AU634386B2 - Treatment of horses using ethylestrenol - Google Patents

Description

6 K,0., S86 S F Ref: 180822

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Biochemical Veterinary Research Pty Ltd 229-233 Hume Highway Mittagong New South NWles 2575

AUSTRALIA

Actual Inventor(s): Hubertus Leonardus Regtop and John Raymond Biffin Address for Service: Spruson Ferguson, Patent Attorneys Level 33 St Martins Towv-r, 31 Market Street Sydney, New South Wales, 2000, Australia Invention Ti tle: "Treatment of horses using ethylestrenol".

ASSOCIATED PROVISIONAL APPLICATION DETAILS [31) Application No(s) [333 Country [323 Application Date PK0242 AU 21 May 1990 The following statemen* is a full description of this invention, including the best methJ of performing it known to me/us:- 5815/4 TECHNICAL FIELD The present invention relates to a method for the treatment of various conditions in mammals and in particular to anabolic replacement therapy in geldings; antagonism of the catabolic effects of corticosteroids, either elevated endogenous cortisol in stress states or as a result of exogenous corticosteroid therapy; adjunctive therapy of diseases resulting in negative nitrogen balance convalescence from surgery, severe infections or fractures; recovery from protein malnutrition; and more particularly to the treatment of those conditions in horses.

BACKGROUND ART Anabolic steroids in horses may be summarized as having the following pharmacological effects: more rapid conditioning increased muscle mass increased bone density positive nitrogen balance and increased protein synthesis enhanced calcium, phosphorus and potassium metabolism improved wound repair and recuperation improved appetite increased bodyweight counteraction of catabolism due to corticosteroids increased blood count by up to 20% enhanced haematopoiesis combned with appropriate diet and vigorous exercise, increased strength Testosterone, boldenone and nandrolone have all been shown to have adverse effects on behaviour and reproductive function (11,12).

It would be highly desirable to administer to a horse a preparation which would have the above beneficial pharmacological effects. However a major disadvantage with anabolic steroids presently in use is the androgenic action of the particular hormone.

Androgenic (masculinising or virilising) effects of anabolic steroids with low anabolic indicies are when administered to fillies or mares behavioural changes development of male sexual behaviour e.g. attempting to mount other females (18); when administered to colts or stallions suppression of normal testicular function, reduction in testicle size, reduced sperm count.

"The residual effects of anabolic steriods contraindicate their use in both female and male horses intended for breeding purposes" (19).

A further disadvantage may be summarized by the advice given by the Australian Equine Veterinary Association which is "Any veterinarian administering or advising the administration of a normal recommended dose of a long acting anabolic steroid closer than 42 days prior to racing is taking the risk of a positive swab resulting." The commonly used anabolic steroids are testosterone, methandriol, boldenone, nandrolone and stanozolol will have little or no therapeutic/ prophylactic effect approaching competition if recommendations to observe Australian Rules of Racing are followed.

Thus, there is a fundamental disadvantage with currently available anabolic steroids in that in order to avoid a positive swab finding, there use needs to be discontinued for a time prior to racing which in turn mitigates against their beneficial effects.

Ethylestrenol is an effective and safe anabolic agent in dogs, cats and humans.

Biffin and Regtop (16) have found Nitrotain oral paste to be safe and effective in trials over 16 months and 16,500 horse doses.

DISCLOSURE OF THE INVENTION According to one broad form of the invention there is provided a method for achievement of a positive nitrogen balance, increased body weight or, increased exercise tolerance; for antagonism to the catabolic effects of corticosteroids; for adjunctive therapy of diseases resulting in negative nitrogen balance; for recovery from protein malnutrition; for enhancement of fibrinolysis; for reduction of thrombosis, or platelet stickiness; for treatment of echinocytosis; and/or for improvement of microcirculation; which method comprises administering to a horse in need of said treatment a composition comprising an effective amount of ethylestrenol together with a pharmaceutically or veterinarially acceptable carrier, diluent and/or excipient.

