USOO581,1451A United States Patent (19) 11 Patent Number: 5,811,451 Minoia et al. (45) Date of Patent: Sep. 22, 1998

54 PHARMACEUTICAL COMPOSITIONS 58 Field of Search ...... 514/816, 823, COMPRISING AN OPIATE ANTAGONST 514/443 AND CALCIUM SALTS, THEIR USE FOR THE TREATMENT OF ENDORPHIN- 56) References Cited MEDIATED PATHOLOGIES U.S. PATENT DOCUMENTS 76 Inventors: Paolo Minoia, Via M. Viterbo 12, 4,241,066 12/1980 Kobylecki et al. . I-70013 Castellana Grotte, (Bari); Rafaele Luigi Sciorsci, Via Positano, FOREIGN PATENT DOCUMENTS 84/B, I-70014 Conversano, (Bari), both 876968 10/1979 Belgium. of Italy 0289070 11/1988 European Pat. Off.. 21 Appl. No.: 737,902 0506468 9/1992 European Pat. Off.. y - - - 9 22 PCT Filed: May 22, 1995 OTHER PUBLICATIONS 86 PCT No.: PCT/EP95/01931 J. of Biological Chemistry, vol. 264, No. 5, 1989, pp. 347-353, Attali et al. S371 Date: Nov. 21, 1996 Peptides, vol. 13, 1992, pp. 947-951, Wang et al. S 102(e) Date: Nov. 21, 1996 Primary Examiner Keith D. MacMillan 87 PCT Pub. No.: WO95/31985 Attorney, Agent, or Firm Bucknam and Archer PCT Pub. Date: Nov.30, 1995 57 ABSTRACT 30 Foreign Application Priority Data The combined use of opiate antagonists and of calcium Salts for the preparation of medicaments for the treatment of May 24, 1994 ITI Italy ...... MI94A1048 endorphin-mediated pathologies is described. (51) Int. Cl." ...... A61K 31/485 52 U.S. Cl...... 514/443; 514/823; 514/816 17 Claims, No Drawings 5,811,451 1 2 PHARMACEUTICAL COMPOSITIONS organs and in free form, in the plasma and in the liquor. The COMPRISING AN OPIATE ANTAGONST ratio between free and bound endorphins may be increased AND CALCIUM SALTS, THEIR USE FOR in relation to the increased production, the reduced catabo THE TREATMENT OF ENDORPHIN lism or the competitive removal from the receptors of the MEDIATED PATHOLOGIES bound endorphins, for instance by the opiate antagonists Such as naloxone, naltrexone and derivatives and analogs. FIELD OF THE INVENTION The free endorphins, if not rapidly removed by catabolic The present invention relates to the combined use of mechanisms, bind again to the respective receptors, inducing opiate antagonists and of calcium Salts for the preparation of a Series of biochemical effects impairing the cellular medicaments for the treatment of endorphin-related patholo metabolism, interfere with the nervous function and induce gies. a pathogenetic action of the affected organs. The invention also relates to pharmaceutical compositions SUMMARY OF THE INVENTION for human and Veterinary use containing as active principle It has now been Surprisingly found that the administration opiate antagonists in combination with calcium Salts and of opiate antagonists in combination with calcium ions is optionally with proteases, prostaglandins and Vitamin C and 15 able to effectively antagonize Said pathogenetic actions, K. The compositions of the invention may optionally be in resulting to be useful, both in human and in Veterinary form of kit-of-parts, consisting of Separate dosage forms for clinical practice, in pathologies characterized by high free the contemporaneous or Sequential administration of the and bound endorphin levels, hereinafter defined endorphin above mentioned active principles. related pathologies. BACKGROUND OF THE PRIOR ART Without any connection to the validity of the invention, the proposed hypothesis is that the high tissue and circulat The neurons of the nigro-striatal System, together with ing level of endorphins, both of physiological and patho many other nervous structures, Synthesize low nuclear logical kind, interacts with the Ca" metabolism and with all weight compounds, endorphins, having actions practically the related or dependent functions. It is in fact presumed identical with that of phenantrene alkaloids of morphine. 25 that, in the event of endorphins increase beyond the physi These endogenous opioids (endorphins) play an essential ological limits, Ca" flow inside and outside the cells is biological role in the Central Nervous System of every Somewhat impaired, resulting in endocellular and endotis animal, man included. Sutal calcium deficit with an increase of calcemia. The endogenous opiate peptides, enkephalins and Contemporaneously, it is probable that the Signal of endorphins, consisting of aminoacid (from 5 to 31) increased endocellular calcium request causes recruitment Sequences, are present at the hypothalamic, cerebral and of external calcium towards the damaged tissues, where spinal level as well as in the endocrine glands (adrenal bound endorphins accumulate. glands, hypophysis, ovaries, testis), and