Abstract:
The invention relates to a process for the solubilzation of forskolin, to the pharmaceutical compositions based on such solubilized forskolin and to the use of these in the treatment of various diseases and disorders and maladies in mammals, including human. Forskolin is 7-beta-acetoxy-8,13-epoxy-1-alpha,6-beta,9-alpha-trihydroxylabd-14-en-11-one.

Description:
This is a continuation of U.S. application Ser. No. 07/826,287, filed Jan. 24, 1992, now abandoned, which is a continuation of application Ser. No. 07/489,961 filed Mar. 6, 1990 now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     The invention relates to solubilized forskolin (hereafter CHRL-1 being forskolin in solubilized form as described herein), and to its preparation. It further relates to pharmaceutical compositions containing such solubilized forskolin ingredient (i.e. CHRL-1). 
     Forskolin is a labdane diterpenoid, obtainable from natural sources by methanol extraction of the roots of coleus forskoli. The chemical name of forskolin is 7-beta-acetoxy-8,13-epoxy-1-alpha,6-beta,9-alpha-trihydroxylabd-14-en-11-one. The synthesis of forskolin is reported by S. D. Baltt, B. S., Bhawa, H. Dornauer, N. J. de Souza, and H. W. Fehlhaber, Tetrahedron Letters (1977); and by N. J. de Souza, A. N. Dohadwalla, and J. Reden Med. Revs., vol. 3 (1983) pp. 201-219 and references cited therein. One commercial source of forskolin is Sigma Pharmaceutical Co., St. Louis, Mo. I believe that forskolin has not previously been used in vivo in the solubilized form described hereinafter. 
     SUMMARY OF THE INVENTION 
     I have found that forskolin is very soluble in dimethylacetate (DMA), and that this solution can be diluted with distilled water or saline for pharmaceutical use in the presence of a further additive, such as a non-ionic surfactant. A solution can also be made up with this solvent and an edible oil, for per os application; and also in non-edible oils. A further effective solvent is dimethyl-sulfoxide (DMSO), used together with a further additive such as a surfactant, as set out in detail hereinafter. A solution of forskolin can also be made up with DMSO and an edible oil for per os application, and also in non-edible oils for its administration by other routes. 
     The invention also relates to potent and long-lasting analgetic compositions which contain CHRL-1, and to pharmaceutical compositions containing CHRL-1 which are long acting and potent in counteracting the respiratory depression, hypothermia and addiction induced by morphine and by related narcotic compounds. Moreover, CHRL-1 has significant anti-depressant activity. CHRL-1 is also very active in inhibiting the psychotropic effects of high doses of delta 1  -tetrahydrocannabinol (THC) and other cannabinoids contained within cannabis. Finally, the invention relates to compositions containing CHRL-1 which counteract the neuronal toxicity (and other toxicity), the behavioral aberrancies and the lethal effects induced by N-methyl-D-aspartate (NMDA) and resulting from NMDA receptor stimulation. Thus, my invention is useful in the treatment of various diseases and disorders and maladies in mammals, including humans. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     CHRL-1 can be prepared from the solvents listed above in the Summary or from other solvents. Amongst other possible solvents for forskolin there may be mentioned dipropylene glycol, ethylene glycol, formamide, dimethyl formamide, ethylene chloride, diethanolamine, triethanolamine, ethyl acetate, ethylmethoxyethanol, methyl chloride, methylene chloride, and dimethylamine. The limiting factor is one of physiological acceptability and possible toxicity. Considering these aspects, the first six members of this list are acceptable and can be used as solvents in the present invention. Such solvents can be used together with diacetylated monoglyglcerides, dihydric alcohols including ethylene glycol, diethylene glycol, methylene glycol, propylene and dipropylene glycol; glyceryl mono-oleate, glyceryl monostearate, mono- or diglycerides; sucrose esters, as well as glycol, glycerol or polyoxyethelene esters; propylene glycol stearate, ethers (macrogol, macrogol lauryl, polyoxyl 20 cetostearyl and polyoxyoxyl 10 oleyl), octoxinols, poloxamers, polyvinyl alcohol, quillaia, saponin polysorbates, 20, 40, 60, 65, 80 and 85); and the sorbitans monolaurate, mono-oleate, monopalmitate, monostearate, sesquiolate trioleate, tristearate; and sorbitan derivatives, all of which may be used by themselves or together with DMA or DMSO. 
