium by glutamate synthetase, glutamine synthetase and
carbamoyl phosphate synthetase. Secondly, given that macrophage
urea production depends upon arginine uptake while hepatic urea
synthesis does not, it may be possible to selectively block the
cachexia-associated nitrogen loss while leaving corresponding
hepatic functions relatively undisturbed.
[0044] In addition it may be advantageous to specifically block the
macrophage form of the arginase enzyme and not the liver form. In
vitro assays are described herein to detect the degree to which
test compounds specifically inhibit each of these metabolic
processes. These assays use the macrophage cell line RAW 264.7.
Twenty compounds, including 15 novel compounds were tested for
inhibition of macrophage urea production in an RAW 264.7 cell line
assay. Six of the compounds display an IC.sub.50 of about 10 .mu.M
or less and a further five compounds have been noted with an
IC.sub.50 of greater than 10 .mu.M but less than about 100 .mu.M
(see Section 7.2).
[0045] Alternative embodiments of the present invention encompass
any known means to inhibit macrophage urea production. Such methods
may include but are not limited to the use of recombinant DNA
methodologies. For example, a vector expressing an antisense
message complementary to the mRNA of the macrophage form of
arginase will be introduced into the macrophages of the cachectic
host. Alternatively, a ribozyme specific for the mRNA of the
macrophage form of arginase could be employed. Specific
introduction into macrophages of vectors appropriate to either will
be obtained by use of liposome carriers.
[0046] Another embodiment of the present invention involves
inhibiting nitric oxide (NO) production and particularly of the
enzyme NO-synthase. NO is produced by activated macrophages and
vascular endothelial cells among other cellular sources. NO has
been implicated as a causative pathological factor in a variety of
inflammatory conditions: particularly in circumstances of shock and
of ischemic necrosis (infarction) of the myocardium and of the
central nervous system. NO-synthase catalyzes the oxidation of
arginine to citrulline with an accompanying release of NO.
Compounds which inhibit arginine uptake will therefore be effective
suppressors of NO-synthase activity at the cellular level. Further,
compounds of the above noted classes may be specific inhibitors of
NO-synthase at the molecular level. The data shown herein
demonstrate that the compounds used in accordance with the
invention inhibit NO-production without inhibiting EDRF
activity.
[0047] In a still further embodiment, the invention may be used to
treat toxic shock, also known as systemic inflammatory response
syndrome (SIRS). The data described herein shows that the compounds
of the invention act by two independent pathways to prevent the
mortality and morbidity associated with SIRS: (a) by preventing
arginine uptake and, thereby, blocking the synthesis of NO by
activated macrophages; and (b) by blocking the secretion of
cytokines such as Tumor Necrosis Factor (TNF).
[0048] In yet another embodiment of the invention, inhibition of
arginine uptake may be used to treat tumors or infections in which
the tumor cells or infectious agent requires arginine. For example,
tumors with arginine requirements include but are not limited to
tumors of the breast, liver, lung and brain; whereas infectious
agents with arginine requirements include but are not limited to
Pneumocystis carinii, Trypanosoma brucei, T. congolense and T.
evansi.
[0049] Examples of inhibitors which could be used in accordance
with the invention include, but are not limited to, analogs of
arginine; more particularly to a class of arylene compounds
substituted with [(aminoiminomethyl)hydrazono]methyl moieties and
[2-(aminoiminomethyl)hyd- razono]ethyl moieties (hereinafter
collectively "guanylhydrazones"); most preferably diphenyl
compounds having 2, 3 or 4 guanylhydrazone moieties. These
inhibitory compounds have one or more non-hydrolyzable analogs of
the guanidino group of arginine.
[0050] Of guanylhydrazone compounds examined for activity in the
present invention, compounds having only a single guanylhydrazone
moiety were either inactive or required mM concentrations to
achieve a 50% reduction in urea output. Benzyl and diphenyl
compounds having 2, 3 or 4 guanylhydrazone moieties were active in
some cases at less than 10 .mu.M. In all cases tested the highly
active compounds inhibited not only urea production but also
inhibited the transport of arginine into the cell. A preferred
embodiment of the present invention are di, tri and tetra
guanylhydrazone substituted phenyl compounds having two phenyl
nuclei linked by an alkanediamide or two phenoxy nuclei linked by
an alkane. A second preferred embodiment are triacetylphenyl or
triformylphenyl tris(guanylhydrazones). Examples of such useful
compounds which were known include monoarylene bisguanylhydrazone,
e.g., 1,3-diacetylpyridine bis(guanylhydrazone) (2), Ulrich, 1982,
a monoarylene tris(guanylhydrazone), e.g., 1,3,5-triacetylbenzene
tris(guanylhydrazone) (1), Ulrich, 1984, and a bisarylene
bis(guanylhydrazone), e.g., 4,4'-diacetyldiphenylurea
bis(guanylhydrazone) (8), Korytnyk, W. et al., J. MEDICINAL
CHEMISTRY 21:507-13, 1978. These compounds inhibit macrophage urea
production in vitro at concentrations of between about 10 .mu.M and
about 50 .mu.M. Further novel compounds of the present invention
have inhibitory activity at five-fold lower concentrations. Such
compounds include a bisarylene tris(guanylhydrazone), e.g.,
3,5,4'-triacetyldiphenylurea tris(guanylhydrazone) (FIG. 17.9), a
bisarylene tetrakis (guanylhydrazone), e.g.,
N,N'-bis(3,5-diacetylphenyl)- decanediamide tetrakis
(guanylhydrazone) (FIG. 17.14) and
3,3'-(ethylenedioxy)dibenzaldehyde bis(guanylhydrazone) (FIG.
17.16).
[0051] Further contemplated within the scope of the invention are
tris arylene guanylhydrazono compounds in which each arylene group
bears 1 or 2 guanylhydrazonoalkyl substituents. Such compounds may
be synthesized using the methods taught herein.
5.1. Assays for Identifying Active Compounds
[0052] The following assays can be used to identify compounds that
are used in the invention. Moreover, the assays can be utilized to
determine the IC.sub.50 ((i.e., the concentration which achieves a
half-maximal inhibition of the parameter assayed) for each compound
tested. When used in vivo, the dose of each compound should be
formulated to achieve a range of circulating concentrations that
include the IC.sub.50 measured in vitro.
[0053] The assays are exemplary and not intended to limit the scope
of the method of the invention. Those of skill in the art will
appreciate that modifications can be made to the assay system to
develop equivalent assays that obtain the same result. In the
working examples described herein, the RAW 264.7 cell line was
used. However, the invention is not limited to the RAW 264.7 cell
line which could be replaced by any macrophage cell line or by
activated non-transformed macrophages in a primary culture.
5.1.1. Whole Cell Assay for Urea and Nitric Oxide Production
[0054] In general, the whole cell assay for urea production may be
conducted as follows: macrophages or endothelial cells are
activated (e.g., using factors including but not limited to
.gamma.IFN and LPS as described in the examples, infra) in the
presence of test inhibitors. After an appropriate time in culture,
e.g., approximately overnight up to 2 days, the culture supernatant
is analyzed for the presence of urea and nitric oxide. The
production of urea and nitric oxide by any cell line can be
measured by the same calorimetric assays used in clinical
laboratories to determine the serum concentrations of these same
compounds. The effects of various concentrations of inhibitor can
be determined by comparison with the supernatant of control
cultures which were not treated with the test inhibitor. A further
control to indentify toxicity at any inhibitory dose may be
included. An assay for the release of the intracellular enzyme
lactate dehydrogenase, as used in the examples described infra, or
an equivalent control, may be employed to such ends.
5.1.2. Whole Cell Assay for Arginine Uptake
[0055] Inhibition of arginine transmembrane concentrating activity
by the compounds of the present invention is measured using
carrier-free radiolabelled arginine. To this end, the cells are
cultured to allow them to adhere (e.g., for 2 hours to overnight)
and activated (e.g., using factors including but not limited to
.gamma.IFN and LPS as described in the working examples infra) in
the presence of the test inhibitor. After an appropriate incubation
time, carrier-free labeled arginine is added to the culture. After
a short time period (e.g., 5 minutes) the cells are washed with a
solution containing unlabeled arginine to displace any radiolabeled
arginine non-specifically bound to the cells in culture. The cells
are then lysed and the cell lysates analyzed for the presence of
radiolabeled arginine. The effects of the test compounds are
determined by comparison of incorporation of radiolabel into
treated cells versus the control cell cultures which were not
treated with the potential inhibitor.
[0056] In the embodiment described in the working example herein, a
test population of cells was cultured for about 3 hours so that the
cells became firmly adherent. Various concentrations of the
potential inhibitors were then added to parallel cultures. One hour
later the macrophage stimulators, e.g. including, but not limited
to LPS and .gamma.IFN, were added. Eighteen hours later the cells
were washed in a warm balanced salt solution supplemented with
glucose. Carrier-free radiolabelled arginine was added; after 5
minutes, active uptake of arginine was stopped by washing the cell
three times with buffer, chilled to 0.degree. C., containing 10 mM
unlabeled arginine to displace any externally bound label. The
contents of the washed cells are solubilized in 100 .mu.l of formic
acid and counted by standard techniques.
5.1.3. Cell Lysate Assay for Arginase Activity
[0057] The cell lysate assay for arginase activity involves
exposing a cell lysate to an arginase activation buffer in the
presence of the test compound. After an appropriate incubation
period, arginine is added and the enzyme activity of arginase is
determined e.g., by measuring the urea concentration in the sample.
Inhibitory activity of the test compound is determined by comparing
the results obtained to control samples which were not exposed to
the test compound. As demonstrated in the working examples, infra,
direct inhibition of arginase is determined by first preparing a
low speed supernatant of a cell lysate at a protein concentration
of between 0.5 and 4 mg/ml. The supernatant is mixed in a 1:4 ratio
with an activation buffer containing MnCl.sub.2 and albumin and
aliquots are incubated with various concentrations of the potential
inhibitory compound. After a 20 minute period of heat activation at
55.degree. C., the solution is made to 0.25 M arginine, then
incubated at 37.degree. C. for 20 minutes. TCA is added to remove
protein by centrifigation, then the urea concentration of the
supernatant determined by colorimetric assay based on
diacetylmonoxime.
5.2. Active Compounds
[0058] By use of the above-noted in vitro bio-assays, compounds
have been identified which are inhibitors of urea and nitric oxide
production and of arginine uptake. The results of these experiments
are summarized in Section 7.2. Of the twenty (20) compounds
examined six (6) are effective inhibitors at concentrations between
1 and 10 .mu.M: Compounds Nos. 1 , 9, 13, 14, 15, 16. A further six
(6) compounds were effective at concentrations of between 10 and
100 .mu.M: Compounds No. 2, 8, 11, 18, and 19. The compound which
was identified as the most active (Compound No. 14) was used in
vivo in animal models of cachexia, NO-mediated inflammation,
endotoxin-induced shock, cerebral infarction and neoplasia. In each
of these models Compound No. 14 proved to be effective (Sections
10, 11, 13, 15 and 16 infra.)
5.2.1. Compounds and their Synthesis
[0059] Hereinafter GhyCH-- .dbd.NH.sub.2(CNH)--NH--N.dbd.CH-- and
GhyCCH.sub.3-- .dbd.NH.sub.2(CNH)--NH--N.dbd.CCH.sub.3--. The
compounds of the invention include the following two major genera.
The first consists of compounds having the formula: 1
[0060] wherein
[0061] X.sub.2=GhyCH--, GhyCCH.sub.3-- or H--;
[0062] X.sub.1, X'.sub.1 and X'.sub.2 independently=GhyCH-- or
GhyCCH.sub.3--;
[0063] Z=--NH(CO)NH--, --(C.sub.6H.sub.4)--, --(C.sub.5NH.sub.3)--
or --A--(CH.sub.2).sub.n--A--, n=2-10, which is unsubstituted,
mono- or di-C-methyl substituted, or a mono or di- unsaturated
derivative thereof; and
[0064] A, independently, =--NH(CO)--, --NH(CO)NH--, --NH-- or --O--
and salts thereof. For ease of synthesis, a preferred embodiment
includes those compounds wherein A is a single functionality.
