Abstract:
The method of treating mammals including humans in need of a drug having analgesic and/or anti-inflammatory activity comprising the administration of a therapeutically effective amount of manoalide, seco-manoalide or dehydro-seco-manoalide. The anti-inflammatory utility includes acute, chronic and delayed sensitization inflammatory processes.

Description:
This invention was made with Government support under Sea Grant No. NA 80 AA-D-00120, Project No. R/MP-21, awarded by the National Oceanic &amp; Atmospheric Administration. The Government has certain rights in this invention. 
    
    
     BACKGROUND OF THE INVENTION 
     Manoalide, from the marine sponge Luffariella variabilis (POLEJAEFF) has been reported by E. D. de Silva and P. J. Scheuer; Tetrahedron Letters Vol. 21, pp 1611-1614 Pergamon Press Ltd. 1980. The structure of this compound is as follows: ##STR1## 
     Seco-manoalide is described in E. D. DeSilva and P. J. Scheuer, Tetrahedron Letters Vol. 22, pp 3147-3150 (1980). Dehydro-seco-manoalide is obtained from the above-identified marine sponge and is recovered from the fraction slightly less polar than and preceding manoalide. 
     Manoalide is a non-steriod and it has now been discovered to be an anti-inflammatory and analgesic agent. Its potency has been found to be greater than that of indomethacin and its site of action in the inflammatory process is different from that of indomethacin. While it is less potent than hydrocortisone, manoalide possesses a pharmacological spectrum different from the glucocorticoids. Uses include treatment of rheumatoid arthritis, osteoarthritis, rheumatic carditis, collagen and/or auto-immune diseases such as myasthenia gravis, allergic diseases, bronchial asthma, and ocular and skin inflammatory diseases. Manoalide appears to be devoid of the endocrine properties of the glucocorticoids yet still appears anti-inflammatory and immunosuppressive. As such, it may also be useful as adjuvant therapy associated with organ and tissue transplants and any neurological disease involving metabolism of nervous tissue phospholipid such as multiple sclerosis. Because of its selective antagonism of chemical irritation (i.e. PMA inflammation) and the fact that it prevents the paralytic action of β-bungarotoxin, manoalide can be useful in the treatment of venoms such as certain snake and insect bites, bee or wasp stings or any venom in which a major constituent is the enzyme phospholipase A 2 . Since manoalide blocks oxazolone induced inflammation this compound would be useful in treating forms of allergic contact dermatitis (such as poison oak or poison ivy). 
     Accordingly, it is believed that this invention makes a significant advance in the pharmaceutical arts. 
     SUMMARY OF THE INVENTION 
     Briefly, the present invention comprises the method of treating mammals including humans in need of a drug having analgesic and/or anti-inflammatory activity comprising the administration of a therapeutically effective amount of manoalide, seco-manoalide or dehydro-seco-manoalide. 
     It is an object of this invention to provide a novel therapeutic method. 
     More particularly, it is an object of this invention to provide an improved therapy involving anti-inflammatory and analgesic properties. 
     These and other objects and advantages of this invention will be apparent from the detailed description which follows. 
     Manoalide, seco-manoalide or dehydro-seco-manoalide are administered to mammals including humans in an effective amount on the order of 10 to 50 mg per day per kilogram of body weight. The drug may be administered orally, parenterally, or by other standard administration routes. The dosage form may be by tablet containing normal acceptable additives, excipients, etc. The parenteral form contains typical aqueous intravenous solution ingredients. 
     The marine natural products seco-manoalide and dehydro-seco-manoalide having the structure shown below have been found to have anti-inflammatory activity in the PMA-mouse ear assay. In preliminary testing, this activity was found to be nearly equivalent in potency to that of manoalide. These comounds had no significant activity in preliminary assays for: inhibition of cell division, effects on cardiac muscle, or effects on skeletal muscle. ##STR2## Spectral characteristics of Dehydro-seco-manoalide: oil; UV (EtOH) 316 nm (ε 12000), 205 nm (ε 10300); UV (EtOH+NaOH) 461 nm (ε 25000), 280 nm (ε 1600), 246 nm (ε 2000);  1  H NMR (CDCl 3 ) δ 0.96 (s, 6 H), 1.56  (s, 3H), 1.60 (s, 3H), 5.11 (bt, 1H, J=7 Hz), 6.14 (s, 1H), 6.32 (s, 1H), 6.82 (d, 1H, J=16 Hz), 6.91 (d, 1H, J=6 Hz), 7.34 (dd, 1H, J=16, 6 Hz), 9.52 (s, 1H);  13  C NMR (CDCl 3 ) δ 194.3, 171.5, 160.0, 146.3, 145.8, 137.8, 136.8, 133.8, 128.3, 126.9, 121.8, 119.5, 97.8, 40.1, 39.7, 34.8, 32.6, 29.5, 28.5, 27.7, 27.2, 24.6, 19.7, 19.4, 16.0. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     A. Acute Toxicity 
     Test compound is injected subcutaneously into mice which are observed for three days for both lethality and any unusual behavior or appearance. This assay may be performed in conjunction with analgesia assays. 
     B. Analgesia--Phenylquinone Writhing Assay 
     Test compound is injected subcutaneously into mice. After 30 minutes, phenylquinone is injected intra-peritoneally to cause pain as indicated by writhing. Absence of or a statistically significant decrease in writhing is considered evidence of analgesia [Hendershot, L. C. and G. Forsaith, Pharmacol. Exp. Ther. 125, 237 (1959)]. 
     C. Analgesia--Haffner Technique 
     Analgesia is measured as the lack of response to a pinch clamp applied to the tail of a mouse 15 to 30 minutes following subcutaneous injection of the test compound [Bianchi, G. and G. Franeschini, Brit. J. Pharmac. 9, 280 (1954)]. 
     D. Electrically Stimulated Guinea Pig Ileum 
     Test compound is administered to an electrically driven section of isolated small intestine and observed for its ability to mimic, antagonize or potentiate the effects of morphine or nalorphine [Gyang, E. A. and H. W. Kosterlitz, Br. J. Pharmac. Chemother. 27, 514 (1966)]. 
