U.S. patent application number 17/496532 was filed with the patent office on 2022-05-19 for antagonists of cb1 receptor.
The applicant listed for this patent is INSERM (Institut National de la Sante et de la Recherche Medicale), UNIVERSITE DE BORDEAUX. Invention is credited to Luigi BELLOCCHIO, Daniela COTA, Francois-Xavier FELPIN, Rafael MALDONADO, Giovanni MARSICANO, Pier Vincenzo PIAZZA, Jean-Michel REVEST, Umberto SPAMPINATO, Monique VALLEE, Sergio VITIELLO.
Application Number | 20220153776 17/496532 |
Document ID | / |
Family ID | |
Filed Date | 2022-05-19 |
United States Patent
Application |
20220153776 |
Kind Code |
A1 |
PIAZZA; Pier Vincenzo ; et
al. |
May 19, 2022 |
ANTAGONISTS OF CB1 RECEPTOR
Abstract
The invention relates to an antagonist of CB1 receptor for use
in the treatment of a pathologic condition or disorder selected
from the group consisting of bladder and gastrointestinal
disorders; inflammatory diseases; cardiovascular diseases;
nephropathies; glaucoma; spasticity; cancer; osteoporosis;
metabolic disorders; obesity; addiction, dependence, abuse and
relapse related disorders; psychiatric and neurological disorders;
neurodegenerative disorders; autoimmune hepatitis and encephalitis;
pain; reproductive disorders and skin inflammatory and fibrotic
diseases.
Inventors: |
PIAZZA; Pier Vincenzo;
(Bordeaux, FR) ; VALLEE; Monique; (Bordeaux,
FR) ; MARSICANO; Giovanni; (Bordeaux, FR) ;
FELPIN; Francois-Xavier; (Nantes, FR) ; BELLOCCHIO;
Luigi; (Bordeaux, FR) ; COTA; Daniela;
(Bordeaux, FR) ; REVEST; Jean-Michel; (Bordeaux,
FR) ; VITIELLO; Sergio; (Bordeaux, FR) ;
SPAMPINATO; Umberto; (Bordeaux, FR) ; MALDONADO;
Rafael; (Barcelona, ES) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSERM (Institut National de la Sante et de la Recherche
Medicale)
UNIVERSITE DE BORDEAUX |
Paris
Bordeaux |
|
FR
FR |
|
|
Appl. No.: |
17/496532 |
Filed: |
October 7, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16169236 |
Oct 24, 2018 |
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17496532 |
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14118420 |
Feb 26, 2014 |
10150793 |
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PCT/EP2012/059310 |
May 18, 2012 |
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16169236 |
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International
Class: |
C07J 7/00 20060101
C07J007/00; A61K 31/57 20060101 A61K031/57; C07J 5/00 20060101
C07J005/00; C07J 11/00 20060101 C07J011/00; C07J 13/00 20060101
C07J013/00; C07J 41/00 20060101 C07J041/00; C07J 31/00 20060101
C07J031/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 20, 2011 |
EP |
11305625.3 |
Claims
1. A method for the treatment of a pathologic condition or disorder
comprising administering to a subject in need thereof a compound of
formula (A), or a pharmaceutically acceptable salt thereof:
##STR00120## wherein: denotes that the bond is a single or a double
bond, R1 denotes that C3 is substituted with --H, halogen, --OH,
C1-8 alkoxy, Bn-O-- Bn- optionally substituted with C1-8 alkyl,
C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen, Ph-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
amino, carboxyl or halogen, .dbd.O, --NR5R6 wherein R5 and R6 each
independently is H, C1-8 alkyl, Bn or Ph, --O--CO--R7 wherein R7 is
alkyl, --O--CO--C.sub.2H.sub.4--COOH, or --N.sub.3, --R2 denotes
that C17 is substituted with --H, --OH, halogen, C1-8 alkyl, C1-8
alkoxy, C2-6 alkenyl, Bn optionally substituted with C1-8 alkyl,
C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen, Ph-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
carboxyl or halogen, or Bn-O--, R3 denotes that C20 is substituted
with --H, --OH, C1-8 alkyl, Bn, --NR8R9 wherein R8 and R9 each
independently is H, C1-8 alkyl or Bn, .dbd.CR10R11 wherein R10 and
R11 each independently is H or C1-7 alkyl, or .dbd.O R4 denotes
that C16 is substituted with --H, --OH, or .dbd.O, with the proviso
that when the bond between C16 and C17 is double, R2 is absent and
the bond between C17 and C20 is single, and when the bond between
C17 and C20 is double, C20 is substituted with --H or --OH and R2
is absent, when the bond between C4 and C5 is double, the bond
between C5 and C6 is single and inversely, wherein the pathologic
condition or disorder is selected from the group consisting of
bladder and gastrointestinal disorders; inflammatory diseases;
cardiovascular diseases; nephropathies glaucoma; spasticity;
cancer; osteoporosis; metabolic disorders; obesity; addiction,
dependence, abuse and relapse related disorders; psychiatric and
neurological disorders; neurodegenerative disorders; autoimmune
hepatitis and encephalitis; pain; reproductive disorders; and skin
inflammatory and fibrotic diseases.
2. The method of claim 1, wherein the compound of formula (A) is a
compound of formula (I), or a pharmaceutically acceptable salt
thereof: ##STR00121## wherein: denotes that the bound is a single
or a double bond, R1 denotes that C3 is substituted with --H,
halogen, --OH, C1-8 alkoxy, Bn-O-- Bn- optionally substituted with
C1-8 alkyl, C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen,
Ph- optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano,
nitro, amino, carboxyl or halogen, .dbd.O, --NR5R6 wherein R5 and
R6 each independently is H, C1-8 alkyl, Bn or Ph, --O--CO--R7
wherein R7 is alkyl, or --O--CO--C.sub.2H.sub.4--COOH, --R2 denotes
that C17 is substituted with --H, --OH, halogen, C1-8 alkyl, C1-8
alkoxy, C2-6 alkenyl, Bn optionally substituted with C1-8 alkyl,
C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen, Ph-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
carboxyl or halogen, or Bn-O--, R3 denotes that C20 is substituted
with --H, --OH, C1-8 alkyl, Bn, --NR8R9 wherein R8 and R9 each
independently is H, C1-8 alkyl or Bn, .dbd.CR10R11 wherein R10 and
R11 each independently is H or C1-7 alkyl, or .dbd.O, R4 denotes
that C16 is substituted with --H, --OH, or .dbd.O, with the proviso
that when the bond between C16 and C17 is double, R2 is absent and
the bond between C17 and C20 is single, and when the bond between
C17 and C20 is double, C20 is substituted with --H or --OH and R2
is absent.
3. The method of claim 1, wherein the compound is pregnenolone, or
a pharmaceutically acceptable salt thereof.
4. (canceled)
5. The method of claim 1, wherein said compound is not
substantially converted into an active pregnenolone downstream
derivative after administration to the subject.
6. (canceled)
7. The method claim 1, wherein the compound of formula (A) is a
compound of formula (B) ##STR00122## wherein R1 denotes that C3 is
substituted with --OH or .dbd.O, --R2 denotes that C17 is
substituted with --H, --OH, C1-8 alkyl, halogen or Bn, 3 denotes
that C20 is substituted with --OH or .dbd.O, R4 denotes that C16 is
substituted with --H,
8. The method of claim 1, wherein the compound of formula (A) is a
compound of formula (C): ##STR00123## wherein R1 denotes that C3 is
substituted with .dbd.O or --OH --R2 denotes that C17 is
substituted with --H R3 denotes that C20 is substituted with
.dbd.O, and R4 denotes that C16 is substituted with --H,
9. (canceled)
10. The method of claim 1, wherein the compound of formula (A) is a
compound of formula (D): ##STR00124## wherein R1 denotes that C3 is
substituted with Halogen, Bn-O or, --N.sub.3, --R2 denotes that C17
is substituted with --H, R3 denotes that C20 is substituted with
.dbd.O, and R4 denotes that C16 is substituted with --H,
11. (canceled)
12. The method of claim 1, wherein the compound of formula (A) is a
compound of formula (D): ##STR00125## wherein R1 denotes that C3 is
substituted with C1-8 alkoxy, halogen or Bn-O--, or N.sub.3 --R2
denotes that C17 is substituted with Bn, --CH.sub.3 or C2-6
alkenyl, R3 denotes that C20 is substituted with .dbd.O, and R4
denotes that C16 is substituted with --H,
13. The method of claim 1, wherein: R1 denotes that C3 is
substituted with --OH --R2 denotes that C17 is substituted with
--OH, halogen, C1-8 alkyl, C1-8 alkoxy, C2-6 alkenyl, Bn-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
amino, carboxyl or halogen, Ph- optionally substituted with C1-8
alkyl, C1-8 alkoxy, cyano, nitro, carboxyl or halogen, or Bn-O--,
R3 denotes that C20 is substituted with .dbd.O, and R4 denotes that
C16 is substituted with --H,
14. The method of claim 1, wherein the compound of formula (A) is a
compound of formula (D): ##STR00126## wherein R1 denotes that C3 is
substituted with --OH, --R2 denotes that C17 is substituted with
C1-8 alkyl, C1-8 alkoxy or Bn-, R3 denotes that C20 is substituted
with .dbd.O, and R4 denotes that C16 is substituted with --H,
15. (canceled)
16. The method of claim 1, wherein the compound of formula (A) is a
compound of formula (D): ##STR00127## wherein R1 denotes that C3 is
substituted with --OH, --R2 denotes that C17 is substituted with
--H, R3 denotes that C20 is substituted with --H, --OH or --NR8R9
wherein R8 and R9 each independently is H or C1-8 alkyl, and R4
denotes that C16 is substituted with --H,
17-19. (canceled)
20. The method of claim 1, wherein the pathologic condition or
disorder is a gastrointestinal disorder.
21. The method of claim 1, wherein the pathologic condition or
disorder is obesity or a metabolic disorder.
22. The method of claim 1, wherein the pathologic condition or
disorder is addiction, dependence abuse, or a relapse related
disorder.
23. The method of claim 22, wherein the pathologic condition or
disorder is cannabis addiction, dependence, abuse, intoxication, or
relapse related disorder.
24. The method of claim 1, wherein the pathologic condition or
disorder is a neurodegenerative or psychiatric disorder.
25. The method of claim 1, wherein the pathologic condition or
disorder is a skin inflammatory or fibrotic disease.
26. A compound of formula (II) ##STR00128## or a pharmaceutical
salt thereof, wherein: denotes that the bond is a single or a
double bond R1 denotes that C3 is substituted with --OH, and --R2
denotes that C17 is substituted with C3-8 alkyl, C2-8 alkoxy, Bn-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
amino, carboxyl or halogen, Ph- optionally substituted with C1-8
alkyl, C1-8 alkoxy, cyano, nitro, carboxyl or halogen, or Bn-O--,
or wherein R1 denotes that C3 is substituted with C1-8 alkoxy,
Bn-O, or Halogen, and --R2 denotes that C17 is substituted with
C1-8 alkyl, C2-6 alkenyl, C1-8 alkoxy, Bn- optionally substituted
with C1-8 alkyl, C1-8 alkoxy, cyano, nitro, amino, carboxyl or
halogen, Ph- optionally substituted with C1-8 alkyl, C1-8 alkoxy,
cyano, nitro, carboxyl, or halogen, or Bn-O--.
27. The compound of claim 26, wherein the compound is
3.beta.-fluoro-17.alpha.-methylpregnenolone, 17.alpha.-benzyl-3
O-fluoropregnenolone,
17.alpha.-benzyl-3.beta.-benzyloxypregnenolone,
3.beta.-benzyloxy-17.alpha.-methylpregnenolone,
17.alpha.-benzylpregnenolone,
3.beta.-methoxy-17.alpha.-methylpregnenolone,
17.alpha.-allyl-3.beta.-methoxypregnenolone, or
17.alpha.-benzyl-3.beta.-methoxypregnenolone.
28. A pharmaceutical composition comprising a compound of claim 26,
or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable carrier.
29-30. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compounds for use in the
treatment of a pathologic condition or disorder selected from the
group consisting of bladder and gastrointestinal disorders;
inflammatory diseases; cardiovascular diseases; nephropathies;
glaucoma; spasticity; cancer; osteoporosis; metabolic disorders;
obesity; addiction, dependence, abuse relapse and related
disorders; psychiatric and neurological disorders;
neurodegenerative disorders; autoimmune hepatitis and encephalitis;
pain; reproductive disorders and skin inflammatory and fibrotic
diseases.
BACKGROUND OF THE INVENTION
[0002] The CB1 receptor is one of the main G-coupled seven membrane
receptor (GCPR) of the body. It is the principal GCPR of the brain
and it is also expressed by most body tissues including, but not
limited to, the adipose tissue, liver, pancreas, muscles, kidney,
bladder and the bones.
[0003] The CB1 is activated by endogenous ligands named
endocannabinoids including, but not limited to, Anandamide and
2-arachidonyl glycerol (2-AG).
[0004] Through activation by endogenous ligands, the CB1 has been
involved in the regulation of a large number of physiological
functions and pathological states. A non exhaustive list of the
functions in which the activation of the CB1 receptor has been
involved include: energy metabolism; inflammation and immunity;
fibrosis, bone homeostasis; lipid storage and accumulation in
various organs, behaviors; self-administration of drugs of abuse,
memory, stress-related adaptation, behaviors mediated by positive
reinforcers; gastrointestinal motility and motility of other
visceral contractile organs; cell proliferation and
differentiation; pain regulation; reproduction and fertility.
(Marsicano et al., J Endocrinol Invest., 2006; 29(3 Suppl):27-46
Review; Pagotto U et al., Int J Obes. 2006, Suppl 1:S39-43 Review;
Pagotto U et al., Endocr Rev., 2006 (1):73-100. Review; Bifulco M,
et al. Mol Pharmacol. 2007, 71(6):1445-56 Review)
[0005] Because of this wide spread physiological role,
over-activation of the CB1 receptor has been involved in a large
number of pathologies, diseases and pathophysiological processes. A
non exhaustive list of examples of diseases and diseases-related
process in which the activation of the CB1 receptor has been
involved include: bladder and gastrointestinal disorders;
inflammatory diseases; cardiovascular diseases; nephropathies;
glaucoma; spasticity; cancer; osteoporosis; metabolic disorders;
obesity; addiction, dependence, abuse and relapse related
disorders; psychiatric and neurological disorders;
neurodegenerative disorders; autoimmune hepatitis and encephalitis;
pain; reproductive disorders and skin inflammatory and fibrotic
diseases. (Di Marzo et al., Nat. Rev., Drug Discov., 2004, 3:
771-784).
[0006] The CB1 receptor is the major target of
.DELTA.9tetrahydrocannabinol (THC), the active principle contained
in the drugs of abuse obtained from Cannabis sativa. It is through
CB1 activation that THC exercises its addictive effects and its
behavioral and physiological disrupting effects. In addition the
CB1 receptor is also involved in mediating the effects of all the
other known drugs of abuse, including, but not limited to,
nicotine, opioids, psychostimulants and alcohol. The CB1 receptor
is also involved in mediating the appetitive properties of non-drug
reinforcing stimuli that are able to induce addiction, including,
but not limited to, food, sexual partners or gambling. The general
effects of CB1 on drugs of abuse and other reinforcing stimuli that
are able to induce addiction is explained by the excitatory control
that activation of the CB1 receptor exercise on the activity of the
dopaminergic transmission. Thus, activation of the dopaminergic
transmission is involved in mediating the appetitive properties and
addictive liability of drug of abuse and non-drug positive
reinforcers. For this reason a blockade of CB1 activity has been
proposed as a method for treating addiction, drug abuse, drug
dependence and relapse (Scherma M et al., CNS Neurol Disord Drug
Targets. 2008; 7(5):468-81. Review; Wiskerke J et al., Addict Biol.
2008; 13(2):225-38. Review; Moreira F A, et al., Addict Biol. 2008;
13(2):196-212. Review; Lopez-Moreno J A et al, Addict. Biol. 2008;
13(2):160-87. Review; Janero D R et al., Curr Psychiatry Rep.,
2007; 9(5):365-73. Review; Laviolette S R et al., Cell Mol Life
Sci., 2006; 63(14):1597-613. Review; Maldonado R, et al. Trends
Neurosci., 2006; 29(4):225-32. Review; Colombo G et al., Pharmacol
Biochem Behav., 2005; 81(2):369-80. Review; Gardner E L. Pharmacol
Biochem Behav., 2005; 81(2):263-84. Review).
[0007] Inhibition of the CB1 receptor has been shown to reduce
weight and enhance improvements in cardiometabolic risk parameters.
Thus, CB1 receptor antagonists have been shown to prophylactically
prevent overweight, to assist in regulating food intake, to assist
as a diet aid, to treat obesity and ameliorate metabolic disorders
often associated with obesity such as diabetes and dislipedimia.
(Bermudez-Silva F J et al., 2010; Lee H K et al. 2009; Xie S et
al., 2007).
[0008] Central CB1 receptor signaling is functionally linked to
monoaminergic neurotransmission. This makes CB1 antagonists
candidates for the treatment of psychosis, affective and cognitive
disorders brought about by disturbances in any of the central
monoaminergic systems. Furthermore, CB1 agonists lead to memory
impairment. CB1 antagonists are therefore good candidate agents for
memory enhancement (see Reibaud M et al., Eur. J. Pharmacol, 1999;
379 (1):R1-2, and Terranova J P et al, Psychopharmacology., 1996;
126(2): 165-72). CB1 activation can also lead to impairment in
movement and movement disorders like Parkinson's disease have been
associated with elevated brain endocannabinoids. CB1 antagonism
would therefore be a good candidate treatment for Parkinson's
disease (see Di Marzo V et al, FASEB J., 2000; 14(10): 1432-8).
Therefore, CB1 antagonists are candidates to treatment of various
psychiatric and neurological diseases.
[0009] CB1 receptor is also involved in spasticity as disclosed by
Pryce G et al., (Br J Pharmacol, 2007,150 (4): 519-525.) and by
Baker D. et al. (FASEB J., 2001, 15: 300-302).
[0010] Chien F Y, et al. have shown that WIN 55212-2, a cannabinoid
agonist at the CB(1) receptor, reduces intraocular pressure in both
normal and glaucomatous monkey eyes. CB1 receptors are expressed in
some peripheral tissues such as nerve endings in the
gastrointestinal tract depress gastrointestinal motility, mainly by
inhibiting ongoing contractile transmitter release. Antagonists of
CB1 receptor could thus find use in pathological states consisting
of decreased intestinal motility such as Paralytic ileus caused by
peritonitis, surgery, or other noxious situations (Mascolo N et al,
FASEB J., 2002 December; 16(14): 1973-5).
[0011] Also about gastrointestinal diseases, CB1 receptors are also
shown to be involved in liver diseases and in particular in liver
steatosis, steatohepatitis (NASH) and cirrosis. The CB1 activation
play a role in these diseases by a double mechanism: 1. Promoting
the accumulation of fat in the liver; 2. Promoting the release of
inflammatory factor such as TNF.alpha.. CB1 inhibitor are
beneficial in these pathologies because they both reduce fat
accumulation and the release of THF.alpha.. 3. In this case For
examples see: 1. Mallat A and Lotersztajn S. Diabetes and Metabolis
34(2008) 680-684; 2. Tam J et al., HEPATOLOGY 2011; 53:346-355; 3.
Soren V. Siegmund S V and Schwabe R F Am J Physiol Gastrointest
Liver Physiol 294: G357-G362, 2008; 4. DeLeve D L et al., The
American Journal of Pathology, 173, No. 4, 2008; 5. Roche M et al.,
Immunology, 2008 125, 263-271; 6. Murumalla R et al., Journal of
Inflammation 2011, 8:33; 7. Croci T, et al., British Journal of
Pharmacology (2003) 140, 115-122.
[0012] CB1 receptors are also expressed in noradrenergic terminals
innervating the bone. CB1 activation is able to inhibit
Noradrenaline release in the bone which in turn increases
osteoclaste activity decreasing bone mass, including, but not
limited to menopause associated osteoporosis. For this reason CB1
antagonists have also been proposed as a treatment for
osteoporosis. (Idris AI Curr Neuropharmacol. 2010 8(3):243-53.
[0013] CB1 receptors also play a role in vascular endothelial cells
where they mediate the hypotensive effects of platelet and
macrophage-derived endocannabinoids. CB1 antagonists would be
useful agents in inhibiting endotoxin-induced or cirrhotic
hypotension (see Batkai S et al, Nat Med., 2001 July; 7(7): 827-32)
both of which are characterized by elevated levels of
endocannabinoids. CB1 also stimulate angiogenesis, as a consequence
blockade of the CB1 receptor has been proposed for the treatment of
diseases in which an increase in angiogenesis plays a
pathophyisiological role as for example in tumor development.
[0014] CB1 receptors have also been involved in pathologies of the
cardiovascular system including cardiomiopathies such as cyrrotic
cardiomiopathy and antideoplastic drugs induced cardiomiopathies,
contractile disfunction, infarction and atherosclerosis. The CB1
receptors play a role in these diseases with multiple mechanisms
that involve control of blood pressure, inflammation, lipid
accumulation, vascularisation and heart contractility. For example
see: 1. Batkai S et al., Am J Physiol Heart Circ Physiol. 2007,
293: H1689-H1695; 2. Seyed Ali Gaskari S A et al., British Journal
of Pharmacology (2005) 146, 315-323; 3. Batkai S and Pacher P.
Pharmacol Res. 2009, 60: 99-106. 4. Nissen S E et al., JAMA. 2008;
299(13):1547-1560. 5. Mukhopadhyay P et al., J Am Coll Cardiol.
2007, 50: 528-536.
[0015] CB1 receptors have also been shown to be involved in
inflammatory diseases and in particular but not limited to in skin
diseases including skin inflammation, skin inflammation and cancer
induced by UV, skin fibrosis and wound healing. In this context an
inhibition or suppression of the CB1 receptor has been shown
beneficial for all these pathological states. For example see: 1.
Marquart S et al., ARTHRITIS & RHEUMATISM, 2010, 62:3467-3476;
2. Zheng D et al., Cancer Res. 2008 May 15; 68(10): 3992-3998.
[0016] Furthermore, endocannabinoid signalling is found in some
human malignancies compared with the corresponding healthy tissues,
as well as in human cancer cells with a high degree of invasiveness
(Sarnataro D et al., 2006; Gazzerro P et al., 2010; Santoro A, et
al. 2009).
[0017] The endocannabinoid signalling is also implied in
fertilization, preimplantation embryo and spermatogenesis and it is
therefore a relevant target to improve infertility and reproductive
health in humans.
[0018] For these reasons the inhibition of the CB1 receptor has
been suggested as a therapy of all these pathological states and
associated diseases.
[0019] Methods aimed at blocking the activity of the CB1 through
inhibition of the orthosteric binding site, the site at which the
endogenous ligands bind to activate the receptor, have been
developed and submitted for clinical trials. One of these
compounds, rimonabant, has even been put on the market with the
brand name Acomplia. Acomplia has been tested and revealed a
beneficial effect for the treatment of metabolic disorders,
diabetes and dyslipidemia, obesity and also in one study for
nicotine addiction.