According to another broad form of this invention there is provided a pharmaceutical or veterinary composition comprising ethylestrenol together with a pharmaceutically or veterinarially acceptable carrier, diluent and/or excipient.

The compositions of the present invention may be administered orally or parenterally containing conventional, non-toxic pharmaceutically or veterinarily acceptable carriers, diluents and/or excipients as desired, BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 represents accumulative total urinary excretion of ethylestrenol following administration for six days.

Fig. 2 represents daily urine nitrogen excretion per horse (grams).

Fig. 3 represents bodyweight after 10 days on anabolic and fixed diet.

Fig. 4 represents heart rate 15 seconds after standardised exercise test.

i BEST MODE AND OTHER MODES FOR CARRYING OUT THE INVENTION SThe composition may be present as a paste which is the preferable presentation when administered to horses.

If the preparation is presented as a paste, the ethylestrenol is preferably mixed in a thickening agent and anti-oxidant. Preferred thickening agent is carbopol and preferred preservatives are hydroxybenzoate, methyl paraben and propyl paraben.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, ethylestrenol may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

When the preparation is presented as a tablet, any well known compound which increases the flow properties of the preparation is suitable and may be disodium phosphate or magnesium stearate, and preferably is disodium phosphate.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, syrups, solutions, suspensions, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents including sugars such as sucrose, sorbitol, fructose etc, glycols such as polyethylene glycol, propylene glycol etc, oils such as sesame oil,olive oil, soybean oil etc, antiseptics such as alkylparahydroxy benzoate etc, and flavours such as strawberry flavour, peppermint etc.

Parenteral as used herein includes subcutaneous injections, intravenous, or intramuscular injection, or infusion techniques.

When present as an injectable preparation, for example, sterile injectable aqueous or oleagenous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic monoor diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

It has been surprisingly found that ethylestrenol is superior to the above anabolic steroids in that while it possesses a potent anabolic action, the androgenic action relative to that anabolic action is decreased.

Ethylestrenol is an orally active anabolic steroid which enhances protein synthesis and diminishes urinary nitrogen excretion. Anticatabolic effects, stimulation of appetite and haematopoiesis, and a positive effect on calcium, phosphorus and potassium metabolism occur when nutrition is adequate. Androgenic effects or gonadotrophic suppression are unlikely at recommended doses.

It has also been surprisingly found that whereas it is necessary to discontinue the use of other anabolic steroids up to 42 days prior to racing, ethylestrenol may be administered up to 48 hours prior to competition.

The composition for this purpose is presented to the horse as a paste. The paste is prepared as follows: Carbopol was dissolved in distilled water to a concentration of 1%.

Methyl paraben and propyl paraben were added to a final concentration of 0.3 and 0.1% respectively; or sodium propyl hydroxybenzoate added to a final concentration of 0.45%.

The ethylestrenol was then added and the composition mechanically mixed to form a smooth paste. Sodium hydroxide was then added to adjust the pH to between about 5.5 to about 6.5. At this pH the thickness and viscosity of the carbopol increased dramatically to form a paste.

When present as a powder, a single dose comprises 15mg of ethylestrenol in calcium gluconate BP.

When present as granules, one dose (12g) consists of dextrose 3g, microcellulose crystalline 4g, starch 3g, magnesium sterate 0.5g, colloidal silica 0.5g, talc 0.5g and ethylestrenol 15mg. The granules can be made by any method known in the art.

The specific dose level for a particular horse will depend on a variety of factors including age, general health, sex, diet, body weight and time of administration.

The dosage rate of ethylestrenol can be between 20 and 70 and preferably between and 35 ilg/kg/day.

The present invention will now be described with reference to the following examples which should not be construed as limiting on the scope thereof.

EXAMPLE 1 ANABOLIC EFFECTS AND DOSE RATES OF SOME ANABOLIC DRUGS Table 1.