     The preferred systems are those of DMSO with a suitable surfactant or DMA with such surfactant. 
     When DMSO is used, the solubilization is effected by contacting a certain quantity of forskolin and the solvent, vortexing at high speed so as to dissolve the forskolin and adding a suitable active agent such as Emulphor (polyoxyethylated vegetable oil); vortexing again until a clear solution is obtained, and diluting same with a desired quantity of water or saline. 
     For example, 50 mg forskolin is mixed with 0.4 mn DMSO, vortexed at high speed for 2 minutes; 0.4 ml Emulphor or another non-ionic surfactant is added followed by further vortexing until a clear solution is obtained, with 1.7 ml water or saline added to result in a clear solution containing about 20 mg/ml containing about 16% DMSO and about 16% Emulphor. 
     As a further example, 2.5-4.5 mg. forskolin is added to 0.5 mn of DMA in a small (4 ml) vial, completely covering all of the drug with the solvent. When the drug is completely dissolved, 0.10 ml of a non-ionic surfactant is added (preferably Tween 20, a polyoxyethylene sorbitan monooleate or Emulphor El-620, a polyoxyethylated vegetable oil). Heating is unnecessary. The forskolin, dissolved in DMA, is thoroughly integrated into the surfactant by turning the vial on its side and rotating slowly, allowing the DMA-containing forskolin and the surfactant to mix together while coating the sides of the vial. The rotation should take 3-3.5 min. Following this, dilution is effected as required with distilled water or saline followed by vortexing at maximum speed for 2-3 min.(The same technique for integrating the forskolin into the surfactant may also be employed when using DMSO as a solvent). 
     The solubilized form of forskolin (i.e. CHRL-1) results in a stimulation of adenylate cyclase activity at dosages 10 to more than 20 times lower than that of the drug in suspension, without any apparent side effects in animals administered 1 μg/kg to 1 mg/kg of CHRL-1 in vivo. When, in contrast, forskolin is administered in vivo om the form of a suspension, the dosage required to stimulate adenylate cyclase is about 20 mg/kg, with behavioral disturbances resulting, including either sedation, or head twitches and motor stereotypies (q.v. Wachtel et al., Neurosci. Lett. 76: 191-196 (1987). 
     CHRL-1 in doses in 1 μg/kg provides very potent analgesia in both inbred (C-57/B-1) and outbred (Sabra) mice, as well as in rats (Sabra; an outbred strain). The analgesia is equivalent to 10-20 mg/kg of morphine but unlike the latter, is not accompanied by sedation, or marked hypothermia and respiratory depression. Moreover, unlike morphine induced analgesia, the antinociceptive activity of CHRL-1 lasts over 24 hours, and is characterized by very low tolerance and addiction potential. These effects are summarized in Tables 1 and 2 below. 
     The same doses of CHRL-1 also completely reverse the respiratory depression induced by a high (25 mg/kg) dose of morphine within 15-20 min. of the administration of CHRL-1; with respiration increasing from about 65% of normal, to slightly over 100% of normal. Moreover, pretreatment with a 50 μg/kg dose of CHRL-1 48 h. prior to the same dose of morphine (25 mg/kg) was seen to completely protect rats so injected from undergoing any significant change in respiration (see Tables 1.1.1. and 1.1.2. below). The same doses of CHRL-1 1 μg/kg to 150 μg/kg also significantly reversed the hypothermia induced by morphine (25.0 mg/kg) within 15 minutes, from about -2° C. to -0.8° C. (the highest dose of 150 μg/kg being less effective than 1 and 50 μg/kg). The hypothermia was completely reversed by all doses of CHRL-1 administered within 1 hr. of its administration at a time when control animals receiving only morphine had a core body temperature 2.2° C. below normal (see Table 2 below). 
