Established Adjuvant Arthritis Research Articles

Anti-Inflammatory and Antioxidant Effects of an Ethanolic Extract of the Aerial Parts of <i>Hilleria latifolia</i> (Lam.) H. Walt. (Phytolaccaceae)

Various parts of the perennial herb Hilleria latifolia (Lam.) H. Walt. (Family: Phytolaccaceae) are used in Ghanaian traditional medicine for the treatment of several inflammatory-related disorders. The present study examined the anti-inflammatory effect of an ethanolic extract of the aerial parts of Hilleria latifolia (HLE) in acute and chronic inflammation models. Since free radicals and reactive oxygen species are implicated in inflammatory diseases, the antioxidant potential of HLE was also investigated in in vitro experimental models. HLE (10-300 mg kg(-1), p.o.), either preemptively or curatively, significantly inhibited carrageenan-induced foot oedema in 7-day old chicks. Similarly, the NSAID diclofenac (10-100 mg kg(-1), i.p.) and the steroidal anti-inflammatory agent dexamethasone (0.3-3 mg kg(-1), i.p.) dose-dependently reduced the oedema in both pre-emptive and curative treatments. In the Freund's adjuvant induced-arthritis model in rats, HLE as well as the positive controls, dexamethasone and methotrexate, showed significant anti-arthritic properties when applied to established adjuvant arthritis. HLE (10-300 mg kg(-1), p.o.) significantly reduced oedema in the ipsilateral paw of rats but failed to prevent systemic arthritic spread. The DMARD methotrexate (0.1-1 mg kg(-1), i.p.) and dexamethasone (0.3-3 mg kg(-1), i.p.) reduced significantly the total polyarthritic oedema as well as the spread of the arthritis from the ipsilateral to the contralateral paws of the treated animals. The extract (0.03-1.00 mg ml(-1)) exhibited Fe(3+) reducing activity, scavenged DPPH and prevented lipid peroxidation. These findings suggest that the extract exerts in vivo anti-inflammatory activity after oral administration and also has antioxidant properties which may contribute to its activity.

Comparative Effects of Azapropazone on Cellular Events at Inflamed Sites. Influence on Joint Pathology in Arthritic Rats, Leucocyte Superoxide and Eicosanoid Production, Platelet Aggregation, Synthesis of Cartilage Proteoglycans, Synovial Production and Actions of Interleukin-1 in Cartilage Resorption Correlated with Drug Uptake into Cartilage In-vitro

Azapropazone (APZ) has been compared with standard NSAIDs in title systems to establish aspects of its mode of action on cellular events at inflamed sites. APZ (150 mg kg-1 day-1) given for 10-13 days exhibited a reduction in joint pathology in established adjuvant arthritis in rats comparable with that of indomethacin (2 mg kg-1 day-1) and clobuzarit (20 mg kg-1 day-1). APZ was shown to be a potent inhibitor of the production of leucocyte superoxide and synovial interleukin-1 (IL-1)-like activity and stimulated articular cartilage proteoglycan synthesis, but was ineffective as an inhibitor of platelet aggregation or IL-1 induced cartilage degradation in-vitro. These in-vitro effects may have relevance to the mode of action of this weak inhibitor of prostaglandin synthesis.

Liposomal encapsulation enhances and prolongs the anti-inflammatory effects of water-soluble dexamethasone phosphate in experimental adjuvant arthritis