DAILY DAILY DRUG ANABOLIC INDEX HUMAN DOSE HORSE DOSE (see note 1) (mg/kg note 2) (mg/kg note 3) Testosterone 1 70-360 14-19 Methandriol 7 140-570 13-18 Boldenone 9-18 Nandrolone 4 30-60 48 Stanozolol 3.8 85 11-18 Ethylestrenol 19 30 1. Ratio of anabolic action to androgenic action relative to testosterone. (1,10) 2. Range of recommended human doses based on a bodyweight of 70 kg. 3. Range of manufacturer's recommended doses. (14) Not published, but known to be between Nandrolone and Testosterone Not used in humans.

The table clearly demonstrates the superior anabolic index uf ethylestrenol.

Human dose rates have been established by laboratory tests, clinical trials, and responses in laboratory animals. There is no evidence that horses respond to lower dose rates than other animals, and a growing amount of evidence that manufacturer's recommended dose rates for many anabolics in horses cause no measurable anabolic response. (17,18) The recommended dose of ethylestrenol is consistent with research in all animals.

EXAMPLE 2 URINARY CLEARANCE OF ANABOLIC DRUGS REGISTERED FOR USE IN

HORSES

Testosterone (phenylpropionate) 19 days (b) Methandriol (dipropionate) 21 days Boldenone (undecylenate) 40 (b)-60 days Nandrolone (phenylpropionate) 21 days (b) Stanozolol 28 days (a) Ethylestrenol 48 hours (16) Manufacturer's recommendation. (14) Example from analytical laboratories. Note that many anabolic preparations available in Australia contain more than one steroid, which may interact to alter excretion patterns. (14) Ethylestrenol excretion following 6 day course of 15 mg/horse/day.

EXAMPLE 3 ETHYLESTRENOL IN HAEMORHEOLOGY Positive effect on the mechanism of blood flow has been demonstrated using ethylestrenol and no other anabolic steroid, although others have been tested. (13) Ethylestrenol reduces the stickiness of platelets, increases the flexibility of erythrocytes, enhances fibrinolysis and reduces thrombosis. (13) Laminar/columnar flow of blood is thus facilitated, The effect results in reduction of blood pressure in microcirculation, improved perfusion, and diminished insult to small vessels and capillaries.

As demonstrated in table 1, Ethylestrenol has by far the greatest anabolic index available. The beneficial effects of anabolic therapy, without the physiological and forensic disadvantages, is available to the equine veterinarian for the first time.

EXAMPLE 4 ABSORPTION METABOLISM EXCRETION Ethylestrenol is absorbed from the gut within 1-3 hours of administration regardless of food intake. (16) Hepatic metabolisation is negligible. Plasma levels are below that which can be measured by Gas Chromatography-Mass Spectrometry (GCMS) within 18 hours.

The drug binds to a cytoplasmic protein receptor, and the hormoncreceptor complex acts by stimulation of RNA polymerase and resultant synthesis of specific RNA and protein. Half life is 16-22 hours.

Renal excretion is particularly rapid and predictable, largely due to the unusual characteristic of no glucuronide conjugation (almost all steroids are excreted conjugated to glucuronides which have variable metabolism). Cumulative total urinary excretion studies 6 Ifollowing administration for 6 days found all drug measurable by GCMS to have been excreted 44 hours after the last dose, Fig. 1.

EXAMPLE URINARY CLEARANCE OF ETHYLESTRENOL I 5 Four non-pregnant mares aged 8-11 years and weighing 420-455 Kg were administered Ethylestrenol 15 mg/day (33-35 ug/Kg/day) for 6 days. Urine was collected for 96 hours following the last dose and analysed by GC/MS for the presence of ethylestrenol. Over 90% of all ethylestrenol excreted was excreted within 24 hours and no drug was present after 44 hours.

EXAMPLE 6 EFFECT OF ETHYLESTRENOL ON BEHAVIOUR AND REPRODUCTION The above 4 mares were dosed at double the recommended anabolic dose i.e. mg/day for 30 days. No behavioural changes were noted. The mares were then paddocked with a pony stallion for 42 days. 9 weeks later the mares were slaughtered and all 4 were found to be pregnant.