[0065] Also included within the invention are compounds having the
same formula wherein X.sub.1 and X.sub.2=H; X'.sub.1 and X'.sub.2
independently GhyCH-- or GhyCCH.sub.3--;
Z=--A--(CH.sub.2).sub.n--A--, n=3-8; and A=--NH(CO)-- or
--NH(CO)NH--, and salts thereof. Also included are compounds
wherein X.sub.1 and X.sub.2=H; X'.sub.1 and X'.sub.2
independently=GhyCH-- or GhyCCH.sub.3-- and
Z=--O--(CH.sub.2).sub.2--O--.
[0066] Further examples of genera of the invention include:
[0067] The genus wherein: X.sub.2=GhyCH--, GhyCCH.sub.3-- or H--;
X.sub.1, X'.sub.1 and X'.sub.2=GhyCH-- or GhyCCH.sub.3--; and
Z=--O--(CH.sub.2).sub.n--O--, n=2-10 and salts thereof; and the
related genus whrein, when X.sub.2 is other than H, X.sub.2 is meta
or para to X.sub.1 and wherein X'.sub.2 is meta or para to
X'.sub.1. A compound having the above formula wherein:
X.sub.2=GhyCH, GhyCCH.sub.3 or H; X.sub.1, X'.sub.1 and X'.sub.21,
=GhyCH-- or GhyCCH.sub.3--; and Z=--NH--(C.dbd.O)--NH-- and salts
thereof; and the related genus whrein, when X.sub.2 is other than
H, X.sub.2 is meta or para to X.sub.1 and wherein X'.sub.2 is meta
or para to X'.sub.1.
[0068] Also included are compounds having the formula: 2
[0069] wherein:
[0070] n=3-8; X.sub.2 and X'.sub.2=GhyCH--, GhyCCH.sub.3-- or H--;
X.sub.1 and X'.sub.1=GhyCH-- or GhyCCH.sub.3--; and salts thereof;
and the related genus wherein, when X.sub.2 or X'.sub.2 or both are
other than H, then X.sub.2 or X'.sub.2 are meta or para to X.sub.1
or X'.sub.1, respectively. Also included are compounds having the
formula: 3
[0071] wherein:
[0072] X.sub.2 and X'.sub.2=GhyCH--, GhyCCH.sub.3--or H--; X.sub.1
and X'.sub.1=GhyCH-- or GhyCCH.sub.3--; and n=2-10 and salts
thereof and the related genus wherein, when X.sub.2 or X'.sub.2 or
both are other than H, then X.sub.2 or X'.sub.2 are meta or para to
X.sub.1 or X'.sub.1, respectively.
[0073] The second major genus consists of compounds of the formula:
4
[0074] wherein,
[0075] X.sub.1, X.sub.2 and X.sub.3, independently=GhyCH-- or
GhyCCH.sub.3--;
[0076] X'.sub.1, X'.sub.2 and X'.sub.3 independently=H, GhyCH-- or
GhyCCH.sub.3--;
[0077] Z=(C.sub.6H.sub.3), when m.sub.l, m.sub.2, m.sub.3=0 or Z=N,
when, independently, m.sub.1, m.sub.2, m.sub.3=2-6; and
A=--NH(CO)--, --NH(CO)NH--, --NH-- or --O-- and salts thereof.
Further examples of genera of the invention include the genus
wherein when any of X'.sub.1, X'.sub.2 and X'.sub.3 are other than
H, then the corresponding substituent of the group consisting of
X.sub.1, X.sub.2 and X.sub.3 is meta or para to X'.sub.1, X'.sub.2
and X'.sub.3, respectively; the genus wherein, m.sub.1, m.sub.2,
m.sub.3=0 and
[0078] A=--NH(CO)--; and the genus wherein m.sub.1, m.sub.2,
m.sub.3=2-6 and
[0079] A=--NH(CO)NH--.
[0080] The compounds of the present invention can be synthesized by
means of two fundamental reactions. Those skilled in the art will
recognize that numerous variants may be synthesized by means of
these reactions and that these variants have properties in common
with the compounds herein disclosed.
[0081] Reaction 1 consists of the reaction of a substituted
aromatic having a primary or secondary amine, e.g.,
3,5-diacetylaniline, and a dioyl dichloride, e.g., glutaryl
dichloride, to yield the corresponding N,N'-diphenylalkanediamide.
"Reversed" diamides can also be prepared. Acetyl and
diacetylbenzoic acid can be prepared by the reaction of the
corresponding substituted toluenes and KMnO.sub.4. The acids may be
then activated bystandard techniques and reacted with the
appropriate .alpha.,.omega.-alkanediamines to yield the reverse
"diamides". Mixed forward and reversed diamides can be synthesized
by methods well known in the field of peptide synthesis. Thus, an
N-t-butyloxycarbonyl amino acid may be reacted with a substituted
aniline, followed by deprotection and reaction of the amino group
with an acitiviated substituted benzoic acid. When used herein the
symbol "--NH(CO)--", unless otherwise indicated, includes the
--(CO)NH-- isomer.
[0082] The method is not limited to dioyl dichlorides. The
trichloride derivatives of trioyl compounds may be used to
synthesize triphenyl alkanetriamides in a similar fashion. Suitable
triacids include cyclic acids, e.g., 1,3,5-cyclohexanetricarboxylic
acid (Aldrich Chem. Co.),
1,3,5-trimethyl,1,3,5-cyclohexanetricarboxylic acid (Kemp's
triacid, Kemp and Petrakis, 1981, J.Org.Chem 46:5140),
1,3,5-benzinetricarboxylic acid (Aldrich Chem. Co.) and linear
tricarboxylic acids such as 1,2,3-propanetricarboxylic acid (Sigma
Chem. Co.). The identical reaction may be performed wherein the
dioyl chloride is replaced by trichloromethyl chloroformate to
yield a diphenylurea condensation product. An alternative to
Reaction 1 can be performed to yield a 1,n-(n-alkanedioxy)
diarylene by reacting the 1,n-dibromoalkane, e.g., 1,2
dibromoethane and a monohydroxylarylene, e.g.,
3-hydroxyacetophenone.
[0083] Further embodiments of the invention include the use of
triamines of the form H.sub.2N-- (CH.sub.2).sub.n--NH--
(CH.sub.2).sub.q--NH.sub.2 wherein (n,q=2-6) and of the form Y--
((CH.sub.2).sub.n--NH.sub.2).sub.3 wherein Y may be one of N
(n=2-6), C(NO.sub.2) (n=3), a C-alkane (n=1), 1,3,5-adamantanetriyl
(n=3) or 1,3,5-benzinetriyl (n=1-3).
[0084] In two further embodiments of the invention, an acetyl- or
diacetylaryl isocyanate is reacted with an alkanediamine or,
alternatively, an acetyl- or diacetylaryl amine is reacted with an
alkanediyl diisocyanate to yield bis-ureido intermediates which may
be reacted with aminoguanidine to form the guanylhdrazono end
products. The requisite isocyanates are either commercially
available or may be synthesized from from the corresponding amines
by reaction with phosgene, trichloromethyl chloroformate, or
bis(trichloromethyl) carbonate in toluene or xylene at elevated
temperature.
[0085] Reaction 2 consists of the reaction of an acetophenone or
benzaldehyde type moiety and an aminoguanidine to yield the
condensation product wherein an imino-bonded (N.dbd.C)
aminoguanidine replaces the ketone or carbonyl moiety of the
arylene thus forming a guanylhydrazone and accompanied by the
release of a water molecule.
5.2.2. Pharmaceutical Formulations
[0086] Because of their pharmacological properties, the compounds
of the present invention can be used especially as agents to treat
patients suffering from cachexia, deleterious NO-mediated
responses, infarction, tumors or infections that require arginine.
Such a compound can be administered to a patient either by itself,
or in pharmaceutical compositions where it is mixed with suitable
carriers or excipient(s).
[0087] Use of pharmaceutically acceptable carriers to formulate the
compounds herein disclosed for the practice of the invention into
dosages suitable for systemic administration is within the scope of
the invention. With proper choice of carrier and suitable
manufacturing practice, the compositions of the present invention,
in particular, those formulated as solutions, may be administered
parenterally, such as by intravenous injection. The compounds can
be formulated readily using pharmaceutically acceptable carriers
well known in the art into dosages suitable for oral
administration. Such carriers enable the compounds of the invention
to be formulated as tablets, pills, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion by a
patient to be treated.
[0088] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
Determination of the effective amounts is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provided herein.
[0089] In addition to the active ingredients these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. The preparations formulated for oral
administration may be in the form of tablets, dragees, capsules, or
solutions.
[0090] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levitating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0091] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0092] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize
starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0093] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0094] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added.
5.3. Uses of the Compounds
[0095] For any compound used in the method of the invention, the
appropriate dose is one which achieves a circulating range of
concentrations which encompass the IC.sub.50 determined to be
effective for that compound as reported herein in Tables I, II and
III or determined in the manner herein described. For example, when
using Compound Nos. 1, 9, 13 or 15, for treating cachexia,
inflammation, endotoxic shock or septic shock, infarction or
neoplasm regardless of the formulation chosen, the amount
administered should be sufficient to achieve a serum concentration
or a circulating plasma concentration of between about 5.mu.M and
100.mu.M. When using Compound No. 14, for treating cachexia,
inflammation, endotoxic shock or septic shock, infarction or
neoplasm regardless of the formulation chosen, the amount
administered should be sufficient to achieve a serum concentration
or a circulating plasma concentration of between about 0.5.mu.M and
10.mu.M. As shown in the working examples, a daily parenteral dose
of Compound No. 14 of about 0.4 mg/Kg, used to treat cachexia, and
a single parenteral dose of 1.0 mg/Kg, to treat LPS-induced
toxicity, are effective in murine models.
[0096] Based on the pharmacokinetic constants reported in Section
18, below, and the time v. concentration curve of FIG. 17, it is
apparent that while single doses of between 0.4-1.0 mg/kg do not
achieve sustained plasma levels of Compound No. 14 in excess of 0.5
.mu.M, doses in this range do achieve peak plasma levels in excess
of about 0.5 .mu.M. Indeed, from the data presented here, it
appears that daily exposures of a subject to the indicated levels
for periods of 10-20 minutes or a single exposure of about an
hour's duration results in a therapeutically significant
effect.
[0097] Those skilled in the art will appreciate that the dose
appropriate to a given route of administration can be determined by
the application pharmacological methods that are well known to
those skilled in the art.
[0098] When the compounds of the present invention are used to
treat chronic inflammation a dose regime should be determined by
application of standard pharmacologic techniques using the
above-noted dose ranges as initial points. To treat acute
inflammatory conditions, a single larger dose may be administered
in an alternative embodiment. As shown in the working examples, a
single parenteral dose of 5.0 mg/Kg of Compound No. 14 was found
effective to treat such an acute event.
6. EXAMPLE
Synthesis of the Active Compounds
[0099] This section describes in detail the synthesis and
purification of useful intermediates and of exemplary compounds of
the present invention.
6.1. Synthesis of Intermediate Products
[0100] In the following it is understood that amidinohydrazone and
guanylhydrazone and (aminoiminomethyl)hydrazono are synonyms.
[0101] The following reactions are used to link substituted arylene
compounds by means of alkane chains of various lengths. The bond
may be an amide, a phenoxyalkane or a urea.
6.1.1. N,N'-bis(3,5-diacetylphenyl)-pentanediamide
[0102] 3,5-Diacetylaniline (531 mg) in dichloromethane (7 mL)
containing pyridine (0.4 mL) was treated with 0.141 mL glutaryl
dichloride. After 1 hr, filtration and washing with water gave 555
mg of N,N'-bis(3,5-diacetylphenyl)pentanediamide, mp 246-7.degree.
C.