[0020] Unfortunately, available orthosteric antagonists such as
rimonabant also act as inverse agonists of the CB1 receptor, i.e.
they not only inhibit the activation of the CB1, but also the basal
activity of the receptor in the absence of the endogenous ligand.
Because of this inverse agonist action and the total inhibition of
the receptor activity, available methods based on the
administration of orthosteric CB1 antagonists also have a series of
serious adverse effects. Because of these adverse effects
commercialization of Acomplia has been suspended and the
development of other methods inhibiting the orthosteric site of the
CB1 stopped.
[0021] Many of the pathologies for which ortostheric antagonists of
the CB1 receptor have demonstrated good therapeutic efficacy are
still in need of new efficient therapies. There is consequently
need to develop methods that can allow to inhibit the CB1 receptor
without interfering with orthosteric binding and have less side
effects than orthosteric antagonists.
[0022] Therefore, there is still a need to develop ligands that
allow an inhibition of the CB1 receptors without modifying the
orthosteric binding or inducing adverse effects.
SUMMARY OF THE INVENTION
[0023] The present invention relates to a compound of formula (A)
or a pharmaceutical salt thereof:
##STR00001##
wherein: denotes that the bound is a single or a double bond, R1
denotes that C3 is substituted with [0024] --H, [0025] halogen,
[0026] --OH, [0027] C1-8 alkoxy, [0028] Bn-O-- [0029] Bn-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
amino, carboxyl or halogen, [0030] Ph- optionally substituted with
C1-8 alkyl, C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen,
[0031] .dbd.O, [0032] --NR5R6 wherein R5 and R6 each independently
is H, C1-8 alkyl, Bn or Ph, [0033] --O--CO--R7 wherein R7 is alkyl,
[0034] --O--CO--C.sub.2H.sub.4--COOH, or [0035] --N.sub.3, --R2
denotes that C17 is substituted with [0036] --H, [0037] --OH,
[0038] halogen, [0039] C1-8 alkyl, [0040] C1-8 alkoxy, [0041] C2-6
alkenyl, [0042] Bn optionally substituted with C1-8 alkyl, C1-8
alkoxy, cyano, nitro, amino, carboxyl or halogen, [0043] Ph-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
carboxyl or halogen, or [0044] Bn-O--, R3 denotes that C20 is
substituted with [0045] --H, [0046] --OH, [0047] C1-8 alkyl, [0048]
Bn, [0049] --NR8R9 wherein R8 and R9 each independently is H, C1-8
alkyl or Bn, [0050] .dbd.CR10R11 wherein R10 and R11 each
independently is H or C1-7 alkyl, or [0051] .dbd.O, R4 denotes that
C16 is substituted with [0052] --H, [0053] --OH, or [0054] .dbd.O,
with the proviso that [0055] when the bond between C16 and C17 is
double, R2 is absent and the bond between C17 and C20 is single,
and [0056] when the bond between C17 and C20 is double, C20 is
substituted with --H or --OH and R2 is absent, [0057] when the bond
between C4 and C5 is double, the bond between C5 and C6 is single
and inversely, for use in the treatment of a pathologic condition
or disorder selected from the group consisting of bladder and
gastrointestinal disorders; inflammatory diseases; cardiovascular
diseases; nephropathies; glaucoma; spasticity; cancer;
osteoporosis; metabolic disorders; obesity; addiction, dependence,
abuse and relapse related disorders; psychiatric and neurological
disorders; neurodegenerative disorders; autoimmune hepatitis and
encephalitis; pain; reproductive disorders and skin inflammatory
and fibrotic diseases.
[0058] The present invention also relates to a compound of formula
(II)
##STR00002##
or a pharmaceutical salt thereof, wherein: denotes that the bound
is a single or a double bond R1 denotes that C3 is substituted with
--OH, and --R2 denotes that C17 is substituted with [0059] C3-8
alkyl, [0060] C2-8 alkoxy, [0061] Bn- optionally substituted with
C1-8 alkyl, C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen,
[0062] Ph- optionally substituted with C1-8 alkyl, C1-8 alkoxy,
cyano, nitro, carboxyl or halogen, or [0063] Bn-O--, or wherein R1
denotes that C3 is substituted with [0064] C1-8 alkoxy, [0065]
Bn-O, or [0066] Halogen, and --R2 denotes that C17 is substituted
with [0067] C1-8 alkyl, [0068] C2-6 alkenyl, [0069] C1-8 alkoxy,
[0070] Bn- optionally substituted with C1-8 alkyl, C1-8 alkoxy,
cyano, nitro, amino, carboxyl or halogen, [0071] Ph- optionally
substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro, carboxyl,
or halogen, or
Bn-O--.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0072] "Agonist" refers to a compound that enhances the activity of
another compound or receptor site.
[0073] The terms "Antagonist" and "Inhibitor" refer to a compound
that diminishes or prevents the activity of another compound at a
receptor site and more generally refer to a compound that
diminishes or prevents the activation and/or the activity of a
receptor. "Treatment or treating" refers to both therapeutic
treatment and prophylactic or preventive measures, wherein the
object is to prevent or slow down the targeted pathologic condition
or disorder. Those in need of treatment include those already with
the disorder as well as those prone to have the disorder or those
in whom the disorder is to be prevented. Hence, the subject to be
treated herein may have been diagnosed as having the disorder or
may be predisposed or susceptible to the disorder.
[0074] As used herein, the term "subject" denotes a mammal, such as
a rodent, a feline, a canine, and a primate. Preferably a subject
according to the invention is a human.
[0075] A "therapeutically effective amount" is intended for a
minimal amount of active agent (e.g.,) which is necessary to impart
therapeutic or a preventive benefit to a subject. For example, a
"therapeutically effective amount" to a mammal is such an amount
which induces, ameliorates or otherwise causes an improvement in
the pathological symptoms, disease progression or physiological
conditions associated with or resistance to succumbing to a
disorder.
[0076] As used therein, the term "cancer" refers to or describes
the physiological condition in subjects that is typically
characterized by unregulated cell growth or death. Examples of
cancer include, but are not limited to, carcinoma, lymphoma,
blastoma, sarcoma, and leukemia or lymphoid malignancies. More
particular examples of such cancers include squamous cell cancer
(e.g. epithelial squamous cell cancer), lung cancer including
small-cell lung cancer, non-small cell lung cancer, adenocarcinoma
of the lung and squamous carcinoma of the lung, cancer of the
peritoneum, hepatocellular cancer, gastric or stomach cancer
including gastrointestinal cancer, pancreatic cancer, glioblastoma,
cervical cancer, ovarian cancer, liver cancer, bladder cancer,
hepatoma, breast cancer, colon cancer, rectal cancer, colorectal
cancer, endometrial or uterine carcinoma, salivary gland carcinoma,
kidney or renal cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as
well as head and neck cancer.
[0077] As used therein, the terms "addiction and dependence" refers
to behavioural alterations towards a reinforcing stimuli, including
but not limited to pharmacological compounds, food, sexual
partners, gambling, risk taking behaviors. This behavioral
alteration is characterized by one or all of the following
characteristics: 1. the inability of the individual to restrain
from consume, intake or be in contact with the above mentioned
stimuli, which results in consumptions of higher quantity of the
stimuli that originally intended and unsuccessful attempts to
discontinue these behaviors for prolonged periods of time; 2. a
strong motivation to obtain, consume, intake or be in contact with
the above mentioned stimuli which become the principal activity of
the subject and that can be associated with the neglecting of other
activities; 3. The appearance of a discomfort, physical or
psychological, at the discontinuation of the consumption or in the
absence of the reinforcing stimuli.
[0078] As used therein, the terms "abuse" refer to a physiological
condition in which the integrity of the organism of an individual
is permanently or transitorily impaired as a results of the
consumption, intake or being in contact with reinforcing stimuli,
including but not limited to, pharmacological compounds, food,
sexual partners, gambling. These impairments include but are not
limited to, cardiovascular complications, respiratory problems,
liver diseases, infection diseases, traumatic injuries. These
impairments of bodily integrity can be associated or not with the
behavioral manifestations that characterize addiction and
dependence as described above.
[0079] As used therein, the terms "relapse" refer to the
reinstatement of addiction, dependence or abuse after a period of
continuous restraint form the consumption, intake or being in
contact with reinforcing stimuli, including but not limited to,
pharmacological compounds, food, sexual partners, gambling.
[0080] As used therein, the terms "metabolic disorders" refer to a
physiological condition in which the normal levels of chemicals
used by the body as energetics or more generally as components that
are necessary for guaranteeing the structural or functional
integrity of the organisms are altered. These chemicals include but
are not limited to: glucides, lipids, aminoacids, and electrolytes.
The pathologies generally resulting from metaboic disorders include
but are not limited to diabetes and dislypidemia. Metabolic
disorder can also facilitate gastrointestinal and cardiovascular
diseases such as atherosclerosis, NASH and cirrhosis. Metabolic
disorders can be associated to obesity or be of idiopathic
nature.
[0081] "Pain" means the more or less localized sensation of
discomfort, distress, or agony, resulting from the stimulation of
specialized nerve endings. There are many types of pain, including,
but not limited to, lightning pains, phantom pains, shooting pains,
acute pain, inflammatory pain, neuropathic pain, complex regional
pain, neuralgia, neuropathy, and the like (Dorland's Illustrated
Medical Dictionary, 28th Edition, W. B. Saunders Company,
Philadelphia, Pa.). The goal of treatment of pain is to reduce the
degree of severity of pain perceived by a treatment subject.
[0082] Skin inflammatory and fibrotic diseases refer to pathologies
of the skin, idiopatic or induced by external agent including UV,
that results in an alteration of the skin, in skin cancer and/or in
a disruption of the wound healing process.
[0083] The other designation of pathologies used therein, including
but not limited to, osteoporosis, neurodegenerative diseases,
Parkinson, Alzheimer, schizophrenia, mood disorders, bladder and
gastrointestinal disorders; inflammatory diseases; cardiovascular
diseases; atherosclerosis, liver steatosis, NASH and cirrhosis
nephropathies; glaucoma; spasticity; autoimmune hepatitis and
encephalitis; reproductive disorders are used in within their
medical inception as defined in any manual of medicine.
[0084] The expression "Ci is substituted with X" means that the
carbon at position i of the chemical formula bears a substituent X,
which may be an atom, such as H or an halogen, or a functional
group.
[0085] "Alkyl" means monovalent linear or branched saturated
hydrocarbon moiety, consisting solely of carbon and hydrogen atoms.
C1-8 alkyl means a linear or branched alkyl having from one to
eight carbon atoms.
[0086] "Alkoxy" means a moiety of the formula --OR, wherein R is an
alkyl moiety as defined herein.
[0087] The term "halogen", refers to a fluorine, chlorine, bromine,
or iodine atom.
[0088] "Amino" means a moiety of the formula --NRR' wherein R and
R' each independently is hydrogen, or alkyl as defined herein.
[0089] The abbreviation Bn refers to a benzyl group.
[0090] The abbreviation Ph refers to a phenyl group.
[0091] Substituents above the plane of the molecule are shown as a
solid line () and are described as .beta.; those below the plane
are shown by a broken line () and are described as .alpha..
[0092] "Optionally" means that the subsequently described event or
circumstance may but need not occur, and that the description
includes instances where the event or circumstance occurs and
instances in which it does not.
Inhibition of CB1 Receptor
[0093] The present invention relates to a compound of formula (A)
or a pharmaceutical salt thereof:
##STR00003##
wherein: denotes that the bound is a single or a double bond, R1
denotes that C3 is substituted with [0094] --H, [0095] halogen,
[0096] OH, [0097] C1-8 alkoxy, [0098] Bn-O-- [0099] Bn- optionally
substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro, amino,
carboxyl or halogen, [0100] Ph- optionally substituted with C1-8
alkyl, C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen,
[0101] .dbd.O, [0102] --NR5R6 wherein R5 and R6 each independently
is H, C1-8 alkyl, Bn or Ph, [0103] --O--CO--R7 wherein R7 is alkyl,
[0104] --O--CO--C.sub.2H.sub.4--COOH, or [0105] --N.sub.3, --R2
denotes that C17 is substituted with [0106] --H, [0107] --OH,
[0108] halogen, [0109] C1-8 alkyl, [0110] C1-8 alkoxy, [0111] C2-6
alkenyl, [0112] Bn optionally substituted with C1-8 alkyl, C1-8
alkoxy, cyano, nitro, amino, carboxyl or halogen, [0113] Ph-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
carboxyl or halogen, or [0114] Bn-O--, R3 denotes that C20 is
substituted with [0115] --H, [0116] --OH, [0117] C1-8 alkyl, [0118]
Bn, [0119] --NR8R9 wherein R8 and R9 each independently is H, C1-8
alkyl or Bn, [0120] .dbd.CR10R11 wherein R10 and R11 each
independently is H or C1-7 alkyl, or [0121] .dbd.O, R4 denotes that
C16 is substituted with [0122] --H, [0123] --OH, or [0124] .dbd.O,
with the proviso that [0125] when the bond between C16 and C17 is
double, R2 is absent and the bond between C17 and C20 is single,
and [0126] when the bond between C17 and C20 is double, C20 is
substituted with --H or --OH and R2 is absent, [0127] when the bond
between C4 and C5 is double, the bond between C5 and C6 is single
and inversely, for use in the treatment of a pathologic condition
or disorder selected from the group consisting of bladder and
gastrointestinal disorders; inflammatory diseases; cardiovascular
diseases; nephropathies; glaucoma; spasticity; cancer;
osteoporosis; metabolic disorders; obesity; addiction, dependence,
abuse and relapse related disorders; psychiatric and neurological
disorders; neurodegenerative disorders; autoimmune hepatitis and
encephalitis; pain; reproductive disorders and skin inflammatory
and fibrotic diseases.
[0128] Indeed, the inventors have shown that pregnenolone and some
of its derivatives are inhibitors of the CB1 receptor and are able
to block the activation of the CB1 receptor induced by natural or
synthetic agonists or endogenous ligands without modifying
orthosteric binding.
[0129] Therefore, the compounds of the invention act similarly to
other antagonists of CB1 receptor such as rimonabant and may be
used in the treatment of pathologies wherein an antagonist of CB1
receptor is required.
[0130] Furthermore, this inhibition is an endogenous mechanism.
Then, it modifies the activity of the receptor in a more
physiological way modulating the response of the receptor to
endogenous or exogenous agonists and not blocking the binding of
the agonists to the receptor. Because of this more physiological
mechanism inhibitors such as pregenolone and its derivatives are
susceptible to presents less side-effect than ortostheric
antagonists.
[0131] Examples of bladder and gastrointestinal disorders that may
be treated with an antagonist of CB1 receptor include but are not
limited to liver fibrosis; liver steatosis; non alcoholic
steatohepatitis (NASH), liver cirrhosis; alcoholic steatosis;
hepatic ischemic reperfusion injury complicated by endotoxaemia;
acute pancreatitis; overactive and painful bladder disorders and
motility alteration of contractile visceral organs.
[0132] Examples of inflammatory diseases that may be treated with
an antagonist of CB1 receptor include but are not limited to
inflammation and arthritis associated with obesity; chronic-immune
inflammatory diseases and ulcer.
[0133] Examples of cardiovascular diseases that may be treated with
an antagonist of CB1 receptor include but are not limited to
cardiomiopathy such as cirrotic cardiomiopathy, anti-neoplastic
drugs induced cardiomiopathy, endothelial dysfunction and cell
death involved in the development of vascular dysfunction
associated with congestive heart failure; hypertension; coronary
artery disease; atherosclerosis; myocardial infarction; diseases
resulting from lipid accumulation such as atherosclerosis,
pathologies derived by increased angiogenesis and diseases
involving angiogenesis.
[0134] Examples of metabolic disorders that may be treated with an
antagonist of CB1 receptor include but are not limited to
dyslipidemia, diabetes and diabetic complications.
[0135] Examples of addiction, dependence, abuse and relapse related
disorders that may be treated with an antagonist of CB1 receptor
include but are not limited to drug dependence; drug abuse; relapse
in drug dependence, abuse and addiction; cannabis and derived
products use; cannabis and derived products abuse; cannabis and
derived products toxicity; cannabis and derived products induced
psychosis.
[0136] Examples of neurodegnerative disorders that may be treated
with an antagonist of CB1 receptor include but are not limited to
Parkinson and Alzheimer
[0137] Examples of psychiatric and neurological disorders that may
be treated with an antagonist of CB1 receptor include but are not
limited to schizophrenia; mood disorders; L-DOPA induced
dyskinesia; memory disorders.
[0138] Examples of reproductive disorders that may be treated with
an antagonist of CB1 receptor include but are not limited to
infertility and recurrent abortion.
[0139] Example of skin inflammatory and fibrotic diseases that may
be treated with an antagonist of CB1 receptor include but are not
limited to skin inflammation, skin inflammation and cancer induced
by UV, skin fibrosis and wound healing.
[0140] The invention relates to a method for the treatment of a
pathologic condition or disorder selected from the group consisting
of bladder and gastrointestinal disorders; inflammatory diseases;
cardiovascular diseases; nephropathies; glaucoma; spasticity;
cancer; osteoporosis; metabolic disorders; obesity; addiction,
dependence, abuse and relapse related disorders; psychiatric and
neurological disorders; neurodegenerative disorders; autoimmune
hepatitis and encephalitis; pain; reproductive disorders and skin
inflammatory and fibrotic diseases
in a subject in need thereof comprising administering to said
subject an effective amount of a compound of formula (A) or a
pharmaceutical salt thereof:
##STR00004##
wherein: denotes that the bound is a single or a double bond, R1
denotes that C3 is substituted with [0141] --H, [0142] halogen,
[0143] --OH, [0144] C1-8 alkoxy, [0145] Bn-O-- [0146] Bn-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
amino, carboxyl or halogen, [0147] Ph- optionally substituted with
C1-8 alkyl, C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen,
[0148] .dbd.O, [0149] --NR5R6 wherein R5 and R6 each independently
is H, C1-8 alkyl, Bn or Ph, [0150] --O--CO--R7 wherein R7 is alkyl,
[0151] --O--CO--C.sub.2H.sub.4--COOH, or [0152] --N.sub.3, --R2
denotes that C17 is substituted with [0153] --H, [0154] --OH,
[0155] halogen, [0156] C1-8 alkyl, [0157] C1-8 alkoxy, [0158] C2-6
alkenyl, [0159] Bn optionally substituted with C1-8 alkyl, C1-8
alkoxy, cyano, nitro, amino, carboxyl or halogen, [0160] Ph-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
carboxyl or halogen, or [0161] Bn-O--, R3 denotes that C20 is
substituted with [0162] --H, [0163] --OH, [0164] C1-8 alkyl, [0165]
Bn, [0166] --NR8R9 wherein R8 and R9 each independently is H, C1-8
alkyl or Bn, [0167] .dbd.CR10R11 wherein R10 and R11 each
independently is H or C1-7 alkyl, or [0168] .dbd.O, R4 denotes that
C16 is substituted with [0169] --H, [0170] --OH, or [0171] .dbd.O,
with the proviso that [0172] when the bond between C16 and C17 is
double, R2 is absent and the bond between C17 and C20 is single,
and [0173] when the bond between C17 and C20 is double, C20 is
substituted with --H or --OH and R2 is absent, [0174] when the bond
between C4 and C5 is double, the bond between C5 and C6 is single
and inversely.
[0175] The invention relates to use of a compound of formula (A) or
a pharmaceutical salt thereof:
##STR00005##
wherein: denotes that the bound is a single or a double bond, R1
denotes that C3 is substituted with [0176] --H, [0177] halogen,
[0178] --OH, [0179] C1-8 alkoxy, [0180] Bn-O-- [0181] Bn-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
amino, carboxyl or halogen, [0182] Ph- optionally substituted with
C1-8 alkyl, C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen,
[0183] .dbd.O, [0184] --NR5R6 wherein R5 and R6 each independently
is H, C1-8 alkyl, Bn or Ph, [0185] --O--CO--R7 wherein R7 is alkyl,
[0186] --O--CO--C.sub.2H.sub.4--COOH, or [0187] --N.sub.3, --R2
denotes that C17 is substituted with [0188] --H, [0189] --OH,
[0190] halogen, [0191] C1-8 alkyl, [0192] C1-8 alkoxy, [0193] C2-6
alkenyl, [0194] Bn optionally substituted with C1-8 alkyl, C1-8
alkoxy, cyano, nitro, amino, carboxyl or halogen, [0195] Ph-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
carboxyl or halogen, or [0196] Bn-O--. R3 denotes that C20 is
substituted with [0197] --H, [0198] --OH, [0199] C1-8 alkyl, [0200]
Bn, [0201] --NR8R9 wherein R8 and R9 each independently is H, C1-8
alkyl or Bn, [0202] .dbd.CR10R11 wherein R10 and R11 each
independently is H or C1-7 alkyl, or [0203] .dbd.O, R4 denotes that
C16 is substituted with [0204] --H, [0205] --OH, or [0206] .dbd.O,
with the proviso that [0207] when the bond between C16 and C17 is
double, R2 is absent and the bond between C17 and C20 is single,
and [0208] when the bond between C17 and C20 is double, C20 is
substituted with --H or --OH and R2 is absent, [0209] when the bond
between C4 and C5 is double, the bond between C5 and C6 is single
and inversely, for the preparation of a medicament for the
treatment of a pathologic condition or disorder selected from the
group consisting of bladder and gastrointestinal disorders;
inflammatory diseases; cardiovascular diseases; nephropathies;
glaucoma; spasticity; cancer; osteoporosis; metabolic disorders;
obesity; addiction, dependence, abuse and relapse related
disorders; psychiatric and neurological disorders;
neurodegenerative disorders; autoimmune hepatitis and encephalitis;
pain; reproductive disorders and skin inflammatory and fibrotic
diseases.
[0210] The present invention also relates to a compound of the
invention, the compound being of formula (I) or a pharmaceutical
salt thereof:
##STR00006## [0211] wherein: [0212] denotes that the bound is a
single or a double bond, [0213] R1 denotes that C3 is substituted
with [0214] --H, [0215] halogen, [0216] --OH, [0217] C1-8 alkoxy,
[0218] Bn-O-- [0219] Bn- optionally substituted with C1-8 alkyl,
C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen, [0220] Ph-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
amino, carboxyl or halogen, [0221] .dbd.O, [0222] --NR5R6 wherein
R5 and R6 each independently is H, C1-8 alkyl, Bn or Ph, [0223]
--O--CO--R7 wherein R7 is alkyl, or [0224] --O--CO--C2H4-COOH,
[0225] --R2 denotes that C17 is substituted with [0226] --H, [0227]
--OH, [0228] halogen, [0229] C1-8 alkyl, [0230] C1-8 alkoxy, [0231]
C2-6 alkenyl, [0232] Bn optionally substituted with C1-8 alkyl,
C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen, [0233] Ph-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
carboxyl or halogen, or [0234] Bn-O--. [0235] R3 denotes that C20
is substituted with [0236] --H, [0237] --OH, [0238] C1-8 alkyl,
[0239] Bn, [0240] --NR8R9 wherein R8 and R9 each independently is
H, C1-8 alkyl or Bn, [0241] .dbd.CR10R11 wherein R10 and R11 each
independently is H or C1-7 alkyl, or [0242] .dbd.O, [0243] R4
denotes that C16 is substituted with [0244] --H, [0245] --OH, or
[0246] .dbd.O, [0247] with the proviso that [0248] when the bond
between C16 and C17 is double, R2 is absent and the bond between
C17 and C20 is single, and [0249] when the bond between C17 and C20
is double, C20 is substituted with --H or --OH and R2 is absent,
for use in the treatment of a pathologic condition or disorder
selected from the group consisting of bladder and gastrointestinal
disorders; inflammatory diseases; cardiovascular diseases;
nephropathies; glaucoma; spasticity; cancer; osteoporosis;
metabolic disorders; obesity; addiction, dependence, abuse and
relapse related disorders; psychiatric and neurological disorders;
neurodegenerative disorders; autoimmune hepatitis and encephalitis;
pain; and reproductive disorders and skin inflammatory and fibrotic
diseases.
