AU670583B2 - Triterpene compound having antiinflammatory activity - Google Patents

Description

,OPI DATE 07/06/93 APPLN. ID 39101/93 jj11111 I11111 AOJP DATE 05/08/93 PCT NUMBER PCT/AU92/00590 III i iII AU9339101 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 93/09129 C07J 63/00, A61K 31/56 Al (43) International Publication Date: 13 May 1993 (13.05 93) (21) International Application Number: PCT/AU92/00590 (81) Designated States: AT, AU, CA, JP, US, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, (22) International Filing Date: 30 October 1992 (30,10.92) MC, NL, SE).

Priority data:- Published PK 9199 30 October 1991 (30,10.91) AU ithd international searchl report.

(71X72) Applicant and Inventor: KWEIFIO.OKAI, George (GH/AU]; 54 Canterbury Street, Richmond, VIC 3121

(AU).

(74) Agents: SLATTERY, John, Michael et al.; Davies Collison Cave, I Little Collins Street, Melbourne, VIC 3000 (AU).

Y' 9 2 2

*G

(54)Tltle: TRITERPENE COMPOUND HAV"ING ANTI INFLAMIMATORY ACTIVI FY (57) Abstract A meti~ord or antiinflamnmatory, and more particulaly antiarthritic, treatment or a human or animal patient comprises administering to the patient an effective amount of a compound of ge 1 :eral formula (11) in substantially pure form, wherein III represents hydrogen or an acyl group derived from a long chain fatty acid, preferably a fatty acid ha% ing (10) or more carbon atoms. Particularly preferred compounds are a-amyrin and a.amyrin palmitate.

WO 93/09129 PCT/A U92/00590 -1- TRITERPENE COMPOUNDS HAVING ANTIINFLAMIATORY ACTIVITY.

This invention relates to triterpene compounds having antiinflammatory activity. More specifically, this invention relates to novel derivatives of the triterpene, a-amyrin, which have been found to have useful antiinflammatory activity, particularly antiarthritic activity, and to the use of a-amyrin itself and of these novel derivatives in antiinflammatory and more especially antiarthritic treatment of human or animal patients.

A water or alcoholic extract of Alstonia boonci root barks, Rauvaoia vomitoria root barks and Elaies guineensis nuts without pericarp is used as a herbal cure for arthritis in Ghana, and previous studies have shown that the extract was effective in reducing swelling in acute and chronic models of inflammation (Kweifio-Okai, 1991a and Alstonia boonci is the major plant constituent of the herbal preparation responsible for the antiarthritic effect (Dalziel, 1937) and some of the triterpene constituents of the root barks (Faparasi and Bassir, 1972) have been shown to be antiinflammatory, antiarthritic and antiproteolytic (Gupta et.al, 1969 and 1971; Chaudhari et.al, 1974; Chaturvedi et.al, 1974). For those reasons, the triterpenes (a-amyrin acetate, p-amyrin acetate, p-amyrin and lupeol acetate) isolated from a petroleum ether extract of Alstonia boonci root barks were tested for their antiarthritic effect in adjuvant rats (Kweifio-Okai and Carroll, 1992). The results demonstrated antiarthritic effects but with varying degrees of toxicity as shown by swellings of the liver, kidney and spleen.

It has now been found that a particular class of a-amyrin derivatives which are not present as such in any of the plants in the herbal preparation, but which WO 93/09129 PCT/A L92/00590 may be present in the combination of plants in the herbal preparation, when prepared and used in substantially pure form have useful antiinflammatory, and more especially antiarthritic activity. In the work leading to the present invention, complete Freund's adjuvant (CFA)-induced arthritis was used as the animal model for human arthritis. As in human arthritis, a granulomatous outgrowth of synovial tissue called pannus invades and destroys joints in adjuvant rats. In rats, pannus formation occurs 5-10 days after adjuvant inoculation (Pearson and Wood, 1963), and therefore testing was performed from days 11 to 19 postadjuvant, i.e.

therapeutically rather than prior to or during the induction of arthritis, It was considered that this mode of testing was more practical and would eliminate drugs which could well inhibit arthritis in the developmental stages but without effect on established arthritis (Glenn et.al, 1977).

Co ouds. c ue been ^o d to V-e properbl es o-re Accoerdingto the-pnt-ve the are providedcomp( unds of the general formula I: CCi H CH I RO I CHI CR3 wherein R represents aa acyl group derived from a long chain fatty acid.

Preferably, the compounds of general formula I are provided in substantially pure form, By "substantially pure" is meant at least 75%, preferably at least 85%, and more preferably at least 95%, pure.

WO 93/09129 PCT/A L92/00590 -3- Suitably, the acyl group is derived from a fatty acid having 10 or more carbon atoms. Such fatty acids include both saturated and unsaturated acids, including for example capric, lauric, myristic, palmitic, stearic, and arachidonic acids as saturated acids, and myristoleic, palmitoleic, oleic, linoleic, linolenic and arachiodonic acids as unsaturated acids. The particularly preferred acyl group of the invention is derived from palmitic acid.