gastrointestinal In other words, when different physiological or patho System, muscle-skeletal System and immunitary System. The logical conditions induce the endogenous increase of circu functions of the up-to-new known endorphins are multiple; 35 lating endorphins, the latter bind to the opiate receptors in the most known are: morphine-like analgesic properties, one or more Structures or organs. While the presence of behavioural effects, neuromodulator functions. normal level of bound endorphins to the nervous receptors These peptides, play also a remarkable role in functions in any organ is physiological, on the contrary the increase of Such as memory, response to StreSS, pain transmission, 40 bound endorphins induces the accumulation of a large regulation of appetite, temperature, respiratory frequency, amount of these neuromodulators which, binding in large libido, immunity etc. amounts to the receptors, form a Sort of "endorphin cloud' The endorphins, ubiquitary present in mammals, inside involving alterations of the membrane potential and perme and outside the central nervous System, derive from at least ability in the nervous, muscular structures or in any cell three different precursors: pre-pro-opiomelanocortine having endorphin receptors. The alteration of the cell per (POMC), pre-pro-enkephaline A and pre-pro-enkephaline B, 45 meability mainly influences the activity and functionality of yielding three classes of peptides related thereto, having calcium channels and consequently all the related and con well defined biological activity. Sequent activities and functions. In particular, pre-pro-opiomelanocortine produces, as a Whenever high endorphin levels persist, the dysmetabolic result of lytic processes, differentiated in the various tissues, 50 processes Start from the nervous terminations. In the acute alpha-, beta- and gamma-endorphins, pre-pro-enkephalin A processes, the block of the calcium entry and the mobiliza yields met-enkephalin and leu-enkephalin whereas pre-pro tion of the intracellular calcium provide a metabolic acco enkephalin B is the precursor of alpha-neo-endorphin, beta modation which may become deadly, by removing the neo-endorphin and dinorphine. The role and distribution of “endorphin cloud' and consequent Sharp change of the these peptides in the various tissues have been widely 55 membrane potential and entry into the bloodstream of cal Studied, with particular reference to their ability of interact cium coming from within the cells previously blocked, in the ing with the opiate receptorS. absence of a Suitable amount of calcium in the bloodstream. The endorphins have been in fact recognized as defense It is presumed that the “endorphin cloud' first decreases agents able to induce analysis and Sedation in organisms the cellular and tissue functionality and reactivity, causing Subjected to StreSS of different kind and aetiology. 60 thereafter an abnormal activity through a kind of block of the For instance, an increased production of endorphins was Ca" channels present on the cell wall. noticed after traumatic injuries, nervous, endocrine, meta The outside and inside calcium block causes the affected bolic or infectious diseases, physical fatigue, delivery, cell to mobilize Ca' from the inner deposits in the endo insomnia, Surgical operations, alimentary or pharmacologi plasmatic reticulum and in the mitochondria, So that its cal intoxication, etc. 65 metabolic activity can be, at least partially, preserved. The The endorphins are found in the organisms both in a form contemporaneous extracellular calcium increase (increased bound to the receptors present in the various tissues and calcemia) causes neuromuscular toxicity. 5,811,451 3 4 The calcium administration according to the invention Also the poSology and the administration route will prevents, in the case of hypocalcemia, the calcium outflow depend on factors (animal species, weight, kind and Seri from cells, already impaired by Ca' deficit, into the ousness of the pathology) which will be evaluated by the bloodstream, with consequent worsening of the cellular Veterinary or by the physician. The dosage will generally be damage and therefore of the pathology. comprised from about 1/10 to about 10 times of that recom In any case, independently on the verification of the above mended for the widely known and classical indications of reported mechanisms, previously never disclosed or these drugs. For instance, in human medicine, naloxone may hypothesized, the present invention allows to achieve Sur be initially administered at doses of 0.1-2 mg daily and prising