     The same doses of CHRL-1 (1 μg/kg to 500 μg/kg) also markedly inhibited the `dispair` of rats in a forced swimming paradigm of anti-depressant activity by as much as 50% relative to control (See Table 3 below). The same doses of CHRL-1 (1 and 50 μg/kg) also significantly inhibited the tremor, seizure and death induced by N-methyl-D-Aspartate (115 mg/kg) with the lower dose of CHRL-1 being totally effective (see Table 4 below). The same doses of CHRL-1 (1 μg/kg) induced a nearly complete supression of naloxone-precipitated jumping in mice addicted to morphine by pellet implantation (75 mg. morphine over 72 h.), in a small group of animals. 
     A 50 μg/kg dose of CHRL-1 also completely reversed the psychotropic effects induced by an extremely high (25 mg/kg) dose of Δ 1  -tetrahydrocannabinol in both inbred (C-57/B-1) and outbred (Sabra) mice, as measured by the Ring Test of Pertwee. CHRL-1 reduced the Δ 1  -THC elicited elevations of the psychotropic index by up to 58-59%, to control levels (see Table 4). CHRL-1 solution stored at ambient temperature for 6 months was used for repeat tests and compared with fresh solutions. No reduction in activity was observed. It should be noted that all of the activities of CHRL-1 discussed above and summarized in the tables below were observed following the administration of a solution prepared with DMSO and emulphor. This solution is clear and essentially odorless, and has a pH of . when made up to a concentration of 50 μg/ml. The solution freezes at 0° C. and has a boiling point of about 94° C. At. 42° C. it is stable and can be stored at room temperature for at least one month without refrigeration. 
     As it may be seen from Tables A and B below, the doses of CHRL-1 at which optimal activity is seen (i.e. 1 and 50 μg/kg) contain about 0.0009% and 0.047% DMSO respectively, and 0.002 and 0.06DMA respectively. DMSO is currently being used as a vehicle for drugs such as idoxuridine and is available for human use as a 50% solution in the USA (as Rimso-50;® Research Industries Corp.) and as a 70% solution in Canada (as Kemsol®, Horner). The safety of DMSO as a solvent or as a drug in its own right for human use is discussed by Mason (in Dimethyl Sulfoxide, Vol. 1, Marcel Dekker, Inc., New York, 1971) and by Swanson (Rev. Clin. Basic Pharmacol. 5: 1-33 (1985). DMA is currently used as a solvent in drugs and other pharmaceutical products (Martindale&#39;s, 28 th  Ed., 1982) and is less toxic than dimethylformamide (Ibid.), which Bristol Myers has included in one of its patents for cis-platinum. 
     TABLES 
     The Tables A and B below give the volume and % of water, solvent and surfactant for various solutions of CHRL-1. In each solution, the absolute amount of solvent (DMSO or DMA) and surfactant remains constant. 