The compounds of general formula I above may be produced synthetically from a-amyrin acetate (Kweifio-Okai and Carroll, 1992) by hydrolysing the acetate to a-amyrin, followed by esterification with the appropriate long chain fatty acid, or a derivative such as an acid halide of the fatty acid, for example palmitic acid or palmitoyl chloride.

~ccorc o> o t0e rese 'ueraor \ere oud.ed Furtherre, in another aspect of-this inven-t-te-ndste-a method of antiinflammatory and more especially antiarthritic treatment of a human or need kcherer.o animal patient which comprises administering to said patient an effective amount Sso.\AcC CoorouncA \~eiN of a compound of the general formula II in substantially pure form: Ch wherein R' represents hydrogen or an acyl group derived from a long chain fatty acid, preferably a fatty acid having 10 or more carbon atoms as described above.

';&RA4 T 0 3a It is to be understood that in order to administer the compound of formula II pharmaceutically acceptable carriers, diluents or adjuvants may be used.

S

S

5*5@55 0 S S S. S

S.

4

S.

0

S

55..

S S S. S 55 S S S 50 S S 55 19 Ali SM"IM11 7111 SPI.. I V A pharmaceutical composition when used in the antiinflammatory and/or antiarthritic treatment of a human or animal patient, comprising a compound of the general formula II said compound being in substantially pure form, together with a pharmaceutically acceptable carrier or diluent.

The preferred compound of the present invention, a-Emirian palmitate, has been shown to reduce ankle swelling caused by the subplantar injection of complete Freund's adjuvant. The drug was given orally daily at 56mg/kg body weight from days 11 to 19 postadjuvant and therefore the effect observed was that on established arthritis. The reduction in ankle swelling was about 32% whether the ankle diameters were compared after 6 days of administration or over the entire period of administration using regression analyses. Pannus would have been present by day 11 of adjuvant (Pearson and Wood, 1963) after the compound was administered, after which the compound caused no further increases in ankle diameter. There is histological evidence that this compound prevented further pannus growth, infiltration and destruction of the joints in adjuvant rats.

Initial confirmation of the antiarthritic effects of a-amyrin palmitate came from measurements of serum hyaluronate. Since it was found that o serum hyaluronate increases i. active human rheumatoid arthritis (Engstrom- Laurent and Hallgren, 1985) and lactobacillus casei, mycobacterium adjuvant and collagen 11 models of aric'itis in rats (Goldberg and Rubin, 1989; Smedegard et.al., 1989), serum hyaluronate has been accepted as a reliable marker of the severity of adjuvant arthritis in rats and in screening potential antiarthritic agents. In this study, the preferred compound significantly reduced adjuvant levels of serum hyaluronate. Serum hyaluronate level therefore confirm the ameliorative effects of this compound on adjuvant swelling.

9; 96VSA'I7719 SPi,4 WO 93/09129 PCT/A U92/00590 a-amyrin palmitate, initially shown to be antiarthritic at the acute stage of adjuvant arthritis (days 11-19), has subsequently been shown to be similarly antiarthritic at the chronic stage of adjuvant arthritis (days 32-50). As at the acute stage, a-amyrin palmitate at the chronic stage returned the increases in serum hyaluronate and circulating granulocytes towards non-arthritic levels and corrected the anaemia of adjuvant arthritis. At the chronic stage, arthritic joint histopathology was improved by the oral administration of a-amyrin palmitate.

This is in agreement with the effect at the acute stage. At both stages, reduced cellular infiltration of bone marrow synovlum and synovial cavity and periarticular tissue were observed. There was also reduced cartilage destruction. At the chronic stage,a-amyrin palmitate appeared to increase new periosteal bone formation. In addition, chronic arthritic rats treated with a-amyrin palmitate showed reduced uptake of radioactive IgG, known to be a useful marker of inflammatory arthritis.

It has also been shown that a-amyrin and lipid esters of a-amyrin are specific inhibitors of 5-HETE synthesis. Coupled with the knowledge that HETE levels increase in the synovial fluid and tissue of patients with rheumatoid arthritis and spondyloarthritis, this finding also suggests that these compounds may be beneficial in antiarthritic therapy. The demonstration of inhibition of lipoxygenase activity in human neutrophils is unique in antiarthritic activity there is no antiarthritic drug in current usage which relies on the inhibition of lipoxygenase activity for antiarthritic effect. Drugs that inhibit only cycloxygenase products of arachidonic acid (the prostaglandins) are limited in their effects because the more potent pro-inflammatory substances are found in lipoxygenase pathways.

Antiarthritic agents are often limited by their deleterious effects (Bonta et.al, 1980). In the present study, liver, kidney and spleen weight changes were followed together with circulating RBC levels to identify possible toxicity of the preferred compound for future studies. The observations that liver and kidney weights did not increase in the presence of this compound would suggest an WO 93/09129 PCT/A U92/00590 -6absence of toxicity. However Barritt and Whitehouse (1977) have shown that liver weight could remain unchanged in the presence of functional impairment.