[0250] The invention also relates to a pharmaceutical composition
comprising a compound of the invention or a pharmaceutical salts
thereof and a pharmaceutically acceptable carrier.
[0251] "Pharmaceutical" or "pharmaceutically acceptable" refers to
molecular entities and compositions that do not produce an adverse,
allergic or other untoward reaction when administered to a mammal,
especially a human, as appropriate. A pharmaceutically acceptable
carrier refers to a non-toxic solid, semi-solid or liquid filler,
diluent, encapsulating material or formulation auxiliary of any
type.
[0252] A pharmaceutically acceptable carrier refers but without
being limited to a non-toxic solid, semi-solid or liquid filler,
diluent, binding agent, disintegrating agent, dissolving agent,
stabilizing agent, salt forming agent, lubricating agent and
encapsulating material or formulation auxiliary of any type.
[0253] The form of the pharmaceutical compositions, the route of
administration, the dosage and the regimen naturally depend upon
the condition to be treated, the severity of the illness, the age,
weight, and sex of the patient, etc.
[0254] The pharmaceutical compositions of the invention can be
formulated for any route of administration including, but without
being limited, oral, intravenous, intramuscular, intraarterial,
intramedullary, intratechal, transdermal, topical, subcutaneous,
intraperitoneal, intranasal, enteral, sublingual, vaginal and
rectal.
[0255] Preferably according to the invention, the active ingredient
is administered by oral route and be presented as a unit dosage
form such as solid dosage form. This unit dosage form could be
either tablets, coated tablets, pills, powders or granules, sachets
or hard gel capsule in order to facilitate product administration
to adult or children.
[0256] For oral administration, in any conventional dosage form,
the compositions are prepared by a classical technique with
pharmaceutically acceptable excipients including, but without being
limited, binding agents (e.g. pregelatinized maize starch,
polyvinylpyrrolidone hydroxypropyl methylcellulose, sodium
carboxymethyl cellulose, gum derivatives as guar gum, carrageenan,
alginic acid or its salt, . . . ); fillers agents (e.g. lactose,
saccharose, microcrystalline cellulose, calcium hydrogen phosphate,
di calcium phosphate, polyol as mannitol, sorbitol or xylitol,
fructose, dextrin, maltodextrin . . . ); lubricants (e.g.,
magnesium stearate, sodium sterayl fumarate, talc or silica);
disintegrating agents (e.g potato starch or sodium starch
glycollate, crospovidone, croscarmellose . . . ), salt forming
agent (e.g n-methyl glucamine, sodium hydroxyde, potassium
hydroxide or chlorydric acid . . . ), or wetting agents (e.g.,
sodium lauryl sulphate). The tablets or hard gel capsules can be
coated by methods known in the art. For example, the tablet or hard
gel capsule can have an enteric or delayed-release coating which
protects the active ingredient until it reaches the colon.
[0257] Another possibility is to sustain release or control release
the active ingredient in order to deliver it during a long period
of time (maximum 24 hours) and limit the number of administration
per day. The tablets or hard gel capsules can be coated with
polymer which ensure the control release of active ingredient or in
the case of tablet, they can be matrixes tablet said tablets
present in their composition components which ensure the control
release of active ingredient; such ingredient are generally,
without being limited to, hydrophilic polymers (e.g.
hydroxypropylmethylcellulose, carboxymethyl cellulose sodium,
xanthan gum, chitosan, polyethylene oxide . . . ) water-insoluble
and hydrophobic polymers (e.g. ethylcellulose, poly(vinylacetate)
cellulose acetate . . . ), fatty acids (e.g. hydrogenated vegetable
oil, glyceryl palmitosterate . . . ), alcohol (e.g., cetyl alcohol,
stearyl alcohol . . . ), waxes (e.g. bees' wax, carnauba wax . . .
). In the specific case of pregnenolone the sustained release
formulation will also serve the function to reduce the production
of downstream active metabolites of pregnenolone.
[0258] When a solid composition in tablet form is prepared, the
tablet can be manufactured either by direct compression, wet
granulation process or dry granulation process. The active
ingredient whatever the manufacturing process used is first mixed
with the entire or a part of the vehicles described above, then
lubricated before being compressed into tablet.
[0259] In another embodiment the present invention could be useful
for buccal delivery of active ingredient; the compositions can take
the form of tablets or lozenges formulated in conventional manner.
The ingredients used are the same for conventional tablet; only the
proportions between ingredients change.
[0260] Preferably, the active ingredient could be delivered only
for local activity; the tablets are presented as muccoadhesive
tablets.
[0261] On the other hand, the active ingredient could be delivered
directly in the mouth, but for systemic absorption, when water is
not available, the dosage form is an orally disintegrating tablet
which presents the advantage to be administered without water . . .
.
[0262] In another embodiment, the present invention could be also
presented as liquid preparations for oral administration. They can
take the form of, for example, solutions, syrups or suspensions, or
they can be presented as a dry product for constitution with water
or other suitable vehicle before use. These pharmaceutical forms
could be either unit dosage form as ampoule or multi dose form
generally filled in vials. Such liquid preparations can be prepared
by conventional techniques with pharmaceutically acceptable
additives including without being limited to, suspending agents
(e.g. sorbitol or manitol syrup, saccharose syrup, cellulose
derivatives as sodium carboxymethyl cellulose or
hydroxypropylcellulose, gum derivatives as guar gum, xanthan gum or
acacia gum . . . or hydrogenated edible fats); emulsifying agents
(e.g. lecithin, polysorbate, polyoxyethylated castor oil, sorbitan
ester or ploxamer . . . ); aqueous and non-aqueous vehicles (e.g
water, monopropylene glycol, polyethylene glycol, glycerol, sesame
oil, cottonseed oil, soybean oil, castor oil, almond oil, oily
esters, ethyl alcohol or fractionated vegetable oils, medium chain
triglycerides . . . ); and preservatives (e.g., generally, methyl
or propyl-p-hydroxybenzoates and their salts, sorbic acid and its
salts, benzoic acid and its salts). The preparations can also
contain buffer salts, stabilizing agent, antioxidant agent,
flavouring, colouring, and sweetening agents as appropriate.
[0263] Another administration route suitable for the invention is
the parenteral route. The active ingredient will be presented as an
injectable product suitable for intravenous route, intramuscular
route or subcutaneous route; the pharmaceutical compositions may
contain vehicles which are pharmaceutically acceptable for a
formulation capable of being injected. These compositions could be
presented as a solution or an emulsion (for example fine emulsion,
microemulsion or nanoemulsion . . . ) and may be sterile. Such
composition could contain saline components (monosodium or disodium
phosphate, sodium, potassium, calcium or magnesium chloride and the
like or mixtures of such salts) in order to be isotonic. In some
cases, when active ingredient is not sufficiently stable to be
presented directly as a solution, the active ingredient is
presented dry, either as a freeze-dried compositions or as powder
form which upon addition, depending on the case, of sterilized
non-aqueous solution suitable for injection, sterilized water or
physiological saline solution, permit the constitution of
injectable solutions suitable for being administered to the
patient.
[0264] The final product is generally presented filled in a vial or
in an ampoule form.
[0265] The doses used for the administration can be adapted as a
function of various parameters, and in particular as a function of
the mode of administration used, of the relevant pathology, or
alternatively of the desired duration of treatment.
[0266] In order to maintain systemic drug level, and in order to
avoid frequent injection, depot-type parenteral formulation could
be used. These pharmaceutical forms are generally, without being
limited to, in the form of microparticles, implants or a liquid
that form in situ a gel or colloid or semi-solid depot after
injection. Such depot formulation can be prepared by conventional
techniques with pharmaceutically acceptable additives including
without being limited to, biocompatible and biodegradable polymers
(e.g. poly(.epsilon.-caprolactone), poly(ethylene oxide),
poly(glycolic acid), poly[(lactic acid)-co-(glycolic acid) . . .
)], poly(lactic acid) . . . ), non-biodegradable polymers (e.g.
ethylene vinylacetate copolymer, polyurethane, polyester(amide),
polyvinyl chloride . . . ) aqueous and non-aqueous vehicles (e.g
water, sesame oil, cottonseed oil, soybean oil, castor oil, almond
oil, oily esters, ethyl alcohol or fractionated vegetable oils,
propylene glycol, DMSO, THF, 2-pyrrolidone. N-methylpyrrolidinone,
N-vinylpyrrolidinone . . . ). In the specific case of pregnenolone
depot formulation will also serve the function to decrease the
production of active downstream metabolite of pregnenolone.
[0267] To prepare pharmaceutical compositions, an effective amount
of the compounds of the invention may be dissolved or dispersed in
a pharmaceutically acceptable carrier or aqueous medium.
[0268] The liquid pharmaceutical forms suitable for injectable use
include, without being limited to, sterile aqueous solutions or
dispersions; or non aqueous formulations including sterile oily
components such as sesame oil, peanut oil, cottonseed medium chain
triglycerides, triacetine oil, sterile propylene glycol, sterile
polyethylene glycol, sterile glycerol or sterile polyol
solution.
[0269] Solutions of the active compounds as free base or
pharmacologically acceptable salts can be prepared in a suitable
solvent, mixed if necessary with solubilizing agent generally as
surfactant, such as, without being limited to, polysorbate
derivatives, polyethoxylated castor oil (cremophor RH40 for example
. . . ), PEG 15 hydroxystearate (solutol HS15), poloxamer (as
lutrol F68), with stabilizing agent for example EDTA and its salts,
with buffering agent or with antioxidant agent (ascorbic acid and
its salt, tocopherol acetate or sodium metabisulfite). Said
preparation can contain preservative agent to prevent the growth of
microorganisms.
[0270] For stability reason the formulation could be presented as a
powder form said powder is sterile and is extemporaneously
solubilized by an aqueous solvent or a non-aqueous solvent. Said
preparation is generally for the extemporaneous administration of
sterile injectable solutions or dispersions.
[0271] In all cases, the form must be sterile and stable under the
conditions of use, manufacture and storage and must be preserved
against the contaminating action of microorganisms, such as
bacteria and fungi.
Compounds of the Invention
[0272] General Formulas:
[0273] The compounds of the invention have the formula:
##STR00007##
wherein: denotes that the bound is a single or a double bond, R1
denotes that C3 is substituted with [0274] --H, [0275] halogen,
[0276] --OH, [0277] C1-8 alkoxy, [0278] Bn-O-- [0279] Bn-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
amino, carboxyl or halogen, [0280] Ph- optionally substituted with
C1-8 alkyl, C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen,
[0281] .dbd.O, [0282] --NR5R6 wherein R5 and R6 each independently
is H, C1-8 alkyl, Bn or Ph, [0283] --O--CO--R7 wherein R7 is alkyl,
[0284] --O--CO--C2H.sub.4--COOH, or [0285] --N.sub.3, --R2 denotes
that C17 is substituted with [0286] --H, [0287] --OH, [0288]
halogen, [0289] C1-8 alkyl, [0290] C1-8 alkoxy, [0291] C2-6
alkenyl, [0292] Bn optionally substituted with C1-8 alkyl, C1-8
alkoxy, cyano, nitro, amino, carboxyl or halogen, [0293] Ph-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
carboxyl or halogen, or [0294] Bn-O--, R3 denotes that C20 is
substituted with [0295] --H, [0296] --OH, [0297] C1-8 alkyl, [0298]
Bn, [0299] --NR8R9 wherein R8 and R9 each independently is H, C1-8
alkyl or Bn, [0300] .dbd.CR10R11 wherein R10 and R11 each
independently is H or C1-7 alkyl, or [0301] .dbd.O, R4 denotes that
C16 is substituted with [0302] --H, [0303] --OH, or [0304] .dbd.O,
with the proviso that [0305] when the bond between C16 and C17 is
double. R2 is absent and the bond between C17 and C20 is single,
and [0306] when the bond between C17 and C20 is double, C20 is
substituted with --H or --OH and R2 is absent, [0307] when the bond
between C4 and C5 is double, the bond between C5 and C6 is single
and inversely.
[0308] In one embodiment, the compounds of the invention have the
formula (I):
##STR00008##
wherein: denotes that the bound is a single or a double bond, R1
denotes that C3 is substituted with [0309] --H, [0310] halogen,
[0311] --OH, [0312] C1-8 alkoxy, [0313] Bn-O-- [0314] Bn-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
amino, carboxyl or halogen, [0315] Ph- optionally substituted with
C1-8 alkyl, C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen,
[0316] .dbd.O, [0317] --NR5R6 wherein R5 and R6 each independently
is H, C1-8 alkyl, Bn or Ph, [0318] --O--CO--R7 wherein R7 is alkyl,
or [0319] --O--CO--C.sub.2H.sub.4--COOH, --R2 denotes that C17 is
substituted with [0320] --H, [0321] --OH, [0322] halogen, [0323]
C1-8 alkyl, [0324] C1-8 alkoxy, [0325] C2-6 alkenyl, [0326] Bn
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
amino, carboxyl or halogen, [0327] Ph- optionally substituted with
C1-8 alkyl, C1-8 alkoxy, cyano, nitro, carboxyl or halogen, or
[0328] Bn-O--, R3 denotes that C20 is substituted with [0329] --H,
[0330] --OH, [0331] C1-8 alkyl, [0332] Bn, [0333] --NR8R9 wherein
R8 and R9 each independently is H, C1-8 alkyl or Bn, [0334]
.dbd.CR10R11 wherein R10 and R11 each independently is H or C1-7
alkyl, or [0335] .dbd.O, R4 denotes that C16 is substituted with
[0336] --H, [0337] --OH, or [0338] .dbd.O, with the proviso that
[0339] when the bond between C16 and C17 is double. R2 is absent
and the bond between C17 and C20 is single, and [0340] when the
bond between C17 and C20 is double, C20 is substituted with --H or
--OH and R2 is absent.
[0341] Pregnenolone:
[0342] In a particular embodiment, the compound of the invention is
pregnenolone or pharmaceutical salt thereof.
[0343] Pregnenolone is a well-known steroid (CAS number 145-13-1).
It is the first step of steroid synthesis in the brain and other
organs.
[0344] As disclosed above, the inventors have shown that
pregnenolone and its pharmaceutical salts such as pregnenolone
acetate or hemisuccinate are inhibitors of CB1 receptor and then
may be used in the treatment of pathologic disorders and diseases
wherein an antagonist of CB1 receptor is required.
[0345] In a preferred embodiment pregnenolone is administered to a
subject in a dosage such that the plasmatic concentration of
pregnenolone in the subject does not exceed 100 ng/ml. Preferably,
the pregnenolone is administered by sustained released
formulation.
[0346] Indeed, when administered at low doses that allow
pregnenolone to be in the range of effective doses (around 100
ng/ml or 100 ng/g of tissue) the conversion of pregnenolone in
downstream active metabolite is diminished. Thus the inventors have
shown that pregnenolone administered at low concentrations that do
not induce the increase of downstream active metabolite is able to
inhibit the effect of the activation of the CB1 receptor. This is a
major difference and innovation compared to previous documents in
which pregnenolone is administered at high dose in order to
increase downstream active metabolite to which the observed
therapeutic effects have been attributed. The administration of
pregnenolone at low doses is advantageous because it allow to act
on CB1-dependent pathologies without the unwanted and undesiderable
effects due to the increase of downstream active steroids
derivatives of pregnenolone that are endowed with progestative,
androgenic, estrogenic, glucocorticoid activity, or neuromodulatory
properties as in the case of others brain steroids derived from
pregnenolone, including but not limited to allopregnanolone,
Testoterone, DHEA.
[0347] Compounds with No or Low Metabolization
[0348] Alternatively, the compound of the invention is not
substantially converted into active pregnenolone downstream
derivatives after administration to a subject.
[0349] Pregnenolone is generally considered an inactive precursor
of downstream active steroids. Active pregnenolone down stream
derivatives including but not limited to pregnenolone-sulphate,
allopregnanolone, DHEA, DHEA-sulfate, have been involved in the
regulation of various behavioural functions.
[0350] However, the inventors have shown that inhibition of CB1
receptor is specific of pregnenolone and does not involve active
downstream pregnenolone derivatives.
[0351] Using derivatives of pregnenolone that are not or not
substantially converted into pregnenolone metabolites avoids side
effects that may be related with metabolites whose pregnenolone is
precursor and that are endowed with progestative, androgenic,
estrogenic, glucocorticoid activity, or neuromodulatory properties
as in the case of others brain steroids derived from pregnenolone,
including but not limited to allopregnanolone, Testosterone,
DHEA.
[0352] The capacity of a compound of the invention to be or not to
be converted into active pregnenolone downstream derivatives may be
evaluated by administering this compound, for example by injecting
50 mg/kg, to a rat, sacrificing the rat 30 min later, measuring the
concentration of allopregnanolone and epiallopregnanolone in
nucleus accumbens of rat by GC/MS and comparing these
concentrations to allopregnanolone and epiallopregnanolone in a rat
whose has been injected only a vehicule or pregnenolone.
[0353] Alternatively the compound can be administered to any cell
line expressing the enzyme that metabolizes pregnenolone in
culture, measuring then the content of allopregnanolone and
epiallopregnanolone within the cell or on the cell culture medium
by GC/MS and comparing these concentrations to allopregnanolone and
epiallopregnanolone in cell cultures that have been received only a
vehicule or pregnenolone
[0354] In one embodiment, the compound of the invention is a
compound of formula I wherein
R1 denotes that C3 is substituted with [0355] --H, -halogen, --OH,
C2-8 alkoxy, Bn-O--, [0356] Bn- optionally substituted with C1-8
alkyl, C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen,
[0357] Ph- optionally substituted with C1-8 alkyl, C1-8 alkoxy,
cyano, nitro, amino, carboxyl or halogen, [0358] .dbd.O, [0359]
--NR5R6 wherein R5 and R6 each independently is H, C1-8 alkyl, Bn
or Ph, [0360] --O--CO--R7 wherein R7 is alkyl, or [0361] --O--CO--
C.sub.2H.sub.4--COOH and --R2, R3, R4 are as defined
previously.
[0362] The inventors have tested a wide range of derivatives of
pregnenolone to find derivatives of pregnenolone that were not
substantially converted into active pregnenolone downstream
derivatives after administration to a subject as they keep an
inhibitor activity on CB1.
[0363] Several of group of derivatives have been found:
[0364] Bonds Between C16 and C17 and C17 and C20 are Single
Bonds.
[0365] In one embodiment, the bond between C16 and C17 and the bond
between C17 and C20 are single bonds.
[0366] Bond Between C4 and C5 is a Double Bond.
[0367] In one embodiment, the bonds between C3 and C4 and C5 and C6
are single bonds and the bond between C4 and C5 is double.
[0368] In this embodiment, the compound of the invention has the
formula B:
##STR00009## [0369] wherein: [0370] R1 denotes that C3 is
substituted with --OH or .dbd.O, [0371] --R2 denotes that C17 is
substituted with --H, --OH, C1-8 alkyl, halogen or Bn, [0372] R3
denotes that C20 is substituted with --OH or .dbd.O, [0373] R4
denotes that C16 is substituted with --H,
[0374] Indeed, the inventors have found that derivatives of
pregnenolone with a double bond between C4 and C5 and substituted
in R1, R2 and/or R3 as disclosed above were not metabolized in
down-stream derivatives of pregnenolone.
[0375] Preferably, the compound is selected from the group
consisting of 4-pregnen-17.alpha.,20.alpha.-diol-3-one,
4-pregnen-3.beta.,20.alpha.-diol, 4-pregnen-20.alpha.-ol-3-one,
17.alpha.-methylprogesterone and 17.alpha.-benzylprogesterone.
[0376] Bonds Between C5 and C6 and Between C4 and C5 are Single
Bonds
[0377] In one embodiment, the bonds between C3 and C4, C5 and C6
and between C4 and C5 are single bonds.
[0378] In this embodiment, the compound of this invention has the
formula (C)
##STR00010##
wherein: R1 denotes that C3 is substituted with .dbd.O or --OH --R2
denotes that C17 is substituted with --H R3 denotes that C20 is
substituted with .dbd.O, and R4 denotes that C16 is substituted
with --H,
[0379] Preferably, said compound is 5.beta.-pregnan-3,20-dione or
5.beta.-pregnan-3.beta.-ol-20-one
[0380] Bond Between C5 and C6 is a Double Bond
[0381] In one embodiment, the bonds between C3 and C4 and C4 and C5
are single and the bond between C5 and C6 is double bond.
[0382] In this embodiment, the compound of the invention has the
formula (D):
##STR00011##
[0383] In this embodiment, the compound of the invention is
preferably a pregnenolone modified at C3, C17 and/or C20.
[0384] Modification at C3:
[0385] In one embodiment, the compound is a pregnenolone modified
at C3.
[0386] In this embodiment, the compound has the formula (D)
and R1 denotes that C3 is substituted with [0387] --H, -halogen,
C1-8 alkoxy, Bn-O--, [0388] Bn- optionally substituted with C1-8
alkyl, C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen,
[0389] Ph- optionally substituted with C1-8 alkyl, C1-8 alkoxy,
cyano, nitro, amino, carboxyl or halogen, [0390] .dbd.O, [0391]
--NR5R6 wherein R5 and R6 each independently is H, C1-8 alkyl, Bn
or Ph, [0392] --O--CO--R7 wherein R7 is alkyl, or [0393]
--O--CO--C.sub.2H.sub.4--COOH, --R2 denotes that C17 is substituted
with --H R3 denotes that C20 is substituted with .dbd.O, and R4
denotes that C16 is substituted with --H,
[0394] Also, in this this embodiment, the compound has the formula
(D) and
R1 denotes that C3 is substituted with [0395] Halogen, Bn-O or,
--N.sub.3, --R2 denotes that C17 is substituted with --H, R3
denotes that C20 is substituted with .dbd.O, and R4 denotes that
C16 is substituted with --H,
[0396] Preferably, the compound of this embodiment is selected from
the group consisting of 3.beta.-benzyloxypregnenolone,
3-azidopregnenolone, and 3.beta.-fluoropregnenolone.
[0397] Modification at C3 and C17:
[0398] In one embodiment, the compound of the invention is a
pregnenolone modified at C3 and at C17.