     E. Mouse Ear Anti-Inflammatory Assay 
     Test compound and phorbol myristate acetate (PMA) or arachidonic acid (ARA) are topically applied simultaneously to the left ears of mice. Three hours 20 minutes (or one hour for ARA) after application, the mouse is sacrificed, left and right ears removed and standard sized bores taken. Edema (inflammation) is measured as the difference in weight between left and right ears [Van Arman, C. G., Clin. Pharmacol. Ther. 16, 900-904 (1974)]. 
     F. Rat Uterus 
     Test compound is administered to the isolated rat uterus and observed for its ability to mimic, antagonize or potentiate the effects of prostaglandins [modified from DeJalon, Bayo and DeJalon, Farmacoter Act. 3, 313 (1945)]. 
     G. Rat Phrenic Nerve--Hemidiaphragm 
     Compound is tested for its ability to inhibit or facilitate the twitch of the isolated diaphragm in response to the electrical stimulation of the phrenic nerve [Bulbring, E., Br. J. Pharmac. Chemother. 1, 38 (1946)]. 
     H. Eight Day Administration 
     Test compound is administered to mice subcutaneously once per day for seven days. On the eighth day, blood, organ and tissue samples are taken from each animal and analyzed for differences relative to controls. 
     I. Histology 
     Mouse ears from the anti-inflammatory assay are fixed in Karnofsky&#39;s fixative and osmium tetroxide and then embedded in araldite epoxy resin for sectioning for light and electron microscopy. 
     Other Methods 
     A. Fertilized Sea Urchin Egg Inhibition of Cleavage Assay 
     Test compound is added to a slurry of eggs (from Lytechinus pictus) within five minutes following fertilization and incubated until the completion of first division in control slurry, approximately 90 minutes. Inhibition is measured as the percent of undivided eggs in the slurry at the end of incubation [Hinegardner, R., In The Sea Urchin Embryo, ed. C. Czihar (Springer-Verlag, Berlin, 1974)]. 
     B. Guinea Pig Auricles 
     Test compound is administered to the spontaneous beating isolated guinea pig auricles and observed for its ability to alter either the rate or the force of contraction or to antagonize the effects of acetylcholine [modified from J. Physiol. 61, 547 (1926); Heffter&#39;s Hanb. Exp. Pharmak. 4, 185 (1937)]. 
     C. Guinea Pig Illeum 
     Test compound is administered to an isolated section of small intestine and observed for its ability to mimic, antagonize or potentiate the effects of either histamine or acetylcholine [Pflugers Arch. ges Physiol. 102, 123 (1940)]. 
     D. Acetylcholinesterase Assay 
     Test compound is incubated with rat brain extract (source of acetylcholinesterase), acetylthiocholine and dithiobis (2-nitrobenzoic acid). Inhibition of acetylcholinesterase activity is measured spectrophotometrically. 
     E. Tumor Promotion 
     An initiating agent, 7,12-dimethylbenzanthracene (DMBA), is applied topically to the ears of mice. Beginning one month later, a tumor promoting agent, phorbol myristate acetate (PMA), and the test compound are applied simultaneously to the ears twice per week for four weeks, then once per week for two weeks. Ears are observed for formation of tumors at the times of application and one week following completion of applications. 
     F. Oxazolone-Induced Sensitization 
     Mice are sensitized by the application of oxazolone to the abdominal area. One week later, test compound and oxazolone are topically applied to the ears of these mice. Six hours after application, the mice are sacrificed, the ears removed and standard sized bores taken and weighed. Edema (inflammation) is measured as the difference in weight between control and treated ears. Without previous sensitization exposure, oxazolone has no inflammatory effect on mouse ears. 
     Results 
     Manoalide was subjected to pharmacological evaluation. Initial results were as follows: 
     Analgesia--Phenylquinone Writhing--100% analgesia at 50 mg/kg, subcutaneous administration. 
     The discovery that manoalide was active against chemically-induced pain led to extensive study of the compound. 
     The onset of action or manoalide was delayed in comparison to other standard analgesics: the peak effect was detected about two hours after administration. The ED 50  for manoalide is approximately 40 mg/kg when administered subcutaneously (FIG. 1). For comparison purposes, the ED 50  for morphine is about 0.6 mg/kg and for indomethacin is about 5 mg/kg under the same conditions. 
     On the basis of these results, experiments were conducted to determine if manoalide&#39;s analgesic action was similar to indomethacin (i.e., anti-inflammatory) or whether it was similar to morphine (i.e., a narcotic analgesic). 
     In studies utilizing the electrically driven ileum, it was found that manoalide neither blocked electrically induced contractions, an effect common to both morphine and the endorphins, nor did it antagonize the morphine block as would be expected if it had properties common to nalorphine. Similarly, high doses of manoalide did not significantly affect mechanically induced pain as tested by the Haffner technique. Thus far, it is only active against chemically induced pain and therefore is pharmacologically distinct from the narcotics. 
     In the presence of manoalide, there was a marked reduction of inflammation due to PMA induction when a total dose of 10 μg was applied to the ear. An approximate ED 50  is 40 μg/ear (FIG. 2) (will vary with PMA dose, FIG. 3). Histological examination of the ears indicated that manoalide qualitatively produced similar effects to both hydrocortisone and indomethacin. Quantitatively, manoalide was more potent than indomethacin and less potent than hydrocortisone (FIG. 2). The combination of indomethacin and manoalide or hydrocortisone and manoalide resulted in a potentiation of the inhibition of edema indicating a novel mechanism of action (Table II). 
     