     
                                           TABLE A__________________________________________________________________________Amt. of CHRL-1    Total vol.          Vol. of Vol. of Drug In Solutionadded to of    water in                  DMSO +  conc.                               % of % ofsolution solution          solution                  surfactant                          (mg/kg)                               DMSO surfactant__________________________________________________________________________50 mg. in    2.5       ml (1.7              ml                + 0.80                     ml)                        = 20.0 16.0 16.0    5.0   4.2     0.80    10.0 8.0  8.0    10.0  9.2     0.8     5.0  4.0  4.0    20.0  19.2    0.8     2.5  2.0  2.0    40.0  39.2    0.8     1.25 1.0  1.0    80.0  79.2    0.8     0.625                               0.5  0.5    160.0 159.2   0.8     0.312                               0.25 0.25    320.0 319.2   0.8     0.156                               0.125                                    0.125    640.0 639.2   0.8     0.078                               0.0625                                    0.0625    liters          liters    1.28  1.2792  0.8     0.039                               0.0312                                    0.0312    2.56  2.5592  0.8     0.0195                               0.0156                                    0.0156    5.12  5.1192  0.8     0.0097                               0.0078                                    0.0078    10.24 10.2392 0.8     0.0048                               0.0039                                    0.0039    20.48 20.4792 0.8     0.0024                               0.0019                                    0.0019    40.96 20.9592 0.8     0.0012                               0.0009                                    0.0009__________________________________________________________________________ 
    
     
                                           TABLE 8__________________________________________________________________________In each solution below, the absolute amount of DMA and surfactant(presentin a 1:2 ratio) remains constant, i.e. 0.05 ml DMA and 0.10 mldetergent.    Total vol.          Vol. of Vol. of DMAAmt of CHRL-1    of    water in                  +        Drug In Solution:added to solution          solution                  surfactant                           conc.                                % of % of sur-solution (ml)  (ml)    (ml)     (mg/kg)                                DMA  factant__________________________________________________________________________3.75 mg in    0.15  (0.00 + 0.15)  = 25.0 33   66    0.30  (0.15 + 0.15)  = 12.5 16   32    0.60  (0.45 + 0.15)  = 6.25 8    16    1.20  (1.05 + 0.15)  = 3.12 4    8    2.40  (2.25 + 0.15)  = 1.56 2    4    4.80  (4.65 + 0.15)  = 0.78 1    2    9.60  (9.45 + 0.15)  = 0.39 0.5  1    19.20 (19.05                + 0.15)  = 0.195                                0.25 0.5    38.40 (38.25                + 0.15)  = 0.0975                                0.125                                     0.25    76.80 (76.65                + 0.15)  = 0.0487                                0.0625                                     0.125    153.60          (153.45                + 0.15)  = 0.0243                                0.0317                                     0.0625    307.20          (307.05                + 0.15)  = 0.0121                                0.0158                                     0.0317    614.40          (614.25                + 0.15)  = 0.0060                                0.0079                                     0.0158    1228.80          (1228.65                + 0.15)  = 0.0030                                0.0039                                     0.0079    2457.60          (2457.45                + 0.15)  = 0.0015                                0.0019                                     0.0039__________________________________________________________________________ 
    
     The TABLES 1 to 6 DIVERSE ACTIVITY OF CHRL-1IN VIVO 
     