[0103] Analogously, the following were prepared from
3,5-diacetylaniline and the corresponding dioyl dichlorides:
[0104] N,N'-bis(3,5-diacetylphenyl) butanediamide, mp 293-6.degree.
C.;
[0105] N,N'-bis(3,5-diacetylphenyl)hexanediamide, mp 269-70.degree.
C.;
[0106] N,N'-bis(3,5-diacetylphenyl)heptanediamide, mp 200-3.degree.
C.;
[0107] N,N'-bis(3,5-diacetylphenyl)octanediamide, mp 183-4.degree.
C.;
[0108] N,N'-bis(3,5-diacetylphenyl)nonanediamide, mp 179-80.degree.
C.;
[0109] N,N'-bis(3,5-diacetylphenyl)decanediamide, mp 196-9.degree.
C.;
[0110] N,N'-bis(3,5-diacetylphenyl)dodecanediamide, mp
178-9.degree. C.;
[0111] N,N'-bis(3,5-diacetylphenyl)(isophthalic acid diamide), mp
283-4.degree. C. Also analogously,
N,N'-bis(3-acetylphenyl)pentanediamide- , mp 174-5.degree. C. was
prepared from 3-aminoacetophenone and glutaryl dichloride.
6.1.2. N-(4-acetylphenyl)-N'-(3,5-diacetylphenyl)Urea
[0112] 4-Aminoacetophenone (1.35 g) in toluene (20 mL) was treated
with trichloromethyl chloroformate (1.2 mL). The mixture was heated
at reflux for 2 hr. 3,5-diacetylaniline (1.77 g) was added and the
mixture was heated at reflux for 1 hr then allowed to stand 16 hr
at room temp. The product was filtered off and washed with ethanol
and dried to give 0.93 g of
N-(4-acetylphenyl)-N'-(3,5-diacetylphenyl)urea, mp 251-20C.
6.1.3. 1.2-bis(3-acetylphenoxy)Ethane
[0113] 3-Hydroxyacetophenone (8.4 g) and 1,2-dibromoethane (5.07 g)
were treated with potassium hydroxide (3.83 g) and heated under
nitrogen at reflux for 2 days. The mixture was cooled and water
(200 mL) was added and the mixture stirred for 1 hr. The
precipitate was filtered out and recrystallized from isopropanol to
give 1.21 g of 1,2-bis(3-acetylphenoxy- )ethane, mp 120-10C
6.1.4. 1,5-bis[([(3,
5-diacetylphenyl)-amino]carbony)lamino]-2-methylpenta- ne
[0114] 1,5- Diisocyanato-2-methylpentane (0.18 ml) was added to a
suspension of 3,5-diacetylaniline (0.531 g) in dichloromethane (7
ml) containing catalytic 4-dimethylaminopyridine (10 mg). The
mixture was refluxed for 2 hr and allowed to stand overnight.
Filtration gave while crystals, 0.30 g, mp 124-1300C.
6.1.5.
tris[2-([(3-acetylphenyl)amino]-carbonylamino)ethyl]amine
[0115] 3-Acetylphenyl isocyanatate (0.60 g) in dichloromethane (10
mL) was reacted with tris(2-aminoethyl)amine (0.146 g). A vigorous
reaction occurred and a copious white turbidity resulted. Methanol
was added and the mixture was re-concentrated to produce
crystalline material which was filtered out. Yield 0.61 g, mp
1930C.
6.1.6. 3,5-diacetylphenyl isocyanate and
N,N'-bis(3,5-diacetylphenyl)urea
[0116] 3,5-Diacetylaniline (3.0 g, 16.9 mmol) was suspended in
toluene (50 mL) with stirring in an ice bath. A solution of
bis(trichlormethyl) carbonate (1.67 g, 5.9 mmol) in toluene (10 mL)
was added. The suspension was allowed to warm to room temp. and was
stirred overnight at r.t. The mixture was then heated at reflux for
4 hr, cooled, and filtered to give 1.1 g of
N,N'-bis(3,5-diacetylphenyl)urea, mp dec 137-8.degree. C. (gas
evol.). The filtrate was evaporated to give 2.2 g of
3,5-diacetylphenyl isocyanate as a white powder, mp 71.degree.
C.
6.2. Conversion of Intermediates to End Products
[0117] The following reactions are examples which illustrate a
general condensation reaction wherein the primary amine of
aminoguanidine displaces the oxygen of an acetophenone or
benzaldehyde or ketone and elaborates an H.sub.2O and forms the
guanylhydrazone. In general, all reactions are carried out at
elevated temperature with acid catalysis in aqueous alcohol. The
products are recovered by crystallization upon cooling and,
optionally, the addition of petroleum ether or isopropanol.
Purification was performed by recrystallization.
[0118] Compound 4, FIG. 7A.4: 4-([(aminoiminomethyl)
hydrazono]methyl)cinnamic acid hydrochloride:
[0119] 4-formylcinnamic acid (1.76 g) and aminoguanidine
hydrochloride (1.22 g) were heated in 83% ethanol (24 mL) for 2 hr.
Cooling and filtration gave 2.56 g of
4-([(aminoiminomethyl)hydrazono]methyl)cinnamic acid hydrochloride,
mp 285-8.degree. C.
[0120] Compound 6, FIG. 7A.6:
2-([(1H-imidazol-1-yl)-1,4-phenylene]ethylid-
yne)hydrazinecarboximidamide hydrochloride:
[0121] 4-(1H-imidazol-l-yl)acetophenone (1.86 g) and aminoguanidine
hydrochloride (1.22 g) were heated in 83% ethanol (12 mL) for 48
hr. Cooling and filtration gave 2.6 g of
2-([(1H-imidazol-1-yl)-1,4-phenylene-
]ethylidyne)hydrazinecarboximidamide hydrochloride, mp
275-6.degree. C.
[0122] Compound 7, FIG. 7A.7:
2-[(3,4-dihydroxyphenyl)-ethylidyne]hydrazin- ecarboximidamide
hydrochloride:
[0123] 3,4-dihydroxyacetophenone (3.04 g) and aminoguanidine
hydrochloride (2.44 g) were heated in 75% ethanol (16 mL) for 4 hr
under nitrogen. Cooling and filtration gave 2.7 g of
2-[(3,4-dihydroxyphenyl)ethylidyne]-- hydrazinecarboximidamide
hydrochloride, mp 242-5.degree. C.
[0124] Compound 9, FIG. 7B.9:
N-(4-acetylphenyl)-N'-(3,5-diacetylphenyl)ur- ea
tris(amidinohydrazone) trihydrochloride:
N-(4-acetylphenyl)-N'-(3,5-dia- cetylphenyl)urea (0.676 g),
aminoguanidine hydrochloride (0.83 g), and aminoguanidine
dihydrochloride (0.01 g) were heated in 83% methanol (12 mL) for 18
hr. Cooling and filtration gave 0.85 g of
N-(4-acetylphenyl)-N'-(3,5-diacetylphenyl)urea
tris(amidinohydrazone) trihydrochloride, mp 247-253.degree. C.
dec.
[0125] Compound 10, FIG. 7B.10:
5-(l-[2-(aminoimino-methyl)hydrazono]ethyl- )salicylic acid
hydrochloride:
[0126] 5-Acetylsalicylic acid (3.6 g) and aminoguanidine
hydrochloride (2.4 g) were heated in 80% ethanol (25 mL) for 2 hr.
Cooling and filtration gave 5.2 g of crude
5-(1-[2-(aminoiminomethyl)hydrazono]ethyl) salicylic acid
hydrochloride. Of this, 0.58 g was purified by dissolving in aq.
NaOH (pH 12.5) and reprecipitation with aq HCl (to pH 2) to give
0.45 g of 5-(1-[2-(aminoiminomethyl)hydrazono]ethyl)-salicylic acid
hydrochloride, mp 312-3.degree. C. (dec).
[0127] Compound 12, FIG. 7B.12:
N,N'-bis(3-acetylphenyl)pentanediamide bis(amidinohydrazone)
dihydrochloride:
[0128] N,N'-bis(3-acetylphenyl)pentanediamide (3.66 g),
aminoguanidine hydrochloride (2.75 g), and aminoguanidine
dihydrochloride (0.05 g) were heated in methanol (35 mL) for 18 hr.
Cooling and filtration gave 5.412 g of
N,N'-bis(3-acetylphenyl)pentanediamide bis(amidinohydrazone)
dihydrochloride, mp 187-191.degree. C.
[0129] Compound 13, FIG. 7B.13:
N,N'-bis(3,5-diacetyl-phenyl)pentanediamid- e
tetrakis(amidinohydrazone) tetrahydrochloride:
[0130] N,N'-bis(3,5-diacetylphenyl)pentanediamide (0.45 g),
aminoguanidine hydrochloride (0.55 g), and aminoguanidine
dihydrochloride (0.01 g) were heated in 91% ethanol (4.4 mL) for 18
hr. Cooling and filtration gave 0.794 g of
N,N'-bis(3,5-diacetylphenyl)pentanediamide
tetrakis(amidino-hydrazone) tetrahydrochloride, mp 299-301.degree.
C. dec.
[0131] Compound 14, FIG. 7C.14:
N,N'-bis(3,5-diacetyl-phenyl)decanediamide
tetrakis(amidinohydrazone) tetrahydrochloride:
[0132] N,N'-bis(3,5-diacetylphenyl)decanediamide (0.65 g),
aminoguanidine hydrochloride (0.691 g), and aminoguanidine
dihydrochloride (0.01 g) were heated in 91% ethanol (5.5 mL) for 18
hr. Cooling and filtration gave 0.87 g of
N,N'-bis(3,5-diacetylphenyl)decanediamide tetrakis
(amidinohydrazone) tetrahydrochloride, mp 323-4.degree. C. dec.
[0133] Compound 15, FIG. 7C.15:
2,2'-[1,2-ethanediylbis(oxy-3,1-phenylenee- thylidyne)]
bishydrazinecarboximidamide dihydrochloride:
[0134] 1,2-bis(3-acetylphenoxy)ethane (0.894 g), aminoguanidine
hydrochloride (0.83 g), and aminoguanidine dihydrochloride (0.01 g)
were heated in aq. 96% methanol (6.25 mL) for 18 hr. Cooling and
filtration gave 1.378 g of
2,2'-[1,2-ethane-diylbis(oxy-3,1-phenyleneethylidyne)]bis-
(hydrazine-carboximidamide) dihydrochloride, mp 303-7.degree.
C.
[0135] Compound 16, FIG. 7C.16: 3,3'-(ethylenedioxy)-dibenzaldehyde
bis(amidinohydrazone) dihydrochloride:
[0136] 3,3'-(ethylenedioxy)dibenzaldehyde (1.08 g), aminoguanidine
hydrochloride (1.105 g), and aminoguanidine dihydrochloride (0.005
g) were heated in 96% ethanol (6.25 mL) for 18 hr under nitrogen.
Cooling and filtration gave 1.428 g of
3,3'-(ethylenedioxy)dibenzaldehyde bis(amidinohydrazone)
dihydrochloride, mp 264-6.degree. C.
[0137] Compound 17, FIG. 7C.17:
4,4'-(ethylenedioxy)di-m-anisaldehyde bis(amidinohydrazone)
dihydrochloride:
[0138] 4,4'-(ethylenedioxy)di-m-anisaldehyde (0.99 g),
aminoguanidine hydrochloride (0.829 g), and aminoguanidine
dihydrochloride (0.01 g) were heated in 95% methanol (10.5 mL)
under nitrogen for 16 hr. Cooling and filtration gave 1.52 g of
4,4'-(ethylenedioxy)di-m-anisaldehyde bis(amidinohydrazone)
dihydrochloride, mp 322-3.degree. C. dec.
[0139] Compound 18, FIG. 7C.18:
3,3'-(trimethylenedioxy)di-p-anisaldehyde bis(amidinohydrazone)
dihydrochloride:
[0140] 3,3'-(trimethylenedioxy)di-p-anisaldehyde (1.032 g),
aminoguanidine hydrochloride (0.829 g), and aminoguanidine
dihydrochloride (0.02 g) were heated in 95% methanol (10.5 mL) for
16 hr. Cooling and filtration gave 1.372 g of
3,3'-(trimethylenedioxy)di-p-anisaldehyde bis(amidinohydrazone)
dihydrochloride, mp 233-5.degree. C.