therapeutic results in endorphin-mediated patholo naltrexone at doses of 5-50 mg daily, whereas doses of gies. 10–20 mg of naltrexone are recommended for the mantein The endorphin receptors, in addition to the Central Ner ance therapy. Vous System, are widespread in the organism, and therefore In Veterinary, 5-50 mg of naloxone i.V. or i.m. may be the pathologies which may be treated or alleviated by the administered to horses and bovine one or more times a day present invention include diseases of the Central Nervous according to the pathology. In dogs, depending on the Size, System Such as paraplegia, nervous conducibility doses of 0.5-1 mg/kg are usually administered. disturbances, Alzheimer's disease, cerebral ischemia, mul 15 In the chronic pathologies in dogs it is preferable the tiple Sclerosis, gastro-intestinal diseaseS Such as ulcers, administration of 5-10-20-50 mg of naltrexone per os, irritable bowel Syndrome, cardiovascular disease Such as considering that the half-life of this drug is by far longer than infarct, Septic Shock, dermatological diseaseS Such as that of naloxone, up to 2-3 days with the active metabolites. Vitiligo, psoriasis, alopecia, dermatitis, traumatic injuries The pharmacological response depends on the used posol and burns, endocrinological and genito-urinary diseases ogy. In fact, minimal doses would induce only partial Such as LUF Syndrome, ovaric micropolycystosis, receptor activation whereas high doses have a complete and impotence, hyperprolattinemia, hypophysary dwarfism, potent effect on the receptors. It is therefore possible to interstitial cystitis, primary amenhorrea. modulate the pharmacological treatment by regulating the The invention may also be advantageously used for the binding of the opiate antagonist to different classes of treatment of inflammatory conditions, infectious diseases, 25 receptor Sites. diseases of the muscle-skeletal System Such as osteoporosis, More precise indications on the dosages may be obtained arthritis, osteitis, perioStitis, myopathies, autoimmune dis from the quantitative determination of the endorphins bound CSCS. to the affected tissues and organs, by means of a dynamic It will be appreciated that the invention generally provides diagnostic method comprising a first radioimmunoassay and beneficial effects in those conditions where the natural one or more Subsequent assays after the administration of a tissue-or cell-repair processes should be preserved or Specific endorphin antagonist, Such as naloxone itself. The re-established. difference between the values of the free endorphins before In Veterinary medicine, in addition to the corresponding and after the antagonist administration yields the value of human pathologies cited above, the invention may be advan bound endorphin and optionally, in the case of more assays tageously used for the treatment of Specific conditions Such 35 after the antagonist administration, the binding kinetics of as puerperal shock, in bovines, Viral diseases in dogs and endorphins. cats (parvovirus infections, distemper), MMA Syndrome The parameters obtainable by Said diagnostic method (metritis-mastitis-agalactia), Mulberry's heart disease, rumi provide guidelines for therapeutic treatments according to nal meteorism, Hoflund Syndrome, Osteo-articular traumas the invention. The calcemia changes induced by the treat Such as fractures, polyarthritis, Osteomalacia, rachitism, hip 40 ment of the invention may also provide useful hints for the dysplasia. therapy to be applied. The invention may also be used for inducing and con AS calcium ion Suppliers, all the Soluble calcium Salts trolling the reproductive activity in mammals, fish and birds, compatible with the pharmaceutical use may be used, Such for inducing the lysis of the corpus luteum, and to improve 45 as ascorbate, gluconate, glucoheptonate, dobesilate, the athletic performance in horses and dogs; it is also useful glucobionate, leVulinate, lactate, lactobionate, pantotenate, for contraception. ketoglutarate, borogluconate, etc. Also the dosage of these The choice of the opiate antagonists will depend on compounds will be determined according to the already Several factorS Such as kinetics, potency, Safety, pharmaco established therapeutic practice. See for instance Goodman logical risks etc. For acute pathologies, for instance, the use 50 & Gilman, “The pharmacological basis of therapeutics”, VII of fast action and short half-life drugs Such as naloxone is ed., Macmillan Pub. Co., p. 1521. preferred whereas for chronic pathologies, long lasting The calcium salt may be administered both