[0399] In this embodiment, the compound has the formula (D) and
R1 denotes that C3 is substituted with C1-8 alkoxy, halogen, Bn-O--
or N.sub.3 --R2 denotes that C17 is substituted with Bn, --CH.sub.3
or C2-6 alkenyl, R3 denotes that C20 is substituted with .dbd.O,
and R4 denotes that C16 is substituted with --H,
[0400] Preferably, the compound of this embodiment is selected from
the group consisting of
3.beta.-fluoro-17.alpha.-methylpregnenolone,
17.alpha.-benzyl-3.beta.-fluoropregnenolone,
17.alpha.-benzyl-3.beta.-benzyloxypregnenolone and
3.beta.-benzyloxy-17.alpha.-methylpregnenolone.
[0401] Modification at C17:
[0402] In one embodiment, the compound is a pregnenolone modified
at C17 only.
[0403] In this embodiment, the compound has the formula (D)
and:
R1 denotes that C3 is substituted with --OH --R2 denotes that C17
is substituted with [0404] --OH, halogen, C1-8 alkyl, C1-8 alkoxy,
C2-6 alkenyl, [0405] Bn- optionally substituted with C1-8 alkyl,
C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen, [0406] Ph-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
carboxyl or halogen, or [0407] Bn-O--, R3 denotes that C20 is
substituted with .dbd.O, and R4 denotes that C16 is substituted
with --H,
[0408] Preferably, --R2 denotes that C17 is substituted with C1-8
alkyl, C1-8 alkoxy or Bn-.
[0409] More preferably, said compound is selected from the group
consisting of 17.alpha.-methylpregnenolone.
17.alpha.-benzylpregnenolone, 17-methoxypregnenolone and
17.alpha.-ethylpregnenolone.
[0410] Modification at C3 and/or C17
[0411] The present invention also relates to a compound of formula
(II)
##STR00012##
or a pharmaceutical salt thereof, wherein: denotes that the bound
is a single or a double bond, R1 denotes that C3 is substituted
with --OH, and --R2 denotes that C17 is substituted with [0412]
C3-8 alkyl, [0413] C2-8 alkoxy, [0414] Bn- optionally substituted
with C1-8 alkyl, C1-8 alkoxy, cyano, nitro, amino, carboxyl or
halogen, [0415] Ph- optionally substituted with C1-8 alkyl, C1-8
alkoxy, cyano, nitro, carboxyl or halogen, or [0416] Bn-O--. or
wherein R1 denotes that C3 is substituted with [0417] C1-8 alkoxy,
[0418] Bn-O, or [0419] Halogen, and --R2 denotes that C17 is
substituted with [0420] C1-8 alkyl, [0421] C2-6 alkenyl, [0422]
C1-8 alkoxy, [0423] Bn- optionally substituted with C1-8 alkyl,
C1-8 alkoxy, cyano, nitro, amino, carboxyl or halogen, [0424] Ph-
optionally substituted with C1-8 alkyl, C1-8 alkoxy, cyano, nitro,
carboxyl, or halogen, or
Bn-O--.
[0425] Preferably, the compound of formula II is selected from the
group consisting of 3.beta.-fluoro-17.alpha.-methylpregnenolone,
17.alpha.-benzyl-3.beta.-fluoropregnenolone,
17.alpha.-benzyl-3.beta.-benzyloxypregnenolone,
3.beta.-benzyloxy-17.alpha.-methylpregnenolone,
17.alpha.-benzylpregnenolone
3.beta.-methoxy-17.alpha.-methylpregnenolone,
17.alpha.-allyl-3.beta.-methoxypregnenolone, and
17.alpha.-benzyl-3.beta.-methoxypregnenolone
[0426] The invention also relates to a pharmaceutically composition
comprising a compound of formula II or a pharmaceutically salts
thereof and a pharmaceutically acceptable carrier.
[0427] Modification at C20:
[0428] In one embodiment, the compound is a pregnenolone modified
at C20.
[0429] In this embodiment, the compound has the formula D,
wherein:
R1 denotes that C3 is substituted with --OH, --R2 denotes that C17
is substituted with --H, R3 denotes that C20 is substituted with
[0430] --H, --OH and C1-8 alkyl, Bn, [0431] --NR8R9 wherein R8 and
R9 each independently is H, C1-8 alkyl or Bn, [0432] .dbd.CR10R11
wherein R10 and R11 each independently is H or C1-7 alkyl, or
[0433] .dbd.O, and R4 denotes that C16 is substituted with --H,
[0434] Preferably, R3 denotes that C20 is substituted with --H,
--OH or --NR8R9 wherein R8 and R9 each independently is H or C1-8
alkyl.
[0435] More preferably, said compound is selected from the group
consisting of 5-pregnen-3.beta.,20.alpha.-diol,
20-deoxypregnenolone and 20-methylamino-5-pregnen-3.beta.-ol.
[0436] Modification at C20 and C16:
[0437] In one embodiment the compound is a pregnenolone modified at
C20 and/or at C16.
[0438] In this embodiment, the compound has the formula D
wherein
R1 denotes that C3 is substituted with --OH, --R2 denotes that C17
is substituted with --H, R3 denotes that C20 is substituted with
--OH or --H, and R4 denotes that C16 is substituted with --OH or
.dbd.O.
[0439] Bond Between C16 and C17 is a Double Bond and the Bond C17
and C20 is a Single Bond.
[0440] In another embodiment, the bond between C16 and C17 is a
double bond and the bond between C17 and C20 is a single bond and
R1 denotes that C3 is substituted with --H, --OH or .dbd.O, R3
denotes that C20 is substituted with --H, --OH or .dbd.O, and R4
denotes that C16 is substituted with --H,
[0441] This embodiment is disclosed in the formula E below.
##STR00013##
[0442] Preferably, said compound is 5, 16 pregnadiene-20-one.
[0443] R1 is in .beta. Position:
[0444] In the most preferred embodiment, when the bonds between C3
and R1 and C3 and C4 are single, R1 is in .beta. position.
[0445] Indeed, the inventors have shown that contrary to the
derivatives with R1 in a position, the derivatives with R1 in
.beta. position have no effect on GABA and glutamate receptors and
avoid the side effects induced by modifications of these receptors,
as for example but not limited to sedation, memory impairments,
motor agitations. Further, the derivatives with C3 in .beta.
position keep their inhibitor activity of CB1 receptor.
Treatment of Pathologic Conditions or Disorders
[0446] The present invention also relates to a compound of the
invention as defined above or a pharmaceutical salt thereof for use
in a method of treatment.
[0447] In one embodiment, the compounds of formula II as defined
above or a pharmaceutical salt thereof is for use in a method of
treatment.
[0448] The present invention also relates to a compound of the
invention as defined above or a pharmaceutical salt thereof is for
the preparation of a medicament.
[0449] In one embodiment, the compounds of formula II as disclosed
above or a pharmaceutical salt thereof is for the preparation of a
medicament.
[0450] The present invention also relates to a method for the
treatment of a pathologic condition or disorder in a subject in
need thereof comprising administering to said subject an effective
amount of a compound of the invention as defined above or a
pharmaceutical salt thereof.
[0451] The present invention also relates to a compound of the
invention as defined above or a pharmaceutical salt thereof for use
in the treatment of a pathologic condition or disorder selected
from the group consisting of bladder and gastrointestinal
disorders; inflammatory diseases; cardiovascular diseases;
nephropathies; glaucoma; spasticity; cancer; osteoporosis;
metabolic disorders; obesity; addiction, dependence, abuse and
relapse related disorders; psychiatric and neurological disorders;
neurodegenerative disorders; autoimmune hepatitis and encephalitis;
pain; reproductive disorders; and skin inflammatory and fibrotic
diseases.
[0452] The invention relates to a method for the treatment of a
pathologic condition or disorder selected from the group consisting
of bladder and gastrointestinal disorders; inflammatory diseases;
cardiovascular diseases; nephropathies; glaucoma; spasticity;
cancer; osteoporosis; metabolic disorders; obesity; addiction,
dependence, abuse and relapse related disorders; psychiatric and
neurological disorders; neurodegenerative disorders; autoimmune
hepatitis and encephalitis; pain; reproductive disorder; skin
inflammatory and fibrotic diseases in a subject in need thereof
comprising administering to said subject an effective amount of a
compound of the invention as defined above or a pharmaceutical salt
thereof.
[0453] The invention relates to use of a compound of the invention
as defined above for the preparation of a medicament for treatment
of a pathologic condition or disorder selected from the group
consisting of bladder and gastrointestinal disorders; inflammatory
diseases; cardiovascular diseases; nephropathies; glaucoma;
spasticity; cancer; osteoporosis; metabolic disorders; obesity;
addiction, dependence, abuse and relapse related disorders;
psychiatric and neurological disorders; neurodegenerative
disorders; autoimmune hepatitis and encephalitis; pain; skin
inflammatory and fibrotic diseases.
[0454] Gastrointestinal Diseases:
[0455] In a preferred embodiment, the compound of the invention is
for use in the treatment of gastrointestinal diseases.
[0456] In a preferred embodiment, the use of a compound of the
invention is for the preparation of a medicament for treatment of
gastrointestinal diseases.
[0457] In a preferred embodiment, the method is for the treatment
of gastrointestinal diseases in a subject in need thereof
comprising administering to said subject an effective amount of a
compound.
[0458] Preferably, the compound of the invention is for use in the
treatment of a liver disease, in particular non alcoholic liver
steatohepatitis (NASH) and cirrosis.
[0459] Indeed, the inventors have shown that pregnenolone and it
derivatives inhibit lipid accumulation in a model of obesity, and
the production of TNF.alpha..
[0460] Obesity or Metabolic Disorders:
[0461] In a preferred embodiment, the compound of the invention is
for use in the treatment of obesity or metabolic disorders.
[0462] In a preferred embodiment, the use of a compound of the
invention is for the preparation of a medicament for treatment of
obesity or metabolic.
[0463] In a preferred embodiment, the method is for the treatment
of obesity or metabolic disorders, in a subject in need thereof
comprising administering to said subject an effective amount of a
compound.
[0464] Preferably, the compound of the invention is for use in the
treatment of diabetes and dislipidemia.
[0465] Indeed, the inventors have shown that pregnenolone and it
derivatives inhibit acute food-intake, fat accumulation in a model
of obesity, and the production of TNF.alpha..
[0466] Addiction, Dependence Abuse and Relapse Related
Disorders:
[0467] In another preferred embodiment, the compound of the
invention is for use in the treatment of addiction, dependence
abuse and relapse related disorders.
[0468] In a preferred embodiment, the use of a compound of the
invention is for the preparation of a medicament for treatment of
addiction, dependence abuse and relapse related disorders.
[0469] In another preferred embodiment, the method is for the
treatment of addiction, dependence, abuse and relapse related
disorders in a subject in need thereof comprising administering to
said subject an effective amount of a compound.
[0470] Preferably, the compound of the invention is for use in the
treatment of cannabis addiction, dependence, abuse, intoxication
and relapse related disorders.
[0471] Indeed, the inventors have shown that, in particular,
pregnenolone and it derivatives inhibit the endocannabinoid tetrad
induced by activation of the CB1 receptor by THC; THC-induced
food-intake; THC-induced memory impairments; THC-induced alteration
of synaptic transmission; Sel-administration of CB1 agonists.
[0472] Preferably, the compound of the invention is also for use in
the treatment of alcohol addiction, dependence abuse and relapse
related disorders.
[0473] Neurodegenerative and Psychiatric Disorders:
[0474] In another preferred embodiment, the compound of the
invention is for use in the treatment of neurodegenerative and
psychiatric disorders.
[0475] In another preferred embodiment, the use of a compound of
the invention is for the preparation of a medicament for treatment
of neurodegenerative and psychiatric disorders.
[0476] In another preferred embodiment, the method is for the
treatment of neurodegenerative and psychiatric in a subject in need
thereof comprising administering to said subject an effective
amount of a compound.
[0477] Preferably, the compound of the invention is for use in the
treatment of Parkinson disease and schizophrenia.
[0478] Thus the inventors have shown that pregnenolone is able to
modulate the activity of the dopaminergic system increased either
by THC or cocaine and the effects of CB1 activation on excitatory
synaptic transmission.
[0479] Skin Inflammatory and Fibrotic Diseases:
[0480] In another preferred embodiment, the compound of the
invention is for use in the treatment of skin inflammatory and
fibrotic diseases.
[0481] In another preferred embodiment, the use of a compound of
the invention is for the preparation of a medicament for treatment
of skin inflammatory and fibrotic diseases. In another preferred
embodiment, the method is for the treatment of skin inflammatory
and fibrotic diseases. Thus the inventors have shown that compounds
of the invention inhibit the production of TNF.alpha..
[0482] Preferably, the compound of the invention is for use in the
treatment of skin inflammation, skin inflammation and cancer
induced by UV, skin fibrosis and wound healing.
[0483] Cardiovascular Diseases:
[0484] In another preferred embodiment, the compound of the
invention is for use in the treatment of cardiovascular
diseases.
[0485] In another preferred embodiment, the use of a compound of
the invention is for the preparation of a medicament for treatment
of cardiovascular diseases.
[0486] In another preferred embodiment, the method is for the
treatment of cardiovascular diseases.
[0487] Thus the inventors have shown that compounds of the
invention decrease lipid accumulation and inhibit the production of
TNF.alpha..
[0488] Preferably, the compound of the invention is for use in the
treatment of cardiomyopathy.
[0489] More preferably, the compound of the invention is for use in
the treatment of cardiomyopathy selected from the group consisting
of cyrrotic cardiomiopathy and antideoplastic drugs induced
cardiomiopathies, contractile disfunction, infarction and
atherosclerosis.
[0490] The invention will be further illustrated by the following
figures and examples. However, these examples and figures should
not be interpreted in any way as limiting the scope of the present
invention.
FIGURES
[0491] FIG. 1 shows diagrams depicting in Wistar rats: (A) Basal
levels of pregnenolone (PREG), allopregnanolone (ALLO),
epiallopregnanolone (EPI), testosterone (T) and dihydrotestosterone
(DHT) in the nucleus accumbens. (B) The effects of the injection of
THC (3 mg/kg, ip), which induces a high and long-lasting increase
of pregnenolone concentrations in the nucleus accumbens. The
effects of other drugs of abuse: cocaine (20 mg/kg, ip), morphine
(2 mg/kg, ip) nicotine (0.4 mg/kg, ip) and ethanol (1 g/kg, ip),
which induce a much smaller increase in pregnenolone in the nucleus
accumbens. The effects of THC and other drugs of abuse on
pregnenolone-derived downstream steroids: allopregnanolone (C),
epiallopregnanolone (D), testosterone (E) and DHT (F), which were
largely lower than the one observed for pregnenolone. Arrows
indicate the time of injection of all drugs. Data are expressed as
mean.+-.SEM (n=6-8 per group).
[0492] FIG. 2 shows diagrams depicting the negative regulation of
pregnenolone on the THC-induced behavioural tetrad in C57Bl/6 mice.
THC dose-dependently (Vehicle group) decreased (A) locomotor
activity [F(3,59)=17.7. P<0.001] and (B) body temperature (delta
T compared to control) [F(3,59)=39.9. P<0.001] and increased (C)
catalepsy (latency to initiate movement) [F(3,59)=47.5. P<0.001]
and (D) analgesia (latency to initiate a nociceptive response in
the hot plate test) [F(3,59)=5.15, P<0.01]. The P450scc
inhibitor, aminoglutethimide (AMG, 50 mg/kg, ip), which block the
synthesis of pregnenolone, amplified all the behavioural effects of
THC: (A) hypolocomotion [F(3,98)=13.8, P<0.001], (B) hypothermia
[F(3,98)=4.7, P<0.01], (C) catalepsy [F(3,98)=2.1, P<0.05],
and (D) analgesia [F(3,98)=2.2, P<0.05]. Pregnenolone (PREG, 6
mg/kg, sc) reduced the effects of THC (10 mg/kg, ip) and completely
rescued the effect of AMG (50 mg/kg, ip) on: (E) locomotion, (F)
body temperature, (G) catalepsy and (H) analgesia. Pregnenolone had
no effects in animals that did not receive THC. Data are expressed
as mean.+-.SEM (n=6-12 per group). *=P<0.05; **=P<0.01;
***=P<0.001 compared to vehicle-treated mice.
[0493] FIG. 3 shows diagrams depicting the inhibition by
Pregnenolone of CB1-mediated food intake. (A) The increase in food
intake induced by THC (0.5 mg/kg, ip) in ad-libitum fed Wistar rats
was dose-dependently inhibited by pregnenolone injections
[F(3,94)=3.65; P<0.02]. (B) The increase in food intake induced
by THC (1 mg/kg, ip) in 24 h-food deprived C57Bl/6 mice was
suppressed by pregnenolone (2 mg/kg, sc). (C) Pregnenolone
dose-dependently reduced food intake in 24 h-food deprived C57Bl/6
mice. (D) The decrease in food intake induced by pregnenolone (PREG
6 mg/kg) in 24 h-food deprived C57Bl/6 mice was reversed by a
pre-treatment with the CB1R antagonist, SR141716A (0.05 mg/kg, ip).
Data are expressed as mean.+-.SEM (n=6-12 per group). *=P<0.05;
**=P<0.01; ***=P<0.001 compared to vehicle treated
animals.
[0494] FIG. 4 shows diagrams depicting the effects of pregnenolone
injections on pregnenolone-derived downstream active steroids in
the brain. (A) Pregnenolone administration (s.c.) dose-dependently
increased pregnenolone levels in frontal cortex and hypothalamus
[F(3,19)=20, P<0001; F(3,19)=23, P<0.001, respectively] of 24
h-food deprived C57Bl/6 mice. Pregnenolone did not modify
concentrations of: (B) allopregnanolone, (C) epiallopregnanolone.
Data are expressed as mean.+-.SEM (n=7-8 per group). *=P<0.05,
***=P<0.001 compared to vehicle-treated animals (PREG 0
mg/kg).
[0495] FIG. 5 shows diagrams depicting the inhibition by
pregnenolone of the self-administration of the CB1 agonist WIN
55,512-2 in CD1 mice. (A) During the acquisition of WIN 55,512-2
self-administration (0.0125 mg/kg/infusion) the number of
nose-pokes was significantly higher in the active hole than in the
inactive hole [F(1,18)=38.3, P<0.001]. (B) After acquisition,
the injection of pregnenolone (2 or 4 mg/kg, sc) decreased the
number of responses in the active hole. (C) Pregnenolone also
decreased the motivation for WIN 55,512-2 as measured by the
reduction in the break-point in a progressive ratio schedule. Data
are expressed as mean.+-.SEM (n=5-8 per group). **=P<0.05, ***.
P<0.001 compared to vehicle-treated animals (PREG 0 mg/kg).
[0496] FIG. 6 shows diagrams depicting the inhibition by
pregnenolone of the adverse effects of THC on memory. As indicated
by the discrimination index in the object recognition test, THC (10
mg/kg, ip) induced a significant amnesia, which was abolished by
pregnenolone (PREG, 6 mg/kg, sc) [F(3,23)=24.6, P<0.001]. Data
are expressed as mean.+-.SEM (n=6-7 per group). ***=P<0.001
compared to vehicle-treated mice.
[0497] FIG. 7 shows diagrams depicting the inhibition by
pregnenolone of the increase in dopaminergic activity induced by
THC. Pregnenolone injection (PREG, 2 mg/kg, sc) in rats decreased
.DELTA..sup.9-tetrahydrocannabinol (THC)-induced increase in (A)
the firing rate of ventral tegmental area (VTA) dopaminergic
neurons [F(4,32)=7.14, p<0.001] and (B) the increase in dopamine
outflow in the nucleus accumbens [F(10,80)=10.80, p<0.001].
Cumulative doses of THC (from 0.15 to 1.2 mg/kg) were administered
i.v. at time 0 over 4 min (1 min recording per dose).
[0498] FIG. 8 Show diagrams depicting the inhibition by
pregnenolone of the modification in dopaminergic activity induced
by cocaine Pregnenolone injection (PREG, 2 mg/kg, sc) in rats
abolished cocaine-induced decrease in (A) the bursting activity of
dopaminergic neurons and the increase in dopamine outflow in the
nucleus accumbens. Cumulative doses of cocaine (from 0.0125 to 0.8
mg/kg) were administered i.v. at time 0 over 4 min (1 min recording
per dose). *=P<0.05**=P<0.01***=P<0.001 compared to
vehicle-treated rats.
[0499] FIG. 9 shows diagrams depicting the effects of pregnenolone
administration on body weight and food intake. (A) Pregnenolone 5
mg/kg (PREGS) injected subcutaneously once a day before the
beginning of the dark phase progressively induced a significant
decrease in body weight [F(1,29)=3.13; p<0.001] in animals fed
with a high fat diet. (B) However, pregnenolone did not modify
food-intake.
[0500] FIG. 10 shows diagrams depicting the effects of pregnenolone
on the accumulation of fat and lean mass in obese mice.
Pregnenolone, injected subcutaneously once a day before the start
of the dark cycle, dose dependently blocked the increase in fat
mass (A,C) and blunted the decrease in lean mass (B,D) observed
during feeding with a high fat diet. Fat and lean mass were
calculated using magnetic resonance in mice. PRE=value obtained
before the start of pregnenolone administration. POST=values
obtained after 30 days of treatment with pregnenolone or vehicle.
*=P<0.05**=P<0.01.
[0501] FIG. 11 shows diagrams depicting the inhibition by
pregenonolone of the increase in TNF alpha induced by LPS. The
bacterial toxin LPS was injected intraperitoneally 30 min after the
injection of Pregnenolone (6 mg/kg subcutaneously) or vehicle
solution. Pregnenolone halved the increase in TNF-alpha induced by
the injection of LPS. *=P<0.5
[0502] FIG. 12 shows diagrams depicting the inhibition by
pregenonolone of THC-induced inhibition of excitatory synaptic
currents in the Nucleus Accumbens of rats. Excitatory post-synaptic
currents (EPSC) induced by electrically stimulating local axons
were recorded using patch clamp in Nucleus Acccumbens principal
neurons in brain slices obtained from adult rats. (A) Bath
application of THC (20 mM) reliably inhibited synaptic transmission
in control slices (34.3.+-.3.7% of inhibition, N=8). The effect of
THC was significantly attenuated when slices were pre-treated with
Pregnenolone 100 nM (15.1.+-.1.8% of inhibition, N=9). (B) Synaptic
current traces from representative experiments averaged during
baseline and after 40 minutes of THC exposure. ***T test: t=4.820,
df=15, p=0.0002.
[0503] FIG. 13 shows diagrams depicting the inhibition by
pregenonolone of the THC-induced inhibition of excitatory synaptic
transmission in mouse Nucleus Accumbens. (A) Field excitatory
post-synaptic potentials (fEPSP) induced by electrically
stimulating local axons were recorded in Nucleus Acccumbens brain
slices obtained from adult mice. Bath application of THC induced a
dose-dependent inhibition of synaptic transmission in control
slices (N=5-9). The effects of THC were reduced when slices were
pre-treated with Pregnenolone 100 nM (N=5-6). (B) Representative
fEPSP average traces recorded during baseline and after 40 minutes
of THC exposure. Two Way ANOVA: Pregnenolone effect p<0.002; THC
effect p<0.0003.
[0504] FIG. 14 shows diagrams depicting that pregnenolone is an
inhibitor of the activation of the CB1 receptor, which does not
modify the orthosteric binding of agonists of the CB1. Pregnenolone
(10-12 to 10-4 M) did not modify the specific binding of the CB1
agonist [3H]CP55,940 to the human CB1 receptor expressed by CHO
cells. Data are expressed as mean.+-.SEM (n=2-3 per
concentration).