[0141] Compound 19, FIG. 7C.19:
1,4-bis[2-(aminoimino-methyl)hydrazono]cyc- lohexane
dihydrochloride:
[0142] 1,4-cyclohexanedione (2.24 g), aminoguanidine bicarbonate
(6.0 g), and concentrated hydrochloric acid (3.67 mL) were heated
in water (50 mL) for 5 min. The solution was cooled and treated
with isopropanol (50 mL). After crystallization was complete,
filtration gave 2.91 g of
1,4-bis[2-(aminoiminomethyl)hydrazono]cyclohexane dihydrochloride,
mp 260.degree. C. dec.
[0143] Compound 20, FIG. 7C.20:
2,2'-(1,4-diphenyl-1,4-butanediylidene)bis-
hydrazinecarboximidamide dihydrochloride:
[0144] 1,2-dibenzoylethane (4.76 g), aminoguanidine bicarbonate
(5.45 g), and concentrated hydrochloric acid (3.33 mL) were heated
in 50% ethanol (60 mL) for 24 hr. Cooling, concentration and
filtration gave 4.3 g of
2,2'-(1,4-diphenyl-1,4-butanediylidene)bis(hydrazinecarboximidamide)
dihydrochloride, mp 285-6.degree. C.
6.3. Further Exemplary Compounds
[0145] Compound 21, FIG. 7D.21:
N,N'-bis(3,5-diacetylphenyl)-butanediamide
tetrakis(amidinohydrazone) tetrahydrochloride:
[0146] N,N'-bis(3,5-diacetylphenyl)butanediamide (0.545 g),
aminoguanidine hydrochloride (0.69 g), and aminoguanidine
dihydrochloride (0.01 g) were heated in 91% ethanol (5.5 mL) for 18
hr. Cooling and filtration gave 0.97 g of
N,N'-bis(3,5-diacetylphenyl)butanediamide tetrakis
(amidinohydrazone) tetrahydrochloride, mp 314.degree. C.
[0147] Compound 22, FIG. 7D.22: N,N'-bis(3,5-diacetylphenyl)
exanediamide tetrakis(amidinohydrazone) tetrahydrochloride:
[0148] N,N'-bis(3,5-diacetylphenyl) hexanediamide (0.58 g),
minoguanidine hydrochloride (0.69 g), and aminoguanidine
ihydrochloride (0.01 g) were heated in 91% 2-mothoxyethanol (5.5
mL) for 18 hr. Filtration while hot gave 0.936 g of
N,N'-bis(3,5-diacetylphenyl)hexanediamide
tetrakis(amidinohydrazone) tetrahydrochloride, mp (chars)
320-330.degree. C.
[0149] Compound 23, FIG. 7D.23:
N,N'-bis(3,5-diacetylphenyl)-heptanediamid- e
tetrakis(amidinohydrazone) tetrahydrochloride:
[0150] N,N'-bis(3,5-diacetylphenyl)heptanediamide (0.478 g),
aminoguanidine hydrochloride (0.553 g), and aminoguanidine
dihydrochloride (0.01 g) were heated in 91% ethanol (4.4 mL) for 18
hr. Cooling and filtration gave 0.739 g of
N,N'-bis(3,5-diacetylphenyl)heptan- ediamide
tetrakis-(amidinohydrazone) tetrahydrochloride, mp 273-7.degree.
C.
[0151] Compound 24, FIG. 7D.24: N,N'-bis(3,5-diacetylphenyl)
(isophthalic acid diamide) tetrakis (amidinohydrazone)
tetrahydrochloride:
[0152] N,N'-bis(3,5-diacetylphenyl) (isophthalic acid diamide)
(0.726 g) and aminoguanidine hydrochloride (0.829 g) were heated in
7:1 2-methoxyethanol/water (11.5 mL) for 18 hr. Cooling and
filtration gave 0.54 g of N,N'-bis(3,5-diacetylphenyl)(isophthalic
acid diamide) tetrakis(amidinohydrazone) tetrahydrochloride,
(chars) mp 322-330.degree. C.
[0153] Compound 25, FIG. 7E.25:
3,3'-(pentamethylenedioxy)di-p-anisaldehyd- e (0.748 g),
aminoguanidine hydrochloride (0.553 g), and aminoguanidine
dihydrochloride (0.01 g) were heated in methanol (5 mL) for 18 hr.
Cooling and filtration gave 0.080 g of
3,3'-(pentamethylenedioxy)di-p-ani- saldehyde bis(amidinohydrazone)
dihydrochloride, mp 195-8.degree. C.
[0154] Compound 26, FIG. 7E.26: A solution of 3,5-diacetylaniline
(0.885 g) in tetrahydrofuran (10 mL) containing 0.45 mL pyridine
was treated with 0.65 mL benzoyl chloride. The mixture was stirred
1 hr, then treated with 1 mL water and stirred 15 min. The mixture
was then diluted with 40 mL water and stirred 30 min. Filtration
and washing with water and isopropanol gave colorless needles of
N-benzoyl-3,5-diacetylaniline, 1.36 g, mp 188-189.degree. C. A
suspension of N-benzoyl-3,5-diacetylaniline (0.844 g) in aq. 87.5%
ethanol (8 mL) containing 0.73 g aminoguanidine hydrochloride and a
trace of HCl was heated at reflux for 18 hr. Cooling and filtration
gave 1.357 g of N-benzoyl-3,5-diacetylaniline bis(amidinohydrazone)
dihydrochloride (Compound 26), mp 268-72.degree. C.
[0155] Compound 27, FIG. 7E.27: A suspension of 3,5-diacetylaniline
(0.531 g) in water (8 mL) was treated with cyanamide (0.143 g) and
conc. HCl (0.25 mL) and heated at reflux. Additional 0.080 g
portions of cyanamide were added at 2 hr and 4 hr. After 6 hr, the
mixture was concentrated in vacuo until crystalline material
separated, then filtered to give 0.110 g of off-white solid, mp
120-2.degree. C. Of this, 0.104 g was treated with aminoguanidine
hydrochloride (0.112 g) in 2.5 mL of aq. 80% ethanol containing
aminoguanidine dihydrochloride (0.01 g). After 18 hr at reflux,
colling and filtration gave 133 mg of (3,5-diacetylphenyl)guanidi-
ne bis(amidinohydrazone) trihydrochloride (Compound 27) as a
slightly off-white solid, mp 270-3.degree. C.
[0156] Compound 28, FIG. 7F.28: A suspension of 3,5-diacetylaniline
(0.531 g) in water (8 mL) was treated with cyanoguanidine (0.285 g)
and conc. HCl (0.25 mL) and heated at reflux. After 6 hr the
mixture was cooled and concentrated and 0.248 g of off-white solid
was filtered out, mp 260-70 (dec). Of this, 0.238 g was heated at
reflux with aminoguanidine hydrochloride (0.221 g) in 5.5 mL of aq.
91% methanol for 24 hr. Filtration gave 0.290 g of
N-(3,5-diacetylphenyl)biguanide bis(amidinohydrazone)
trihydrochloride (Compound 28) as fine white needles, mp
294-7.degree. C.
[0157] Compound 31, FIG. 7G.31: 4-acetylphenyl isocyanate (1.2 g)
and tris(2-aminoethyl)amine (0.300 mL) in methylene chloride (10
mL) were stirred at r.t. for 30 min. Filtration gave 1.35 g of
tris(2-[([(4-acetylphenyl)amino]carbonyl)-amino]ethyl)amine, mp
189-90.degree. C. This triketone (1.0 g) and aminoguanidine
dihydrochloride (0.77 g) were heated in methanol (5 mL) for 4 hr.
Addition of ethanol (5 ml) and filtration gave 1.08g of
tris(2-[([(4-acetylphenyl)-amino]carbonyl)amino]ethyl)amine
tris(amidinohydrazone) trihydrochloride (Compound 31) as the
dihydrate, mp 224-5.degree. C. (dec.).
[0158] Compound 32, FIG. 7G.32: 3'-aminoacetophenone (0.446 g) and
1,3,5-benzenetricarbonyltrichloride (0.266 g) in tetrahydrofuran (5
mL) were stirred at r.t. for 30 min. Filtration gave 0.500 g of
N,N',N"-tris(3-acetylphenyl)-1,3,5-benzenetricarboxamide, mp
270.degree. C. The triketone (1.0 g) and aminoguanidine
dihydrochloride (0.87 g) were heated in 2-methoxyethanol (5 mL) for
4 hr. Cooling and filtration gave 1.4 g of
N,N',N"-tris(3-acetylphenyl)-1,3,5-benzenetricarboxamide
tris(amidinohydrazone) trihydrochloride (Compound 32) solvated with
one molecule of 2-methoxyethanol, mp 270-5.degree. C. dec.
[0159] Compound 33, FIG. 7G.33, was prepared analogously from
4'-aminoacetophenone via the triketone
N,N',N"-tris(4-acetylphenyl)-1,3,5- -benzenetricarboxamide, mp
310.degree. C., to give N,N',N"-tris(4-acetylph-
enyl)-1,3,5-benzenetricarboxamide tris(amidinohydrazone)
trihydrochloride (Compound 33) solvated with three molecules of
2-methoxyethanol, mp 295-300.degree. C. (dec) (slow heating).
[0160] Compound 34, FIG. 7H.34: 3,5-diacetylphenyl isocyanate (0.6
g) and tris(2-aminoethyl)amine (0.13 g) in methylene chloride (5
mL) were stirred at r.t. for 30 min. Filtration gave 0.6 g of
tris(2-[([(3,5-diacetylphenyl)amino]carbonyl)-amino]ethyl)amine, mp
197-8.degree. C. This hexa-ketone (0.47 g) and aminoguanidine
dihydrochloride (0.61 g) were heated in methanol (5 mL) for 4 hr.
Cooling and filtration gave 0.86 g of
tris(2-[([(3,5-diacetylphenyl)amino]carbony- l)-amino]ethyl)amine
hexakis(amidinohydrazone) heptahydrochloride (Compound 34) as the
dihydrate hemi-ethanolate, mp 245-6.degree. C. dec.
[0161] Compound 35, FIG. 7H.35: 4-acetylphenyl isocyanate (3 g) and
tris(3-aminopropyl)amine (1.06 g) in tetrahydrofuran (50 mL) were
stirred for 30 min. Ethanol (100 mL) was added and the mixture was
left at r.t. for overnight. Filtration gave 2.8 g of
tris(3-[([(4-acetylphenyl)amino]c- arbonyl)-amino]propyl)amine, mp
184-5.degree. C. This diketone (0.4 g) and aminoguanidine
dihydrochloride (0.29 g) were heated in methanol (3 mL) for 4
hours. Addition of ethanol (3 mL) and filtration gave 0.5g of
tris(3-[([(4-acetylphenyl)-amino]carbonyl)amino]propyl)amine
tris(amidinohydrazone) trihydrochloride (Compound 35) as the
dihydrate ethanolate, mp 209-10.degree. C. dec.
[0162] Compound 36, FIG. 7H.36: 3,5-Diacetylphenyl isocyanate (0.4
g) and tris(3-aminopropyl)amine (0.12 g) in tetrahydrofuran (5 mL)
were stirred at r.t. for 30 min. Filtration gave 0.32 g of
tris(3-[([(3,5-diacetylphen- yl)-amino]carbonyl)amino]propyl)amine,
mp 147-8 C. This hexaketone (0.4 g) and aminoguanidine
dihydrochloride (0.49 g) were heated in methanol (3 mL) for 4
hours. Addition of ethanol and filtration gave 0.58 g of
tris(3-[([(3,5-diacetylphenyl)amino]carbonyl)amino]propyl)amine
hexakis(amidinohydrazone) heptahydrochloride (Compound 36) as the
dihydrate hemi-ethanolate, mp 250-1.degree. C. (dec).