by oral and drugs. Such as naltrexone will be preferably used. parenteral route, according to the Specific therapeutic indi Other opiate antagonists which may be used according to cation. the invention comprise: dipre morphine, nalbuphine, 55 According to a preferred embodiment of the invention, betachloronaltrexonine, naltrexonazine, naloxazone, the combination of an opiate antagonist and calcium may be nal me fene, beta-funaltrexamine, ICI 174.864, added with proteases which, decomposing the free 7-benzylide ne naltrexone (BNTX), naltrindole, endorphins, increase the efficacy of the combination itself. norbinal torphimine, norbinal torphammine, naltribene Examples of Suitable proteases, which may be administered (NTB), profadol, quadazocine, naloxonazine, D-Pen-Cys 60 at doses ranging form 40 to 160 U.P.F.U., include bromeline, Tyr-D-Trp-Orn-Thr-Pen-NH2 (CTOP), MR-2266, papaine, chymotrypsine, trypsine, pepsine, Subtilisine, pro naltrindole-5'-isothiocyanate(5'-NTII), N-methyl-D- teinase A and K, kallicreine, elastase, chymopapaine, aspartate (NMDA), dextrorphane, methylnaltrexone cloStripaline, collagenase, metalloendo-peptidase, ficines. (MNTX), DALCE(D-Ala2, Leu5, Cys6-enkephalin), The combination may also comprise other active methylnaloxonium, bremazocine and LY 274614. 65 principles, namely prostaglandins, phorbol, ATP, Vitamin C, It is anyhow possible to use any compound having opiate levamisol, always at the dosage already known for these antagonist activity. Substances. 5,811,451 S 6 The preparation of the compositions of the invention, in The therapy induced the remission of Symptoms already combined or in "kit' form, is carried out using conventional in the second day and the full restitution ad integrum in 3-5 excipients, Such as those disclosed in "Remington's Phar days. maceutical Sciences Handbook', Mack Pub.Co., NY, USA, XVII ed. EXAMPLE 4 The following Examples further illustrate the invention. Treatment of parenchymatous mastitis by colibacilli in cows The parenchymatous mastitis is a Serious inflammation of EXAMPLE 1. a mammary Section induced by colibacteria. Treatment of cows affected by milk fever 10 cows affected by this disease were treated with nalox The hypocalcemic milk fever in cows provides an effec one hydrochloride at the dose of 0.5 mg/100 kg body weight, tive experimental model Since the bovine Species has a calcium gluconate (50 g) and protease (Endozim'). The particularly complex calcium metabolism. antibiotic or Sulfamidic specific for the pathology was The milk secretion involves, in fact, the need to fix in the contemporaneously administered. The treated Subjects mammary glands, Starting from the circulating liquids, about recovered their normal organic functions already at the first 1 g of calcium per kg of produced milk, whereas the total 15 administration, with complete remission of the Symptoms. amount of calcium in the blood flow is 1.5 g. AS a The therapy lasted 2-3 days. consequence, it is evident that, particularly at the beginning of lactation, the calcium turnover in cows should be par EXAMPLE 5 ticularly efficient and that in Some cases there are block and Treatment of distemper in the dog interaction mechanisms, more Serious in respect with what 8 Animals affected by distemper were treated with 0.5-1 occurs in other Species. This happens for instance during mg of naloxone hydrochloride daily for one week, Vitamin delivery when, as in every mammal, the maximum physi C (0.5-1 g/die of one week), calcium gluconate i.V. (0.5 ological increase of beta-endorphin and Serious impairments g/die for one week), Endozym' and vitamin B (500-1000 of the Ca" metabolism occur. mg) parenterally for 1 week and antibiotics 30 Cows affected by milk fever were treated with 5 mg of 25 (cephalosporins+aminoglycosides i.m. for 1 week). naloxone, 50 g of calcium borogluconate i.V., trypsine 100 In each case, the forms were remarkably advanced, with uFu and chymotrypsine 27.7 uFu, i.m. manifest nervous Symptoms. All the animals readily recovered and no fatal exitus The subjects improved after two-three days. The complete occurred. recovery even from nervous Symptoms occurred after 5-15 days. EXAMPLE 2 Treatment of cows affected by milk fever with meteorism EXAMPLE 6 The milk fever in cows is sometimes combined by the Effect of naloxone administration on healing process contemporaneous block of the forestomachs motility and of The oral administration of naloxone to a clinically healthy the eructation refleX with consequent meteorism. 