Spleen weight increase may primarily account for the moderate anaemia of adjuvant arthritis because the anaemia was normocytic in the absence of liver and kidney weight increases, The basis for the correction of anaemia by this compound is unclear but it is important to note that the compound halved the spleen weight increase in adjuvant rats. More detailed study is required to elicit any latent toxicity of the preferred compound but the results obtained here, compared with the toxic manifestations of triterpenes from Alstonia boonei only (Kweifio-Okai and Carroll, 1992), confirm the rationale behind the combination of plants in the herbal preparation used in antiarthritic therapy in Ghana, In this combination, Alstonia boonei is responsible for the antiarthritic effect, Rauvolfia vomitoria provides sedative cover and Elaiesguineensis reduces toxicity (Kweifio- Okai, 1991a). Elaies guineensis could reduce toxicity by rendering a triterpene of Alstonia boonci as prodrug to limit gut destruction or in a form that enables gut absorption to bypass the usual metabolic routes (Van Arman, 1976).

Further details of the present invention, and in particular of the production and activity of the preferred compound a-amvrin palmitate, are set out in the following Examples, and in the accompanying Figure, which shows: Figure 1 Percent change in right ankle diameter after 11 days of adjuvant (CFA) and in the presence of the triterpene lipid ester, a-amyrin palmitate. Significance of difference of slope from adjuvant control: *P<0.05; 0.001 EXNAMPLE I Preparation of a-amyrin palmitate Triterpene isolation from Alstonia boonei root barks.

The triterpenes, a-amyrin acetate, p-amyrin acetate, p-amyrin and lupeol acetate were isolated from the petroleum ether extract of Alstonia WO 93/09129 PCT/A U92/00590 -7boonei de Wild root bark. The crude petroleum ether extract (8g) obtained from a batch of powdered root bark (300g) was separated by vacuum liquid chromatography on silica using petroleum ether/ethyl acetate The terpene acetate fraction (4.5g) was further purified by reverse phase h,p.l.c. {elution with dichloromethane/acetonitrile (30:70)} n yield lupeol acetate (438mg), a-amyrin acetate (1260mg) and p-amyrin ar -ate (200mg), The more polar terpene alcohol fraction (Ig) from the initial vacuum liquid chromatography was recrystallised to yield pure pamyrin (990mg), From a proton NMR spectrum of the crude petroleum ether extract it was estimated that the root bark contains 1,116% a-amyrin acetate, 0,186% p-amyrin acetate, 0.254% p-amyrin and 0,186% lupeol acetate.

WO 93/09129 WO 939129PT/A U92/00590 -8

CH

3

OH

3 COHq C11 3 J'N CH 3 0-amvrin acetate

OH

3 C Hj R =l 11 amyrin R Aor~-amyrin zwetate 1upeol acetate .0 H i ell C 0113 SU Tr I UT s[E: WO 93/09129 WO 9309129PCT/AUL92/00590 -9- (ii) Preparation of a-amyrin palmitate.

a-amr'in acetate isolated from Aistonia boonci root barks as described above was hydrolysed with ethanolic sodium hydroxide to aamyrin, and the a-amyrin esterified wvith pairnitoyl chloride to a-amyrin palmitate, according to the reaction scheme: heat in NaOH/ethanol 1. a-amyrin acetate a- amyri n 0 11

CH

1

(CHZ)

14

CC'

2, a-anlyrin a a myrin palmitate Dichloromethane ENANIPLE 2 EffectoCL_-arn)vrin pamtt naueaj~narthrfitis AL Miaterials and Niethods Animal stock, Five-and-a-half-week-o iouthred Wistar rat,, v~eighting between 107 to 130g (Monash University Animial Hous., -iayton, Australia) were w~ed. Animals were kept in groups of 5 at 23 I C on a 12:l2hr ]light, dark cyclo and had free access to food and water throughout the experiment.

Induction of arthritis.

rats were inoculated with complete Freund's adjuvant (CFA) containing additional 10mg/mi of MYcobacteriwn tuiwrculosis H37 Ra (Difco Laboratories, Detroit, Ml), Each rat received a suhplantar injection of 150p] of CFA in the rmidline mid-metatarsal region of the right hindfoot pad. Five control rats receivedI an equivalent amount of saline. From days 'I to 19, 5 adjuvant rats were fed orally daily with the triterpene, W"aMyrin palmitate (also referred to herein as CK091 (56mg/Kg bodY weight in I rn of drinking water). 5 control rats and the other 5 adiuvant rats received eq~uivalent amounts of drinking water each timne.

WO 93/09129 PCr/AU92/00590 Anteroposterior diameters of the ankles on injected and contralateral limbs were measured with sliding vernier scale on days 0 and 11 and thereafter every 2 days through to day 19, On day 19 blood was removed by cardiac puncture under ether anaesthesia at two stages. Firstly 1 ml syringe containing heparinized beads and 18" gauge needle was used to withdraw 500-750l of blood for measurement of blood cells by routine techniques. Then an additional 2 to 3 ml of blood was withdrawn with a 5ml syringe (non-heparanised), allowed to clot and the serum separated by centrifugation. 100pl of serum was used for duplicate measurements of serum hyaluronate (Pharmacia HA Test 50, Pharmacia Diagnostics AB, Uppsala, Sweden). The liver, spleen and kidneys were removed for weighing and right and left limbs were cut above the ankles for histological study. Data was analysed by multiple t-test in which unpaired Student's t-test was used to compare-the drug group with either the single adjuvant control group or the single non-adjuvant control group (Sinclair, 1988). Linear regression analysis by the least square fit was used to assess significant correlation of ankle swelling against time, Significance of difference between regression coefficients (slopes) was also assessed (Petrie, 1987). A P<0.05 was considered significant for the above tests.