                       TABLE I______________________________________Summary of Manoalide StudiesAssay                Remarks______________________________________Initial Screening:Fertilized Sea Urchin Egg                ActiveInhibition of CleavageGuinea Pig Auricles  ActiveMouse Toxicity and Analgesia                ActiveGuinea Pig Ileum     ActiveRat Phrenic Nerve -  ActiveHemidiaphragmDose Response Studies:Analgesia (Phenylquinone Writhing)                ED.sub.50 = 40 mg/KgFertilized Sea Urchin Egg                ED.sub.50 = 1.0                microgram/mlPMA - Mouse Ear Anti-inflammatory                ED.sub.50 = 40                microgram/earInvestigative Assays:Electrically Stimulated Guinea                InactivePig IleumAnalgesia (Haffner Tail-Clip)                InactiveRat Uterus (Prostaglandin Assay)                InactivePMA - Mouse Ear Potentiation                Active(with Hydrocortisone andIndomethacin)Acetylcholine Antagonism in                InactiveGuinea Pig AuriclesArachadonic Acid - Mouse Ear                InactivePMA - Mouse Ear Time Course                Optimum activity at lessof Action            than 5 minutes                post treatmentAcetylcholinesterase InactivePMA - Mouse Ear Competition                Does not competefor binding          directly with PMAEight day administration                Non-toxic;                no glucocorticoid                (steroid-like) propertiesRat Phrenic Nerve-Hemidia-                Inactivates thephragm with β-Bungarotoxin                irreversible neurotoxic                action of β-BungarotoxinHistology (methodology)                Decreased lymphocyte(summary master degree                and PMN infiltrationpaper)PMA = Mouse Ear anti-                ActiveinflammatoryInhibition of purified                Activephosplolipase A.sub.2______________________________________ 
    