EXAMPLE 7 EFFECT OF ETHYLESTRENOL ON NITROGEN RETENTION, BODYWEIGHT AND EXERCISE TOLERANCE Six thoroughbred geldings aged 6-9 years in regular riding school work were placed on a program of fixed exercise and nutrition. Horses were ridden for the following daily exercise: 800 metres walking, immediately followed by 1600 metres trotting, immediately followed by 1600 metres cantering at approximately 8 metres/second then followed by 400 metres walking.

Each horse received the same daily diet consisting of the following: 4.7 Kg "Racegro" [Australian Feed Co, complete-without-roughage ration, 14% protein, min 75% NRC for all vitamins and minerals.] Kg Lucerne chaff Kg Oaten chaff 2.0 Kg red clover hay After 7 weeks on this program, the horses each underwent a 4-day nitrogen balance study while still receiving exercise, and had bodyweight and response to a standardised exercise test measured. The standardised exercise test on treadmill inclined at 4 degrees consisted of: 1 Km at 8 Km/hr 2 Km at 14 Km/hr 2 Km at 22 Km/hr 1 Km at 14 Km/hr seconds after the treadmill stopped, heart rate was measured.

All horses were then placed on daily administration of 15 mg Ethylestrenol (29-34 mg/Kg), and maintained on the same diet and exercise program. After 10 days horses were weighed and a second 4-day nitrogen balance study commenced, and the standardised exercise test repeated.

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RESULTS

Exercise tolerance improved in 5 of 6 horses, bodyweight increased in all 6 horses, and urine nitrogen decreased markedly in all 6 horses.

1 MEAN DAILY URINE NITROGEN EXCRETION PER HORSE (GRAMS) (Fig. 2) LESTERSON MAX MALCOLM HAYDON MERV NORM gmN pre-drug 90.8 116.7 94.7 170.6 110.1 121.5 gmN post-drug 77.7 52.9 52.1 59.3 50.8 70.0 added retention 13.1 63.8 42.6 111.3 59.3 51.5 %improvement 16.9% 120.6% 81.8% 187.7% 116.7% 73.6% mean improvement all horses 99.55% 2 BODYWEIGHT AFTER 10 DAYS ON ANABOLIC AND FIXED DIET (Fig. 3) LESTERSON MAX MALCOLM HAYDON MERV NORM BWpre-Drug 510 496 483 512 451 443 BWpot-Drug 520 517 488 532 471 453 10 da' gain Kg 10 21 5 20 20 3 HEART RATE 15 SECONDS AFTER STANDARDISED EXERCISE TEST (Fig. 4) LESTERSON MAX MALCOLM HAYDON MERV NORM HRpre-Drug 98 92 128 82 92 96 HRpost-Drug 104 82 94 74 76 improvement -6 10 34 8 16 16 %improvement -6.12% 10.87% 26.56% 9.76% 17.39% 16.67% mean improvement 12.52% EXAMPLE 8 EFFECT OF ETHYLESTRENOL ON LIVER FUNCTION Six thoroughbred horses in work were administered a 4x normal dose of ethylestrenol, i.e. 60 mg/day, for 60 days, then serum biochemistry for liver function examined for the following: Serum Alkaline Phosphatase (SAP) Total Bilirubin (Bili) Serum Glutamic Oxaloacetic Transaminase (SGOT) Glutamate Dehydrogenase (GLDH) HORSE SAP Bill SGOT GLDH Norm 131 18 227 3.7 Max 128 23 205 5.1 Cinnamon 80 25 219 5,1 Bliss 95 16 201 6.3 Haydon 152 24 225 2.8 Major 114 16 218 8 Lab, lower normal 70 17 150 Lab. upper normal 200 45 250 DISCUSSION OF EXPERIMENTS a) Toxicity and Side effects: No toxic effects have been seen administering ethylestrenol to horses. The lack of behavioural or reproductive side effects was to be expected, since none had been recorded with "Filybol" with an anabolic index of 8 and ethylestrenol has an index of 19 b) Efficacy: The lack of anabolic effect recorded by authors examining other drugs could have been due to a number of factors: i. The dose rate recommended by manufacturers for horses is lower than that found effective in other animals. There is no evidence that horses should respond to a lower dose rate.