[0163] Compound 37, FIG. 7I.37: 3-Acetylphenyl isocyanate (1 g) and
4'-aminoacetophenone (0.84 g) in methylene chloride (5 mL) were
stirred at r.t. for 30 min. Filtration gave 2.0 g of
3,4'-diacetyl-N,N'-diphenylu- rea, mp 252-3.degree. C. (dec). This
diketone (0.6 g) and aminoguanidine dihydrochloride (0.65 g) were
heated in ethanol (5 mL). Cooling and filtration gave 0.8g of
3,41-diacetyl-N,N'-diphenylurea bis(amidinohydrazone)
dihydrochloride (Compound 37) as the hemihydrate, mp 243-4.degree.
C. dec.
[0164] Compound 38, FIG. 7I.38: 3-Acetylphenyl isocyanate (0.27 g)
and 3,5-diacetylaniline (0.3 g) were stirred in methylene chloride
(5 mL) at r.t. for 30 min. Filtration gave 0.5g of 3,3
,5'-triacetyl-N,N'-diphenylu- rea, mp 223-4.degree. C. This
triketone (0.34 g) and aminoguanidine dihydrochloride (0.49 g) were
heated in ethanol (5 mL) for 4 hr. Cooling and filtration gave 0.6g
of 3,3,5'-triacetyl-N,N'-diphenylurea tris(amidinohydrazone)
trihydrochloride (Compound 38) as the hydrate, mp 245.degree. C.
dec.
[0165] Compound 39, FIG. 7I.39: N,N'-Bis(3,5-diacetylphenyl)-urea
(0.38 g), aminoguanidine hydrochloride (0.44 g) and aminoguanidine
dihydrochloride (59 mg) were heated at 90-100.degree. C. in
2-methoxyethanol (5 mL) for 6 hours. Cooling and filtration gave
0.66g of N,N'-bis(3,5-diacetylphenyl)urea
tetrakis(amidinohydrazone) tetrahydrochloride (Compound 39) as the
2.5-hydrate hemi-methanolate, mp 263-4.degree. C. dec.
[0166] Compound 40, FIG. 7I.40:
N,N'-bis(3,5-diacetylphenyl)-nonanediamide (0.100 g),
aminoguanidine hydrochloride (0.115 g), and aminoguanidine
dihydrochloride (3 mg) were heated at reflux in 95% ethanol (2.5
mL) for 5 hr. Cooling and filtration gave 0.18 g of
N,N'-bis(3,5-diacetylphenyl)-- nonanediamide
tetrakis(amidinohydrazone) tetrahydrochloride (Compound 40) as the
dihydrate, mp 295-6.degree. C.
[0167] Compound 41, FIG. 7J.41:
N,N'-bis(3,5-diacetylphenyl)dodecanediamid- e (0.100 g),
aminoguanidine hydrochloride (0.115 g), and aminoguanidine
dihydrochloride (3 mg) were heated in 95% ethanol (2.5 mL) for hr.
Cooling and filtration gave 0.070 g of
N,N'-bis(3,5-diacetylphenyl)dodeca- nediamide
tetrakis(amidinohydrazone) tetrahydrochloride (Compound 41) as the
tetrahydrate, mp 268-9.degree. C.
[0168] Compound 42, FIG. 7J.42: A suspension of 3,5-diacetylaniline
(0.354 g) in methylene chloride (7 mL) containing
4-dimethylaminopyridine (5 mg) was treated with
2-methyl-1,5-pentanediyl diisocyanate (0.18 mL). After heating at
reflux for 2 hr, the mixture was cooled. An aliquot was treated
with t-butyl methyl ether to give seed crystals which were added to
the reaction mixture. After stirring several hr, filtration gave
0.120 g of
1,5-bis[([(3,5-diacetylphenyl)-amino]carbonyl)amino]-2-methylpentane-
, mp 128.degree. C. This tetraketone (0.100 g), aminoguanidine
hydrochloride (0.115 g), and aminoguanidine dihydrochloride (3 mg)
were heated in 95% ethanol (2.5 mL) for 18 hr. Cooling and
filtration gave 0.16 g of
1,5-bis[([(3,5-diacetylphenyl)amino]carbonyl)amino]-2-methylpen-
tane tetrakis(amidinohydrazone) tetrahydrochloride (Compound 42) as
the tetrahydrate, mp 258-9.degree. C.
[0169] Compound 43, FIG. 7J.43:
N,N'-bis(3,5-diacetylphenyl)octanediamide (0.100 g), aminoguanidine
hydrochloride (0.115 g), and aminoguanidine dihydrochloride (10 mg)
were heated in 95% ethanol (2.5 mL) for 20 hr. Cooling and
filtration gave 0.17 g of N,N'-bis(3,5-diacetylphenyl)octaned-
iamide tetrakis(amidinohydrazone) tetrahydrochloride (Compound 43)
as the 2.5-hydrate, mp 308-9.degree. C.
7. EXAMPLE
Whole Cell Inhibition Assays for Urea and no Output
[0170] This section describes in detail the methods and the results
of a tissue culture assay to determine the ability of the compounds
of the invention to inhibit urea synthesis in activated
macrophages.
7.1. Material and Methods
[0171] RAW 264.7 cells are plated in microculture wells at
1.times.10.sup.6/ml in RPMI 1640 with 10% FBS and otherwise
standard culture conditions and allowed to adhere for 5 hours. They
are then activated with 25 U/ml .gamma.IFN and 0.1 .mu.g/ml LPS in
the presence of test inhibitors. The urea concentration present in
the supernatant media, after 18 hours of culture, is determined by
a colorimetric blood urea nitrogen diacetylmonoxime assay performed
on an aliquot of the culture supernatant (Sigma Chem. Co., St.
Louis, Mo.) and inhibition is expressed as the percentage urea
compared to that of a parallel control culture treated identically
except that the concentration of added inhibitor is zero.
[0172] The production of nitrite is determined by assay of the same
tissue culture supernatant by means of a calorimetric assay.
Briefly, 4 parts of test solution containing 1% (w/v)
sulfanilamide, 0.1% naphthylethylenediamine di-HCl (Griess
Reagent), 2.5% H.sub.3PO.sub.4 and one part media are mixed,
incubated for 10 minutes and the absorbance at 560 nm determined.
The nitrite concentrations are interpolated from reference curves
prepared using NaNO.sub.2.
[0173] Various concentrations of the test compounds are included in
the media of the stimulated RAW 264.7 cells in culture. The
concentration of urea and/or nitrite after 18 hours of activated
culture is determined and compared to the uninhibited control value
obtained from at least one parallel culture. The fractional
reduction is calculated for each concentration of inhibitor and the
IC.sub.50 (concentration giving 50% reduction) is interpolated.
Compounds were dissolved in media with mild heat (60.degree. C.).
Those with limited solubility were dissolved in base. The
concentration of stock solutions of those compounds which were not
completely soluble was determined by OD.sub.280.
[0174] The culture supernatants are tested for the presence of an
intracellular enzyme, lactate dehydrogenase (LDH) to determine the
degree, if any, to which the test compound has caused cell death.
In the examples reported hereinafter no such cellular toxicity due
to the test compounds was observed.
7.2. Results
[0175] The results of the examination of guanylhydrazone compounds
to measure their capacity to inhibit urea production are presented
in Tables I, II and III. The most active compounds, reported in
Table I, displayed IC.sub.50 of less than 10 .mu.M. The most active
compound was an tetraguanylhydrazone decanediamide (Compound No.
14). Also highly active were the pentanediamide homolog of the
above (Compound No. 13); the ethanedioxy bis(guanylhydrazone),
(Compound No. 16); a mixed urea bis(guanylhydrazone) (Compound No.
9); and triacetylbenzine tris(guanylhydrazone) (Compound No.
1).
[0176] Compounds having either one or two phenyl nuclei were
effective. When two nuclei were present their linkage by means of a
urea, diamide or alkanedioxy functionality was effective. Test
compounds having a single guanylhydrazone functionality were much
less effective.
1TABLE I RESULTS OF IN VITRO ASSAYS FOR COMPOUNDS DEMONSTRATING
HIGH LEVELS OF ACTIVITY arginine dose urea no.sub.2/no.sub.3
transport evaluated % suppression % suppression % suppression # Of
(I.C..sub.50 of of of Compound Guandino Dissolved In induced
induced induced # Groups In parenthesis) production production peak
1 3 5 mM 25 .mu.M 100 72 40 solution in 10 .mu.M 60 58 20 5 mM
NaOH; 1 .mu.M 0 precipita- (10 .mu.M) tion when diluted into RPMI 9
3 5 mM 25 .mu.M 100 71 68 solution in 10 .mu.M 60 53 32 5 mM NaOH;
1 .mu.M 0 0 14 precipita- (10 .mu.M) tion when diluted into RPMI 13
4 5 mM 500 .mu.M 100 52 93 solution in 25 .mu.M 100 43 19 5 mM
NaOH; 50 .mu.M 87 7 precipita- 25 .mu.M 77 tion when 10 .mu.M 45
diluted into (10 .mu.M) RPMI 14 4 5 mM 200 .mu.M 100 100 100
solution in 50 .mu.M 100 100 86 5 mM NaOH, 10 .mu.M 78 69 19 forms
a kind 2 .mu.M 68 0 9 of clot when (1 .mu.M) cold, precipita- tion
when diluted into RPMI 15 2 Not soluble; 150 .mu.M 100 100 100
treat 15.0 .mu.M 100 9 35 as #11, 12 5.0 .mu.M 8 0 10 (10 .mu.M) 16
2 Not soluble; 250 .mu.M 100 100 100 treat 25 .mu.M 100 90 44 as
#11, 12, 15 5 .mu.M 40 17 26 (1-10 .mu.M)
[0177]
2TABLE II RESULTS OF IN VITRO ASSAYS FOR ACTIVE COMPOUNDS UREA
NO.sub.2/NO.sub.3 # OF DOSE TESTED % SUPPRESSION % SUPPRESSION
COMPOUND GUANIDINO DISSOLVED (I.C..sub.50 IN OF INDUCED OF INDUCED
# GROUPS IN PARENTHESIS) PRODUCTION PRODUCTION 11 2 Not soluble;
163 .mu.M 100 100 dose 16.3 .mu.M 0 0 defined by OD 5.4 .mu.M 0 0
determination (100 .mu.M) (280 mM) of SN of 5 mM NaOH -solution-
+heat+ spin the suspension used as St. curve the ODs of #1, 9, 13
12 2 Not soluble, 100 .mu.M 18-32 20 dose defined by (250 .mu.M)?
OD determin- ation (at 280 mM) using as St. curve the ODs of #1, 9,
13 17 2 Not soluble 177 .mu.M 54-100 71-87 treated as (150 .mu.M)
#11, 12, 15, 16 18 2 Not soluble 233 .mu.M 100 100 treated as 23.3
30 13 #11, 12, 15, 16 and 7.7 9 0 17 (25 .mu.M) 19 2 Well dissolved
1 mM 100 in PBS 500 .mu.M 100 250 .mu.M 100 100 .mu.M 50 50 .mu.M
30 10 .mu.M 0 (100 .mu.M) 20 2 5 mM solution in 400 .mu.M 100 84 5
mM NaOH + heat 200 .mu.M 38 15 50 .mu.M 19 0 10 .mu.M 16 0 (300
.mu.M)
[0178]
3TABLE III RESULTS OF IN VITRO UREA PRODUCTION ASSAYS FOR COMPOUNDS
DEMONSTRATING ACTIVITY UREA # OF DOSE TESTED % SUPPRESSION COMPOUND
GUANIDINO DISSOLVED (I.C..sub.50 IN OF INDUCED # GROUPS IN
PARENTHESIS) PRODUCTION 2 2 Heat (60.degree. C.-2 hrs) 200 .mu.M
100 200 mM solution 20 .mu.M 0-17 in RPMI (50 .mu.M) 3 2 Heat
(60.degree. C.-2hrs) 100 .mu.M 100 1 mM solution in 50 .mu.M 42
RPMI 40 .mu.M 21 1 .mu.M 9 (500 .mu.M) 4 1 Heat (60.degree.