B. Results Ankle diameter changes and serum hyalironate Control rats did not shw an\ significant changes in inected (ipsilateral) limb ankle diameters during the 19 days of the experiment (Table Similarly there were no changes in contralateral ankle diameters. Adjuvant increased ipsilateral ankle diameters to 94( of preniection values by day 19 but there was no transfer of adjuvant swelling to the contralateral ankle, The use of older Wistar rats ensured a stable ankle diameter from the beginning of this experiment and a greater response to admuvan t at he ips.ateral limb but as previously (Kweifio'Okai, 1991b) contralateral limb was not nearly as responsive. By day 11, ankle swelling was statistically similar in the two adjuvant groups. 67±c6%C in the adjuvant control group and 5U±6G in the group which subsequently received the triterpene (Table Histological sections would show that in the range of 9ai 3/0129 PCI /A U92/0059() -11 adjuvant swellings observed in the 2 groups, there was pannus formation and the infiltration of joint (Kweifio-Okai and Bird, unpublished observations), In Table 1 CK091 reduced adjuvant swelling by 20% after 2 days of administration and then after 6 days by an average of 32%, Regression analyses of the rate of ankle diameter change from days 11 to 19 postadjuvant (Fig,l) showed that only in the absence of CK091 was statistical significance of change observed (P<0,001) and the slopes were significantly different (P From the regression analyses, ankle diameter increased by 34% in adjuvant rats and by 3% in the presence of CK091 reduction of 31% by CK091, In Table 2, adjuvant caused an apparent but non-significant increase in serum hyaluronate, CK091 significantly reduced serum hyaluronate of adjuvant rats by 48% (P<0.01).

White blood cell count.

In Table 3, granulocytes and monocytes increased significantly in adjuvant rats but the increase in lymphocytes did not reach statistical significance. The apparent increase in total WBC in adjuvant rats (+14.700/mm was achieved by a greater increase in granulocytes (+8535, dt S, P<0,05) than agranulocytes (+6165, In the presence of CK091 white blood cells were lower than adjuvant levels but were neither significantly different from them, nor from nonadjuvant control levels. Also in Table 3, total neutrophils and the segmented types as percent of total WBC count increased significantly in adjuvant rats but the increases in the presence of CK091 did not reach statistical significance.

Percent banded neutrophils, the less mature neutrophils, remained unchanged in adjuvant rats with and without CK091.

Organ weight changes and red blood cell counts.

In Table 4, neither adjuvant nor CK091 had any effect on liver and kidney weights. Spleen weight increased in adjuvant rats in the presence and absence of CK091. Adjuvant significantly reduced total RBC count WO 93/09129 PCTF/A U92/00590 12 but RBC volume was unchanged. CK091 returned RBC count to nonadjuvant control levels (Table 4).

TABLE I RIGHTANKLE DIAMETER CHANGES IN CONTROL AND ADJUVANT RATS WITH AND WnTIOUT TRITERPENE Tatwlar vatues represent mean S.E.M. changes from 0 time readings, N-S/group.

TTeatmnent TYime Ankle diameter ankle diameter (mm) +11 +13 +15 417 +19 days Control 6.57 ±0.07 6 ±2 10 ±3 11 t2 7 ±2 9 ±3 CFAonly 6.25±t0.16 67 ±6 81 ±6 a! f 5 8 8 ±8 9 4 ±7 CFA CK091 6.57 t 0.12 50 6 61 5 64 7 53 9 6 6 7 (b -0.32 +61)x +71lx +7 0" +8lx +0.32 -17 -20x 17 -35x -28x Statistically significant for comparison specified: xP<cO.05. xx<.0 CKO9I. a triterpene lipid ester present in Aistonia boonei. Rauvolfia vomnitoria and Elalegs guneensis. was given orally daily (56mng/Kg wi) from days 11 to 19 postadjuvant.

WO 93/09129 PCT/A U392/00590 14 TABLE 2 SERUM F{YALURONATE IN CONTROL AND ADJUVANT RATS WITH AND WrTHOUTf TRITERPENE Data at day 19 of adjuvant, 8 days after administration of triterpene.

Treamn Mean serum hyaluronate NP S.E.M. (jig/I) Co;t-roF- 181-±-34 -5 >01 0 CFA only 2 87 4 9 4 CFA 40K091 14 9 3 5 5 C0.01 om-parison with adjuvant control.

TABLE 3 DIFFERENTIAL WBC AND NEUTROPHIL COUNT IN CONTROL AND ADJUVANT RATS WITH AND WITHOUT TRFUERPENE TABULAR VALUES REPRESENT MEAN S.E.M. Data at day 19 of adjuvant, 8 days after administration of triterpene.