                                           TABLE 1__________________________________________________________________________ANALGESIA: ACUTE EFFECTAnalgesia was assayed by means of the two most widely used tests ofanti-nociception extant; viz. the tail flick and hot plate tests ofanalgesia.1.1. Tail Flick A photocell beam interrupted by a flick of the tail automaticallystopped a digital timer to within a tenth of a second of the time of thefirst tailflick. The intensity of the nociceptive stimulus was adjusted to a meanlatency of5 ± 0.5 sec. in control (vehicle injected) animals. Each animal wastested only onceto either the first tail flick or to a cut-off of 15 sec. (greaterlatencies beingunduly traumatic to the tail). Data from animals with ambiguous tailmovements (i.e.not a crisp, well-defined flick) were discarded. Data presented as meanlatency insecs. ± S.E.M.1.1.1. Statistical AnalysisDOSE OF              FORSKOLIN                        MORPHINE   DOSE OF                                          MORPHINEDRUG  SPECIES       STRAIN   LATENCY LATENCY    MORPHINE                                          LATENCY  SEDATION(mg/kg) TESTED       TESTED             N  .sup.-- X                   ±                     SEM                        .sup.-- X                           ±                             SEM                                N  (mg/kg)                                          .sup.-- X                                             ±                                                SEM                                                   PRESENT__________________________________________________________________________0.00  MOUSE C-57/B             7  4.3  0.3                        5.2  0.3                                7  2.50   12.2  1.2                                                   +       SABRA 7  4.8  0.6                        4.8  0.6                                7         --    -- + RAT   SABRA 7  5.1  0.5                        5.1  0.5                                6         12.7  1.1                                                   +0.001 MOUSE C-57/B             5  14.2 0.6                        5.3  0.7                                5  6.25   14.0  1.0                                                   ++       SABRA 5  14.4 0.5                        --   --           --    -- RAT   SABRA 5  14.9 0.1                        5.2  0.6                                50.05  MOUSE C-57/B             10 14.9 0.1                        7.2  1.1                                5  10.00  14.9  0.1                                                   +++       SABRA 8  14.5 0.5                        --   -- RAT   SABRA 6  14.2 0.4                        7.0  1.0                                5         14.6  0.3                                                   + ++0.20  RAT   SABRA 5  13.2 0.7                        10.2 1.1                                51.00  MOUSE C-57/B             5  13.6 0.6                        10.8 1.0                                5       SABRA 5  13.8 0.7                        11.0 0.9                                5__________________________________________________________________________ NOTE: Doses of morphine &gt;10.0 mg/kg induced no greater analgesia but only increased the intensity of sedation. No dose of CHRL1 induced any sedatio 
    
     
         __________________________________________________________________________DOSE OF               FORSKOLIN                         MORPHINEFORSKOLIN   SPECIES         STRAIN  LATENCY LATENCY(MG/KG) TESTED         TESTED               N .sup.-- X                    ±                      SEM                         .sup.-- X                           ±                             SEM                                N__________________________________________________________________________0.00    MOUSE C-57/B               6  9   1   9  1  6         SABRA 6 12   1   RAT   SABRA 6 12   1  11  1  60.001   MOUSE C-57/B               5 19   3  10  1  5         SABRA 5 35   4   RAT   SABRA 5 27   1  12  1  50.05    MOUSE C-57/B               5 24   2  12  1  5         SABRA 5 38   2   RAT   SABRA 5 28   1  11  1  50.20    RAT   SABRA 5 26   2  16  2  51.00    MOUSE SABRA 5 28   2  22  2  5__________________________________________________________________________ 
    
     
                                           TABLE 2__________________________________________________________________________ANALGESIA: CHRONIC EFFECT (OVER 24 HOURS)In addition to measuring the acute effects of forskolin on analgesia(within 1 h.of its administration), the solublized form of forskolin described hereinwas alsoused in chronic tests of analgesia, wherein C-57/B and Sabra mice wereadministeredforskolin and tested only once either 1 h. or 24 h. subsequently.                           FORSKOLIN                                   MORPHINEHOURS POST DOSE OF              LATENCY LATENCYADMINISTRATION      FORSKOLIN              SPECIES                    STRAIN                         N .sup.-- X&#39;                              ±                                SEM                                   .sup.-- X                                      ±                                        SEM                                           N__________________________________________________________________________2.1.   