C.-2hrs) 400 .mu.M 100 400 .mu.M solution 200 .mu.M 0 in RPMI 100
.mu.M 0 50 .mu.M 0 20 .mu.M 0 (300 .mu.M) 5 2 Well dissolved 1 mM
73-100 in PBS; 5 mM 500 .mu.M 64 solution 250 .mu.M 52 125 .mu.M 48
60 .mu.M 37 30 .mu.M 24 (250 .mu.M) 6 1 Heat (60.degree. C.-2hrs) 1
mM 100 1 mM solution in 100 .mu.M 40 RPMI 50 .mu.M 0 (250 .mu.M) 7
1 Heat (60.degree. C.-2hrs) 10 mM 100 10 mM solution 2 mM 55 in
RPMI 1 mM 40 100 .mu.M 35 10 .mu.M 23 (2 mM) 8 2 Heat (60.degree.
C.-2hrs) 500 .mu.M 100 5 mM solution 50 .mu.M 76 in RPMI (50 .mu.M)
10 1 Heat (60.degree. C.-2hrs) 400 .mu.M 0 400 .mu.M solution 200
.mu.M 0 20 .mu.M 0 (No effect)
8. EXAMPLE
Whole Cell Inhibition Assay for Arginine Uptake
[0179] This section describes in detail the methods and the results
of a tissue culture assay to determine the ability of the compounds
of the invention to inhibit the uptake of arginine by activated
macrophages.
8.1. Materials and Methods
[0180] RAW 264.7 cells are plated in standard 96-well microculture
plates at a concentration of 10.sup.5/well and allowed to adhere
for from 2 hours to overnight. The medium is then replaced with
medium containing the compound to be tested. One hour later, the
medium is supplemented so as to contain 25 U/ml .gamma.IFN and 0.1
.mu.g/ml LPS and the cultures incubated a further. The cells are
then rinsed twice in HEPES- buffered Krebs salt solution with 0.1%
glucose. Carrier-free tetra-.sup.3H-arginine is added to each well
(2.5 .mu.Ci/well with specific activity of 69 mCi/mmol) and the
active uptake of arginine is allowed to continue for a 5 minute
period after which the cells are rapidly cooled to 0.degree. C. by
lavage with iced saline containing 10 mM unlabeled arginine to
displace any externally bound label. The contents of the washed
cells are solubilized in 100 Al of formic acid and counted by
standard techniques. The amount of tetra-.sup.3H-arginine uptake as
a function of time incubation time is shown in FIG. 6. In
subsequent experiments to determine the acitivity of the
inhibitors, incubations were performed for 8 hours.
8.2. Results
[0181] The compounds of the present invention which were most
active in suppressing urea production in activated macrophages were
tested to determine their effects on the uptake of arginine. The
results, shown in Table I of Section 7.2, indicate that each of the
active compounds were effective inhibitors of uptake at a dose
similar to that which effectively inhibited urea production. These
data suggest that an arginine transport protein is a target of
action of these compounds.
9. EXAMPLE
Arginase Inhibition Assay
[0182] This section describes in detail the methods and the results
of a cell-free assay to determine the ability of the compounds of
the invention to inhibit the activity of argininase obtained from
macrophages.
9.1. Materials and Methods
[0183] A 1200 g.times.2 minutes supernatant is obtained from a cell
lysate of washed RAW 264.7 cells made by the addition of a lysis
buffer containing 50 mM Hepes, 1% NP-40, 0.1 mg/ml
phenylmethylsulfonylfluoride (PMSF), and aprotinin at 1 .mu.g/ml.
The volume of supernatant is adjusted so that the protein
concentration is between 2 and 4 mg/ml. A 1:4 mixture of the
supernatant in activation buffer (30 mM MnCl.sub.2, 0.3 M glycine,
1% BSA, pH 9.8) containing various concentrations of-the test
inhibitor is incubated at 550C for 20 minutes. Arginase activity is
determined by mixing a 1:2 the above solution and 0.375 M arginine,
pH 9.8 for 10 minutes at 370C. The urea concentration after this
incubation is determined as above.
9.2. Results
[0184] The six compounds most active in the suppression of cellular
urea production were tested to determine whether any could inhibit
the activity of arginase in the above-described cell lysate assay.
The results do not indicate arginase to be sensitive to inhibition
by the compounds of the present invention at the concentrations and
under the conditions employed.
10. EXAMPLE
Treatment of Cachexia in vivo
[0185] An animal model of tumor-associated protein catabolic
illness (cachexia) was employed to directly test the efficacy of
Compound No. 14 in reducing whole-body nitrogen losses. Tumors were
induced in the appropriate groups by intramuscular inoculation with
15.times.10.sup.6 AtT-20 cells; this model is known to cause a
protein catabolic illness characterized by the consumption of
normal quantities of food, but 20% net whole-body losses of protein
within 14 days. Compound No. 14 was administered to the "treated"
groups in a daily dose of 0.4 mg/Kg, intraperitoneal. Nude mice
(nu/nu) were housed in metabolic cages (4 per cage) to facilitate
collection of daily urine samples for 14 days. Food intake was
measured daily. There were four groups of animals: 1) untreated,
non-tumor bearing controls; 2) untreated, tumor-bearing; 3)
treated, tumor-bearing, and 4) treated, non-tumor-bearing. Urine
was collected and urinary urea losses quantified.
[0186] The results are summarized in Table IV (expressed as mg
urea/group):
4TABLE IV IN VIVO ACTIVITY OF COMPOUND #14 IN SUPPRESSING
WHOLE-BODY NITROGEN LOSS URINARY UREA LOSS (mg/group) Day After
Tumor, Tumor, Controls, Tumor Controls Untreated Treated Treated 2
189 240 195 193 4 252 342 368 285 6 256 508 295 397 7 302 456 312
247 8 287 300 275 257 10 177 259 206 216 11 240 279 219 239 12 230
230 115 203 13 192 186 201 242 14 161 238 187 157 Sum totals 2286
3038 2373 2436
[0187] The groups consumed similar quantities of food (16.+-.2
g/day) during this experiment.
[0188] The data demonstrate that Compound No. 14 prevented excess
urinary urea excretion normally associated with cachexia.
[0189] Since urea loss represents 80% of whole-body nitrogen
losses, and the nitrogen intakes were similar in all groups, these
data further suggest that Compound No. 14 augments whole-body
nitrogen retention.
[0190] The significance of these observations is demonstrated by
estimating the impact of these nitrogen losses to muscle mass.
Compound No. 14 prevented the loss of approximately 200 mg
urea/mouse over the 14 day period. This is equivalent to
approximately 100 mg of nitrogen, 625 mg of protein, or 2.7 g of
wet muscle mass that is retained by treatment with Compound No.
14.
11. EXAMPLE
Treatment of Inflammation in vivo
[0191] A standard animal model of inflammation was utilized to
directly test the efficacy of Compound No. 14 as an
anti-inflammatory. Paw swelling induced by injection of the
irritant carrageenan into murine foot pads has been used to detect
clinically useful anti-inflammatory drugs since the early
1960's.
[0192] Paw edema was induced by injection of 50 microliters of 1%
Lambda-carrageenan in HEPES 25 mM, pH 7.4, into the planter surface
of the left hindpaw of C3H/HeN mice, the right paw was injected
with 50 microliters of HEPES alone. The animals were divided into
two groups: controls (n=3) received vehicle only, i.p. 1.5 hour
before paw injection; the experimental group (n=4) was treated with
Compound No. 14, 5 mg/Kg, i.p. 1.5 hour before paw injection. Three
hours after paw injection, paw thickness was measured using a
caliper, and the data expressed as change of carrageenan paw vs
HEPES paw thickness.
[0193] The results are summarized in Table V which contains the
carrageenan-induced increase in paw thickness in mm of three
control and three Compound No. 14 treated mice. The inhibition can
also be reported as percent inhibition of swelling as defined by
the standard formula: Percent
inhibition=(1-(treated/control)).times.100. Calculation of this
parameter for treated animals in which Compound No. 14 suppressed
swelling yields a 70% inhibition.
5TABLE V EFFECT OF COMPOUND NO. 14 ON INFLAMMATION CHANGE IN PAW
THICKNESS Untreated Treated With Controls Compound #14 1.47 0.54
1.50 0.41 1.24 0.4
[0194] FIG. 8 presents the effects of various doses of Compound No.
14 in the same assay. The data show that doses of between about 1
and 10 mg/Kg of body weight are effective at inhibiting paw
swelling.
[0195] These data demonstrate that Compound No. 14 prevented
inflammation, which is believed to be mediated by the inhibition of
arginine-transport dependent nitric oxide production in
inflammatory cells.
12. EXAMPLE
Compound No. 14 has no Effects on Endothelial Derived Relaxing
Factor Mediated Vasodilatory Responses
[0196] An important requirement of drug used to control the
vascular collapse and hypostension caused by macrophage NO
production is that it not interfere with the activity of EDRF in
vivo. Such interference can cause an uncontrolled hypertension and
has prevented the effective clinical use of NO-synthase inhibitors.
We measured the effect of Compound No. 14 on EDRF activity in an
animal model and found that compound 14 inhibits NO production yet
does not inhibit EDRF activity.
[0197] Female Sprague-Dawley rats (225-250 g body weight) were
anesthetized with nembutal (50 mg/Kg, i.p.), a tracheostomy tube
was inserted, and the carotid artery and jugular vein cannulated by
standard methods using polyethylene tubing (PE 50). Tracey, K. J.,
et al., 1986, SCIENCE 234, 470-474. Blood pressure was recorded
continuously with a pressure transducer and recorder (Model
RS-3200, Gould Inc.,). In the experiment shown here, animals
received a single sterile intra-arterial dose of either
N.sup.G-methyl-L-arginine (NMA; Sigma; 10 mg/Kg), Compound No. 14
(50 mg/Kg), or vehicle (0.4 ml). Acetylcholine (ACh) diluted in
LPS-free sterile water was administered via the jugular vein
cannula at the doses indicated. The solutions were diluted to
produce a constant injectable volume of 1 ml/Kg body weight.
[0198] The hypotensive (EDRF) response was measured as the decline
in mean arterial blood pressure recorded 30 sec after
administration of ACh. The number of animals studied at each dose
of acetylcholine was 4-6 for each experimental condition; data are
expressed as the mean +s.e.m.
[0199] EDRF activity was inhibited by NMA, evidenced by attenuated
blood pressure responses as compared to vehicle-treated controls
(FIG. 9; see also, Kilbourn, R. G., et al., 1990, PROC NATL. ACAD
SCI. 87, 3629-3632). In contrast, Compound No. 14 did not suppress
acetylcholine-induced EDRF activity in vivo, indicating that
Compound No. 14-treated animals retained the functional capacity
for endothelial-derived NO activity.
13. EXAMPLE
Compound No. 14 Prevents Fatal Endotoxic Shock
[0200] Experiments were undertaken to evaluate the effects of
Compound No. 14 in preventing the lethal toxicity of
lipopolysaccharide (LPS). LPS was administered to induce 50%
lethality within 72 hr in BALB/c mice (FIG. 10). BALB/c mice (19-21
g) were given LPS (E. coli 0111:B4; Sigma) in a dose of 13.75 mg/Kg
by intraperitoneal injection (0.2 ml/mouse). Stock LPS solutions
(10 mg/ml) were sonicated initially for 20 min, diluted in LPS-free
water (1.375 mg/ml), then sonicated again for 10 min immediately
prior to injection. Compound No. 14 (1 mg/Kg, i.p.) was
administered 1.5 hr before LPS. Compound No. 14 injectate was free
of LPS as measured by quantitative chromogenic Limulus amebocyte
lysate test (BioWhittaker, Walkersville, Md.). Data points are from
two groups consisting of 10 mice per group.