[0505] FIG. 15 shows diagrams depicting the lack of effects of
pregnenolone on anxiety like behaviors. Anxiety-like behaviors were
measured by the % of entries and time spent in the open arms of an
elevated plus maze. Pregnenolone did not induce anxiety like
behavior even at the highest doses (10 mg/kg) well above its
effective behavioral doses between (1 and 6 mg/kg) corresponding to
its maximal behavioral effects. The orthosteric antagonist of the
CB1 receptor rimonabant at 10 mg/kg induced an increase in anxiety
as shown by the decrease of the entries and time spent in the open
arms. P1, P6, P10=pregnenolone 1, 6, 10 mg/kg. Rimo10=rimonabant 10
mg/kg. V=vehicle. *=P<0.05; ***=P<0.001.
[0506] FIG. 16 depict diagrams showing that the lack of effect of
pregnenolone on GABA-A receptors-mediated currents. mIPSC where
recorded from adult mouse NAc PN voltage-clamped at -80 mV. A.
Summary of amplitudes (ANOVA f=5.39, df=4.66, p<0.001) and decay
times (ANOVA f=24.7, df=4.66, p<0.0001) of mIPSC from controls
(N=16) and slices pre-treated with pregnenolone (100 nM: N=15; 1
.mu.M: N=11) or allopregnanolone (100 nM: N=18; 1.quadrature.M
N=11). Post hoc tests: *p<0.05, **p<0.01, ***p<0.001. B.
representative traces of mIPSC recording. C. average mIPSC traces
normalized to the peak. Note that only allopregnanolone 1 .mu.M
substantially affected mIPSC decay phase. cont=controls;
preg=pregnenolone; allo=allopregnanolone.
[0507] FIG. 17 depict diagrams showing that Pregnenolone does not
modify AMPAR nor NMDAR-mediated currents. (A) mEPSC recorded from
adult mouse Nucleus accumbens principal neuorons voltage-clamped at
-80 mV. a) mEPSC recorded from control (N=16) and pregnenolone (100
nM) pre-treated (N=15) slices showed similar amplitude (T test:
t=1.16, df=29, p=0.25) and decay time (T test: t=1.28, df=29,
p=0.21). b) Average mEPSC traces normalized to the peak showing
that its kinetics was not affected by pregnenolone. c)
Representative traces of mEPSC recording. B. Whole cell currents
recorded in NAc PN induced by bath application of NMDA 25 .mu.M for
1 min. a) NMDAR-induced currents were comparable between controls
(N=17) and slices pre-treated with pregnenolone 100 nM (N=12) and 1
.mu.M (N=7) (ANOVA: f=0.09, df=2.33, p=0.91). b) Representative
experiments showing the effect of NMDA on holding currents of NAc
PN voltage-clamped at -30 mV. cont=controls; preg=pregnenolone.
[0508] FIG. 18 shows diagrams depicting the effects of the
injection of high doses (50 mg/kg, sc) of pregnenolone and of the
C3 and C17 synthetic derivatives 3-Fluoropregnenolone (CP1),
17-methylpregnenolone (CP2) and 3-fluoro-17-methylpregnenolone
(CP3) on nucleus accumbens steroids content. Pregnenolone but not
3-Fluoropregnenolone, 17-methylpregnenolone or
3-fluoro-17-methylpregnenolone increased allopregnanolone (A) and
epiallopregnanolone (B) levels in the accumbens of Wistar rats.
Data are expressed as mean.+-.SEM (n=5-7 per group).
[0509] FIG. 19 depicts diagrams showing that the C3 and C17
synthetic derivatives 3-Fluoropregnenolone (CP1),
17-methylpregnenolone (CP2) and 3-fluoro-17-methylpregnenolone
(CP3) reduced food intake. (A) The increase in food intake induced
by THC (0.5 mg/kg, sc) in ad libitum fed Wistar rats was
significantly reduced by 17-methylpregnenolone (8 mg/kg, sc), a non
statistically significant trend to decrease was also observed after
3-Fluoropregnenolone and 3-fluoro-17-methylpregnenolone. (B)
Pregnenolone [(F4,28)=5.5; P<0.01], 3-Fluoropregnenolone
[(F3,20)=3; P<0.05], 17-methylpregnenolone [(F3,20)=5.3;
P<0.01] and 3-fluoro-17-methylpregnenolone[(F3,20)=4; P<0.02]
dose-dependently decreased food intake in 24 h-food deprived
C57Bl/6 mice. (C) Pregnenolone, 3-Fluoropregnenolone,
17-methylpregnenolone and 3-fluoro-17-methylpregnenolone (2 mg/kg,
sc) decreased THC-induced hyperphagia in 24 h-food deprived C57Bl/6
mice. (D) Full dose response effects of pregnenonolone and
17-methylpregnenolone on THC-induced hyperphagia in 24 h-food
deprived C57Bl/6 mice. For both compound the first effective dose
was 1 mg/kg. Data are expressed as mean.+-.SEM (n=6-8 per group).
*=P<0.05, ***=P<0.001 compared to vehicle-treated animals.
##=P<0.01; ###=P<0.001, compared to THC treated animals.
[0510] FIG. 20 shows diagrams depicting the effects of a steady
state administration of pregnenolone with Alzet minipumps on the
level of pregnenolone and down stream metabolite Allopregnanolone.
The subcutaneous injection of pregnenolone induced an increase in
pregnenolone levels (A) that was short lasting (<1 h) and also
increased allopregnanolone levels over at least 1 hour (D). The
administration of pregnenolone through Alzet minipumps (B,C) dose
dependently increased plasmatic levels of pregnenolone but not the
ones of allopregnanolone. (E,F).
EXAMPLES
[0511] Examples of Synthesis of Derivatives of Pregnenolone
[0512] Pregnenonole is well-known and commercially available and
can be used as precursor for the synthesis of it derivatives.
[0513] Example of Synthesis of a Derivative of Pregnenolone Having
C17 Substituted with an Alkyl:
[0514] As shown below, to synthetise a compound substituted with an
alkyl at C17 position, in a first step, the corresponding enol
acetate is formed. Then, it is treated with a Grignard reagent to
generates an enolate which is subsequently trapped with an
electrophile. The electrophile would be preferentially an iodo- or
bromo-alkyl, -allyl, -benzyl or -aryl.
##STR00014##
[0515] Example of Synthesis of a Derivative of Pregnenolone Having
C17 Substituted with --OR
[0516] The figure below shows how to obtain a C17 substituted with
an alkoxy-, benzyloxy- and aryloxypregnenolones thank to
copper-mediated functionalization with alcohols.
##STR00015##
[0517] Example of Synthesis of a Derivative of Pregnenolone Having
C3 Substituted with Halogen and C17 Substituted with R:
[0518] As shown below, the derivative of pregnenolone having C17
substituted with R is treated with DAST in order to change the
alcohol function at C3 with a fluorine atom.
##STR00016##
[0519] Example of Synthesis of a Derivative of Pregnenolone Having
a C3 Substituted with an Alkoxy:
[0520] As shown below, the formation of an ether function at C3
position requires, in a first step, the transformation of alcohol
into the corresponding tosylate as leaving group. Then, it is
treated with the suitable alcohol in order to lead to the formation
of C3-alkoxy pregnenolone.
##STR00017##
[0521] Example of Synthesis of a Derivative of Pregnenolone Having
C3 Substituted with .dbd.O and C17 Substituted with R:
[0522] As shown below, to obtain a derivative of pregnenolone
having a C3 substituted with .dbd.O and C17 substituted with R,
C17-substituted pregnenolone is treated with oxidants to lead to
the oxidation of alcohol function into the corresponding ketone
followed by the spontaneous isomerization of the double bond to
give modified progesterones.
##STR00018##
[0523] Examples of Role of Pregnenolone and its Derivative in the
Inhibition of CB1 Receptor
[0524] Material and Methods:
[0525] Animals
[0526] Animals were individually housed in a temperature
(22.degree. C.) and humidity (60%) controlled animal facility under
a constant light-dark cycle (light on, 8:00-20:00 h). Except for
food intake experiments and during the experimental sessions of WIN
55,212-2 self administration, food and water were freely available
throughout the experiments. After arrival animals were handled
periodically for two weeks before experiments. Most of the
experiments were performed during the light phase except for the
food intake experiments in rats and WIN 55,212-2 self
administration sessions test in CD1 mice that were conducted during
the dark phase. All the experiments were conducted in strict
compliance with the recommendations of the European Union
(86/609/EEC).
[0527] Adult male Wistar rats (3-4 months), C57Bl/6N mice (2-3
months) C57Bl/6j mice (2-3 months) and CD1 mice (weighing 25-30 g
at the beginning of the experiments) were purchased from Charles
River Laboratories (France). CB1-deficient (CB1-/-) and D1-CB1
mutant (D1-CB1-/-) mice were produced in our laboratory as
described (Marsicano et al., Nature. 2002 418:530-4; Monory et al.,
PLoS Biol. 2007 5(10):e269).
[0528] Drugs
[0529] .DELTA.9-tetrahydrocannbinol (THC, Sigma-Aldrich, France)
was purchased as a 30 mg/ml (w/v) solution in 100% ethanol. Before
injection this solution was dissolved with Tween 80 (1 drop/3 ml)
and dimethylsulfoxide (DMSO) diluted 1:40 with saline (2.5%).
Vehicle solution contained all ingredients (1 drop/3 ml of Tween
80, DMSO (2.5%) and ethanol diluted with saline to obtain a final
concentration of 1.8% of ethanol). Cocaine HCl (Cooperation
Pharmaceutique Francaise, France), morphine sulfate (Francopia,
France), nicotine bitartrate (Sigma-Aldrich, France) and USP
alcohol (95%, Sigma-Aldrich, France) were dissolved in saline.
HU210, JWHI 33 and AM251 were purchased from Tocris, UK and WIN
55,212-2, aminoglutethimide (AMG) pregnenolone
(5-Pregnen-3.beta.-ol-20-one) lipopolysaccharides from E. Coli
0111:B4 (LPS) from Sigma-Aldrich (France) and rimonabant
(SR141716A) from Cayman Chemical (Interchim, Montlucon,
France).
[0530] The synthetic compounds 3-Fluoropregnenolone;
17-methylpregnenolone and 3-fluoro-17-methylpregnenolone were
synthesized by AtlanChimPharma (France). Drug solutions were
dissolved in Tween 80 (1 drop/3 ml) and DMSO (2.5%) or NMP (2.5%)
and diluted in saline solution. THC, cocaine, morphine, nicotine,
ethanol. HU210, JWH133, AM251, WIN 55,212-2, AMG and SR141716A were
injected intraperitoneally (ip) and pregnenolone or pregnenolone
derivatives were injected subcutaneously (sc). The injection
volumes were 1 ml/kg of body weight for rats and 10 ml/kg for
mice.
[0531] In SA experiments, WIN 55,212-2 (Sigma Chemical Co., Madrid,
Spain) was dissolved in one drop of Tween 80 and diluted in saline
solution. Ketamine hydrochloride (100 mg/kg) (Imalgene 1000; Rhone
Merieux, Lyon, France) and xylazine hydrochloride (20 mg/kg)
(Sigma, Madrid, Spain) were mixed and dissolved in ethanol (5%) and
distilled water (95%). This anesthetic mixture was administered
intraperitoneally prior to catheter implantation in an injection
volume of 20 ml/kg of body weight. Thiopental sodium (5 mg/ml)
(Braun Medical S.A. Barcelona, Spain) was dissolved in distilled
water and delivery by infusion of 0.1 ml through the intravenous
catheter. For in vitro human CB1 receptor functional assay,
pregnenolone and CP 55940 were dissolved in DMSO to final
concentration of 100 mM and stored at -20.degree. C.
[0532] Neurosteroid Quantification
[0533] Blood and Brain Sampling.
[0534] The animals were sacrificed by decapitation and trunk blood
was collected in EDTA-coated tubes, centrifuged at 2000.times.g for
10 min, and the supernatant was stored at -20.degree. C. Brains
were quickly harvested, the brain areas were dissected on ice and
samples were rapidly frozen in cold ice and stored at -80.degree.
C.
[0535] Measurement of Steroid Levels by GC/MS.
[0536] Plasma, brain and culture medium levels of pregnenolone
(5-Pregnen-3.beta.-ol-20-one), allopregnanolone
(3.alpha.-hydroxy-5.alpha.-pregnan-20-one), epiallopregnanolone
(3.beta.-hydroxy-5.alpha.-pregnan-ol-20-one), testosterone, and 5a
dihydrotestosterone were determined by GC/MS according to the of
estraction, purification and quantification protocol described
previously (George O, et al., Biol Psychiatry. 2010 68: 956-63,
Vallee M, et al., Anal Biochem. 2000, 287:153-66).
[0537] THC Behavioral Tetrad
[0538] Body Temperature.
[0539] Body temperature was measured using a rectal probe (RET3
probe, Physitemp instruments, USA) in conscious mice and was
monitored by a thermalert monitoring thermometer (TH-5, Physitemp
instruments, USA).
[0540] Locomotor Activity.
[0541] Locomotion was measured by an automated open field system
(box size 100.times.100.times.30 cm, illumination of 10 lux,
videotracking system: Viewpoint, Lyon, France) or Plexiglas cages
(19 cm long.times.11 cm wide.times.14 cm high) mounted with
computer-monitored photocell beams (Imetronic, France). Animals
were individually tested for 15 min. The cumulative horizontal
distance the animals moved within the box was recorded.
[0542] Catalepsy.
[0543] Catalepsy was measured by the bar catalepsy test. The
forepaws of mice were placed on a 1-cm-diameter bar fixed
horizontally at 3.5 cm from the bench surface. The latency to
descend was recorded.
[0544] Analgesia.
[0545] Analgesia was measured using a hot plate analgesia meter
(BIO-HC1.00, Bioseb, France). The plate was heated to 52.degree.
C..+-.0.1.degree. C. and the time until mice showed the first sign
of discomfort (licking or flinching of the paws or jumping on the
plate, here defined as escape latency) was recorded. A cut-off time
of 60 s was set to prevent tissue damage.
[0546] Object Recognition Task
[0547] Object recognition was measured in a two arms L-maze (size
of each arm 30 cm length.times.4.5 cm width) in dim light condition
(50-60 lux). Animals were individually tested for three consecutive
days for 9 min-session each day, corresponding to habituation,
training and test sessions. On day 1 (habituation session), mice
were let explore the L maze with no object. On the second day
(training session), two identical objects were presented at the end
of the each arm of the maze. Although no preferences for arm of
object appeared (data not shown) object and arms were randomized
for each mouse/condition. On the 3rd day (test session), one of the
familiar objects was replaced with a novel object and the total
time spent exploring each of the two objects (novel and familiar)
was computed. The time spent in each arm and the time spent
exploring an object (familiar or novel) were recorded. Object
exploration was defined as the orientation of the nose to the
object at a distance of less than 2 cm. During the test session, a
discrimination index was calculated as the difference between the
times spent exploring either the novel or familiar object divided
by the total time exploring the two objects. A higher
discrimination index is considered to reflect greater memory
retention for the familiar object (Puighermanal et al., 2009).
[0548] Food Intake Measurements
[0549] Food intake was evaluated by measuring consumption of food
in the home cages of the animals. For each animal, 50-100 g of
standard laboratory chow (U.A.R., France) was placed in the cleaned
home cage. The remaining amount of food was weighted 1 h later and
the amount of food consumed calculated.
[0550] WIN 55,212-2 Self-Administration
[0551] The intravenous self-administration experiments where the
animals learned to self-infuse WIN 55,212-2 (WIN) were conducted in
mouse operant chambers (Model ENV-307A-CT, Medical Associates,
Georgia, VT, USA) using procedures previously described (Mendizabal
V, et al., Neuropsychopharmacology. 2006, 31:1957-66).
[0552] Coupled In Vivo Microdialysis and Electrophysiology
[0553] General procedures. Surgery and perfusion procedure were
performed to allow concomitant electrophysiological and
microdialysis monitoring. Briefly, rats were anesthetized using a
2% mixture of isoflurane/air, and a catheter was inserted into the
femoral vein for intravenous drug administration. Thereafter,
animals were placed in a stereotaxic frame (David Kopf Instruments,
Phymep, Paris, France) equipped with a nose mask for constant
delivery of the gas anesthesia (2% isoflurane during surgery, 1.5%
isoflurane during electrophysiology and microdialysis experiment),
and their rectal temperature was monitored and maintained at
37.+-.1.degree. C. by a heating pad (CMA 150, Carnegie Medecin,
Phymep). A microdialysis probe (CMA/11, 2 mm long, 240 .mu.m outer
diameter, Cuprophan; Carnegie Medicin, Phymep) and a recording
electrode (glass micropipette TW150E-4, 2-3 .mu.m outer diameter,
WPI-Europe, Aston Stevenage, UK) were implanted respectively in the
medio-ventral part of the right nucleus accumbens corresponding to
the shell subdivision [coordinates, in mm relative to bregma:
anteroposterior (AP)=+1.7, lateral (L)=1, ventral (V)-8], and in
the right ventral tegmental aerea (VTA) (coordinates, in mm
relative to bregma: AP=-5.4-5.8, L=0.4-0.8, V=-7.0-8.5], according
to the Paxinos and Watson atlas. Probes were perfused at a constant
rate of 2 .mu.L/min by means of a microperfusion pump (CMA 111,
Carnegie Medicin, Phymep) with artificial cerebrospinal fluid
(aCSF) containing (in mM): 154.1 Cl.sup.-, 147 Na.sup.+, 2.7
K.sup.+, 1 Mg.sup.2+, and 1.2 Ca.sup.2+, adjusted to pH 7.4 with 2
mM sodium phosphate buffer. Perfusion was then maintained during 2
h to allow the stabilization of dopamine (DA) levels in the
perfusates.
[0554] Single unit recording of DA neuronal firing and monitoring
of DA extracellular levels were started 2 h after the beginning of
probe perfusion (stabilization period). Dialysates (30 .mu.L) were
collected on ice every 15 min, and immediately analyzed to
determine the baseline values of DA extracellular levels, defined
by three consecutive samples in which DA content varied by less
than 10% (9). Search of DA neurons for electrophysiological
recording was performed during the 30 min preceding the drug
treatment (THC or cocaine) administration. DA neuron firing rate
was recorded for 3-5 min to obtain the firing baseline, defined by
a variation of less than 10% of the average frequency discharge of
the DA neuron. Pharmacological treatments were performed once
obtained a stable baseline for DA neuron firing and DA
extracellular levels in the perfusate.
[0555] DA neuron recording. Single unit activity of neurons located
in the VTA was recorded extracellularly with glass micropipettes
filled with 1% Fast Green dissolved in 0.5 M sodium acetate
(impedance, 2-5 M.OMEGA.). Signals were filtered (bandpass, 0.4-1
kHz) and amplified by a high-impedance amplifier (Dagan 2400A,
Dagan Corporation, USA) and individual spikes were isolated by
means of a window discriminator (WD-2, Dagan Corporation, USA),
displayed on an analog-digital storage oscilloscope (HM507, Hameg,
Frankfurt, Germany). Then, the experiments were sampled on line
with Spike2 software (Cambridge Electronic Design, Cambridge, UK)
by a computer connected to CED 1401 interface (Cambridge Electronic
Design, Cambridge, UK). VTA DA neurons were identified according to
the already published criteria (12-14). The firing rate was defined
as the number of spikes/sec.
[0556] DA assay. dialysates were injected into an HPLC apparatus
equipped with a reverse phase Equisil BDS column (C18; 2.times.250
mm, particle size 5 .mu.m; Cluzeau Info Labo, Ste Foy la Grande
France), and an amperometric detector (Antec Leyden DECADE II,
Alpha-mos, Toulouse, France) with a glassy carbon electrode set at
+450 mV versus Ag/AgCl, in order to quantitate DA. The composition
of the mobile phase was (in mM) 70 NaH2PO4, 0.1 Na.sub.2EDTA, and
0.1 octylsulfonic acid plus 10% methanol, adjusted to pH 4.8 with
orthophosphoric acid. The sensitivity for DA was 0.3 pg/20 .mu.L
with a signal/noise ratio of 3:1.
[0557] Histology. At the end of each experiment, a direct
continuous current (-20 .mu.A for 15 min) was passed through the
electrode to eject Fast Green dye, allowing the identification of
the recording site. Afterwards, brains were removed and fixed in
NaCl (0.9%)/paraformaldehyde solution (10%). The location of the
electrodes in the VTA and the microdialysis probes in the nucleus
accumbens was determined by microscopic examination on serial
coronal sections (60 .mu.m) stained with Neutral Red.
[0558] Diet Induced Obesity and Evaluation of Fat Accumulation
[0559] 2-months old male C57BL/6J mice were ad libitum fed a 60%
high-fat diet (HFD; Catalog #D12492, Research Diets, New Brunswick,
N.J.) for 8 weeks and subsequently treated either with pegnenolone
or vehicle. Homogeneous distribution of the animals in the 3
treatment groups was guaranteed by matching their body weight, fat
mass and fasting glucose levels before the start of the
pharmacological treatments.
[0560] Body composition analysis. Fat mass and lean mass were
assessed in vivo using an EchoMRI analyzer (EchoMedical Systems,
Houston, Tex.) before the mice were placed on the HFD, as well as
immediately before and at the end of the chronic treatment with
pregnegnolone or its vehicle.
[0561] Plasma free fatty acids measurement. Trunk blood from DIO
mice was collected at the end of the 4 weeks of treatment and
plasma free fatty acids (FFA) were measured by a colorimetric
reaction kit, following the manufacturer's instructions (Abeam,
catalog #65341).
[0562] Plasma TNFalpha measurement. Trunk blood was collected and
centrifuged at 3000 rpm for 15 min at 4.degree. C. Plasma was
stored at -80.degree. C. until measurement of TNF.alpha. was
carried out. Plasma TNF.alpha. levels were assessed using an ELISA
kit, following the manufacturer's instructions (Fisher Scientific,
Catalog #E6473C).
[0563] Electrophysiology on Brain Slices
[0564] Slice Preparation. Animals were deeply anesthetized with
Isoflourane and transcardially perfused with a sucrose-based
physiological solution at 4.degree. C. (in mM: 23 NaHCO.sub.3, 70
Choline Cl, 75 Sucrose, 25 Glucose, 2.5 KCl, 1.25
NaH.sub.2PO.sub.4, 7 MgCl.sub.2 and 0.5 CaCl.sub.2). The brain was
removed and sliced (250-300 .mu.m) in the coronal plane using a
vibratome (Campden Instruments, Loughborough, UK). During the
slicing process, the brain was maintained in the sucrose-based
solution. Immediately after cutting, slices were stored at
32.degree. C. for 40 minutes in a low-calcium artificial
cerebrospinal fluid (low-Ca.sup.2+ ACSF) that contained (in mM): 23
NaHCO.sub.3, 120 NaCl, 11 Glucose, 2.5 KCl, 1.2 NaH.sub.2PO.sub.4,
2.4 MgCl.sub.2, 1.2 CaCl.sub.2. Slices were then stored in
low-Ca.sup.2+ ACSF at room temperature until recording.