Tail Flick   2.1.1. Statistical Analysis1.0        0.00    MOUSE C-57/B                         7 5.2  0.3                                   5.2  0.3                                           71.0        0.05    MOUSE C-57/B                         5 14.4 0.4                                   5.0  0.5                                           524.0       0.05    MOUSE C-57/B                         5 14.5 0.5                                   5.1  0.4                                           524.0       10.00   MOUSE C-57/B    --   6.4  0.9                                           52.2.   Hot Plate   2.2.1. Statistical Analysis1.0        0.00    MOUSE SABRA                         6 12   1  12   1  61.0        0.05    MOUSE SABRA                         5 38   2  12   1  524.0       0.05    MOUSE SABRA                         5 33   5  11   1  524.0       10.00   MOUSE SABRA  --      13   1  5__________________________________________________________________________ 
    
     
                                           TABLE 3__________________________________________________________________________INHIBITION OF NARCOTIC INDUCED RESPIRATORY DEPRESSION,HYPOTHERMIA AND WITHDRAWAL SYMPTOMS/ADDICTION BY FORSKOLIN__________________________________________________________________________CHRL-1, the solublized form of forskolin described herein, provides aprompt, prolonged andpotent inhibitory action against the major side effects of narcotics suchas morphine while notinterfering with or actually potentiating their analgesic activity.1. RESPIRATORY DEPRESSION1.1. Respiratory Frequency1.1.1. Short-Term ExperimentsSabra rats were randomly assigned to one of 4 sub-groups, receivingmorphine alone(25.0 mg/kg), or morphine followed 30 min. subsequently by one of 3 dosesof CHRL-1 (viz.0.001, 0.05, or 0.15 mg/kg, s.c.). All animals were observed for 90 min.The basal respiratoryrate of each animal (R.sub.f) was determined 60 min. following theirhabituation in a metal cage ata time when the animals were seen to lie quietly in their cages. R.sub.fwas measured as distinct,individual displacements of the thorax in a 15 sec. interval asdetermined by visual observation,with each interval measured by a stopwatch accurate to 0.1 sec. Therespirations observed per15 sec. interval were multiplied by a factor of 4 to give an R.sub.f inrespirations per minute.       TIME POST MORPHINE PRETREATMENTDOSE OF     0 MIN.            30 MIN.  45 MIN.                          90 MIN.FORSKOLIN   TIME POST FORSKOLIN ADMINISTRATION(MG/KG)     0 MIN.            0 MIN.   15 MIN.                          60 MIN.  (N)__________________________________________________________________________0           125 ± 4             91 ± 13                      87 ± 12                           80 ± 8                                   60.001       128 ± 9            90 ± 8                     122 ± 6                          133 ± 6                                   60.05        126 ± 5            93 ± 6                     124 ± 5                          129 ± 4                                   60.15        129 ± 5            96 ± 4                     127 ± 5                          135 ± 8                                   6__________________________________________________________________________1.1.2.   Long-Term Experiments Sabra rats were pretreated with saline (0.9%, s.c.), and subsequentlyadministered CHRL-1(0.05 mg/kg, s.c.; 1 h. later). 48 h. following the injection of CHRL-1,the animals wereadministered morphine (25.0 mg/kg) at the same time that the animals inthe acute studydescribed above (q.v. 1.1.1) received morphine. Consequently, the controlgroup from theacute study served as the control group of the chronic study. Bothcontrol and forskolininjected groups were then examined for 90 min.      TIME POST FORSKOLIN PRETREATMENTDOSE OF    0 MIN           2880 MIN.FORSKOLIN  TIME POST MORPHINE ADMINISTRATION(MG/KG) 0 MIN.        30 MIN.             60 MIN                  90 MIN.                       (N)__________________________________________________________________________0       125 ± 4         91 ± 13              87 ± 12                   80 ± 8                       60.05    128 ± 7        110 ± 11             121 ± 4                  116 ± 7                       6__________________________________________________________________________1.2.   