ii. The time and body compartment distribution of drug from a depot injection may have a further deleterious effect on actual daily dose.

iii, A deficient diet may prevent an anabolic effect i.e. improved protein utilization should be impossible if a single essential amino acid is deficient.

iv. Anabolic effects may not occur in the absence of a stimulus e.g. regular strenuous work.

The experiments described above have aimed to eliminate these confounding problems. The results indicate that administration of ethylestrenol at a dose rate of 29-34 mg/Kg/day, combined with adequate diet and strenuous exercise, does in fact produce an anabolic response, The experiments further demonstrate that the drug is safe at a dose rate well above the effective dose, EXAMPLE 9

PRECAUTIONS

Complete dissociation of anabolic from androgenic effects has not been achieved.

Ethylestrenol approaches dissociation much more than any other steroid, Increased dosage in attempt to obtain an anabolic response which is not possible, e.g. in the presence of dietary deficiencies, may result in androgenic effects.

Anabolic steroids may cause an increased response to anticoagulants. Prothrombin time may be maintained with a decreased dose of anticoagulant if the patient is on concomitant anabolic therapy.

Animals with a history of liver dysfunction should have regular liver function tests during prolonged anabolic therapy.

Renal or hepatic dysfunction may alter the pharmacokinctics of ethylestrenol. In cases where urinary clearance is important, it should be established that normal urinary function exists.

Ethylestrenol should not be administered within 28 days of slaughter for human consumption.

Presentation Stable, colourless (opalescent), tasteless oral paste, in 60 g dial-a-dose syringe with 15 x 4 g graduations on plunger.

Dosage Administration Gonadal replacement, anti-catabolic, appetite and haematopoietic applications: 4 g (one graduation on plunger) daily per 450 kg horse.

Convalescence and repair applications: 8 g (2 graduations) daily per 450 kg horse.

As with most anabolic steroids, a detectable response should not be expected until 5-8 days after commencement of treatment.

Note 1: When used to combat the catabolic effects of stress-induced endogenous cortisol, it is recommended that administration occur at the usual time of peak cortisol levels i.e. early in the morning.

Note 2: Where regulatory authorities require all horses presented for competition drug-free, administration should cease in time for all drug to be excreted. In a normal horse this will be 48 hours before competition. Administration should cease sooner than this if hepatic or renal/urinary function is abnormal.

Note 3: For optimal response it is essential that the diet be adequate in vitamins, minerals and amino acids.

INDUSTRIAL APPLICABILITY It should be clear that the preparation and method of treatment will find wide use in the veterinary and medical fields.

The foregoing describes only some embodiments of the present invention and modifications obvious to those skilled in the art can be made thereto without departing from the scope of the invention.

REFERENCES

1. Tausk, M (1975) "Pharmacology of Hormones". George Theime, Stuttgart 2, Sykes, P.E. (1988) Anabolic Steroids. Aust. Eq. Vet 6(1):22-25 3. Tobin, T, (1981) "Drugs and the Performance Horse". Charles C. Thomas, Springfield Ill 4. Tobin, T (1989) Anabolic Steroids: Use and excretion data. In: Proceedings of Eleventh Bain-Fallon Memorial Lectures "Equine Pharmacology and Therapy" Ed.

Dyke, T.M. Australian Equine Veterinary Assn, Sydney.