[0565] Electrophysiology. Recordings were performed with an
Axopatch-1D amplifier (Molecular Devices, Sunnyvale, Calif.). Data
were filtered at 1-2 kHz, digitized at 10 kHz on a Digidata 1332A
interface (Molecular Devices), collected on a PC using Clampex 9,
and analyzed using Clampfit 10 (Molecular Devices).
[0566] Whole cell patch-clamp recordings were performed from NAc
core principal neurons (PNs). Cells were identified using
differential interference contrast infrared videomicroscopy (Leica
DM LFSS microscope, Leica Microsystems, Germany; Camera Till
Photonics, Germany).
[0567] For recording AMPAR and NMDAR-mediated currents, glass patch
clamp electrodes (resistance 4-6 MOhms) were filled with a
cesium-based solution as follows (in mM): 125 Gluconic acid, 125
CsOH, 10 HEPES, 10 NaCl, 0.3 EGTA, 0.05 Spermine, 10 TEA-Cl, 2
MgCl.sub.2, 0.3 CaCl.sub.2, 4 Na.sub.2ATP, 0.3 NaGTP, 0.2 cAMP. For
recording inhibitory currents, a similar solution was used with the
exception of (in mM): 80 Gluconic acid, 80 CsOH, 30 CsCl, 20 NaCl.
The higher chloride concentration favor a stronger driving force
when recorded at hyperpolarized potentials. Throughout the
experiments access resistance, (R.sub.a) was evaluated with a 2-mV
hyperpolarizing pulse. R.sub.a was not compensated and cells were
rejected if R.sub.a was >25 M.OMEGA. or changed >20% during
the experiment. The potential reference of the amplifier was
adjusted to zero prior to breaking into the cell.
[0568] For fEPSPs, extracellular glass recording and stimulating
electrodes were filled with ACSF. Synaptic potentials were evoked
by means of two electric stimuli (0.1-0.25 mA, 200 .mu.sec
duration) delivered at 20 Hz every 10 seconds. Stimulating
electrode was placed at a distance >150 .mu.m in the dorsomedial
direction from the recoding electrode.
[0569] Data Acquisition and Analysis. AMPAR-mediated synaptic
currents: GABA-A receptors were blocked by adding picrotoxin 50
.mu.M to the superfusion medium. The contribution of NMDAR was
ruled out by recording at hyperpolarized potentials. For evoked
synaptic currents and mEPSC cell were voltage clamped at -70 mV and
-80 mV, respectably. mEPSC were recorded in the presence of TTX 0.5
.mu.M.
[0570] NMDAR-mediated currents: Changes in holding currents induced
by NMDA were measured in cells voltage clamped at -30 mV, to
relieve NMDAR from the voltage-dependent magnesium block. AMPAR and
GABA-A receptors were blocked with DNQX 20 .mu.M and picrotoxine 50
.mu.M, respectably.
[0571] GABA-A receptors-mediated currents: mIPSC were recorded in
the presence of the AMPAR and NMDAR antagonists (DNQX 20 .mu.M and
AP-5 50 .mu.M, respectably).
[0572] mEPSC-mIPSC analysis: Typically, after breaking in, cells
were left to equilibrate for 20 minutes and the subsequent 10
minutes recording of mEPSC-mIPSC was used to analysis. mEPSC-mIPSC
were detected using a template generated from averaging several
typical events (Clampfit 10, Molecular Devices). The template was
slid along the data trace one point at a time. At each position,
the template was optimally scaled and offset to fit the data. A
lower-amplitude threshold of 7 pA and 10 pA were applied for mEPSC
and mIPSC, respectably, equivalent to 2.5 SD of baseline noise. For
each cell, the kinetics of mEPSC-mIPSC was measured from the
average event using Clampfit 10. To estimate decay times, a two
exponentials curve was fitted between 5 and 95% of the decay phase
of the current given by the following equation:
y(t)=A1e.sup.(-t/.tau.1)+A2e.sup.(-t/.tau.2), where A is the
amplitude, t is the time and .tau. is the decay time constant. The
weighted tau was then calculated.
[0573] In Vitro Human CB1 Receptor Assays
[0574] The binding assay for orthosteric binding of pregnenolone
has been evaluated using Chinese hamster ovary (CHO) cells
expressing the human CB1 receptor.
[0575] The CB1 binding of pregnenolone has been evaluated by the
affinity of pregnenolone for the agonist site of the human CB1
cannabinoid receptor expressed in CHO cells, determined in a
radioligand binding assay of the CB1 agonist [3H] CP 55940. The
experiments were performed by CEREP France, using the standard
procedures of this provider.
[0576] Elevated Plus Maze
[0577] The elevated plus maze was made by a central platform
(10.times.10 cm) from which departed 4 arms (45.times.10 cm) at a
90.degree. angle from each other. Two opposite arms, named closed
arms had peripheral wall 50 cm high. The two other arms, named open
arms had no walls. The maze was suspended at 66 cm from the floor
of the room and was brightly lighted (120 lux). The testing
consisted in placing the animal in the central platform and the
number of entry and the time spent in each compartment of the maze
recorded for 5 min. The number of entry and time spent in the open
arms are considered and index of anxiety-like behaviors, whilst the
total number of entry are and index of locomotor activity.
[0578] Study of Metabolism In Vitro
[0579] The CHO-K1 cell line (#CCL-61, ATCC-LGC, USA) derived as a
subclone from the parental CHO cell line initiated from a biopsy of
an adult Chinese hamster ovary was used. The CHO-Kl cells were
seeded on 24-well plates (#353047, BD Biosciences, USA) to the
appropriate concentration (25.times.10.sup.4 cells/well) in fresh,
antibiotic-free medium constituted by 90% DMEM-Glutamax;
(#31966-21, Life technologies, USA) and 10% Fetal Bovine Serum
(#10270-106, Life technologies, USA).
[0580] Steady State Administration of Pregnenolone
[0581] Micro-osmotic pumps (Alzet Osmotis Pumps, Charles River,
France) with a pump rate of 0.15 .mu.l/hr (model 2006) were filled
with pregnenolone dissolved in PEG300 (85%) and ethanol (15%) at a
concentration of 125 or 250 mg/ml (corresponding respectively to a
daily dose of 12 or 24 mg/kg body weight) and implanted
subcutaneously after light anesthesia. All pumps were primed by
soaking them in saline at 37.degree. C. for 60 h before
implantation.
[0582] Statistical Analysis
[0583] Statistical analysis were performed using: Two-way or one
way analysis of variance (ANOVA), Newman-Keuls, Student's T-tests.
All results were expressed as mean.+-.S.E.M. Statistical tests were
performed with GraphPad Prism (GraphPad Software Inc., La Jolla,
Calif., USA) or Statistica 5.0.COPYRGT. (StatSoft Inc, Tulsa,
Okla., USA).
[0584] Results:
[0585] I. CB1 Activation Increases Pregnenolone Synthesis and
Concentrations.
Example 1: THC Increases Pregnenolone Concentrations in the Brain
More than Other Drugs of Abuse
[0586] In this example the inventors analyzed the effects of the
injection of the principal drugs of abuse on the production of
pregnenolone in male Wistar rats. In all tissues, the first step of
steroid synthesis is the production of pregnenolone that has been
largely considered as an inactive precursor of downstream active
molecules. For example, in the brain, starting from pregnenolone
two parallel enzymatic cascades allow producing on one hand
allopregnanolone and its stereoisomer epiallopregnanolone and on
the other testosterone and its metabolite DHT. These brain steroids
were quantified using GC-MS, the only technique able to
differentiate their subtle structural differences. The major
classes of drugs of abuse were injected subcutaneously or
intraperiotenally at doses corresponding to the ED50 for most of
their unconditioned behavioural effects: the psychostimulant
cocaine (20 mg/kg), the opioid morphine (2 mg/kg), nicotine (0.4
mg/kg), alcohol (1 g/kg) and the active principle of Cannabis
sativa .DELTA.9 tetrahydrocannabinol (THC) (3 mg/kg).
Concentrations of neurosteroids were analyzed 15, 30 and 120 min
after the injection in several ascending brain structure, the
ventral midbrain the hypothalamus, the striatal complex and the
frontal cortex.
[0587] Very similar results were obtained in all the brain
structures studied (frontal cortex, striatum, accumbens, ventral
midbrain). As shown in the example for the ventral striatum (the
nucleus accumbens) basal level of steroids (FIG. 1A) ranged from
approximately 1 ng/g of tissue for pregnenolone and testosterone to
0.2 ng/g for epiallopregnanolone. DHT and allopregnanolone had
intermediate levels around 0.4 ng/g. All drugs of abuse increased
brain concentrations of pregnenolone between 15 and 30 min after
the injection (FIG. 1B). Strikingly, the increase in pregnenolone
induced by THC was several time higher than the one induced by the
other drugs of abuse: around 1500% increase for THC compared to
approximately 200% increase for the other drugs of abuse.
Example 2: THC Increases Pregnenolone Concentrations in the Brain
of Rats in a Dose Dependent Manner
[0588] In this example the inventors further characterized the
effects of the administration of different concentrations of THC
(0.3, 0.9, 1.5, 3, 6 and 9 mg/kg) or vehicle to male Wistar rats on
body concentrations of pregnenolone measured at the pick of the
drug effects that, as shown in the previous example, was observed
30 min after the injection. These experiments demonstrated that the
increase in pregnenolone observed in the brain was dose dependent,
with an ED50 of approximately 3 mg/kg. The example provides data
obtained in the plasma and in several brain structures: frontal
cortex (FCX); nucleus accumbens (ACC); striatum (STR); hypothalamus
(HYP). After THC administration, pregnenolone increased in all the
studied brain structures in a comparable manner. Pregnenolone also
increased in the plasma but this increase was several times lower
than the one observed in the brain.
Example 3: THC Increases Pregnenolone Concentrations in the Brain
of Mice in a Dose Dependent Manner
[0589] In this example the inventors further characterized the
effects of the administration of THC on concentrations of
pregnenolone by studying the effects of THC in the mouse.
Pregnenolone was measured at the pick of the drug effects, 30 min
after the injection. These experiments demonstrated that THC
induced a dose-dependent increase in pregnenolone also in mice. The
example provides data obtained from the several brain structures:
frontal cortex (FCX); nucleus accumbens (ACC); striatum (STR). THC
induced a similar increase in pregnenolone concentration in all
these brain structures.
Example 4: Agonists of the CB1 Receptor Induce an Increase in
Pregnenolone Concentrations Similar to the One Induced by THC
[0590] The effects of THC in the brain are mediated by a family of
G-protein-coupled seven membrane receptors (GCPR) and principally
by the CB1 and CB2 receptors. THC increased pregnenolone synthesis
via the activation of the CB1 receptor. Thus, the injection to
independent groups of rats (n=6-12 per group) of both a synthetic
mix CB1/CB2 agonists Win55,212 and of an agonist that have a higher
affinity for the CB1 than for the CB2 receptor (HU210) induced a
significant increase in pregnenolone. In contrast, an agonist with
a higher affinity for the CB2 than the CB1 receptor (JWH, 133) had
a much lower and not significant effects on pregnenolone
concentrations
Example 5: THC-Induced Increase in Pregnenolone is Suppressed by an
Antagonist of the CB1 Receptor
[0591] The dependence of THC effects on CB1 activation were further
demonstrate by the observation that the increase in pregnenolone
concentrations induced in the nucleus accumbens of Wistar rats by
the administration of THC (3 mg/kg, i.p) was blocked by the
administration of a CB1 selective antagonist (AM251, 8 mg/kg, i.p.)
that was injected 30 min before the injection of THC (n=6 per
group).
Example 6: THC-Induced Increase in Pregnenolone is Suppressed in
Mutant Animals Lacking the CB1 Receptor
[0592] In this example the inventors further analyzed the
dependence from the CB1 receptor of the increase in pregnenolone
induced by THC. For this purpose the inventors studied the effects
of THC on pregnenolone in mutant mice (n=6-8 per group) in which
the expression of the CB1 receptor was constitutively deleted. The
examples show data obtained in the nucleus accumbens. THC-induced
increase in pregnenolone was completely suppressed in mutant
animals in which the CB1 receptor had been deleted in all types of
cells or selectively in the neuronal population expressing the
dopaminergic DI receptor. In this mutant the CB1 receptor is
deleted in most GABA neurons in the accumbens. The latest
experiment indicates that in the brain, THC increases pregnenolone
by acting on the CB1 receptor expressed by neurons.
[0593] Discussion:
[0594] The data presented in the previous examples converge in
supporting the hereby disclosed discovery that: "activation of the
CB1 receptors in mammals induces the synthesis of pregnenolone and
increase the concentration of this steroid in the body". The data
presented in the previous examples converge then in providing a
generalized method for increasing pregnenolone concentrations in
the body.
[0595] The converging evidence presented in the examples can be
summarized as follow: First, pregnenolone is dose-dependently
increased by the administration of three different agonists of the
CB1 receptor: THC, HU210 and Win55,212. In contrast, pregnenolone
was not significantly increased by an agonist that has a higher
affinity for the CB2 than for the CB1 receptor. The increase in
pregnenolone concentrations induced by CB1 agonists was confirmed
in two different species (the mouse and the rat) and was found both
in the brain and in the plasma.
[0596] Second, the increase in pregnenolone induced by THC was
suppressed by administration of a CB1 antagonist and was abolished
in mutant animals lacking the CB1 receptor.
[0597] II. Pregnenolone Exerts a Negative-Feedback on the CB1
Receptor and Inhibits the Effects of CB1 Receptor Activation.
Example 7: THC-Induced Increase in Pregnenolone Provides a Negative
Feed-Back on the Activation of the CB1 Receptor
[0598] In these examples the inventors analyzed the potential
functional role of the increase in pregnenolone induced by THC
activation of the CB1 receptor. The inventors found that
pregnenolone exerts a negative feed-back on the effects that are
mediated by the stimulation of the CB1 receptor.
[0599] The activation of the CB1 receptor is usually identified by
four effects, generally called the cannabinoid tetrade, which
include: 1. hypolocomotion, 2. hypothermia, 3. catalepsy (impaired
ability to initiate movements), and 4. analgesia. Accordingly, the
injection of THC (3, 10, 15 mg/kg) to C57Bl/6N (n=7-8 per group)
induced a dose-dependent: i) decrease of locomotor activity in the
open-field; ii) decrease of body temperature; iii) increase of the
latency to initiate movement (increased catalepsy); and iv) an
increase in the nociceptive threshold (FIG. 2A-D).
[0600] Since the dose of THC at which the tetrad is observed
(between 3 and 15 mg/kg of THC) induces a strong increase in
pregnenolone concentrations, the inventors analyzed the effects of
the inhibitor of pregnenolone synthesis aminogluthetimide (AMG, 50
mg/kg, ip) injected 30 min before THC, behaviors were sequentially
measured 30 min after THC injection. AMG strongly increased all the
behavioural effect of THC (FIG. 2 A-D) and this enhancement was
completely reversed by the exogenous injection of pregnenolone (6
mg/kg) (FIG. 2 E-H), demonstrating the dependence from pregnenolone
of the observed effects of AMG administration. These data
demonstrate that the secretion of pregnenolone induced by the
activation of the CB1 receptor serves the function of inhibiting
with a negative feedback loop the effects resulting from such an
activation of the CB1.
Example 8: Pregnenolone Inhibits the Endocannabinoid Tetrad Induced
by Activation of the CB1 Receptor by THC
[0601] In these examples the inventors analyzed if the exogenous
administration of pregnenolone could also inhibit the cannabinoid
tetrad induced by THC. Pregnenolone administration (6 mg/kg) before
THC and in the absence of AMG decreased all the behaviours of the
THC-induced cannabinoid tetrad: locomotor activity, body
temperature, catalepsy and pain threshold (FIG. 2 E-H). However,
administration of pregnenolone per se in the absence of THC had no
effect on locomotor activity, body temperature, catalepsy and pain
threshold (FIG. 2 E-H).
Example 9: Pregnenolone Inhibits the Increase in Food Intake
Induced by THC
[0602] To provide further examples of the ability of pregnenolone
to inhibit the effects resulting from the activation of the CB1
receptors, the inventors then studied if pregnenolone (injected 30
min before THC) could also inhibit THC-induced increase in food
intake.
[0603] THC has been shown to increase food intake in sated rats
(0.5 mg/kg, n=7-8 per group) and in 24 hours food-deprived mice
(n=7-8 per group 1 mg/kg). In sated rats (FIG. 3A) pregnenolone
dose-dependently decreased THC-induced food intake with a
statistically significant effect at 2 mg/kg. This dose also
suppressed the increase in food intake induced by the injection of
THC in mice (FIG. 3B). At this dose pregnenolone did not
significantly modified basal food intake (FIG. 3 A, B).
Example 10: Pregnenolone Inhibits the Increase in Food Intake
Induced by Food-Deprivation
[0604] CB1 activation by endogenous endocannabinoid has been
involved in the regulation of physiological food intake, i.e. food
intake not stimulated by exogenous CB1 agonists such as THC. The
inventors then further investigated if pregnenolone administration
was able to modify food intake in food deprived animals that did
not received THC. The inventors found that pregnenolone dose
dependently decreased food intake in food-deprived mice (FIG. 3C),
however the first statistically significant dose (6 mg/kg) was
higher than the one (2 mg/kg) able to block THC-induced food
intake.
Example 11: Pregnenolone Inhibits the Increase in Food Intake
Induced by Food-Deprivation Through a CB1-Dependent Mechanism
[0605] Many physiological systems regulate food intake. For this
reason in this example the inventors verified if the reduction in
food intake induced by pregnenolone in food-deprived animals that
were not treated with THC was dependent on the CB1 receptor. The
inventors studied the effects of a pre-treatment with the CB1
antagonist SR141716A (0.05 mg/kg, ip) on the reduction in food
intake induced by pregnenolone in food-deprived animals. The
inventors found that the inhibition induced by pregnenolone on food
intake in food deprived animals was dependent on the CB1 receptors.
Thus, the CB1 antagonist SR141716A administered to food-restricted
mice 30 min before the administration of Pregnenolone suppressed
the reduction in food intake induced by pregnenolone administration
(FIG. 3D).
Example 12: Pregnenolone Inhibits Self-Administration of CB1
Agonists
[0606] In order to analyse the effects of pregnenolone
administration on the positive reinforcing effect of CB1 activation
that are related to the ability of THC to induce addiction, the
inventors used the intravenous self-administration model performed
in accordance to protocols previously described (Soria et al.,
2006; Mendizabal et al., 2006). Intravenous self-administration is
considered the best behavioural model of addiction. In this model
animals learn to produce an operant response, in our case pocking
the nose in a hole, in order to obtain an intravenous infusion of
the drug. Mice readily self-administer the CB1 agonist WIN 55,212
(12.5 .mu.g/kg per injection), showing a clear preference for the
device in which responding trigger the infusion of this compound
(active) in comparison to an inactive device in which responding
had no scheduled consequences (inactive) (FIG. 4A). Administration
with a Latin square design of 2 or 4 mg/kg of pregnenolone=before
the self-administration session profoundly reduced the
self-administration of WIN55,212 (FIG. 4B). In addition
pregnenolone administration also decreased the motivation to
self-administer WIN55,212, as shown by the reduction in the
break-point in a progressive ratio (PR) schedule (FIG. 4C). In this
schedule animals are required to produce an increasing number of
responses (ratio) to obtain one drug infusion, the break-point
being the last ratio completed and is considered a reliable measure
of the motivation for the drug. On PR session the response
requirement to earn an injection escalated according to the
following series:
1-2-3-5-12-18-27-40-60-90-135-200-300-450-675-1000.
Example 13: Pregnenolone Inhibits Memory Loss Induced by THC
Administration
[0607] In this example the inventors further analyzed the ability
of pregnenolone to inhibit the effects of CB1 activation. A
supplementary effect of CB1 activation is the induction of memory
impairments. This effect is related to one of the adverse effects
of cannabis use: a cognitive impairment characterized by the loss
of recent memories. CB1 receptor activation by THC (10 mg/kg)
injected 10 min after training strongly impairs memory retention in
an object recognition task in mice (FIG. 6).
[0608] Pre-treatment with pregnenolone injected immediately after
training (6 mg/kg) strongly blunted the amnesic effect of 10 mg/kg
of THC. However, pregnenolone (6 mg/kg) did not induce any change
in memory retention when administered in the absence of THC (FIG.
6).
Example 14: Pregnenolone Inhibits the Increase in Dopaminergic
Activity Induced by THC Administration
[0609] In this example the inventors further analyzed the ability
of pregnenolone to inhibit the effects of CB1 activation. Cannabis
is thought to exercise its addictive properties by activating the
CB1 receptor that in turn increase the release of the
neurotransmitter dopamine in the nucleus accumbens, a brain region
that regulate the shift from motivation to action. The effects of
pregnenolone on the increase in dopaminergic activity produced by
THC (FIG. 7) were studied recording two parameters in parallel: 1.
the release of dopamine at the level of the dopaminergic terminals
in the nucleus accumbens; and 2. the electrical activity of the
dopaminergic neurons at the level of their cell body in the ventral
tegmental area (VTA). Pregnenolone (2 m/kg injected subcutaneously,
30 min prior to THC) strongly blunted the increase in dopamine
release and in the activity of the dopaminergic neurons induced by
THC THC or cocaine was administered intravenously at exponentially
increasing cumulative doses (0.15 to 1.2 mg/kg). After each dose,
DA neuronal firing was recorded for 1 minute before the subsequent
administration. (FIG. 7).
Example 15: Pregnenolone Inhibits the Increase in Dopaminergic
Activity Induced by Cocaine Administration
[0610] In this example the inventors further analyzed the ability
of pregnenolone to inhibit the activation of the dopaminergic
system. In the previous example pregnenolone was able to antagonize
the hyperactivity of the dopaminergic system induced by THC. In the
present example (FIG. 8) it is shown the pregnenolone (2 mg/kg
injected subcutaneously, 30 min before cocaine) is also able to
antagonize the increase in activity of the dopaminergic system
induced by psychostimulants such as cocaine. Cocaine was
administered intravenously at exponentially increasing cumulative
doses (0.0125 to 0.8 mg/kg). After each dose, DA neuronal firing
was recorded for 1 minute before the subsequent administration.
This results are relevant to schizophrenia because the increase in
dopaminergic activity induced by psychostimulants is considered one
of the experimental models of psychosis. Thus, to the increase in
dopamine induced by psychostimulants is attributed the development
of acute psychosis that can occur after the use of these drugs in
humans.
Example 16: Pregnenolone Inhibits Body Weight Gain and Fat
Accumulation in Animals Submitted to a High Fat Diet
[0611] In this example the inventors analyzed the ability of
pregnenolone to inhibit the effects of CB1 activation in the
context of obesity. The effects of pregnenolone on metabolic
disorders were studied using the model of diet induced obesity
(DIO) in mice. In this procedure animals are maintained on a high
fat diet (60% of fat) which progressively induce obesity. After
eight weeks on this diet which already induced overweight and
excessive fat accumulation in these animals the treatment with
pregnenolone was started for thirty days (once a day 2 mg/kg or 5
mg/kg, n=8). Pregnenolone decreased body weight with a delayed
effect that appeared after 15 days of treatment (FIG. 9A) but did
not modify food intake (FIG. 9B).