[0201] Compound No. 14 administered 1.5 hours before LPS reduced
lethality. Data were subjected to statistical analysis using the z
test for independent proportions. The difference bewteen control
and Compound No. 14 is significant (one-tailed p value<0.05;
z=1.95).
[0202] There were no gross signs of systemic toxicity in animals
receiving Compound No. 14 alone. After LPS, controls were
ill-kempt, had decreased mobility, and huddled together. These
visible signs of LPS toxicity were markedly suppressed by Compound
No. 14. Diarrhea occurred in all animals, and was not suppressed by
Compound No. 14.
[0203] Previously available NOS inhibitors have had limited success
in improving survival from endotoxemia, in part because they
indiscriminately suppress EDRF. Cobb, J. P., 1992, J. EXP. MED.
176:1175-1182; Minnard, E. A., 1994, ARCH SURG. 129:142-148;
Billiar, T. R., 1990, J. LEUKOCYTE. BIOL. 48:565-569. Suppression
of EDRF during endotoxemia may impair survival by causing
vasoconstriction and a diminution of blood flow to critical
vascular beds. The present data now indicate that inhibiting
cytokine-inducible macrophage NO with an agent that preserves
endothelial-derived NO responses can confer a survival advantage
during septic shock.
14. EXAMPLE
Compound No. 14 Prevents the Production of Cytokines and No
[0204] This section describes in detail the methods and the results
of a tissue culture assay to determine the ability of the compounds
of the invention to inhibit the production of TNF by activated
macrophages, and shows, for purposes of explanation and not
limitation, that Compound No. 14 is effective in blocking the
secretion of TNF by a mechanism that is independent of the
inhibition of arginine uptake.
[0205] RAW 264.7 cells were plated in standard 6-well culture
plates at a density of 10.sup.6/well and allowed to adhere for from
2 hr to overnight. The medium was then replaced with medium
containing the compound to be tested. One hour later, the medium
was supplemented so as to contain 25 U/ml .gamma.IFN and 0.1
.mu.g/ml LPS and the cultures incubated a further 18 hr. The medium
was then collected, and cell debris removed by centrifugation. This
conditioned supernatant was then assayed for the presence of TNF
using standard methodologies: L929 cell cytotoxicity bioassay,
radioimmunoassay or ELISA, and Western blotting with antibodies
against murine TNF.
[0206] FIG. 11 shows that in the presence of increasing amounts of
Compound No. 14, the production of bioactive TNF as determined by
L929 cell bioassay by RAW 264.7 cells was prevented. The data show
that at 10 .mu.M Compound No. 14 there was a reduction of more than
99% of the TNF accumulation in the culture medium. FIG. 12 shows a
Western blot that demonstrates the absence of TNF protein in the
medium of cells cultured with 5 and 25 .mu.M concentrations of
Compound No. 14. The failure of Compound No. 14-treated cells to
secrete TNF can not be simply attributed to the inhibition of NO
synthesis by Compound No. 14. The NOS inhibitor N-methyl-arginine
(NMA), even at concentrations of 10 .mu.M, did not inhibit the
production of TNF in this system. The inhibition of TNF production
was also not attributable to the effects of arginine depletion
brought on by the blockage of arginine transport. FIG. 13A shows
that Compound No. 14, at a 5 .mu.M concentration, functioned as an
inhibitor of NO synthesis, but that this inhibition could be
partially overcome by the presence of between about 50 and 100
.mu.M arginine in the medium. By contrast the data in FIG. 13B
clearly show that the effects on TNF secretion of Compound No. 14
at 5 .mu.M were not reduced even by as much as 1.0 mM extracellular
arginine. These results show that Compound No. 14 specifically
blocked the production of TNF from activated macrophages by a
mechanism that does not critically depend on arginine.
[0207] Qualitatively similar data has been obtained by measurement
of the serum TNF levels in rats stimulated to produce TNF by
parenteral LPS administration. Rats were given Compound No. 14,
i.p., 2.0 hours prior to LPS stimulation. The serum levels of TNF
were measured at between 2 and 3 hours after LPS stimulation.
Compound No. 14-treated animals showed only about half the level of
serum TNF as found in the untreated controls.
[0208] Similar results concerning a variety of cytokines in
addition to TNF, e.g., IL-6 and Macrophage Inflammatory
Proteins-1.alpha. and -1.beta. (MIP-1.alpha. and MIP-1.beta.) were
obtained using human peripheral blood as a source of monocytes.
Human peripheral blood mononuclear cells (PBMC) were isolated using
"FICOLL.RTM."-based methods and plated in standard 6-well culture
plates at a concentration of 10.sup.6/well and allowed to adhere
for from 2 hr to overnight whereupon non-adherent cells were washed
out and the medium was then replaced with medium containing the
compound to be tested. One hour later, the medium was supplemented
so as to contain 25 U/ml .gamma.IFN and 0.1 .mu.g/ml LPS and the
cultures incubated a further 18 hr. The medium was then collected,
and cell debris removed by centrifugation. This conditioned
supernatant was then assayed for the presence of cytokines using
the standard methodologies of L929 cell cytotoxicity bioassay and
immunoassay (ELISA). The results indicated that, when tested in
this system, Compound No. 14 effectively inhibited the production
of TNF, IL-6, MIP-1.alpha., and MIP-1.beta. by human PBMC cells at
a concentration of about 10-20 .mu.M. FIGS. 14A-D show that in the
presence of increasing amounts of Compound No. 14 from 1 to 20
.mu.M, the production of these cytokines by human PBMC cell
cultures was prevented.
15. EXAMPLE
Compound No. 14 Confers Protection from Focal Cerebral
Infarction
[0209] This section describes in detail the methods and the results
of an animal model to determine the ability of Compound No. 14 to
treat cerebral infarction (also known as brain infarction or
"stroke").
[0210] Lewis rats (male, 270-300 g) were given food and water ad
libi tum before and after surgery. Animals were anesthetized with
ketamine (120 mg/Kg i.m.), allowed to breathe spontaneously, and
body temperature maintained at 35.5-36.5.degree. C. with a heating
blanket. The ventral neck and area between the right eye and ear
was shaved. A midline ventral cervical incision was used to expose
the left common carotid artery (CCA) which was dissected free from
surrounding tissue with preservation of the vagus nerve. A loop of
4-0 silk was then placed around the artery for future manipulation.
The right common carotid artery was then exposed and permanently
occluded with double 4-0 silk ligatures.
[0211] To perform the craniotomy, a 1 cm incision was made
orthogonal to the line joining the external auditory canal and the
lateral canthus of the right eye. With the aid of a dissecting
microscope, the right middle cerebral artery was exposed through a
1 mm burr hole drilled approximately 2 cm rostral to the fusion of
the zygoma with the temporal bone. Drilling was performed under a
continuous drip of normal saline to avoid transmission of heat to
the underlying cortex. The bone was thinned with the drill, leaving
a thin shell which was removed with a micro-hook and micro-forceps.
The dura mater was then cut and reflected with a 30 gauge needle,
exposing the right middle cerebral artery (MCA) approximately 1 mm
from the rhinal fissure.
[0212] Using a micromanipulator and a 20 micron tungsten wire hook,
the right middle cerebral artery was elevated 0.5 mm above the
cortical surface and divided by application of an electrocautery
tip to the tungsten hook above the vessel. The application of heat
quickly cauterized and severed the artery which fell back onto the
cortex with no underlying cortical injury. To cause a reproducible
stroke the left CCA must be temporarily occluded for 30-60 minutes.
Accordingly, in the present model the left CCA was occluded for 30
minutes. Surgical gelfoam was placed over the craniotomy defect,
and the skin incisions closed with a vicryl sutures. The animals
were then returned to their cages, where they were allowed free
access to food and water for 24 hours. After surgery, animals were
somewhat clumsy but resumed activities including walking, eating,
and drinking.
[0213] Twenty-four hours after MCA severing, animals were
anesthetized and decapitated and the brains were quickly removed
without perfusion and coronally sectioned at 1 mm intervals with a
brain slicer for analysis. Freshly prepared slices were immersed
and incubated in 2,3,5-triphenyltetrazolium (TTC) (2% in NaCl, 154
mM) for 30 minutes at 37.degree. C. in the dark to stain for
mitochondrial dehydrogenase activity. Brain infarctions were
visualized as areas of unstained (white) tissue which were easily
contrasted with viable tissue which stained red. Slices were then
placed in buffered 10% formalin and infarct area determined by
planimetry on projected images of photographed brain slices.
Infarct size for an individual animal was calculated by summing the
infarct area present on each brain slice and dividing by the sum of
hemisphere area for all slices for that animal (expressed as a
percentage of hemisphere area for each slice). Animals were studied
in groups of 10, and the data expressed as average stroke volume
for the group.
[0214] The mean infarct size of controls (not treated with Compound
No. 14) was observed to be 3.1%.+-.0.5%. Animals that received
Compound No. 14 (1 mg/Kg, i.v.) one hour before the artery was
severed developed smaller stroke size (1.7%.+-.0.2%). These
differences were statistically significant (p<0.05) by Student's
t-test for unpaired data. These experiments indicated that Compound
No. 14 effectively reduced the size of focal cerebral
infarction.
16. EXAMPLE
Anti-neoplastic Activity of Compound No. 14
[0215] This section describes in detail the methods and results of
an animal model of tumor growth to determine the ability of the
compounds of the invention to inhibit tumor growth, and cause
regression of tumors. Cells are plated in standard tissue culture
flasks in DMEM supplemented with fetal calf serum (10%). Chinese
hamster ovary (CHO) cells stably transfected with a mammalian
expression vector constitutively secrete human TNF (CHO-TNF). The
experiments with the CHO-TNF tumor were performed as follows: on
the day of injection into nude mice, cells are harvested and
injected intramuscularly (10-15.times.10.sup.6 cells per animal)
into the hindlimb of nu/nu nude mice (20 g body weight). Animals
are housed and provided food and water ad libitum. Tumor growth is
monitored, and when established tumors are present (during week
six) the test compounds are administered daily (0.4 mg/Kg
intraperitoneal, once daily). After two weeks the animals are
euthanized, the tumor weighed, and measured with a caliper. The
CHO-TNF tumor also metastasizes to the skin of these animals; the
number of metastases is scored.
[0216] Parenteral administration of Compound No. 14 caused a
reduction in tumor size in four out of five animals. The tumors of
these animals were weighed and their size determined. Examination
of the mice for skin metastases revealed that an average of
2.5.+-.1 skin metastases developed in 4 out of 5 controls; skin
metastases were not present in any of the treated animals. These
data show that Compound No. 14 has anti-tumor activity.
6 # of TUMOR WEIGHT TUMOR DIMENSION METAS- (g) (mm.sup.2) TASES
CONTROL 2.686 .+-. 0.77 343.58 .+-. 64.1 2.5 .+-. 1.1 (n = 5) No.
14 1.628 .+-. 1.07 207.99 .+-. 119.90 0 (n = 4)
17. Inhibitory Effects of Compound No. 14 on Arginine Uptake and no
Output of Previously Quiescent Versus Activated Cells
[0217] This section describes experiments that measure the
concentration of Compound No. 14 needed to inhibit arginine uptake
and NO output. The techniques employed were described in section
7.1 supra.
[0218] FIG. 13A shows the effects of Compound No. 14 on the output
of NO by RAW 264.7 cells that have been continuously exposed to
Compound No. 14 from one hour prior to .gamma.-IFN/LPS stimulation
until completion of the assay 18 hours after stimulation. The data
show that Compound No. 14 was a inhibitor of NO output that was
competitively antagonized by extracellular arginine. These data
indicate that the I.C..sub.50 for Compound No. 14 in this assay, at
physiologic arginine concentrations (100 .mu.M), was between 3 and
5 .mu.M.
[0219] As shown in FIG. 6, the peak level of arginine uptake by RAW
264.7 cells occured at about 8 hours after .gamma.-IFN/LPS
stimulation. When RAW 264.7 cells were exposed to Compound No. 14
prior to stimulation, as in FIG. 13B, and the level of arginine
uptake measured at 8 hours, the I.C..sub.50 for arginine uptake of
Compound No. 14 was very similar that observed for NO output, 7.5
.mu.M (data not shown).