     
                       TABLE II______________________________________Mouse Ear Edema Potentiation Studies______________________________________%          Manoalide  Hydrocortisone                             IndomethacinInhibition of      0.04 mg/ear                 0.009 mg/ear                             0.09 mg/earEdema Alone      A          B           C______________________________________      28.24 ± 6.58                 16.59 ± 7.00                             31.42 ± 12.72______________________________________Inhibition ofEdema Additive      A + B      B + C       A + C______________________________________Expected   44.83      48.01       59.66Observed   66.83 ± 5.06                 75.84 ± 7.01                             83.31 ± 2.99______________________________________ (Eight animals in each experiment) 
    
     The next question pursued was whether manoalide had a mechanism of action similar to hydrocortisone or to indomethacin. In this case, the working hypothesis was based on the mechanism of action of PMA induced inflammation. 
     PMA causes the release of arachidonic acid (ARA) from lysosomes. The released ARA is the substrate for the synthesis of prostaglandins which are believed to contribute to pain and inflammation. It is thought that hydrocortisone exerts its anti-inflammatory effect in part by &#34;stabilizing lysosomes&#34; and thereby preventing release of ARA. On the other hand, indomethacin acts to inhibit prostaglandin synthetase and by this mechanism prevents formation of prostaglandins. 
     ARA induced inflammation was effectively blocked by indomethacin over the same dosage range as PMA induced inflammation, as would be expected if prostaglandin synthetase was inhibited. Neither manoalide nor hydrocortisone affected the ARA induced inflammation at any of the doses employed, suggesting that manoalide acts at a site prior to prostaglandin synthesis. Direct interaction of manoalide with prostaglandins (PGE 2 , PGE 1  and PGF 2 ) was ruled out when it was shown to be inactive against these agents in the rat uterus preparation. Time course studies showed that manoalide was most effective when applied within five minutes of PMA application (FIG. 4). 
     To differentiate between the spectrum of activity of manoalide versus that of hydrocortisone, a seven day systemic administration of manoalide (50 mpk) or hydrocortisone (25 mpk) was given to immature mice. (Table III) 
     
                                           TABLE III__________________________________________________________________________7 Day Manoalide Treatment 50 mpk          Group A                Group B Group CTreatment      Control                Manoalide                        Hydrocortisone__________________________________________________________________________Dose           0.1 ml/10 g                50 mg/kg                        25 mg/kg          sesame oilLethality      0     3.8-37.5%                        0N              7     4       7__________________________________________________________________________          Mean  Mean    Mean__________________________________________________________________________Body Weightday 0 (g)      17.9  18.3 (+2.2)                        18.1 (+1.1)day 7 (g)      25.7  26.3 (+2.3)                        22.3 (-13.2)Acute Inflamation with          13.5  15.5 (+14.8)                        --PMA (mg)Organ Wts % of Body wgt.Liver          5.80  6.32 (+8.9)                        5.67Spleen         0.48  0.83 (+71.7)                        0.17 (-63.8)Kidney         1.41  1.28 (-9.2)                        1.63 (15.8)Testes         0.53  0.49 (-6.5)                        0.62 (+18.1)Thymus         0.45  0.37 (-18.4)                        0.08 (-81.6)Heart          0.42  0.35 (-17.7)                        0.56 (+32.4)Adrenal (mg % pair)          13.9  18.41 (+32.3)                        11.01 (-20.9)Food Consumptionmean g/mouse/day          5.89  5.47 (-7.1)                        6.69 (+13.6)H.sub.2 O Consumptionml/mouse/day   8.1   6.1 (-25.7)                        9.8 (+21.0)Blood Analysiswbc/mm.sup.3 mean          1667  8025 (+381.4)                        2850 (+71.0)__________________________________________________________________________ 
    
