AU620567B2 - Prevention of soft tissue damage - Google Patents

Description

Dated this 3 day of December 1991 SHARPE RESEARCH PTY. LIMITED By: (42974) Registered Patent Attorney fr.

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COMMONWEALTH OF AUSTRAL 162056 7 Patent Act 1952 CO0M P L ET E -S PEC

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(ORIGINAL)

F I C A TIO N Class Int. Class Application Number :PI 4743 Lodged :6 October 1987 Acomplete Specification Lodged 04 0 0 0 Accepted Published Y riority: ,,*elated Art ,0"Name of Applicant Address of Applicant Actual Inventor Address for Service SHARPE RESEARCH PTY. LIMITED 12 Hope Street, Ermington, New South Wales, Commonwealth of Australia MICHAEL SHARPE PAUL PEMBERTON F.B. RICE CO., Patent Attorneys, 28A Montague Street, BALMAIN. 2041.

Complete Specification for the invention entitled: "PREVENTION OF SOFT TISSUE DAMAGE" The following statement is a full description of this invention including the best method of performing it known to Us:- Complete with Associated Provisional

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V The present invention relates to novel uses for branched-chain amino acids and mixtures thereof.

It has been suggested that the branched-chain amino acids (BCAA), leucine, isoleucine, valine and mixtures thereof, have a part to play in improving liver cell function when administered to patients with liver cirrhosis. It has also been suggested that the BCCAs may be effective in relieving hepatic encephalopathy.

It has also been known that the treatment of dogs and other animals for heartworm (Dirofilaria immitis) with adulticides such as arsenical compounds, principally thiacetarsamide, can cause renal and hepatic complications.

I It is believed that such adulticides cause altered membrane permeability or necrosis in the kidney, liver and other soft ,15 and peripheral tissue.

The present inventors have found that the onset of toxic symptoms following the administration of adulticides to animals for the purpose of heartworm treatment may be *4 .o prevented to a large extent by the administration of BCAAs to the dogs in association with the arsenical compound.

In one aspect the present invention consists in a method for reducing the toxic effect of compounds which cause 0 I increased membrane permeability or necrosis in soft or peripheral tissues comprising administering the said compound in association with an effective amount of at least one branched-chain amino acid.

In a further aspect the present invention consists in a method for the treatment of heartworm in dogs and other mammalian animals, comprising administering to the animal an effective amount of either an adulticide selected from the group comprising thiacetarsamide and other arsenical compounds or levamisole, its derivatives or another microfilaricide, in association with an effective amount of at least one branched-chain amino acid.

In a further aspect the present invention consists in a 1$ -2method for repairing damaged soft or peripheral tissue in an animal which tissue has been damaged by the administration to the animal of a compound which causes increased membrane permeability or necrosis in the soft or peripheral tissue, which method consists in administering to the animal effective amounts of at least one branched-chain amino acid.

In a further aspect of the present invention the present inventor has found that when applied topically the branched-chain amino acids, individually or in combination, are effective in the promotion of wound healing in mammals, particularly dogs. The BCAAs are preferably applied to the wound surface in dry powder form either alone or in conjunction with carriers, antiseptics and the like.

As used in this specification the administration of one 00 ,°15 compound "in association with" another is taken to mean that 0" oi the two compounds are administered to the animal such that both compounds are at least for a period simultaneously present within the animal. The compounds may be administered 0° simultaneously, sequentially or spaced apart in time, by the 0 00 same or different routes of administration. The administration of one compound may commence before and/or 00 0 continue after the administration of the other. It is S: preferred that the administration of the BCAA should commence before or on the same day as the administration of the *25 arsenical compound and should continue for some days after the administration of the arsenical compound has ceased.

In preferred embodiments the BCAA is selected from the group comprising L-leucine, L-valine and mixtures thereof.

The BCAAs are preferably administered orally to the animals in amounts of from 0.1 to 1 g/kg body weight per day. Most preferably the administration of the BCAA commences 4 days before the day of administration of the arsenical compound.

The most preferred adulticide is thiacetarsamide sodium (sold under the Registered Trade Mark "Caparsolate"). The most preferred microfilaricide is levamisole hydrochloride.

-3 thiacetarsamide sodium is preferably administered at dose rates of 2.5 mg/kg body weight twice daily for the first two or three days followed by levamisole preferred at rates increasing from 2.5 mg/kg daily to 10 mg/kg body weight bi-daily over a period up to about 35 days.

The prior art protection of liver damage in dogs treated intravenously with thiacetarsamide sodium has consisted of the suggested use of a meal two hours before each treatment; a diet high in carbohydrate; calcium and protein of High Biological Value (Blood), the thiacetarsamide o given in 5% dextrose in water equal to 5 ml/lb of body S weight/Vitamin C; Vitamin E; Vitamin B12; Vitamin Bl; 'furfural compounds and steroids.

S The need for protection for the liver and organs has been shown by studies of the effects of T.S. on the liver I enzymes. They treated both heartworm infected and Snon-infected dogs (control) by intravenous administration of T.S. at the rate of 0.1 mg/lb body weight, twice a day for two days. Liver damage was determined by measuring the .'20 concentrate of serum glutamic-pyruvic transaminase (SGPT).