52 years old Subject, appendicectomized 6 months before, The administration of 5 mg of naloxone dissolved in a 35 affected by liponecrosis in the healing phase, induced a solution of 50 g of calcium gluconate in 500 ml of sterile localized itching within 2-3 hours from the drug water in one cow affected by the above mentioned compli administration, at the laparotomy Seat. In the following dayS, cation with very marked tympanism induced a positive the healing process induced the formation of a Small fistula effect both on the milk fever and on the tympanism, after from which Some non-reabsorbed Suture residues were slow i.v. infusion of 250 ml of the calcium-naloxone, with 40 eliminated. It is presumed that endorphins were responsible recovery of the eructation refleX and expulsion of the exceSS of the block of the healing process. gas in the rumen. At the end of the infusion (500 ml), the cow stood up with EXAMPLE 7 remission of the Symptoms. The administration of proteases Treatment of the LUF syndrome (Endozym) finally induced the decrease of free endorphins 45 A particular form of anoVulation, in human medicine, is concentrations. known as luteinized unrupted follicle or LUF, characterized by regular menstrual flows and by a normal luteinization EXAMPLE 3 without ovulation. The LUF syndrome is considered to be Treatment of parvovirus-induced haemorrhagic gastro responsible of unexplained Sterility. enteritis in dogs 50 A woman affected by LUFSyndrome, who was previously Parvovirus gastroenteritis in dogs is a virulent contagious treated with gonadotropine Since more than one year without disease which, if not treated, generally causes the animals ovulating had plasma concentration of beta-endorphin of 50 death. Even when a Suitable therapy is applied, this disease pg/ml, usually accepted as normal. The patient, after oral has often an unfavourable prognosis. The disease is frequent treatment with oral naloxone (25 mg), calcium (1g), Vitamin in pups less than 1 year old. After incubation period of 3-4 55 C (2 g) had a double ovulation after 4 days of therapy, days, the Subject presents: anorexia, Sensory depression, conceived and a normal child was delivered at term. Vomit, heamorrhagic diarrhoea, Serious dehydratation, shock. The disease results in the subject's death in 2–5 days EXAMPLE 8 in 70% of the cases. Treatment of pathologies of the muscolo-skeletal System in Recovery may occur in animals Surviving after the fifth 60 the dog day only after complex therapies consisting in infusion of 1 Dog affected by hip dysplasia and two dogs with bone electrolytes, large amounts of Vitamins C and K, antibiotics, fractures of the limbs were treated. The animals, after cortisone, etc. pharmacological treatment according to the invention 40 dogs affected by parvovirus gastroenteritis were (0.2-0.5 mg of naloxone or 5-10 mg of naltrexone every 48 treated i.v. daily with a sterile aqueous Solution containing 65 h for 2-4 weeks, 250-500 mg/die of calcium for 1 month, naloxone (0.5-1 mg), calcium gluconate (0.5 g), Vitamin C optionally proteases and Vitamin C) readly improved (in 2-3 (500-1000 mg), vitamin K (1 g). days) their pain and functional situation. 5,811,451 7 8 The bone callus rapidly formed in the fractured subjects naltrindole-5'-isothiocyanate(5'-NTII), N-methyl-D- and the consolidation times were about half of the usual aspartate (NMDA), dextrorphane, methylnaltrexone OCS. (MNTX), DALCE(D-Ala2, Leu5, Cys6-enkephalin), methylnaloxonium, bremazocine and LY 27614. EXAMPLE 9 3. The method according to claim 1 wherein Said com Induction of apoptosis of corpus luteum in cyclic bovines position additionally contains at least one member Selected 5 cyclic bovines in diestral phases were treated, for two from the group consisting of proteases, prostaglandins, consecutive days, with 5 mg of naloxone +2 g of phorbol, Vitamin C and Vitamin K. Ca-borogluconate i.v. per 100 kg body weight. 4. The method according to claim 3 wherein Said com The progressive damage of the corpus luteum was 1O position contains a protease and the protease is a member observed by ecography. All the treated bovines had estruS Selected from the group consisting of bromeline, papaine, after 4-5 days from the end of the treatment. The level of chymotrypsine, trypsine, pepsine, Subtilisine, proteinase A circulating progesterone progressively approached Zero. The and K, kallicreine, elastase, chymopapaine, cloStripaline, results show that the endorphins mediate the calcium influx collagenase, metalloendopeptidase and ficines. into the luteinic cells, influencing the apoptosis process of 15 5. The method according to claim 1 wherein said admin the corpus luteum. istration is carried out intravenously, parenterally or orally. 