Blood Gas Analysis Basal R.sub.f, measurements were taken for Sabra rats subsequentlypretreated with morphine (25.0 mg/kg, s.c.), followed 1 h. later byCHRL-1 (0.05 mg/kg,s.c.). Subsequently, all animals were administered 0.3 ml per 100 g. ofan anesthetic solution.Polyethylene catheters were introduced into the carotid artery for bloodsamples. Subsequentexperiments (now in progress) will also include cannulation of theexternal jugular vein formeasurements of pulse and blood pressure using a polygraph and calibratedpressure tran-sducers. Each blood sample was analyzed by an automatic continuouslyrecalibrated blood gasanalyzer, for pH, pCO.sub.2, pO.sub.2, HCO.sub.3.sup.-, and B.E. (baseexcess). Typical values arepresented.MIN.TIME POST   TIME POSTFORSKOLIN   MORPHINE          R.sub.f  pCO.sub.2                        pO.sub.2                             HCO.sub.3.sup.-                                  B.E.INJECTION   INJECTION          (per min.)                pH (mm Hg)                        (mm Hg)                             (mmol/l)                                  (mmol/l)__________________________________________________________________________0        0     134   7.22                   20.3 133.5                             8.5  -16.70       30     102   7.32                   26.0 127.1                             13.6 -9.80       60      91   7.29                   32.8 127.4                             15.8 -8.920      70     136   7.24                   28.1 129.3                             11.3 -14.530      90     139   7.20                   18.2 128.5                             6.5  -21.3120     180    136   7.21                   20.8 128.1                             8.0  -18.2__________________________________________________________________________2. HYPOTHERMIA Sabra rats were randomly divided into one of 4 groups. The core (rectal)temperature ofeach animal was determined, whereupon each was pretreated with 25 mg/kgmorphine HCl(s.c.). 30 min. following morphine, animals were administered CHRL-1 indoses of 0.00(controls), 0.001, 0.05 or 0.15 mg/kg), and their core temperaturesmeasured over a 90 min.period from the time of their first injection. Results below are given asthe mean temperature(in °C.) per time period ± SEM.          TIME POST MORPHINE ADMINISTRATION          0 MIN. 30 MIN.                        45 MIN.                               90 MIN.DOSE OF        TIME POST FORSKOLIN ADMINISTRATIONFORSKOLIN N    0 MIN. 0 MIN. 15 MIN.                               60 MIN.__________________________________________________________________________0.        6    37.6             .3  35.5                    .6  35.7                           .8  35.4                                  .50.001     6    37.5             .2  35.2                    .4  36.7                           .3  37.0                                  .30.05      6    37.7             .3  35.6                    .3  36.9                           .1  37.5                                  .20.15      6    37.5             .1  35.7                    .5  36.4                           .2  37.3                                  .1__________________________________________________________________________3. ADDICTION In a pilot study, Sabra mice were implanted subcutaneously with pelletsof morphine HCl,each containing 75 mg. A constant slow release of the morphine waseffected by the body heatof the animal over a 72 h. period. The presence of addiction wassubsequently determined bythe appearance of the withdrawal symptoms of precipitated abstinance(jumping and vocal-izing) induced by naloxone (0.8 mg/kg). Preliminary results with CHRL-1(0.05-1 mg/kg)indicate a very marked-to nearly complete supression of morphinewithdrawal. Data are notpresented due to the small number of animals per group (n = 3), and theseresults are to beregarded as tentative albeit extremely encouraging. A full-scale,protracted study using dosesof CHRL-1 ranging from 0.001 to 1.0 mg/kg before, during and/or afterpellet implantationwill be implemented shortly.__________________________________________________________________________ 
    
     
                       TABLE 4______________________________________INHIBITION OF THE CANNABIMIMETIC (PSYCHO-TROPIC) EFFECT OF Δ.sup.9 -TETRAHYDROCANNABINOL(THC)______________________________________The Mouse Ring Test of Pertwee (1972) was used to assess theeffect of CHRL-1 on the psychotropic activity of an extremelyhigh dose of THC (25.0 mg/kg). One hour following the admini-stration of either vehicle, CHRL-1 (0.05 mg/kg), THC (25.0mg/kg) or CHRL-1 (0.05 mg/kg) + THC (25 mg/kg)Animals were randomly assigned to one of 4 treatment groupsreceiving either vehicle alone, CHRL-1 (0.05 mg/kg), THC(25 mg/kg), CHRL-1 (0.05 mg/kg) + THC (25 mg/kg)administered simultaneously, or CHRL-1 (0.05 mg/kg) followed1 h. later by THC (25 mg/kg). 