This enzyme is a leakage enzyme, and indicates either altered SC,° membrane permeability or necrosis.

The results were:- Uninfected Infected I c 25 SGPT (SF) Units SGPT (SF) Units Pretreatment 18.8 23.6 1-4 days post-treatment 490.0 658.0 17 days post-treatment 236.1 312.0 At necropsy 18.0 31.0 From the results, it is concluded that T.S. is hepatoxic Sirrespective of the presence of heartworm in the dog treated Swith T.S.

The Executive Board of the American Heartworm Society and the American Veterinary Medical Association's Council on Veterinary Service has endorsed the use of T.S. as the only approved adulticide drug for heartworm.

There is no direct diagnostic procedure to predict adverse reactions to but the dogs general health status should be assessed prior to the decision being made to treat the dog with T.S.

The evaluation procedures for dogs with heartworm disease can include history, physical examination, complete blood counts, thoracic radiograph, electrocardiagram, urinalysis, serum nitrogen level, liver enzyme concentrations, Bromsulphalein (BSP) clearance, transtracheal S0o° wash (TWW), nonselective angiogram and immunodiagnostic coca .o o tests. Additional procedures, such as platelet counts, °ooo coagulagraphy and echocardiography, may be performed on a selected dogs.

o 15 It has also been found that signs of toxicity and some °o deaths occurred in dogs considered suitable for the o 00 prescribed treatment with T.S. It was difficult to attribute these effects solely to the dying worms, as signs often *0 develop within hours of the first injection, but worms do not 0o. 020 normally begin to disintegrate until some ten days after completion of treatment.

0000 0oo 0 It is noteworthy from this review of the art that there has been no suggestion that BCAAs may be used to improve the condition of animals following heartworm treatment let alone o25 that the administ:ation of BCAAs in association with heartworm treatment can contribute to the prevention of the toxic effects usually found with such treatment.

Hereinafter given by way of example only is a preferred embodiment of the present invention.

MATERIALS METHODS 0.33 g of L-Leucine and 0.33 g of L-Valine per kg body weight per day, mixed with artificial beef or bacon flavourings was administered by mouth, to animals in Group C, from Day 1 to Day 4 of their treatment, with thiacetarsenimide sodium at 0.2 mg/kg. The branch-chain amino acid mixture was continued until ten days after the T.S. ceased.

GROUP A Ten dogs with no heartworms, no treatment and no amino acids arranged in increasing order of their naturally occurring S.G.P.T. and S.A.P. levels added together.

GROUP B Naturally occurring cases of adult heartworm infestation given standard heartworm treatment with T.S. and nothing else arranged in similar order.

S. GROUP C Naturally occurring cases of adult heartworm disease given T.S. and branch-chain amino acids. Similarly arranged.

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PROCEDURES

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C

S* 15 Three measurements were made: Weight, S.G.P.T. and o S.A.P. The latter two are enzymes produced by damage to general liver cells and liver cells surrounding the bile ducts (Hepatocytes) respectively.

Dogs have been organised in increasing order of liver 0 0 damage, as judged by the sum of S.G.P.T. and S.A.P. figures.

S.G.P.T. and S.A.P. are given in standard international units and the weight to the nearest kilogram.

Juju, the Sheltie, was deemed to have died of pulmonary Sembolism as a result of too many fragments of dead adult '25 lungworms entering the pulmonic arterial system at once, combined with encephalopathy due to liver damage because she was comatose for two days before death (Day 16).

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6 CONTROL GROUP A C C C C Ii '2 0 Cc.

C t

C

Cc C aC C t 25 Cf c C IC

CL

@1CC C C o I .25 STRATUM WT. DAY 1 WT. DAY 4 WT. DAY 14 DOG Kgs. SGPT SAP Kgs. SGPT SAP Kgs. SGPT SAP 1 HARTON 2.5 10 2.5 2.5 12 2.5 2.5 15

POMERANIUM

2 GREYHOUND 25 12 5.0 25 12 5.0 26 14 3 GREYHOUND 26 15 3.0 26 15 3.0 26 14 4 GREYHOUND 25 15 4.5 25 18 5.0 26 14 5 GREYHOUND 27 15 6.0 26 15 7.0 26 15 6 GREYHOUND 26 18 3.0 26 20 4.0 26 20 7 GREYHOUND 29 30 4.5 29 30 5.0 29 32

BENNY

8 BOXER 25 40 4.5 26 30 4.5 27 30

MAHONY

9 WHIPPET 10 50 1.0 10 50 4.0 10 58

CLOWN

This group:- 1) Did not 2) Did not 3) Did not have heartworm receive T.S. treatment receive BCAA treatment I 7 TABLE 1 GROUP B HEARTWORM TREATMENT ONLY o e: 10 o 1 c 0 «ec

C

o 0 C a o S€ e 00t o 0 o 00 o o NAME WT/ DAY 1 DAY 4 DAY 14 KGS. SGPT SAP SGPT SAP SGPT SAP

NORWEGIAN

ELKHOUNDS

BUFFY 24 14 30 4280 365 1420 340 BELLA 24 12 32 1230 149 960 580 CATTLE DOGS REBO 21 16 60 390 940 186 888 TIMMY 23 18 40 480 690 142 362 TULIP 20 40 82 4120 1260 1420 629