N-S/group except data for CK091 -4 CCNTRCL CFA CFA CK091 Total WBC/mm 3 9820 t 589 24520 6367 18325 4006 Gran ulocytes/m m 3 1292 t 280 9827 t 3429x 5861 2097 Lymphocytes/mm 3 7477 t 775 10725 3690 10317 1255 Monocytes/mM 3 1051 8 5 3968 1148x 2147 1002 Total neutrophils (%WBC) 12-8 3-6 36.2 8_3x 26.5 6-6 Segmented neutrophils (%WBC) 8.8 32.0 7_ 3 xx 22-5 5.6 Banded neutrophils (%WBC) 4-0 t 1.2 4.2 1.2 4-0 1 Statistically significant for comparison with non-adjuvant control: xP< 0 -0 5 XXP<00 2 0 TABLE 4 ORGAN WET WEIGHTS AND RBC IN CONTfROL AND ADJUVANT~ RATS WITH AND WITHOUT TRITERPENE Tabular values represent mean t S.E.M. Data at day 19 of adjuvant. 8 days after administration of triterpene Liver Spleen (goo Kidney (go/) RBC (I0 6 /rnm 3 j MCV (f!q Mean S.E.M.

3.7±0.08 0.28 t 0.01 0.80 t 0.01 7.18 0.10o 57-6 1.0

CFA

3.71 0.18 0.53 0.06 0.84:t 0.04 6-47 ±0.26 54-9 ±1.3 Chainge -4 +89xX +5 -1 0o' -5 CFA +CK091 3.75 0.09 0.41 0.04 0-87 ±L 0.04 7-02 0.06 57-9 0.6 Change(% -3 +46xx +9 -2 0 Statistically significant for comparison with non-adjuvant control: XP< 0 0 5 xxP<~ 0 0 except RBC data for CK091 -4.

WO 93/09129) IPCI/AU92/00590 17- EXAMPLE 3 Effect of a-amyrin palmitate on chronic adjuvant arthritis A. Materials and Methods, Rats and treatment.

Fifty 8-9 week old outbred male Wistar rats weighing between 150-215g (Monash University Animal Centre, Clayton, Australia) were kept at room temperature of 23 C on a 12:12 light/dark cycle and had free access to water and food (KMM mouse and rat ration, Pakenham, Australia). 34 rats were inoculated with 150 pl of complete Freund's adjuvant containing additional 10mg/ml of Mycobacteriun tuberculosis H37 Ra (Difco Laboratories, Detroit, MI) (right hindfoot subplantar), 32 days later, those animals with a satisfactory level of arthritis (n 24) were randomly assigned to cages in groups of 4. The criteria for successful induction of arthritis (Jacka et al., 1983) were: an ipsilateral (injected) paw diameter increase of 3.4 mm minimum and ipsilateral ankle diameter increase of 3,2 mm minimum by day 11 of adjuvant maintained up to day 32, (ii) severe restriction of movement in affected limbs and pain responses when handled. The arthritic animals receiked 66 mg/kg body weight of a-amyrin palmitate, synthesised as described in Example 1, or sham fed by oral gavagefor days, 48 hours apart, from days 32 to 40 postadjuvant. Body weights, anteroposterior diameters of ankles and dorsoventral heights of food pads were measured on days 0, 11, 18, 32, 34, 36, 38, 40, 42, 46 and On day 50, 8 each of non-arthritic, treated and untreated arthritic rats were sacrificed by ether anaesthesia, blood removed by cardiac puncture before death and analysed for serum calcium, phosphate, alkaline phosphate, SGOT, SGPT, total bilirubin, glucose, urea and creatinine. Biochemical measurements were made with the KODAK Ektachem DT analyser system using the appropriate slides (Eastman Kodak Company, NY). Blood removed in heparinised syringes into heparinised tubes were analysed for blood cells using routine techniques.

Serum hyaluronate was measured in duplicate using the Pharmacia HA 50 test kit WO 93/09129 PCT/AL192/00590 -18- (Pharmacia Diagnostics, AB). The liver, kidney and spleen were removed for weighing. Data was analysed using the unpaired student t-test and a p<0.05 was considered significant.

B. Results and Discussion.

Ipsilateral and contralateral ankle and paw diameters of control rats remained unchanged throughout the experiment. Ipsilateral ankle and paw diameters in adjuvant rats significantly increased and peaked on day 11 after adjuvant inoculation and +113% respectively) and the increases were maintained up to day 50. a-amyrin palmitate made no difference to ipsilateral ankle and paw diameter changes in adjuvant rats during and after administration up to day 50, Contralateral ankle and paw diameters reinained unchanged in adjuvant rats with and without the drug. Body weight increases in normal control, treated and untreated arthritic rats were identical over the duration of the experin'rnt, In Table 5, both treated and untreated arthritic rats had identical serum alkaline phosphatase and Ca+ Ho\ever serum inorganic phosphate was 17% greater in treated arthritic rats than in control rats and 11%' greater than in untreated arthritic rats. Serum hyaluronate increased b\ 77'c in untreated arthritic rats but the 47"i increase in treated rats did not reach statistical significance.