90 min. following the injectionof either vehicle, CHRL-1 alone or THC (in the last two groupswith CHRL-1, or by itself). C-57 mice were placed upon the rimof a laboratory ring stand 6 cm in diameter situated 25 cm abovea horizontal surface.The movement of each mouse on the metal ring was thenconstantly monitored for each episode of complete dormancy(excluding slight head movements due to breathing) over a 5 min.period. The total time of dormancy on the ring was calculated asan index expressed as the % of total time of complete dormancyobserved (T.sub.D) where T.sub.D = duration of dormancy (in sec.)/300 sec. × 100. When Sabra mice were used, the same procedureas above was followed with the sole exception of THC beingadministered 1 h. prior to CHRL-1 (and hence the animals weretested 90 min. following CHRL-1).            DOSE      INDEXSTRAIN DRUG      (MG/KG)   (%)    N______________________________________C-57/B Vehicle   --        11 ± 1                             10  CHRL-1    0.05      11 ± 2                             17  Δ.sup.1 -THC            25.00     72 ± 5                             10  CHRL-1    0.05      13 ± 1                             10  ←                                     CHRL-1  +                                  pretreat-  Δ.sup.1 -THC            25.00                    ment 1 h.                                     pre-THC  CHRL-1    0.05      16 ± 3                             8   ←                                     CHRL-1  +                                  injected  Δ.sup.1 -THC            25.00                    simul-                                     taneously                                     with                                     THCSABRA  Vehicle   --        12 ± 1                             10  CHRL-1    0.05      14 ± 2                             6  Δ.sup.1 -THC            25.00     73 ± 6                             6  Δ.sup.1 -THC            25.00     15 ± 3                             7  +  CHRL-1    00.05______________________________________ 
    
     
                       TABLE 5______________________________________ANTI-DEPRESSANT PROPERTIES OF CHRL-1______________________________________ Recently, Borsini and Meli (Psychopharmacology  --97: 183 (1988;see also Psychopharmacology  --94: 147) have reported considerableevidence of their own supported by that of a number of otherinvestigators that the `despair` induced by the forced swimmingtest originally proposed by Porsolt et al. (Eur. J. Pharmac.  --47:379 (1978) is a valid and reliable test of anti-depressant activity,provided that (1) a pretest of 15 min. duration is included and (2)the test is conducted solely in rats.The paradigm consists of immersing rats (in the present caseSabra) in a plexiglass cylinder, 40 cm high and 18 cm in diameter,containing 15 cm of water heated to 25° C. After a 15 min.pre-test,the animals are dried for 15 min. in a heated enclosure (32° C.)and exposed again to the same conditions 24 h. later, for 5 min.Total immobility time is then recorded, where immobility isdefined as the animal making only those minimal movementsrequired to keep its head above water.FORCED SWIMMING TEST OF PORSOLT:Statistical AnalysisCHRL-1 DOSE (mg · kg)          N      % DISPAIR (OVER 5 MIN)______________________________________0.00 (Vehicle) 7      69 ± 50.001          8      36 ± 90.05           9      32 ± 20.20           9      26 ± 30.50           5      19 ± 4______________________________________ 
    
     
                       TABLE 6______________________________________EFFECT OF CHRL-1 ON NMDA______________________________________ When N-Methyl-D-Aspartate is injected subcutaneously intomice a distinct syndrome is induced, consisting initially ofsedation, followed by tremor, unilateral rotation and jumping,convulsion, and finally, death. The syndrome is highly specificto NMDA receptors and is unaffected by drugs exerting nosignificant inhibitory effect on these receptors. The experimentsbelow are an extention of a pilot experiment and differ from itin using a lower dose of NMDA (115 mg/kg rather than 150mg/kg as before), and using 9 animals per group rather than 5.C-57/B Mice; All injections s.c.    VEHICLE    CHRL-1    (TIME      TIME FROM    FROM NMDA  NMDA INJECTIONPARAMETER  INJECTION    0.05 mg/kg                             0.001 mg/kg______________________________________Latency to Tremor       5.7 ± 1.1                   11.5 ± 1.2                             --Latency to 1.sup.st      12.4 ± 1.0                   --        --SeizureTime from  17.9 ± 1.3                   --        --NMDA Injectionto Death______________________________________