[0220] All effects of Compound No. 14 on macrophage NO output,
however, could not be entirely attributed to an acute blockade of
arginine transport. FIG. 15 shows the results observed when RAW
264.7 cells were .gamma.-IFN/LPS-stimulated in the absence of any
inhibitor and, 8 hours thereafter, the cells were exposed for 10
minutes to Compound No. 14, whereupon arginine uptake was measured.
An I.C..sub.50=60.+-.15 .mu.M (mean.+-.std. err., n=3) was measured
when RAW 264.7 cells, that had been .gamma.-IFN/LPS-stimulated 8
hours previously were exposed to Compound No. 14 in arginine-free
buffer for 10 minutes prior to assay of arginine uptake. The
results of these acute exposure experiments differed from the
previously discussed, continuous exposure results in two ways.
Firstly, approximately one third of the total arginine uptake
occured through a Compound No. 14-independent mechanism which
appeared to be present in unstimulated as well as stimulated RAW
264.7 cells. Secondly, the concentration of Compound No. 14 needed
to 50% inhibit arginine uptake after .gamma.-IFN/LPS-induced
arginine transport has been established for a period of hours was
greater than that needed to prevent 50% of the increased arginine
uptake or NO output when Compound No. 14 was introduced prior to
stimulation.
[0221] An intermediate level of sensitivity to Compound No. 14 was
observed when .gamma.-IFN/LPS-stimulated RAW 264.7 cells were
exposed to inhibitor from 8 hours after the initial stimulation
until the end of the experiment. In these experiments, NO output
was measured during hours 12-24. FIG. 16 demonstrates that under
such experimental conditions an I.C..sub.50 of 20.+-.2 .mu.M
(mean.+-.std. err., n=3) was observed. Together these data show
that when RAW 264.7 cells were exposed to Compound No. 14 prior to
activation by .gamma.-IFN/LPS, NO output and inducible arginine
uptake were equally sensitive to low levels of Compound No. 14,
while the arginine uptake of unstimulated RAW 264.7 was insensitive
to Compound No. 14. By contrast, the arginine uptake of RAW 264.7
cells, stimulated with .gamma.-IFN/LPS, and exposed briefly to
Compound No. 14 after a delay was less sensitive to of Compound No.
14 than when Compound No. 14 was given prior to stimulation.
Likewise the NO output of cells treated with Compound No. 14 hours
after stimulation was shown to be less sensitive to inhibition by
Compound No. 14 than was the induction of NO production by
previously quiescent cells.
[0222] These data indicate that higher levels of Compound No. 14
will be required, in vivo, to block the ongoing production of NO by
activated macrophages than would be needed to prevent the
initiation of NO production by non-activated macrophages and that
Compound No. 14 will be more effective when chronically applied
than when acutely applied to cells.
18. High-performance Liquid Chromatography Method and Determination
of Pharmacokinetic Constants Thereby
[0223] A high-performance liquid chromatographic (HPLC) method has
been developed for a series of aromatic guanylhydrazones that have
demonstrated therapeutic potential as anti-inflammatory agents. The
compounds were separated using octadecyl or diisopropyl-octyl
reverse-phase columns, with an acetonitrile gradient in water
containing heptane sulfonate, tetramethylammonium chloride, and
phosphoric acid. The method was used to reliably quantify levels of
analyte as low as 78.5 ng per injection, and the detector response
was linear to at least 5000 ng per injection. The compounds could
be extracted and concentrated from biological samples using
octadecyl-silane solid-phase extraction columns. The assay system
was used to determine the basic pharmacokinetics of a lead
compound, Compound No. 14, from plasma concentrations following a
single intravenous injection in rats.
18.1. Experimental Materials and Methods
[0224] Chemicals
[0225] Heptane sulfonate (HS), tetramethylammonium chloride (TMAC),
and phosphoric acid were obtained from Aldrich (Milwaukee, WI,
USA), and pentamidine isethionate from May and Baker (now
Rhone-Poulenc; Dagenham, UK). HPLC-grade acetonitrile was acquired
from Fisher (Fairlawn, N.J., USA) and all water was filtered and
deionised by a Picopure system (Hydro Service and Supplies;
Research Triangle Park, N.C., USA) All guanylhydrazones were
synthesized as described (Ulrich, P. & Cerami, A., 1984,
J.Med.Chem. 27:35; Ulrich, P. et al., 1982, Drug Dev.Res. 2:219)
and the purity confirmed by elemental analysis, proton NMR, and
melting point.
[0226] Chromatographic Conditions
[0227] A Hewlett-Packard model 1090 liquid chromatograph
(Wilmington, Del., USA) equipped with an autosampler, photodiode
array detector, and Chemstation operating software was used for all
analyses. The columns used were either a Supelcosil LC-18
250.times.4.6 mm octadecylsilane column with 5 mm particle size
(Supelco; Bellefonte, Pa., USA) or a Zorbax RX-C8 250.times.4.6 mm
column with 5 mm particle size (Mac Mod Analyticals; Chadds Ford,
PA, USA) kept at room temperature. Buffer A was 10 mM HS/10 mM
TMAC/4.2 mM H.sub.3PO.sub.4/H.sub.2O and buffer B 10 mM HS/10 mM
TMAC/4.2 mM H.sub.3PO.sub.4/75% CH.sub.3CN/25% H.sub.2O. Using a
flow rate of 1.5 ml/min, runs were initiated at 10% B and a linear
gradient to 90% B was performed over 30 min. The column was then
returned to 10% B over 7 min, followed by 3 min re-equilibration.
The compounds were detected by absorbance at 265 nm, with 540 nm
used as a reference wavelength.
[0228] Sample Preparation
[0229] The test compounds and the internal standard, pentamidine,
were dissolved in distilled water to make 1 mg/ml stock solutions.
To determine the relative retention times and peak shapes, a single
test compound and the internal standard were diluted to 10 .mu.g/ml
in distilled water, and 100 .mu.l injected onto the HPLC.
[0230] To extract the compounds, an equal volume of HPLC buffer A
was added to the test sample (to which pentamidine had been added
to 5 .mu.g/ml) before being loaded onto conditioned Supelclean C-18
solid-phase extraction cartridges. The cartridges were then washed
with 1.0 ml of distilled water and eluted with 1.0 ml of 10 mM
HS/10 mM TMAC/4.2 mM H.sub.3PO.sub.4/95% CH.sub.3CN/5% H.sub.2O. In
some experiments, 100 .mu.l of this eluate was injected onto the
HPLC, and, in others, the elution buffer was removed in vacuo and
the pellet resuspended in HPLC buffer A before injection of 100
.mu.l. Standard addition curves were constructed in distilled
water, human urine, and mouse plasma by the addition of various
amounts of test compound and 5 .mu.g/ml pentamidine. These samples
were either injected directly or subjected to the solid-phase
extraction system previously mentioned.
[0231] Pharmacokinetic Studies
[0232] Male Sprague-Dawley rats (Harlan Sprague Dawley,
Indianapolis, Ind., USA) were anesthetized with ketamine and the
right carotid artery cannulated with polyethylene tubing (PE-50).
The animals were given 10 mg/kg of Compound No. 14 in a single
intra-arterial injection of 380 .mu.l. At 0, 5, 15, 30, 60, 90,
120, 180, 240, 300, 360 minutes 400 .mu.l of blood was removed,
stored at 4.degree. C. for 4 hr, and then centrifuged at 15,000
.times.g for 10 min, and the serum layer collected. Sodium azide
was added to 0.0l v/v to prevent microbial growth and pentamidine
was added to 5 .mu.g/ml. Twenty-five .mu.l of the resulting sample
was analyzed by the HPLC method described.
18.2. Results
[0233] In designing a separation system for the aromatic
guanylhydrazones, ion-pair buffers were chosen which contained 10
mM heptane sulfonate, 10 mM tetramethylammonium chloride, and 4.2
mM phosphoric acid, as these buffers had been used successfully to
separate aromatic diamidines, B. J. Berger, et al., 1991, J.
PHARMACOL. EXPER. THER. 256:883, which bear some structural
similarity to the present compounds. Use of these buffers with a
reverse-phase C-18 column was found to be ideal for the elution of
Compound No. 14. Elimination of either of the ion-pair reagents
from the buffer led to a complete retention of Compound No. 14 by
the HPLC column (data not shown). Separation of Compound No. 14 and
the internal standard, pentamidine, was performed with a Zorbax
RX-C8 column.
[0234] While Compound No. 14 and pentamidine could easily be
detected in raw serum samples, an extraction system was developed
to allow for concentration of the analytes from samples containing
trace amounts and also to minimise the amount of protein injected
on the column. Using reverse-phase, solid-phase extraction columns
(SPEC) and an elution buffer consisting of 10 mM TMAC/10 mM HS/4.2
MM H.sub.3PO.sub.4/95% CH.sub.3CN, the recovery of Compound No. 14
and pentamidine was found-to be 75.60.+-.13.77% and 92.27.+-.6.52%
respectively from C-18 SPEC (n=6). This recovery was superior to
that found for the compounds on C-8, cyanopropyl, or phenyl SPEC
(data not shown). In addition, to optimize recovery, it was found
beneficial to add an equal volume of HPLC A buffer to the sample
before loading onto the SPEC. This step led to a 20-fold increase
in recovery from C-18 SPEC.
[0235] Further studies with Compound No. 14 and the solid-phase
extraction system demonstrated that the limit of detection was 78.5
ng per injection, and that the compound could be efficiently
extracted from urine and plasma samples (data not shown). The assay
was found to be linear from the limit of detection up to at least
5000 ng per injection, and gave the following regression for a plot
of Compound No. 14 peak area vs. amount injected: y=267.507x-45.251
(r.sup.2=0.99). The method was also found to be accurate, with an
intraday variation of 1.5% on samples of 1000 ng Compound No. 14
injected (n=4), and an interday variation of 9.5% on samples of 625
ng injected (n=3).
[0236] The HPLC method was applied towards estimating the
pharmacokinetic parameters of Compound No. 14 in adult rats
receiving a 10 mg/kg dose as a single intra-arterial injection. In
these experiments, the solid-phase extraction step was omitted due
to the small volume of each sample, and the relatively large amount
of Compound No. 14 which was recovered. Typical serum decay curves
were obtained (FIG. 17, solid line), and the method of residuals,
Gibaldi, M., & Perrier, D., 1982, PHARMACOKINETICS (Marcel
Dekker, New York) pp. 433-444, was used to calculate the
pharmacokinetic parameters (FIG. 17, dashed lines). The
distribution rate constant (.alpha.) was found to be 0.31.+-.0.09
min.sup.-1, the elimination rate constant (.beta.) 0.0023.+-.0.0000
min.sup.-1, the initial distribution concentration (A)
63.01.+-.43.78 mg/ml, the initial elimination concentration (B)
1.57.+-.0.14 mg/ml, the distribution half-life (t.sub.1/2.alpha.)
2.41.+-.0.69 min, the elimination half-life (t.sub.1/2.beta.)
5.02.+-.0.00 hr, the volume of distribution (V.sub.d) 2.45.+-.0.21
L, and the total clearance (C.sub.L) 5.62.+-.0.47 ml/min (n=3 for
all). These values show that the compound persists in the serum for
some time after a single i.a. injection. Experiments performed via
intraperitoneal or oral dosing routes indicate that the drug is not
rapidly absorbed, and may have a low bioavailability (data not
shown). The choice of a 10 mg/kg dose is applicable, as the
compound was found to have an LD.sub.50 of 50 mg/kg when given
intraperitoneally and one that exceeds 1 g/kg when given
orally.
[0237] The present invention is not to be limited in scope by the
specific embodiments described which were intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components were within the
scope of the invention. Indeed, various modifications of the
invention, in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and accompanying drawings. Such modifications are
intended to fall within the scope of the appended claims.
* * * * *