In Table 6, granulocytes were the only white blood cells which increased significantly in untreated arthritic rats. The increase was 242% compared with an increase of 171% in treated rats. The latter increase however, was barely significant (df 14; p<0.10). Table 6 also shows that neutrophils and the mature (segmented) and immature (banded) types expressed as percent total WBC count increased in both treated and untreated arthritic rats, but treated rats showed a greater increase in immature neutrophils and a lesser increase in mature neutrophils than untreated rats. Anaemia of the microcytic and hypochromic type was present in untreated arthritic rats (Table In treated rats WO 93/09129 PC/A L'92/00590 19 RBC count and haemotocrit were normal but the RBCs remained microcytic and hypochromic. Reticulocyte count would suggest that bone marrow production was stepped up significantly in treated rats to avert anaemia but not in untreated rats Table 7 shows that by day 50 of adjuvant arthritis, liver and kidney function were preserved both in the treated and untreated arthritic rats. Serum urea was however reduced by 14% in both treated and untreated arthritic rats but only the decrease in treated rats reached statistical significance. Spleen weight increased in both treateu and untreated arthritic rats to about the same extent (30-35%), Various parameters have been used to assess the potential of a-amyrin palmitate as an antiarthritic drug. Physical measurements of local ankle and paw diameters after 32 days of adjuvant inoculation failed to reflect the increasing severity of adjuvant arthritis or its curtailment by a-amyrin palmitate. This is in contrast to previous studies over 17 or 19 days of adjuvant arthritis (see Example During the period of drug administration in the present study, there would be significant joint destruction and some fibrous ankylosis (Pearson and Wood, 1963) and at this chronic inflammatorN stage local limb sizes indicate nothing about joint activities.

Biochemical indices of bone metabolism suggested reduced joint inflammation and osteoclastic activity in treated arthritic rats. It has been shown that blood leucocytes, agents for adjuvant joint destruction, and serum hyaluronate which result from articular cartilage degradation increase in clinical and experimental arthritis and are reduced by antiarthritic agents. In the present experiment significant joint inflammation or destruction is implied by the 77% increase in serum hyaluronate on day 50 postadjuvant. Treated rats however, showed reduced and insignificant increases in serum hyaluronate. Blood granulocytes also increased in arthritic rats but the increase in treated rats was not statistically significant. Glenn and others (1977) suggested that drugs inhibited arthritis primarily by reduction of granulocytes even if agranulocytes WO 93/09129 PCT/AUL92/00590 were reduced as well. Circulating agranulocytes (lymphocytes) form a greater proportion of white blood cells in the rat than in humans and therefore response to infection in the rat is primarily by increase in circulating granulocytes (Glenn et al., 1977; Van Arman, 1976). The reduced joint inflammatory activity is consistent with observations of reduced uptake of radioactive labelled IgG at the joints of a-amyrin palmitate-treated arthritic rats (Griffiths et al,, 1992), The present data would also suggest that joint repair took place in treated arthritic rats the 17% increase in serum phosphate levels would favour Ca deposition in bone, The hyperphosphataemia of treated arthritic rats cannot be attributed to haemolysis or destruction of RBC in vivo or in vitro (normal bilirubin and RBC numbers) not to phosphate leaching from bone (normal Ca+ nor to phosphate retention in renal impairment (normal glucose and creatinine), Nor could it be attributed to metabolic effec:s since the changes in serum Ca and PO'" were inconsistent with the known effects of the major calciotropic hormones parathormone, calcitonin and active vitamin D3. Alkaline phosphatase, an index of osteoblastic activity in bone disease in the absence of hepatobiliary disease, did not increase in treated rats. However, it has been suggested that increased osteoblastic activity at local bone sites may not necessarily translate into increases in serum alkaline phosphatase levels.

The results suggest that a-amyrin palmitate is less toxic compared with aamyrin acetate in previous studies, a-amyrin acetate increased kidney weight by SGOT by 67% and had no effect on the anaemia of adjuvant arthritis (Kweifio-Okai and Carroll, 1992). In the present experiments, a-amyrin palmitate had no effect on kidney weight and the serum liver enzymes SCOT and SGPT and corrected the anaemia of adjuvant arthritis.

WO 93/09129 WO 9309129PCF/A U92/00590 21 TABLE 5 Biochemical indices of bone turnover in adjuvant arthritic rats treated wNith a7-amyrin palmitate.

Control Untreated Treated arthritic arthritic Total Ca++(mmol/L) Inorganic P0 4 =(mmol/L) Alkaline phosphatase (U/L) Serum hyaluronate (ug/L) 2,61 0,03 2,24 0.06 2 74 10 100 4 4(6) 2.54 ±0.02 2.35 ±0.07 2 66 2 2 177 ±2 2 ~xx 2 63 0.05 2.61 0.08xxx 2 82 2 2 147 26(6) Values are mean SEM of 8 rats except where indicated in parenthesis. Significance of difference from normal rats was calculated by students 1-test for unpaired variates and given as: xp<0.O5: xxp<0,O2; x~xp.<,0l; xx~xp<.00,O2; xxxzxp<O,O0i, Treated rats were orally administered with 66mg/Kg body weight of a-amyrin palmite every 46 hrs from days 32 to 40 postadjuvant and assessed on day WO 93 109129 WO 9309129PCT/A U92/00590 22.