SHELTIES

JUJU 4 60 20 5670 420 3290 400 LULU 7 14 30 3720 490 1739 390 BASIL 7 18 90 600 970 650 980 AUSSIE 6 24 40 2960 840 490 602

BELGIAN

GROENENDALES

LUVVY DUWVVY 18 18 18 865 510 700 620 JASMINE 22 20 30 870 402 520 869

GERMAN

SHEPHERDS

MAXIS MATE 34 20 42 3010 680 496 296 SHEBA 31 22 30 4120 942 560 502 This group:- 1) Had heartworm 2) Received T.S. treatment for heartworm 3) Did not receive BCAA treatment 000o 0 0 0 S C C OC C t I'1 -7

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8 TABLE 2 GROUP C HEARTWORM TREATMENT BCAA TREATMENT o 00 000 o 0.

o oo 0 0 0000 0 0 o 00 00 00 0 .o o25 00 00 0 0000 o 0* Q 0 0 oo 025 NAME WT/ DAY 1 DAY 4 DAY 14 KGS. SGPT SAP SGPT SAP SGPT SAP

NORWEGIAN

ELKHOUNDS

FLAIR 22 14 32 16 40 14 MICK 29 16 40 30 42 22 28 CATTLE DOGS TRESCA 19 14 34 14 32 12 32 CATTLE KING 25 14 34 14 32 14 32

SHELTIES

KELLY 9 14 32 19 40 19 36 MICKY 7 30 20 23 40 40 RICKY 8 16 34 14 30 18 34

BELGIAN

GROENENDALES

JACQUIE 21 14 26 70 85 60 MINTY 19 16 19 90 100 75 92

GERMAN

SHEPHERDS

BAMBI 32 14 32 60 62 14 32 CAESAR 35 14 32 50 42 23 34 MAX 38 16 30 48 40 49 This group:- 1) Had heartworm Received T.S. treatment for Did receive BCAA treatment heartworm i::i ;I :x 9 TABLE 3 Inspection of measurements in the control group (Group A) indicated that the levels of both SGPT and SAP were stable over the 14 day period.

The means of the three groups, with standard errors in brackets, are given in the attached table.

Natural logarithms of the SGPT and SAP levels were taken for groups B and C because of the large variation in the data. The retransformed means are given in the table below each logarithmic value. These are weighted means and so will differ slightly from the arithmetic means.

There were no significant differences between 'the two treatment groups B and C on Day 1. However, on both Day 4 and Day 14 the levels of both SGPT and SAP in the dogs in 15 treatment group B were higher than those in group C. These differences were very highly significant (P .001).

A summary of the results obtained is set out hereunder.

Group A: Control 0 00 0 0 0 9000 ;0 @0 0 0 00 00 0 0000 00 0 0 C 0 0 0OD O 9 0 00 O* 0 0 0 0 00 o *0.

O *BO* 0 0000( 0 °20 a o 6 Day 1 Day 4 Mean (SE) Mean (SE) SGPT 22.78 (4.67) 22.44 (4.13) SAP 3.78 (0.51) 4.44 (0.44) Group B: Heartworm treatment (carparsolate) only Day 14 Mean (SE) 23.56 (4.90) 4.50 (0.28) Natural Logarithms 0 00 .o o025 SGPT 2.97 (0.11) 7.46 (0.20) 19.49 1737.1 SAP 3.57 (0.11) 6.31 (0.09) 35.52 550.0 Group C: Heartworm treatment BCAA treatment

SGPT

SAP

Natural Logarithms 2.73 (0.11) 3.39 (0.21) 15.33 29.67 3.44 (0.11) 3.83 (0.09) 31.19 46.06 6.52 678.6 6.27 528.5 3.21 24.78 3.64 38.09 (0.20) (0.09) (0.20) (0.09)

Claims (2)

1. A method for the treatment of heartworm in dogs and other mammalian animals, comprising administering to the animal an effective amount of either an adulticide selected from the group comprising thiacetarsamide and other arsenical compounds or levamisole, its derivatives or another microfilaricide, in association with an effective amount of at least one branched-chain amino acid.

2. A method as claimed in claim 1 in which the animal is Soo 10 administered thiacetarsamide sodium for two or three days 0 0 followed by levamisole for up to 35 days and is 0 0® 00.. administered at least one branched-chain amino acid .A throughout this period. A 0o 00 0 rOr.--A-HtG-- -eetpoi&itian for the prevoention or o 0o c 15 of soft or peripheral tissue of an an iaJ- altially as 00 0o hereinbefore described wi r-e erence to the group C .0oj animals of he-m~ erials and methods section of this 00 0 0 e* 0 o. o DATED this 31 day of October 1991 0000 o 0 SHARPE RESEARCH PTY. LTD. 0 o0o00a Patent Attorneys for the 0.q Applicant: otb 0 a ov 00 ou 00 0 00. F.B. RICE CO. 0o o o