[0612] As a consequence pregnenolone effects on body weight seemed
not due to a primary metabolic effect and not to a behavioral
effect on food intake. This was confirmed by an analysis of body
composition performed with magnetic resonance which revealed that
under pregnenolone treatment there was a different effect on the
fat and lean mass of the animals (FIG. 10). In control animals
during the high fat diet the percentage of the fat mass
progressively increased whilst the one of the lean mass decreased.
When animal were treated with the highest dose of pregnenolone (5
mg/kg) the increase in fat mass was suppressed, whilst the decrease
in lean mass was blunted.
[0613] The lack of effect on food intake on pregnenolone in the DIO
model seems in contradiction with what observed using the
fasting/refeeding model in which pregnenolone decreased food intake
(FIG. 3). This could be due to the feeding condition, a high fat
diet in the DIO model versus standard chow in the
fasting/re-feeding model, or to a potential specific effect of
pregnenolone on the burst of eating that is induced in fasted
animals during the first hour of re-exposure to food. For this
reason in the fasting/refeeding model food intake is classically
evaluated during one hour, whilst in the DIO model food-intake is
evaluated over 24 hours. In a subsequent experiment the effects of
pregnenolone were studied over 24 hours also in the
fasting/refeeding model. The results obtained confirmed the effects
of pregnenolone during the first hour of re-feeding but no
significant effect was seen over 24 hours. These data indicate a
specific effect of pregnenolone on the burst of eating induced by
fasting that strongly activates the endocannabinoid system and the
CB1 receptor. This lack of effects of pregnenolone on 24 hours food
intake is quite different from the known action of the reference
CB1 orthosteric receptor antagonist rimonabant that has been
previously shown to profoundly reduce food-intake over 24 hours.
Similarly, during a high fat diet, rimonanbant has also been shown
to reduce food-intake during the first week of treatment, whilst
pregnenolone did not (FIG. 9A).
Example 17: Pregnenolone Inhibits the Increase in TNF-Alpha Induced
by LPS
[0614] In this example the inventors further analyzed the ability
of pregnenolone to inhibit the effects of CB1 activation in the
context of inflammation and fibrosis. The activation of the CB1
receptor is involved in inflammatory and fibrotic process as shown
but the fact that the inhibition of this receptor by ortosteric
antagonists such as rimonabant decreases the increase in TNF-alpha
induced by proinflammatory stimuli such as LPS. TNF-alpha is one of
the cellular responses to inflammatory stimuli more involved in
promoting fibrosis. Administration to mice of pregnenolone (6
mg/kg, subcutaneously) 30 min before the administration of LPS
halved the increase in TNF-alpha measured 90 min after LPS
administration (FIG. 11).
Example 18: Pregnenolone Inhibits the Effects of CB1 Activation on
Synaptic Transmission
[0615] In this example the inventors further analyzed the ability
of pregnenolone to inhibit the effects of CB1 activation in the
context of synaptic transmission. It is widely documented that
activation of CB1 receptors suppresses synaptic transmission by
inhibiting neurotransmitters release. This has been observed in
many regions of the brain at both excitatory and inhibitory
synapses. We assessed whether pregnenolone alters the ability of
THC to inhibit excitatory synaptic transmission in the nucleus
accumbens (NAc). Whole cell patch clamp recording were performed in
the adult NAc and AMPAR-mediated EPSC were induced by electrical
stimulation of local axons. Bath perfusion of THC reliably
decreased EPSC amplitude in control slices (34.3.+-.3.7% of
inhibition). The effect of THC was significantly attenuated when
slices were pre-treated with Pregnenolone 100 nM (15.1.+-.1.8% of
inhibition, p<0.001) (FIG. 12).
[0616] In order to test the effect of pregnenolone on a wider range
of THC concentrations, we recorded fEPSP in NAc slices. Due to the
possibility to achieved stable fEPSP measurements for several
hours, this technique is ideally suited to perform dose-response
curves and has previously been used to address CB1 receptor
function (Robbe et al., 2001; Mato et al., 2004). Thus,
AMPAR-mediated evoked fEPSP were recorded by electrically
stimulating local axons. Bath perfusion of THC to control slices
inhibited fEPSP in a dose-dependent manner (10 .mu.M: 23.9.+-.6.0%;
20 .mu.M: 35.3.+-.4.7%; 40 .mu.M: 48.6.+-.3.6%). Conversely, THC
induced lesser inhibition of synaptic transmission in slices
pre-incubated with pregnenolone 100 nM (10 .mu.M: 11.1.+-.3.2%; 20
.mu.M: 22.7.+-.2.7%; 40 .mu.M: 34.6.+-.3.1%; two way ANOVA
neurosteroid factor p=0.001) (FIG. 13).
[0617] Altogether, these data demonstrate that the neurosteroid
pregnenolone impairs the ability of THC to activate a
CB1receptors-dependent modulation of excitatory synaptic
transmission.
[0618] Discussion:
[0619] The data presented in the previous examples converge in
demonstrating the hereby disclosed discovery that: "the increase in
pregnenolone concentrations induced in the body by the activation
of the CB1 provides an endogenous negative feedback on the activity
of the CB1 receptor. This negative feed-back is materialized by the
fact that pregnenolone endogenously produced or exogenously
administered antagonizes the effects of CB1 activation".
[0620] The data presented in the previous examples converge then in
providing a general method for inhibiting the effects of the
activation of the CB1 receptor by the administration of
pregnenolone.
[0621] These converging evidences can be summarized as follow:
[0622] First, the inhibitory action of pregnenolone on CB1-mediated
effects were of physiological relevance, since the endogenous
increase in pregnenolone induced by CB1 activation, provided an
endogenous negative feed-back that served the function of
decreasing the effect of CB1 activation. Thus, when the production
of pregnenolone induced by CB1 activation was blocked the
behavioral effects of THC were increased. Second, exogenous
administration of pregnenolone was able to inhibit a large number
of effects induced by the activation of the CB1 receptors: 1.
hypolocomotion; 2. catalepsy; 3. hypotermia; 4. analgesia; 5. food
intake in fastig-refeeding model; 7. Food intake induced by THC; 6.
intravenous self-administration of a CB1 agonists; 7. memory loss
induced by THC;_8. activation of the dopaminergic system by THC or
cocaine; 9. fat accumulation and body weight gain in a model of
obesity; 9. The production of TNF-alpha; 10. The inhibition of
synaptic transmission induced by THC. Third, inhibitory action of
pregnenolone on CB1-mediated effects was found in two different
species: the rat and the mouse.
[0623] The conversing effects on these multiple parameters
demonstrated here by the inventors are unique and non-predictable
on the basis of previous knowledge of the effects of other known
steroids that quite at the opposite are predicted to increase and
not decrease the effects of the activation of the CB1 receptor. For
example many steroids, such as pregnanolone, allopregnanolone and
their derivatives have been described to facilitate the activation
of the GABA receptor. These steroids should then increase the
effects of CB1 activation since compounds that potentiate the
activity of the GABA receptor have been shown to increase the
effects of CB1 activation by THC (Bellocchio L et al., Nature
Neurosci 2010, 13:281-3; Pertwee R G and Wickens A P.
Neuropharmacology. 1991, 30:237-44 Pertwee R G, et al.,
Neuropharmacology. 1988 27:1265-70). Similarly on the basis of
current knowledge also progesterone and progestinic compounds,
other sex steroids and glucocorticoids are predicted to increase
the effects of CB1 activation (Anaraki D K et al., Europ. J.
Pharmacol, 2008, 586, 186-196; Rdriguez de Fonseca F, et al., Life
Sci. 1993, 54: 159-170; Becker J B, Rudick C N. Pharmacol Biochem
Behav. 1999, 64:53-7; Piazza P V and Le Moal M Brain Res Rev 1997,
25:359-72).
[0624] III. Pregnenolone is an Inhibitor of the CB1 Receptor with
Less Side Effects and Less Non-Specific Actions than Orthosteric
Antagonists of the CB1 and Other Neuroactive Steroids.
Example 19: Pregnenolone does not Modify the Orthosteric Binding to
the CB1 Receptor
[0625] The previous examples indicate that pregnenolone is able to
inhibit all the studied effects of CB1 activation. Based on these
observations in this example the inventors studied the potential
interactions between pregnenolone and the CB1 receptor. The
inventors analyzed if pregnenolone could act as an orthosteric
antagonist of the CB1. This was not the case, since pregnenolone
did not displace the orthosteric binding of the CB1 agonist
[3H]CP55,940 to the CB1 receptor present on the plasma membrane of
CHO cell (FIG. 14). Although pregnenolone does not act as an
orthosteric antagonists preliminary evidence produced by the
inventors indicate that it could act as an allosteric
inhibitor.
Example 20: Pregnenolone does not Induce Anxiety Like Behaviors
[0626] One of the major side effects of orthosteric antagonists of
the CB1 is the induction of behavioral side effect and in
particular an axiodepressive state. These effects have been judged
serious enough by regulatory authorities to suppress the market
approval of the first CB1 orthosteric antagonist rimonabant. To
substantiate the different safety profile of pregnenolone, its
effects have been compared to the ones of the CB1 orthosteric
antagonist/inverse agonist rimonabant (both drugs given
subcutaneously) using an animal model of anxiety the elevated plus
maze. This test has been chosen because an increase in anxiety was
the principal undesirable side effect of this compound in humans.
Mice were injected subcutaneously with either pregnenolone (1, 6,
10 mg/kg), rimonabant (10 mg/kg) or vehicle (at least n=7 per
group) 30 min later they were placed in the central platform of the
plus maze and the time spent and the number of entry in the open
and closed arms recorder for 5 mins. This study (FIG. 15) confirmed
anxiogenic effects of rimonabant as shown by the decrease in the
number of entries and in the time spent in the open arm of the plus
maze. In contrast pregnenolone induced no increase in anxiety (FIG.
15).
Example 21: Pregnenolone does not Modify the Activity of GABA-A
Receptors
[0627] In this example the inventors tested the specificity of
pregnenolone effects on other neurotransmitter receptors and in
particular the GABA-A receptors. Thus, other active steroids such
as allopregnanolone and pregnenolone have profound behavioral
effects facilitating the activation of the GABA receptors.
[0628] In order to evaluate the effect of pregnenolone on the
function of post-synaptic GABA we recorded mIPSC and compared its
amplitude and decay time between groups. We observed that mIPSC
amplitude and decay time were similar between controls (amplitude:
17.9.+-.0.8 pA; decay time: 11.1.+-.0.3 ms) and slices pre-treated
with pregnenolone (100 nM and 1 .mu.M, respectively; amplitude:
17.3.+-.0.4 pA, 15.9.+-.0.4 pA; decay time: 10.2.+-.0.4 ms,
10.8.+-.0.5 ms). As opposed to the lack of effect of pregnenolone,
the neurosteroid allopregnanolone, known for being a modulator of
GABA-A receptors, significantly modified mIPSC properties (100 nM
and 1 .mu.M, respectively; amplitude: 15.9.+-.0.5 pA, 20.2.+-.1.2
pA, p<0.001; decay time: 13.6.+-.0.7 ms, 22.7.+-.2.3 ms,
p<0.0001) (FIG. 16).
[0629] In conclusion, this data suggest that GABA-A-mediated
synaptic currents are not modulated by the neurosteroid
pregenenolone. This data then confirm, as previously shown (U.S.
Pat. No. 5,232,917), that the C3 beta position of pregnenolone
suppresses the effect on the GABA receptor. The inventor discover
here that the C3 beta position confer instead the property to
inhibit the activation of the CB1 receptor.
Example 22: Pregnenolone does not Modify the Activity of the
Glutamate Receptors
[0630] In this example the inventors tested the specificity of
pregnenolone effects on other neurotransmitter receptors and in
particular the NMDA and AMPA receptors. Thus, other active steroids
such as DHEA and DHEA sulphate are supposed to induce behavioral
effect by modifying the activity of the glutamate receptors. To
evaluate the effect of pregnenolone on AMPA receptors currents, we
decided to record action potentials independent mEPSC. In this
case, synaptic currents arise from stochastic quantal release of
neurotransmitters and assuming that neurosteroids do not change the
content of synaptic vesicles, the amplitude and kinetics of the
recorded mEPSC depends on the function of the post-synaptic
receptors.
[0631] We found that pregnenolone did not modify AMPAR function in
the adult NAc (FIG. 17A). Thus, AMPAR-mediated mEPSC from controls
and pregnenolone (100 nM) treated slices showed similar amplitudes
(controls: 14.4.+-.0.8 pA, pregnenolone: 15.7.+-.0.7 pA, p=0.25)
and decay times (controls: 4.76.+-.0.08 ms, pregnenolone:
4.62.+-.0.06 ms, p=0.21).
[0632] To quantify NMDA receptors-mediated currents we used a
different strategy. Because of its slow kinetics and voltage
dependence, the isolation of NMDAR-mediated mEPSC is not as
reliable as for AMPAR-mediated currents. Thus, we decided to record
whole cell currents in response exogenously applied NMDA. In NAc PN
voltage-clamped at -30 mV, bath perfusion of NMDA 25 WA for 1
minute induced an inward current of similar magnitude in control
slices (115.+-.15.5 pA) and in slices pre-treated with pregnenolone
(100 nM: 107.+-.9.6 pA; 1 .mu.M: 108.9.+-.12.5 pA; p=0.91) (FIG. 17
B).
[0633] Overall, these experiments show that pregenolone does not
affect the function of the main post-synaptic ionotropic
glutamatergic receptors in the rodent adult NAc. These data also
indicate that the substitution of the ketone in position 17 of the
steroid ring with an ethanone (methylketone) suppress activity on
the NMDA and AMPA receptors and confer the property to inhibit the
activation of the CB1 receptor.
[0634] Discussion:
[0635] The data presented in the previous examples converge in
demonstrating the hereby disclosed discovery that: "pregnenolone
act as a inhibitor of the human CB1 receptor with a pharmacological
profile different from orthosteric antagonis and from other
neuroactive steroids that indicate that pregnenolone will have less
unspecific and undesiderable effects than orthosteric antagonists
of the CB1 and other neuroactive steroids.
[0636] The data presented in the previous examples converge then in
providing a general method for inducing an inhibition of the
activity of the CB1 receptor by the administration of pregnenolone.
Consequently the data presented in the previous examples converge
in providing a general method to treat or alleviate all pathologies
that are related to the activation of the CB1 receptor and/or
pathologies that can benefit from the inhibition of the CB1
receptors by administration of pregnenolone without the potential
side effects of orthosteric antagonists of the CB1 and of other
active steroids including but not limited to DHEA, Allopregnanolone
and pregnanolone
[0637] These converging evidences can be summarized as follow:
[0638] First, pregnenolone does not modify the binding of an
orthosteric agonist, whilst it inhibits the effects resulting from
the activation of the CB1 receptor. This profile could correspond
to the one of an allosteric inhibitor.
[0639] Second, pregnenolone differently from orthosteric antagonist
does not induce anxiety (example 20) nor reduce food-intake in an
obesity model although it reduce fat accumulation (example 18). The
pure metabolic effects of pregnenolone devoid of a modification of
food intake also predict fewer side effects for pregnenolone. Study
on the effects of ortostheric antagonists of the CB1, such as
rimonabant, on body weight and metabolism have shown that these
compounds act on metabolism with a double action the first due to
the weight loss that results from a decrease in food intake
(approximately 50% of the effects) and the second from a direct
metabolic effect. The side effects of CB1 orthosteric receptor
antagonists, and in particular the increase in depression, likely
involve their behavioral effects on food intake. Thus, it is well
known that in the obese population all manipulations,
pharmacological, surgical, or behavioral treatment that reduce food
intake also induce in up to 5% of the subjects serious behavioral
disturbances and in particular depression.
[0640] Finally, pregnenolone differently from other neuroactive
steroids is devoid of effects on the GABA and glutamate receptors.
This is important in predicting that pregnenolone will not have the
side effects of some of these steroids which can induce important
modifications of weakfulness inducing sedation, impairment of
memory performances and motor behavior.
[0641] IV. Pregnenolone Derivatives for which the Transformation in
Other Active Steroids Derived from Pregnenolone has been Limited
Act as Inhibitors of the Effects of CB1 Receptor Activation.
Example 23: Derivatives of Pregnenolones for which the
Transformation in Downstream Active Steroids is Limited do not
Produce Allopregnanolone and EpiAllopregnanolone In Vivo
[0642] As examples of the compound described in the general formula
A, the inventors tested herein three compounds that were obtained
by:
[0643] 1. The substitution of the OH group at C3 by a fluorine
atom, which generated the compound named 3-Fluoropregnenolone
(CP1).
[0644] 2. The quaternization of C17 with a methyl group, which
generated the compound named 17-methylpregnenolone (CP2).
[0645] 3. The substitution of the OH group at C3 by a fluorine atom
and the quaternization of C17 with a methyl group, which generated
the compound named 3-fluoro-17-methylpregnenolone (CP3).
[0646] None of the modified pregnenolone derivatives (compound:
CP1, CP2, CP3), injected at high doses (50 mg/kg, n=6-7 per group))
to wistar rat, induced the production of allopregnanolone and
epiallopregnanolone (FIG. 18), whilst allopregnanolone and
epiallopregnanolone increased after the injection of pregnenolone
(50 mg/kg) (FIG. 18). The concentrations of allopregnanolone and
epiallopregnanolone were measured in nucleus accumbens of
individual animal by GC/MS 30 min after the injections.
Example 24: Derivatives of Pregnenolones for which the
Transformation in Downstream Active Steroids In Vitro is
Limited
[0647] In this example the inventors further analyzed the
metabolism of pregnenolone derivatives using an in vitro test in
CHO cells. These cells derived from the ovary have all the enzymes
needed to metabolize pregnenolone in down stream steroids. In
particular administration of pregnenolone (1 .mu.M) to these cells
for 48 hours produced a significant increase in Allopregnanolone,
Epiallopregnanolone and a much smaller increase in THDOC in the
culture medium (Table 1A). A total of 55 compounds plus
pregnenolone were tested, of these compounds only 25 had an absent
or significantly reduced metabolism in down stream active steroids
(Table 1B-D, Table 2A, B). The remaining compounds were more or
less metabolized in one or several of the downstream steroids of
pregnenolone, such as Allopregnanolone, Epiallopregnanolone, THDOC,
testosterone and DHEA.
TABLE-US-00001 TABLE 1 A Pregnenolone metabolism ALLO EPIALLO THDOC
PREG DHEA TESTO Control cell cultures Steroid levels 0.00 0.00
27.96 96.92 0.00 0.00 Pregnenolone (1 .mu.M) pg/ml 3529.99 16963.84
77.47 11440.66 0.00 0.00 treated cells
TABLE-US-00002 TABLE 1 B Not detectable metabolism % changes from
Pregnenolone treated cells pg/ml Comp. N.sup.o Name Structure ALLO
EPIALLO THDOC PREG DHEA TESTO 14 4-Pregnen-
17.alpha.,20.alpha.-diol-3- one ##STR00019## -100.00 -100.00
-100.00 -100.00 0.00 0.00 24 20- Deoxypregneno- lone ##STR00020##
-100.00 -100.00 -100.00 -100.00 0.00 0.00 74
17.alpha.-Benzyl-3.beta.- fluoropregnano- lone ##STR00021## -100.00
-100.00 -100.00 -100.00 0.00 0.00 77 5.beta.-Pregnan-3.beta.-
ol-20-one (Epipregnano- lone) ##STR00022## -100.00 -100.00 -100.00
-100.00 0.00 0.00
TABLE-US-00003 TABLE 1C Decrease in Allo and Epiallo >99% %
changes from Comp. Pregnenolene treated cells pg/ml N.sup.o Name
Structure ALLO EPIALLO THDOC PREG DHEA TESTO 40 17.alpha.-
Methylpro- gesterone ##STR00023## -99.70 -99.83 -100.00 -100.00
0.00 0.00 42 3.beta.- Benzyloxy- 17.alpha.- methyl- pregnen- olone
##STR00024## -99.87 -99.94 -94.10 -100.00 0.00 0.00 69 17.alpha.-
Allyl-3.beta.- methoxy- preg- nenolone ##STR00025## -99.59 -100.00
-100.00 -100.00 0.00 0.00 73 17.alpha.- Benzylpro- gesterone
##STR00026## -99.28 -99.93 -100.00 -100.00 0.00 0.00 67 20- Methyl-
amino- 5-pregnen- 3.beta.-ol ##STR00027## -99.20 -99.79 -100.00
-99.79 0.00 0.00
TABLE-US-00004 TABLE 1 D Decrease in Allo and Epiallo >97% %
changes from Comp. Pregnenolone treated cells pg/ml N.sup.o Name
Structure ALLO EPIALLO THDOC PREG DHEA TESTO 41 3.beta.- Benzyl-
oxy- preg- neno- lone ##STR00028## -98.82 -99.88 -100.00 -99.35
0.00 0.00 12 4- Pregnen- 3.beta.,20.alpha.- diol ##STR00029##
-98.54 -97.17 -100.00 -98.80 0.00 0.00 18 4- Pregnen- 20.alpha.-
ol-3- one ##STR00030## -98.16 -96.80 -100.00 -100.00 0.00 0.00 65
17.alpha.- Benzyl- preg- nenolone ##STR00031## -97.26 -99.72
-100.00 -100.00 0.00 0.00 72 3- Azido- preg- neno- lone
##STR00032## -97.76 -99.58 -100.00 -100.00 0.00 0.00
[0648] Table 1. Pregnenolone derivatives with reduced metabolism in
CHO cells. Results are expressed as percentage changes from CHO
cells treated with pregnenolone (table 2A) or as pg/ml
(0=concentrations below the detection limit).
ALLO=Allopregnanolone. EPIALLO=epialiopregnandone.
PREG=pregnenolone, TESTO=testosterone.
TABLE-US-00005 TABLE 2A Decrease in Allo, and Epiallo, % changes
from >97% and/or decrease in THDOC >29% Pregnenolone treated
cells pg/ml Comp. N.sup.o Name Structure ALLO EPIALLO THDOC PREG
DHEA TESTO 32 17.alpha.- Ethylpreg- nenolone ##STR00033## -97.54
-98.72 -88.33 -100.00 0.00 0.00 1 3.beta.- Fluoropreg- nenolone
##STR00034## -99.38 -99.93 -81.87 -100.00 0.00 0.00 3
3.beta.-Fluoro-17.alpha.- methylpreg- nenolone ##STR00035## -99.56
-100.00 -71.63 -100.00 0.00 0.00 39 5,16- Pregnadien- 20-one
##STR00036## -100.00 -100.00 -29.14 -100.00 0.00 0.00
TABLE-US-00006 TABLE 2B Decrease in Allo and Epiallo >96% and no
decrease in THDOC % changes from Pregnenolone Comp. treated cells
pg/ml N.sup.o Name Structure ALLO EPIALLO THDOC PREG DHEA TESTO 60
5.beta.- Pregnan- 3,20- dione ##STR00037## -100.00 -100.00 78.80
-99.72 0.00 0.00 36 17- Methoxy- preg- nenolone ##STR00038##
-100.00 -100.00 86.37 -100.00 0.00 0.00 35 3.beta.- Methoxy-
17.alpha.- methylpreg- nenolone ##STR00039## -99.98 -99.96 14.83
-100.00 0.00 0.00 20 5-Pregnen- 3.beta.,20.alpha.-diol ##STR00040##
-97.11 -96.41 35.54 -97.11 0.00 0.00 63 17.alpha.- Benzyl- 3.beta.-
benzyloxy- pregnen- olone ##STR00041## -99.01 -99.84 46.63 -99.87
0.00 0.00 70 17.alpha.- Benzyl- 3.beta.- methoxy- pregen- olone
##STR00042## -99.75 -100.00 12.76 -99.76 0.00 0.00 2 17.alpha.-
Methyl- preg- nenolone ##STR00043## -99.26 -98.89 34.17 -95.70 0.00
0.00
[0649] Table 2. Pregnenolone derivatives with reduced metabolism in
CHO cells. Results are expressed as percentage changes from CHO
cells treated with pregnenolone (table 2A) or as pg/ml
(0=concentrations below the detection limit).