TABLE 6 Blood cell status of adjuvant arthritic rats treated with a-amyrin palmitate, Control Untreated arthritic Treated arthritic Total WB1C (106/L) Granulocytes (10 6

/L)

Lymphocytes (10 6

/L)

Monocytes (10 6

/L)

Total Neutrophils (%WBCj Segmented Neutrophils

(%WBC)

Banded Neutrophils

(%WBC)

6713 668 5866 179 9.1 7.6 1,5 t 810 7 72 ±72 1 4 42 ±0.8 ±0.8 ±0.3 8088 2287 5581 220 26.8 21,9 4,9 ±150 8 5 592 ).x 4 4.

2 xxxx I 1.2x 6225 1813 4228 183 24.5 17.5 1369 580 796 39 2.G~xxxx 1 .2 xx Total RBC (10 12 1L) Reticulocytes, (%RBC) Hb (g/dL) Hct MCV (fl.) M CH (pg) MCHC (g/dL) 8.36 11.00 16 4 45 2 54.0 19 6 36.2 ±0O.06 t1 10(6) ±0 10 z0.2 ±04 0,2 ±0.2 7,97 14.00 14.6 41.5 51.9 18.3 35.2 ).l15 X 1.15 0 3 xxxxx 1 lXx 0.

3 x x 8.22 16.38 15 1 42.9 52.1 18.3 35.2 0.18 1.l24)1 ±0 4A*A z1.2 0

X

1 x xx 2 xx Platelets (10 9 786 t 23 8 26 4 1 816 t 43 See legend for Table WO 93/09129 WO 9309129PCT/A U92/0059) -23 TABLE 7 Organ status of adjuvant arthritic rats treated with caxnyrin p~i1mitate, Control Untreated Treated arthritic arthritic Liver weight Kidney weight Spleen weight SGQT (U/L) 3.47 t 0.06 0.66 0.01 0.20 0,001 141 13 3.53 ±0.09 0.70 ±0.02 0.27 ±0.O1xxx 128 ±15 3.64 ±0.14 0.71 ±0.03 0.26 ±0.02XX 18 3 ±2 1 SGPT (U/L) 9 3 3 9 5 4 9e 6 Glucose (mrnol/L) Urea (mmol/L) Creatirine (pmnol/L) 4,63 0.4 6 7,88 0,50 6.61 0,3 6 3.8 8 0.4 8 7.36 t 0 68 5.71 t 0.26 5.50 ±0.85 8.4 6 ±0.7 3 5.71 ±0,17x 3 5 2 33 t 1 3 2 1 See legend for Table EXAMPLE 4 Effect of a-amyrina and -a-mrn s n,:io.

in huanpttrophils.

A. Materials and Methods, Drug preparation.

amyrin was purchase~d from ICN (USA) and the chlorides of lauric, myristic, palmitic, stearic, oleic and linolele acids were purchased from Sigma (USA). In the preparation of lipid esters, cz'amyrin was dissolved in dichloromethane (DCM) and equivalent moles of pyridine and the lipid chloride were added under dry nitrogen atmosphere. The DCMI extract was dried over anhydrous sodium sulphate, concentrated to thf. oil ard the oil applied to a Si0 2 WO 93/09129 WO 9309129PCI/AUL92/0059o 24 preparative plate run on petroleum ether spirit. The appropriate band was then separated and identified by NMR spectroscopic analysis, Lipoxygenase test method, The method described elsewhere (Cleland et al, 1990) was based on calcium lonophore (A231 87) -induced synthesis of 5-lipoxygenase products (Table 8) by human neutrophils in the presence of exogenous arachidonir acid. I ml of 2.7x1 0 6 neutrophils was used. Measurements were done in quadruplicate on 1:100 dilution of approximately 3.6 mg/mI stock solution of test compound, Data was analysed by multiple t-test in which unpaired Student's t-test was used to compare the tes( compounds with thet single ethanol control (Sinclair, 1988). A P< 0.05 ws considered significant.

B. R=ults and DiscussjLon.

The 5-lipoxygenase enzyme in human nieutrophils catalyses the oxidation of membrane arachidonic acid to 5-H PETE from which 5-H ETE and ihe unstable precursor of the leukotrientes, LTA,,, are tormed (Lewis et (41, 1990; Samuelsson and Funk, 1989). Inhibitionl of5-11ipoX~g(=nae xNoUld be expCted to inhibit all the proditcts mea~i,,led hle: InI thle preent stuld%, inhibition of H-ETE synthesis vas observed to ;igdegrees, without inhibition of L.TB 4 synthesis. As shown in Tablv S. xanimi-in and a-min~ linoieate showed the greatest and most significaiii iedluctions ii 5-I-ETE synthesi and -63% r spectively). And while all the other triterpienes tended to increase the synthcsis, of the leukotrimns. a-amyrin am) amiin ioleate reduced the levels of isomer 1 and -25%7 respectively. latter only- apparently, P and isomner 11 39% and -37% respectively) but had no eftect on LTB, In rheumatoid arthr'tis, LTB. anid 54-11TT increase rn the synovial fluid and svnovial tissue fRhklick n ct 19SO. [tLiidsorn at. 1982:. Davidson et al. 1983) but the rclatt.e importate ot the tmo lipoxygeniase products is unknownf. The knowledge that intra-articular iniectior of corticosteroids redu~ce LTI3, but nut S5H8TE~ (KlccksteOn (T a41. l9I!'4 atd thaut LT8{1 IS thC MOSt WO 93/09129 WO 9309129PCT/A U92/ 00590 25 significant proinflammatory substance among the 5-lipoxygenzse products, (Goetzl and Sun, 1979; Palmblad eltal., 1981; Ford-Hutchinson el al,, 1981; Goetzl et al, 1980) would suggest that inhibition of LTB 4 is more important in the curtailment of chronic inflammation. Evidence is accumulating however that HETE primes neutrophils for the expression of cellular functions associated with inflammation such w, augmenting cytosolic calcium and mobilising protein kinase C from cytosol to membrane (Lewis et al., 1990; Sam'ielsson and Funk, 1989). Therefore, it is suggested that inhibition of 5-H-ETE synthesis may be relevant in antiarthritic therapy.