5. Murad, F Haynes, R.C. (1985) Androgens. In: "The Pharmacological Basis of Therapeutics" 7th Ed. Eds Gilman, Goodman, Rail, and Murad, F. MacMillan, New York 6. Keenan, Bruce, IJ., Allardyce, C.J. (1987) The effect of breed, date of birth and anabolic steroids on bodyweight of foals. Aust. Vet. J. 64(1):32 7. Beroza, G.A. (1980) Anabolic steroids in the horse. J.A.V.M.A. 179:278- 280 8. Snow, D.H. et al (1978) Anaolic steroids in equine practice. Proc. 23rd Ann. Conv. AAEP 411-488 9. Tobin, T. (1978) The anabolic steroid androgen group of drugs. J. Equine Med Surg 2:163-166 Overbeek G.A. (1962) Anabolic steroids. Acta Endocr. 63:7-16 11. Squires, Todter, Berndtson, W.E. Pikett, B.W. (1982) Effect of anabolic steroids on reproductive function of young stallions. J. Animal Sci.

54(3):576-582 12. Schumacher E.M.A. et al (1987) The behavioural outcomes of anabolic steroid administration to female horses. Equine Pract. 9(6):11-15 13. Chakrabarti, Evans, J.F. Fearnley, G.R. (1970) Effect on platelet stickiness and fibrinolysis of ethylestrenol or stanozolol. Lancet, Mar 21:591-593 14. Wilkinson, B (Ed) IVS annual 1989/90. IMS Publishing, Crow's Nest NSW Dyke, T.M. (1989) Pharmacokinetics of therapeutic substances in racehorses. Aust. Equine Vet. 7:(Supp. 1):22-26 16. Biffin, J.R. Regtop, H.L. (1989) Biochemical Veterinary Research Pty Ltd Unpublished data 17. Snow, DH., Munro, C.D. Nimmo, M.A. (1982) Effects of Nandrolok~n Phenylpropionate in the horse. Equine Vet J 14:224-228 18. Dowsett, K.F. University of Queensland. Personal communication 19. Stewart, G.A, (1988) Routine Administration of Anabolic Steroids in Racehorses, Aust. Eq. Vet 6(1):25-28.

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Claims (6)

1. A method for achievement of a positive nitrogen balance, increased body weight or, increased exercise tolerance; for antagonism to the catabolic effects of corticosteroids; for adjunctive therapy of diseases resulting in negative nitrogen balance; for recovery from protein malnutrition; for enhancement of fibrinolysis; for reducton of thrombosis, or platelet stickiness; for treatment of echinocytosis; and/or for improvement of microcirculation; which method comprises administering to a horse in need of said treatment a composition comprising an effective amount of ethylestrenol together with a pharmaceutically or veterinarially acceptable carrier, diluent and/or excipient.

2. The method of claim 1 wherein the method of treatment achieves a positive nitrogen balance; achieves increased body weight; achieves increased exercise tolerance.

3. The method of claim 1 or claim 2 wherein the horse is a gelding or mare.

4. The method of any one of claims 1 to 3 wherein the composi'don is administered orally or parenterally.

5. The method of any one of claims 1 to 4 wherein the dosage rate of ethylestrenol is 20-70 tg/kg/day.

6. The method of claim 5 wherein the dosage rate is 25-35 jg/kg/day. DATED this the SEVENTEENTH day of MAY 1991 Biochemical Veterinary Research Pty Ltd Patent Attorneys for the Applicant SPRUSON FERGUSON L METHOD OF TREATMENT OF HORSES WITH ETHYLESTRENOL Abstract A method for achievement of a positive nitrogen balance, increased body weight or, increased exercise tolerance; for antagonism to the catabolic effects of corticosteroids; for adjunctive therapy of diseases resulting in negative nitrogen balance; for recovery from protein malnutrition; for enhancement of fibrinolysis; for reduction of thrombosis, or platelet stickiness; for treatment of echinocy :osis; and/or for improvement of microcirculation; which method comprises administering to a horse in need of said treatment a composition comprising an effective amount of ethylestrenol together with a pharmaceutically or veterinarially acceptable carrier, diluent and/or excipient. (Figure I) WPI12OC.doc