6. The method according to claim 1 wherein Said pathol EXAMPLE 10 ogy is rachitism, said bioavailable calcium Salt is calcium Treatment of rachitic pups gluconate and Said opiate antagonist is naloxone. 12 Dogs affected by rachitism were treated i.m. with 0.1 7. The method according to claim 1 wherein said pathol mg/kg of naloxone and 50 mg/kg of calcium gluconate at ogy is an inflammatory condition, an infectious disease, alternate days for 1 month and Vitamin C (250mg) for 1 Osteoporosis and an autoimmune disease. month. All the animals finally recovered after 2 months from 8. The method according to claim 1 wherein said calcium the beginning of the treatment. Pain disappeared already Salt is a Soluble calcium Salt compatible with the pharma after the third day of treatment. 25 ceutical use and is a member Selected from the group The results are particularly Surprising Since the adminis consisting of calcium ascorbate, gluconate, glucoheptonate, tration of naloxone alone to rachitic pups induce in a few dobesilate, glucobionate, leVulinate, lactate, lactobionate, minutes acute hypocalcemia with tetanic crisis, whereas the pantotenate, ketoglutarate, and borogluconate. administration of calcium Salts alone induces vomit with 9. The method according to claim 1 wherein said living marked tachycardia. Subject is a cow affected by milk fever, Said opiate antago The treatment according to the invention is on the con nist is naloxone and Said calcium Salt is calcium boroglu trary free from side-effects and causes the full recovery of COnate. the treated Subjects. 10. The method according to claim 1 wherein Said living Subject is a cow affected by milk fever meteorism and Said EXAMPLE 11 35 opiate antagonist is naloxone and Said calcium Salt is cal Therapy of the cholic syndrome in horses cium gluconate. 11 Horses affected by cholic syndrome were treated i.v. 11. The method according to claim 1 wherein Said living with 0.6 g of calcium gluconate +1.2 mg of naloxone /100 Subject is a dog affected by parvovirus gastroenteritis, Said kg body weight. The ready recovery of the good general opiate antagonist is naloxone and Said calcium Salt is cal conditions, pain disappearance and re-establishment of 40 cium gluconate. eminction and defecation occurred already after 15-30' 12. The method according to claim 1 wherein Said living from the treatment. Subject is a cow affected by parenchymatous mastitis, Said opiate antagonist is naloxone hydrochloride, Said calcium EXAMPLE 12 Salt is calcium gluconate. Treatment of hypertropic osteodistrophy in the dog 45 13. The method according to claim 1, Said living Subject A two months-old dog affected by hypertrophic osteodis is a dog affected by distemper, Said opiate antagonist is trophy was treated i.m. with Calcium borogluconate (1 g) naloxone hydrochloride and Said calcium Salt is calcium and naloxone (1 mg) die for 30 days. The dog showed a gluconate. remarkable clinical and functional recovery, confirmed by radiological examination showing the normalization of peri 14. The method according to claim 3 wherein said living osteum and disappearance of the Winberger Sign. 50 Subject is a woman affected by luteinized unrupted follicle, We claim: Said opiate antagonist is naloxone, and Said composition 1. The method of treatment of a living subject suffering contains Vitamin C. from an endorphin-mediated pathology which consists of 15. The method according to claim 1 wherein the living administering to Said living Subject a composition compris Subject is a cow Suffering from damage of the corpus luteum, 55 Said opiate antagonist is naloxone and Said calcium Salt is ing an opiate antagonist (a) and a bioavailable calcium Salt calcium borogluconate. (b). 16. The method according to claim 1 wherein said living 2. The method according to claim 1 wherein Said opiate Subject is a horse affected by cholic Syndrome, Said calcium antagonist is a member Selected from the group consisting of Salt is calcium gluconate and Said opiate antagonist is naloxone, naltrexone, dipre morphine, nalbuphine, 60 naloxone. betachloro-naltrexonine, naltrexonazine, naloxazone, 17. The method according to claim 1 wherein said living nal me fene, beta-funaltrexamine, ICI 174.864, Subject is a dog affected by hypertrophic osteodistrophy, Said 7-benzylide ne naltrexone (BNTX), naltrindole, norbinal torphimine, norbinal torphammine, naltribene calcium Salt is calcium borogluconate and Said opiate (NTB), profadol, quadazocine, naloxonazine, D-Pen-Cys antagonist is naloxone. Tyr-D-Trp-Orn-Thr-Pen-NH2 (CTOP), MR-2266, k k k k k