TABLE 8 Effect of aoamyrin esters on neutrophil synthesis of products.

ng/106 neutrophils Isomer I Isomer 11 LT13 4 Ethanol Control 278.3 2 8.2 13.4 1.3 13.6 1.4 9.4 A 202.1 7,5x 7.0 0.61- 8.3 0.7xx 9.8 0,4 LU 243.0 t 6.3 20.4 0,51"1 19.3 0.5-2 11.6 0. 1" IVA251.1 t 14.9 15.3 t 1,9 14.2 1.9 10.1 0.7 P A 261.1 2.6 2,2 0.91x 22.5 0.9111,1 15.0 ±t 0.4x1112 SA 234.6 3.2 1 5.Y 0.6 15.1 0.8 11.4 ±0.01 OA 253.4 4.9 17.0 0.32 15.4 0.4 1110 ±0.2" LA 103.2 4.9" 10.1 t 0.8 8.5 0,61- 10.4 ±0.4 Values are mean t S.E.M. of 4 experiments.

Significance of difference from control was calculated by tho Student t-test for unpaired varitos and given as- xP<0.05; i"P.O2; 111P.001; AP<0, 0 02, X1111p.<0.001, A, a-amy,-. LU. a-amyrin laurato; MA, a-amyrin myristate; PA. a.amyrin painmitato-, SA, ct.

amyrin stearate; OA, cz-amyrin oleate;- LA, ci-amyrmn linoleate; S.HETE t- S(S)-Hydroxy.

(E,Z,ZZ)-6.8,1 1, 14.olcosatetraefloic acid; LT84 3, (S,1 2R).5.12.dlhydroxy.(Z,e, EZ)- 6,8,10,1 4-eicosatetraenolc acid: Isomer I x 6'trins-leukotriene B 4 or (S12)5 2 dihyrroxy(E.E.E,Z)-6,8,10,14.eicosatettaencic acid. Isomer 11 6.trans-12-epi-teu)kotriene B4 or (5S.12 I 2.dihydreoxy-(E. E. EZ).6,8,1 0,14.olcosa tetra enoic: acid WO 93/09129 WO 9309129PCr/AU92/00590 -26

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93/09129 WO 9309129PCT/A U92/00590 27 Ford-H-utchinson, Bray, Cunningham, Davidson, E.M. and Smith, M.J.H. (1981). Prostaglandins 21, 143-152.

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Claims (7)

1. A method of antiinflammatory, and more particularly antiarthritic, treatment of a human or animal patient in need thereof, which comprises administering to said patient an effective amount of a compound of the general formula II said compound being in substantially pure form: CHIi *o* H CHI 0R 1 o I nm*.ta care a C.e wherein R 1 represents hydrogen or an acyl group derived from a long chain fatty acid, *o

2. A method of antlinflammatory, and more particularly antiarthritic, treatment of a human or animal patient in need thereof which comprises S administering to said patient an effective amount of a composition comprising a compound of the general formula II as defined in claim 1, said compound being in substantially pure form, together with a diluent, carrier and or adjuvant.

3. A method according to claim 1 or claim 2 wherein in the compound of the general formula II, R 1 represents an acyl group derived from a saturated or unsaturated acid having 10 or more carbon atoms.

4. A method according to claim 3 wherein in the compound of the general rqmula II, R 1 represents an acyl group derived from capric, lauric, myristic, palmitic, stearic, arachidonic, myristoleic, palmitoleic, oleic, linoelic, linolenic or arachiodonic acid.

A method according to claim 1 wherein the compound of the general formula II is a-amyrin palmitate.

6. A pharmaceutical composition when used in the antiinflammatory and/or antiarthritic treatment of a human or animal patient, comprising a compound of the general formula II as defined in any one of claims 1 to 5, said compound being in substantially pure form, together with a pharmaceutically acceptable carrier or diluent.

7. A method according to claim 1 substantially as hereinbefore described with reference to any one of the examples. o DATED this 22nd day of April, 1996. S GEORGE KWEIFIO-OKAI By His Patent Attorneys: CALLINAN LAWRIE