ALLO=Allopregnanolone. EPIALLO=epiallopregnanolone.
PREG=pregnenolone, TESTO=testosterone.
[0650] As can be seen in table 3 current knowledge on steroid
metabolism does not allow to fully predicting which modifications
of pregnenolone will reduce notably metabolism and which will not.
For example an alpha-hydroxyl group in C20 reduced metabolism
whilst a beta-hydroxyl group in C20 did not. Similarly compounds
with a beta-hydrogen in C5 had a reduced metabolism whilst C5 alpha
compounds were strongly metabolized. Also selective groups in C3
and C17 or some specific combination blocked metabolism.
TABLE-US-00007 TABLE 3A Modifications in position C3 No Name
Structure Reduced metabolism 41 3.beta.- Benzyloxy- pregnenolone
##STR00044## 72 3- Azidopregnenolone ##STR00045## 1 3.beta.-
Fluoropregnenolone ##STR00046## Metabolized 33
3.beta.-Methoxypregnenolone ##STR00047## 66
3.beta.-Aminopregnenolone ##STR00048## 34
3.beta.-Ethoxypregnenolone ##STR00049## 55
3.beta.-Acetoxypregnenolone ##STR00050## 25 Pregnenolone
hemisuccinate ##STR00051## 37 3- Dehydroxypregnenolone ##STR00052##
53 Pregnenolone sulfate sodium ##STR00053##
TABLE-US-00008 TABLE 3B Modifications in position C20 alpha or beta
No Name Structure Reduced metabolism 14
4-Pregnen-17.alpha.,20.alpha.- diol-3-one ##STR00054## 12
4-Pregnen-3.beta.,20.alpha.- diol ##STR00055## 18
4-Pregnen-20.alpha.-ol-3- one ##STR00056## 20
5-Pregnen-3.beta.,20.alpha.- diol ##STR00057## Metabolized 13
4-Pregnen-3.beta.,20.beta.- diol ##STR00058## 15
4-Pregnen-17.alpha.,20.beta.- diol-3-one ##STR00059## 19
4-Pregnen-20.beta.-ol-3- one ##STR00060## 21
5-Pregnen-3.beta.,20.beta.- diol ##STR00061##
TABLE-US-00009 TABLE 3C Modifications in position C5 No Name
Structure Reduced metabolism 77 5.beta.-Pregnan-3.beta.-ol-20- one
(Epipregnanolone) ##STR00062## 60 5.beta.-Pregnan-3,20- dione
##STR00063## Metabolized 8 5.alpha.-Pregnan-3.beta.,20.alpha.-diol
##STR00064## 10 5.alpha.-pregnan-3.alpha.-ol-20-one hemisuccinate
(Allopregnanolone hemisuccinate) ##STR00065## 11
5.alpha.-Pregnan-3.beta.-ol-20-one (Epiallopregnanolone)
##STR00066## 17 4-Pregnen-3.beta.-ol-20-one ##STR00067## 29
5.alpha.-Pregnan-3,20-dione ##STR00068## 46
5.alpha.-Pregnan-3.alpha.-ol-20- one (Allopregnanolone)
##STR00069## 47 Progesterone ##STR00070##
TABLE-US-00010 TABLE 3D Modifications of the bound C16-C17 or of
position C16 No Name Structure Reduced metabolism 39
5,16-Pregnadien- 20-one ##STR00071## Metabolized 6 5,16-Pregnadien-
3.beta.-ol ##STR00072## 7 5,16-Pregnadien- 3.beta.-ol-20-one
##STR00073## 38 5,16-Pregnadien- 3.beta.,20-diol ##STR00074##
TABLE-US-00011 TABLE 3E Modifications in position C20 & C21 No
Name Structure Reduced metabolism 24 20- Deoxypregnenolone
##STR00075## 67 20-Methylamino-5- pregnen-3.beta.-ol ##STR00076##
Metabolized 52 4-Pregnen-21-ol-3, 20- dione ##STR00077## 26
5-Pregnen-3.beta.,21-diol-20- one ##STR00078## 76 5.alpha.-Pregnan
3.beta.,21-diol-20- one ##STR00079## 75
5.alpha.-Pregnan-3.alpha.,21-diol- 20-one ##STR00080## 50
5-Androsten-3.beta.-ol-17- one (DHEA) ##STR00081##
TABLE-US-00012 TABLE 3F Modifications in position C17 and C3 No
Name Structure Reduced metabolism 2 17.alpha.- Methylpregnenolone
##STR00082## 65 17.alpha.- Benzylpregnenolone ##STR00083## 32
17.alpha.- Ethylpregnenolone ##STR00084## 36 17-
Methoxypregnenolone ##STR00085## 74 17.alpha.-Benzyl-3.beta.-
fluoropregnenolone ##STR00086## 73 17.alpha.- Benzylprogesterone
##STR00087## 63 17.alpha.-Benzyl-3.beta.- benzyloxypregnenolone
##STR00088## 70 17.alpha.-Benzyl-3.beta.- methoxypregenolone
##STR00089## 42 3.beta.-Benzyloxy-17.alpha.- methylpregnenolone
##STR00090## 40 17.alpha.-Methylprogesterone ##STR00091## 3
3.beta.-Fluoro-17.alpha.- methylpregnenolone ##STR00092## 35
3.beta.-Methoxy-17.alpha.- methylpregnenolone ##STR00093## 69
17.alpha.-Allyl-3.beta.- methoxypregnenolone ##STR00094##
Metabolized 71 17- Ethoxypregnenolone ##STR00095## 22 17.alpha.-
Hydroxypregnenolone ##STR00096## 64 17.alpha.-Allylpregnenolone
##STR00097## 23 17.alpha.- Hydroxypregnenolone hemisuccinate
##STR00098##
[0651] Table 3. Comparison of the modifications of pregnenolone
derivatives that reduced or maintained significant metabolism in
downstream active steroids.
Example 25: Derivatives of Pregnenolones for which the
Transformation in Downstream Active Steroids is Limited Inhibit the
Effects of CB1 Receptor Activation
[0652] As examples of the compound described in the general formula
A, the inventors tested in the hereby presented examples the effect
on food intake of compounds 3-Fluoropregnenolone,
17-methylpregnenolone, 3-fluoro-17-methylpregnenolone in rat or
mice after stimulation with THC and/or after food deprivation. The
compounds 3-Fluoropregnenolone, 17-methylpregnenolone,
3-fluoro-17-methylpregnenolone were able to inhibit the effects of
CB1 activation on food intake. Compound 17-methylpregnenolone
seemed more effective than pregnenolone and 3-Fluoropregnenolone
and 3-fluoro-17-methylpregnenolone in inhibiting the effects of the
activation of the CB1 receptor (FIG. 19). Compound
17-methylpregnenolone was able to decrease significantly the
increase in food intake induced by THC in rats, whilst only a
tendency to decrease food intake was observed for the other two
compounds (FIG. 19A). In food-restricted mice all compounds
decreased food intake. However, a statistically significant effects
was obtained at the lowest dose (4 mg/kg) for compound
17-methylpregnenolone, whilst 6 mg/kg were needed for reaching
statistical significance with pregnenolone and 3-Fluoropregnenolone
and 3-fluoro-17-methylpregnenolone (FIG. 19B). Finally, THC-induced
increase in food intake in mice was decreased by all compounds at
(2 mg/kg) in mice (FIG. 19C). A dose response function for
THC-induced increase in food-intake showed that both pregnenolone
and 17-methylpregnenolone inhibited this behavior at 1 mg/kg, whist
0.5 mg/kg dose was ineffective.
[0653] Other pregnenolone derivatives (Table 4) for which the
metabolism in downstream active steroids was reduced were tested
for their ability to inhibit: 1. effects of the THC-induced
cannabinoid tetrade (decrease in body temperature and in locomotor
activity, (THC 10 mg/kg, compounds 6 mg/kg 15-30 min before THC)
that is recognized as a sound method to evaluate the activation of
the CB1 receptor; 2. THC-induced increase in food intake a typical
effect of CB1 activation (THC between 0.5 and 1 mg/kg, compounds
between 2 and 4 mg/kg 30 min before THC); 3. the increase in
TNFalpha induced by LPS, another effects typical of CB1 antagonists
(compounds 6 mg/kg 15 min before LPS). For each compound and each
test independent groups of animals were used (at least n=6 per
compound/per test).
TABLE-US-00013 TABLE 4A Compounds with a beta-hydroxyl group in
position C3 CB1 Antagonism % Inhibition % increase % Inhibition of
% Compounds with reduced metabolism temperature in motor
THC-Induced Inhibition No Name Structure decrease activity food
intake of TNF-.alpha. 77 5.beta.-Pregnan-3.beta.-ol- 20-one
(Epipregnanolone) ##STR00099## 70%, P < 0.0001 177%, P < 0.02
.gtoreq.100%, P < 0.01 76%, P < 0.003 2 17.alpha.-
Methylpregnenolone ##STR00100## 43%, P < 0.001 240%, P < 0.01
.gtoreq.100%, P < 0.001 60%, P < 0.02 65 17.alpha.-
Benzylpregnenolone ##STR00101## 53%, P < 0.003 142%, P < 0.03
.gtoreq.100%, P < 0.001 65%, P < 0.01 36 17-Methoxy-
pregnenolone ##STR00102## 54%, P < 0.001 326%, P < 0.001
.gtoreq.100%, P < 0.003 37%, P < 0.04 12
4-Pregnen-3.beta.,20.alpha.- diol ##STR00103## 42%, P < 0.004
100%, P < 0.16 .gtoreq.100%, P < 0.001 81%, P < 0.002 20
5-Pregnen-3.beta.,20.alpha.- diol ##STR00104## 34%, P < 0.04
123%, P < 0.04 85%, P < 0.03 65%, P < 0.01
TABLE-US-00014 TABLE 4B Modifications in position C17, C20 that
decrease activity CB1 Antagonism % Inhibition % increase %
Inhibition of % Compounds with reduced metabolism temperature in
motor THC-induced Inhibition No Name Structure decrease activity
food intake of TNF-.alpha. 32 17.alpha.- Ethylpregnenolone
##STR00105## 12%, P > 0.25 -2%, P > 0.25 nt 64%, P < 0.02
24 20- Deoxypregnenolone ##STR00106## 25%, P < 0.08 33%, P >
0.20 nt 34%, P > 0.14 67 20-Methylamino-5- pregnen-3.beta.-ol
##STR00107## 28%, P < 0.05 -20%, P > 0.35 nt nt
TABLE-US-00015 TABLE 4C Modifications in position C3 that maintain
activity CB1 Antagonism % Inhibition % increase % Inhibition of %
Compounds with reduced metabolism temperature in motor THC-Induced
Inhibition No Name Structure decrease activity food intake of
TNF-.alpha. 41 3.beta.- Benzyloxy- pregnenolone ##STR00108## 38%, P
< 0.001 106%, P < 0.03 nt 62%, P < 0.01 72 3-
Azidopregnenolone ##STR00109## 34%, P < 0.05 145%, P < 0.05
.gtoreq.100%, P < 0.001 61%, P < 0.02 1 3.beta.-
Fluoropregnenolone ##STR00110## nt nt .gtoreq.100%, P < 0.01
58%, P < 0.02 3 3.beta.-Fluoro-17.alpha.- methylpregnenolone
##STR00111## nt nt .gtoreq.100%, P < 0.01 nt 39
5,16-Pregnadien-20- one ##STR00112## 56%, P < 0.001 309%, P <
0.001 .gtoreq.100%, P < 0.02 46%, P < 0.03
TABLE-US-00016 TABLE 4D Modifications in position C3 that decrease
activity CB1 Antagonism % Inhibition % increase % Inhibition of %
Compounds with reduced metabolism temperature in motor THC-Induced
Inhibition No Name Structure decrease activity food intake of
TNF-.alpha. 70 17.alpha.-Benzyl-3.beta.- methoxypregenolone
##STR00113## 8%, P > 0.49 2%, P > 0.48 nt nt 35
3.beta.-Methoxy-17.alpha.- methylpregnenolone ##STR00114## 33%, P
< 0.01 -32%, P > 0.24 nt 57%, P < 0.05 69
17.alpha.-Allyl-3.beta.- methoxy- pregnenolone ##STR00115## 4%, P
> 0.39 -49%, P > 0.1 nt nt
TABLE-US-00017 TABLE 4E Compounds with a ketone in position C3 CB1
Antagonism % Inhibition % increase % Inhibition of % Compounds with
reduced metabolism temperature in motor THC-Induced Inhibition No
Name Structure decrease activity food intake of TNF-.alpha. 14
4-Pregnen-17.alpha.,20.alpha.- diol-3-one ##STR00116## 45%, P <
0.001 277%, P < 0.004 70%, P < 0.06 65%, P < 0.01 18
4-Pregnen-20.alpha.-ol-3- one ##STR00117## 25%, P < 0.04 146%, P
< 0.1 30%, P > 0.25 65%, P < 0.01 40 17.alpha.-
Methylprogesterone ##STR00118## 18%, P < 0.02 57%, P < 0.21
74%, P < 0.07 57%, P < 0.02 60 5.beta.-Pregnan-3,20- dione
##STR00119## 66%, P < 0.0001 70%, P < 0.05 .gtoreq.100%, P
< 0.002 77%, P < 0.003
[0654] Table 4 Inhibition of CB1 activation by pregnenolone
derivatives with reduced metabolism. Mice (at least n=6 per group)
treated with pregnenolone derivatives (between 2 and 6 mg/kg) were
compared to the appropriate controls treated with vehicle. Changes
in body temperature and locomotor activity were studied after
injection of 10 mg/kg of THC, food intake after injection of THC
between 0.5 and 1 mg/kg, TNF.alpha. after systemic injection of
LPS. nt=not tested. Statistics were performed using Student's
t-test.
[0655] For compounds that maintained a beta-hydroxyl group in
position C3, the substitution in C17 with a methyl or a benzyl or a
methoxyl group generated pregnenolone derivatives that maintained a
good level of antagonism of CB1 activity (Table 4A). Also a good
level of activity was observed when position C20 is substituted
with an alpha-hydroxyl group and/or the C5-C6 double bound was
shifted to the C5-C4 position or was substituted with a
beta-hydrogen in C5 (Table 4A). In contrast an ethyl group in
position C17, the suppression of the ketone in position C20 or its
substitution with a methylamino group profoundly reduced antagonism
of CB1 activity (Table 4B). The suppression of the alcohol function
in position C3 or its substitution with a fluor or an azide or a
benzyloxyl group generated compounds with a good antagonism of CB1
activity (Table 4C). In contrast the substitution of the alcohol in
C3 with a methoxyl group induced a profound decrease of CB1
activity (Table 4D). When the alcohol in C3 was substituted with a
ketone there was also a general decrease in the antagonism of CB1
activity (Table 4E). However, the reduced activity of the compounds
with a ketone in C3 could be ameliorated by modifications in
position C5, C20 and C17. An alpha-hydroxyl group in position C20
and C17 or the replacement of the C5-C6 double bond with a
beta-hydrogen in position C5 ameliorated the CB1 antagonists of the
ketone compounds (Table 4E).
[0656] Discussion:
[0657] These converging data can be summarized as follow:
[0658] First, the general formula A allows producing derivatives of
pregnenolone for which the transformation in active steroids
derived by pregnenolone is limited. This formula is original
because the ability of chemical modification of pregnenolone to
reduce or not metabolism in downstream active steroids could not
have been predicted by previous knowledge or by an expert of the
art.
[0659] Second, the general formula I and/or II allows producing
derivatives of pregnenolone that are able to inhibit the effects of
CB1 activation in different system models: 1. food intake induced
by THC in both mice and rats; 2. food intake induced by food
restriction in mice; 3. Behaviors belonging to the cannabinoid
tetrade induced by THC; 4. Increase in TNFalpha induced by LPS.
[0660] V. Inhibition of the Effects of CB1 Receptors Activation is
Specific of Pregnenolone and does not Involve Downstream
Metabolites.
Example 26: THC Increased Pregnenolone Concentrations in the Brain
of Male Wistar Rats More than the Ones of Pregnenolone-Derived
Downstream Active Steroids
[0661] In this example the inventors show that administration of
THC (3 mg/kg sc) to male Wistar rats induced over time a
significant increase of some of the pregnenolone-derived steroids
and in particular of allopregnanolone and epiallopregnanolone.
However, the effect of THC on pregnenolone was of several orders of
magnitude higher than any of the effects observed on the downstream
steroid derived from pregnenolone (FIG. 1C-F).
[0662] When THC was administered at various doses to male Wistar
rats, the inventors found that THC increased the concentrations of
the pregnenolone downstream derived steroids, allopregnanolone and
epialopregananolone, whilst there was no significant increase in
testosterone and DHT. However, even after the highest dose of THC,
the increase in the concentrations of the other steroids were much
smaller than the ones observed for pregnenolone.
Example 27: THC Did not Increase Pregnenolone-Derived Downstream
Active Steroids in Mice
[0663] When THC was administered at various doses to male mice, a
strong dose-dependent increase in pregnenolone concentrations was
observed. However, in mice pregnenolone-derived downstream active
steroids allopregnanolone and epialopregananolone, testosterone and
DHT did not increase significantly in the brain.
Example 28: Doses of Pregnenolone that Inhibit Food Intake do not
Increase the Concentrations of Pregnenolone-Derived Downstream
Active Steroids in the Brain
[0664] In this example the inventors studied the effects of the
doses of pregnenolone (between 2 and 8 mg/kg) that were able to
inhibit the effects of CB1 activation in mice. Pregnenolone
injections between 2 and 8 mg/kg increased brain levels of
pregnenolone; however they did not modify the concentrations of
downstream metabolites, such as epiallopregnanolone and
allopregnanolone in the brain of mice (FIG. 4).
[0665] Discussion
[0666] These converging data can be summarized as follow:
[0667] First, CB1. activation by THC administration in rats induces
a much smaller increase in allopregnanolone and epiallopregnanolone
than in pregnenolone. In mice, THC administration did not increase
allopregnanolone and epiallopregnanolone significantly.
Consequently the negative feed-back on the activity of the CB1,
exercised by the endogenous increase in pregnenolone
concentrations, which was studied in mice, cannot be due to a
subsequent increase of downstream active steroids derived from
pregnenolone.
[0668] Second, the exogenous administration of pregnenolone in the
range of doses at which the inhibition by pregnenolone of the
effect of CB1 activation were observed (2-8 mg/kig) in mice did not
increase either allopregnanolone or epiallopregnanolone in the
brain (FIG. 2). Consequently, the inhibition of the CB1 activity
observed after pregnenolone administration cannot be attributed to
a subsequent increase of downstream active steroids derived from
pregnenolone.
[0669] Finally, derivatives of pregnenolone obtained following the
formula I and/or II that cannot be transformed in pregnanolone and
allopregnanolone and other downstream active steroids are still
able to inhibit the effects of CB1 activation. Consequently, the
inhibition of the CB1 receptor and/or the inhibition of CB1 effects
observed after pregnenolone administration cannot be attributed to
downstream active steroids derived from pregnenolone.
[0670] VI. Methods for Administering Pregnenolone without Inducing
and Increase in Downstream Neuroactive Metabolites.
Example 39. Administration of Pregnenolone with Methods that
Simulate a Slow Release Formulation Allow to Reduce the Metabolism
in Down Stream Active Steroids
[0671] Here the inventors exemplify a method to administer
pregnenolone at doses that are able to inhibit the CB1 activation
but that do not increase downstream neuroactive steroids.
[0672] The effects of pregnenolone administration on plasmatic
levels of pregnenolone were compared (n=5 per group) when
pregnenolone was administered subcutaneously (6 mg/kg) or by Alzet
micro-osmotic pumps (Alzet Osmotis Pumps, Charles River, France,
model 2006) that simulate an extended release formulations of
pregnenolone. Thus, these mini pump implanted subcutaneously
provide a steady release of pregnenolone. Two pregneolone
concentrations were used 0.6 mg/kg/hour and 1 mg/kg/hour (10 times
and six times lower than the dose administered subcutaneously).
Although pregenonolone administration at 6 mg/kg subcutaneously in
mice des not increase the concentration of allopregnenolone in the
brain (FIG. 4) a significant increase in this downstream active
steroid is observed in the plasma (FIG. 20). Pregnenonolone
administered subcutaneously at 6 mg/kg increased the plasmatic
levels of pregnenolone (around 100 ng/ml) but also induced an
increase in allopregnanolone. The half life of pregnenolone
administered subcutaneously was quite short (half an hour) and the
increase in allopregnanolone was maintained over one hour (FIG.
20). On the contrary when pregnenolone was steadily administered
through Alzet minipump at 0.6 mg/kg/hour, pregnenolone increased in
the range of the maximum increase observed after the subcutaneous
injection but did not increased allopregnanolone (FIG. 20). Also
pregnenolone levels remained in the range of effective therapeutic
doses over two weeks. Even when pregnenolone was administered at 1
mg/kg/hour with increased pregnenolone plasmatic concentrations at
the double of what observed after 6 mg/kg allopregnanolone did not
increase (FIG. 20).
[0673] Discussion
[0674] Pregnenolone has been described in previous documents as a
method to treat psychiatric diseases certain type of inflammation
and metabolic disorders and certain type of addiction and in
particular nicotine addiction and alcohol. However in all this
methods pregnenolone was used at high concentrations with the
explicit goal to increase down stream neuroactive steroids to which
the therapeutic effects of the administration of pregnenolone was
attributed. Here we show that pregnenolone in itself without the
involvement of down stream neuroactive steroids at doses much lower
the ones used in previous documents can be useful to treat the
pathologies that involve an activation of the CB1 receptors. In
this context since pregnenolone has a very short half-life
(approximately 30 min) extended release formulations can be useful
in order to maintain pregnenolone levels in the therapeutic range.
By simulating such formulations using Alzet minipump we show that a
steady administration of pregnenolone at low hourly concentrations
allow to reach two objectives: 1. Induce stable concentration of
pregnenolone that are in the range of the ones able to block all
the effects of CB1. activation (around 100 ng/ml); and 2. Reduce
the increase in downstream active steroids such as
allopregnanolone. These results then provide a methods, trough the
use of extended release formulations of pregnenolone, to administer
pregnenolone at low doses to treat diseases involving the CB1
taking advantage of the pharmacological effects of pregnenolone
itself and reducing the unwanted effects of down stream
steroids.
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