ANTAGONISTS OF CB1 RECEPTOR
20220153776 · 2022-05-19
Inventors
- Pier Vincenzo Piazza (Bordeaux, FR)
- Monique Vallee (Bordeaux, FR)
- Giovanni Marsicano (Bordeaux, FR)
- Francois-Xavier Felpin (Nantes, FR)
- Luigi Bellocchio (Bordeaux, FR)
- Daniela Cota (Bordeaux, FR)
- Jean-Michel Revest (Bordeaux, FR)
- Sergio Vitiello (Bordeaux, FR)
- Umberto Spampinato (Bordeaux, FR)
- Rafael Maldonado (Barcelona, ES)
Cpc classification
A61P1/04
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61P25/18
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
A61P9/10
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
A61P9/04
HUMAN NECESSITIES
A61K31/57
HUMAN NECESSITIES
A61P1/18
HUMAN NECESSITIES
C07J11/00
CHEMISTRY; METALLURGY
A61P1/16
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
C07J41/005
CHEMISTRY; METALLURGY
A61P1/00
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
International classification
C07J7/00
CHEMISTRY; METALLURGY
A61K31/57
HUMAN NECESSITIES
C07J11/00
CHEMISTRY; METALLURGY
C07J13/00
CHEMISTRY; METALLURGY
C07J31/00
CHEMISTRY; METALLURGY
C07J41/00
CHEMISTRY; METALLURGY
C07J5/00
CHEMISTRY; METALLURGY
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.
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, ═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, ═CR10R11 wherein R10 and R11 each independently is H or C1-7 alkyl, or ═O
R4 denotes that C16 is substituted with —H, —OH, or ═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, ═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, ═CR10R11 wherein R10 and R11 each independently is H or C1-7 alkyl, or ═O,
R4 denotes that C16 is substituted with —H, —OH, or ═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 ═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 ═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 ═O or —OH —R2 denotes that C17 is substituted with —H
R3 denotes that C20 is substituted with ═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 ═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 ═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 ═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 ═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 3β-fluoro-17α-methylpregnenolone, 17α-benzyl-3 O-fluoropregnenolone, 17α-benzyl-3β-benzyloxypregnenolone, 3β-benzyloxy-17α-methylpregnenolone, 17α-benzylpregnenolone, 3β-methoxy-17α-methylpregnenolone, 17α-allyl-3β-methoxypregnenolone, or 17α-benzyl-3β-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
FIGURES
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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 ═O and C17 Substituted with R:
[0522] As shown below, to obtain a derivative of pregnenolone having a C3 substituted with ═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° 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] Δ9-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 Française, 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-3β-ol-20-one) lipopolysaccharides from E. Coli 0111:B4 (LPS) from Sigma-Aldrich (France) and rimonabant (SR141716A) from Cayman Chemical (Interchim, Montluçon, 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) (Imalgène 1000; Rhône Mèrieux, 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° 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×g for 10 min, and the supernatant was stored at −20° C. Brains were quickly harvested, the brain areas were dissected on ice and samples were rapidly frozen in cold ice and stored at −80° C.
[0535] Measurement of Steroid Levels by GC/MS.
[0536] Plasma, brain and culture medium levels of pregnenolone (5-Pregnen-3β-ol-20-one), allopregnanolone (3α-hydroxy-5α-pregnan-20-one), epiallopregnanolone (3β-hydroxy-5α-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 100×100×30 cm, illumination of 10 lux, videotracking system: Viewpoint, Lyon, France) or Plexiglas cages (19 cm long×11 cm wide×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° C.±0.1° 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×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° C. by a heating pad (CMA 150, Carnegie Medecin, Phymep). A microdialysis probe (CMA/11, 2 mm long, 240 μm outer diameter, Cuprophan; Carnegie Medicin, Phymep) and a recording electrode (glass micropipette TW150E-4, 2-3 μm 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 μL/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 μL) 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 MΩ). 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; 2×250 mm, particle size 5 μm; 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 μL with a signal/noise ratio of 3:1.
[0557] Histology. At the end of each experiment, a direct continuous current (−20 μA 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 μm) 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° C. Plasma was stored at −80° C. until measurement of TNFα was carried out. Plasma TNFα 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° 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 μm) 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° 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 MΩ 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 μsec duration) delivered at 20 Hz every 10 seconds. Stimulating electrode was placed at a distance >150 μm 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 μM 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 μM.
[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 μM and picrotoxine 50 μM, respectably.
[0571] GABA-A receptors-mediated currents: mIPSC were recorded in the presence of the AMPAR and NMDAR antagonists (DNQX 20 μM and AP-5 50 μM, 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)=A1.Math.e.sup.(−t/τ1)+A2.Math.e.sup.(−t/τ2), where A is the amplitude, t is the time and τ 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 (10×10 cm) from which departed 4 arms (45×10 cm) at a 90° 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 (25×10.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 μl/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° 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© (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 Δ9 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 (
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 (
[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 (
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 (
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 (
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 (
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 (
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 μg/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) (
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 (
[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 (
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 (
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 (
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 (
[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 (
[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 (
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 (
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) (
[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 μM: 23.9±6.0%; 20 μM: 35.3±4.7%; 40 μM: 48.6±3.6%). Conversely, THC induced lesser inhibition of synaptic transmission in slices pre-incubated with pregnenolone 100 nM (10 μM: 11.1±3.2%; 20 μM: 22.7±2.7%; 40 μM: 34.6±3.1%; two way ANOVA neurosteroid factor p=0.001) (
[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 (
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 (
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 μM, 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 μM, 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) (
[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 (
[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 μM: 108.9±12.5 pA; p=0.91) (
[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 (
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 μM) 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 μM) 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- 17α,20α-diol-3- one
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 17α- Methylpro- gesterone
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 3β- Benzyl- oxy- preg- neno- lone
[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 17α- Ethylpreg- nenolone
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 5β- Pregnan- 3,20- dione
[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 3β- Benzyloxy- pregnenolone
TABLE-US-00008 TABLE 3B Modifications in position C20 alpha or beta No Name Structure Reduced metabolism 14 4-Pregnen-17α,20α- diol-3-one
TABLE-US-00009 TABLE 3C Modifications in position C5 No Name Structure Reduced metabolism 77 5β-Pregnan-3β-ol-20- one (Epipregnanolone)
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
TABLE-US-00011 TABLE 3E Modifications in position C20 & C21 No Name Structure Reduced metabolism 24 20- Deoxypregnenolone
TABLE-US-00012 TABLE 3F Modifications in position C17 and C3 No Name Structure Reduced metabolism 2 17α- Methylpregnenolone
[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 (
[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-α 77 5β-Pregnan-3β-ol- 20-one (Epipregnanolone)
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-α 32 17α- Ethylpregnenolone
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-α 41 3β- Benzyloxy- pregnenolone
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-α 70 17α-Benzyl-3β- methoxypregenolone
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-α 14 4-Pregnen-17α,20α- diol-3-one
[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, TNFα 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 (
[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 (
[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 (
[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 (
[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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