     Following this treatment, blood samples were taken and autopsies were performed. In brief, hydrocortisone produced the expected thymus involution and reduction of adrenal gland, spleen weights and body weight. In contrast, treatment with manoalide only slightly decreased thymus weight and size, did not alter adrenal gland weight and markedly enlarged the spleen. The animals grew to normal body weight. Manoalide appeared to be acutely immunosuppressive in that injection wound sites did not heal. Finally, manoalide produced a 400% increase in white blood cell count (sbc) over control whereas hydrocortisone did not. Thus, there is evidence that manoalide is inducing an immune response in mice, different than that induced by hydrocortisone. 
     A concurrent line of investigation concerned another effect of the phorbol esters, tumor promotion. Of the phorbols which cause inflammation and promote tumor formation, PMA is the most potent inflammatory agent (FIG. 5). Manoalide not only blocks this inflammation, but also significantly decreases the incidence of tumors formed on mouse ears using PMA as promoter in conjunction with the initiator DMBA (FIG. 6). 
     Manoalide produces a modest contracture in the rat hemidiaphragm preparation as well as mildly inhibiting the directly stimulated twitch. To investigate a possible phospholipase A 2  inhibition by manoalide, competition against β-bungarotoxin action on the diaphragm was investigated. In these studies it was found that manoalide inactivated the irreversible neurotoxic action of β-bungarotoxin (Table IVa). This is a direct reaction with the toxin requiring pre-incubation (Table IVb). 
     
                       TABLE IVa______________________________________Inactivation of β-BuTX paralysis by manoalide(pre-incubation one hour)ConcentrationManoalide β-BuTX        Mean× 10.sup.-5     × 10.sup.-7                  n     TI.sub.50 ± S.E.______________________________________0.0       2.4          (4)   36.0 ± 4.20.6       2.4          (4)   45.2 ± 7.0*1.2       2.4          (4)    75.7 ± 16.5*2.4       2.4          (5)   137.0 ±  22.2*______________________________________ *statistically significant difference relative to BuTX alone, P &lt; 0.05 unpaired students&#39; ttest. 
    
     
                       TABLE IVb______________________________________Effect of incubation time on β-BuTX.sup.+  inactivation       Mean       TI.sub.50 ± S.E.Pre-incubation time             manoalide        mepacrine(min.)      n     2.4 × 10.sup.-5 M                          n   2.1 × 10.sup.-5______________________________________                              M0           (3)   64.60 ± 6.03                          (2) 43.50 ± 2.5015          (4)   83.20 ± 6.65*30          (5)   79.10 ± 3.46*                          (2) 49.00 ± 4.0060          (7)   140.50 ± 18.90*                          (3) 46.60 ± 7.54120         (5)   156.60 ± 22.50*                          (3) 35.66 ± 4.67______________________________________ *Statistically significant difference relative to controls (zero), P &lt; 0. unpaired students&#39; ttest .sup.+ 5 μg/ml BuTX. 
    
     Another study involves the effect of manoalide on an inflammatory response which requires previous sensitization. Manoalide inhibits local, topical inflammation and the possibility that it also may inhibit sensitization has been investigated. It was found that manoalide blocks the oxazolone induced inflammation (Table V). 
     Addendum to Activity of Manoalide 
     Purified phospholipase A 2  from bee venom was incubated with phosphatidylcholine (1.36 mM). Initial velocity of hydrolysis was measured via a pH state (radiometer) as the amount of 0.005 N NaOH consumed per minute. Varying concentrations of manoalide were preincubated with PLA 2  for one hour and compared to solutions preincubated with no manoalide. Manoalide caused a concentration dependent decrease in enzyme activity (FIG. 8). Mepacrine, a PLA 2  antagonist, at concentrations as high as 10 -2  M was ineffective in this protocol. Thus, it appears that manoalide is a potent inhibitor of the hydrolysis of phosphatidylcholine. 
     In experiments designed to explore the mechanism of action of manoalide in vitro, it was found that this compound is a non-competitive inhibitor of the enzyme phospholipase A 2  (FIG. 9). 
     
                       TABLE V______________________________________INHIBITION OF OXAZOLONE-SENSITIZATIONINFLAMMATION                           EARCOMPOUND      DOSE       N      WEIGHT (MGS)______________________________________Oxazolone alone              5    17.7 ± 2.0Ox. + Manoalide         50     μg/ear                        5    17.5 ± 3.7Ox. + Manoalide         100    μg/ear                        5     13.1 ± 1.4*Ox. + Indomethacin         50     μg/ear                        15   16.6 ± 5.7Ox. + Indomethacin         100    μg/ear                        10    11.1 ± 1.2*Ox. + Hydrocortisone         50     μg/ear                        10    9.7 ± 1.2*Ox. + Hydrocortisone         100    μg/ear                        10    9.2 ± 0.4*______________________________________ *Statistically significant difference relative to oxazolone control, p &lt; .05 one tailed student&#39;s Ttest. 
    
     Having fully described the invention it is intended that it be limited only by the lawful scope of the appended claims.