IntroductionThe objective of this study was to evaluate the efficacy of intravenous (i.v.) injection of liposomally encapsulated dexamethasone phosphate (DxM-P) in comparison to free DxM-P in rats with established adjuvant arthritis (AA). This study focused on polyethylene glycol (PEG)-free liposomes, to minimize known allergic reactions caused by neutral PEG-modified (PEG-ylated) liposomes.MethodsEfficacy was assessed clinically and histologically using standard scores. Non-specific and specific immune parameters were monitored. Activation of peritoneal macrophages was analyzed via cytokine profiling. Pharmacokinetics/biodistribution of DxM in plasma, synovial membrane, spleen and liver were assessed via mass spectrometry.ResultsLiposomal DxM-P (3 × 1 mg/kg body weight; administered intravenously (i.v.) on Days 14, 15 and 16 of AA) suppressed established AA, including histological signs, erythrocyte sedimentation rate, white blood cell count, circulating anti-mycobacterial IgG, and production of interleukin-1beta (IL-1β) and IL-6 by peritoneal macrophages. The suppression was strong and long-lasting. The clinical effects of liposomal DxM-P were dose-dependent for dosages between 0.01 and 1.0 mg/kg. Single administration of 1 mg/kg liposomal DxM-P and 3 × 1 mg/kg of free DxM-P showed comparable effects consisting of a partial and transient suppression. Moreover, the effects of medium-dose liposomal DxM-P (3 × 0.1 mg/kg) were equal (in the short term) or superior (in the long term) to those of high-dose free DxM-P (3 × 1 mg/kg), suggesting a potential dose reduction by a factor between 3 and 10 by liposomal encapsulation. For at least 48 hours after the last injection, the liposomal drug achieved significantly higher levels in plasma, synovial membrane, spleen and liver than the free drug.ConclusionsThis new PEG-free formulation of macrophage-targeting liposomal DxM-P considerably reduces the dose and/or frequency required to treat AA, with a potential to enhance or prolong therapeutic efficacy and limit side-effects also in the therapy of rheumatoid arthritis. Depot and/or recirculation effects in plasma, inflamed joint, liver, and spleen may contribute to this superiority of liposomally encapsulated DxM-P.

Antiarthritic and antioxidant effects of the leaf extract ofFicus exasperataP. Beauv. (Moraceae)

Leaf extracts of Ficus exasperata P. Beauv. (Moraceae) are commonly used in Ghanaian traditional medicine for the treatment of several pathological states including inflammatory disorders. The present study was undertaken to evaluate the antiarthritic effect of an ethanolic extract of F. exasperata (FEE) in the Freund's adjuvant-induced arthritis model in rats. Since free radicals and reactive oxygen species are implicated in inflammatory joint diseases such as rheumatoid arthritis, the antioxidant potential of the extract was investigated in in vitro experimental models. FEE as well as the positive controls, dexamethasone and methotrexate, showed significant dose-dependent antiarthritic properties when applied to established adjuvant arthritis. Oral administration of FEE (30–300 mg/kg p.o.) significantly reduced the arthritic edema in the ipsilateral paw of rats with a maximal inhibition of 34.46 ± 11.42%. FEE (30–300 mg/kg p.o.) also significantly prevented the spread of the edema from the ipsilateral to the contralateral paws indicating inhibition of systemic spread. The disease-modifying antirheumatic drug methotrexate (0.1–1 mg/kg i.p.) and the steroidal anti-inflammatory agent dexamethasone (0.3–3 mg/kg i.p.) also reduced very significantly the total polyarthritic edema as well as the spread of the arthritis from the ipsilateral to the contralateral paws of the treated animals. The extract also exhibited reducing activity (EC50 = 8.105 ± 18.49), scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH, EC50 = 0.499 ± 0.302) and prevented lipid peroxidation (IC50 = 1.283 ± 0.923) in rat brain homogenates. Phenols were detected in the extract. These results suggest that ethanolic extract of the leaves of F. exasperata exerts antiarthritic activity after oral administration and also has antioxidant properties which may contribute to its activity.

Folate-targeted immunotherapy effectively treats established adjuvant and collagen-induced arthritis

Activated macrophages express a cell surface receptor for the vitamin folic acid. Because this receptor is inaccessible or not measurably expressed on other normal cells, folic acid has been recently exploited to selectively deliver attached radio-emitters to sites of activated macrophage accumulation, allowing scintigraphic imaging of inflamed joints and organs of arthritic rats. We demonstrate here that folate-linked haptens can also be targeted to activated macrophages, decorating their cell surfaces with highly immunogenic molecules. Under conditions in which the rodent has already been immunized against keyhole limpet hemocyanine-(fluorescein isothiocyanate) FITC, activated macrophages are eliminated. Administration of folate-FITC conjugates to rodents with experimental arthritis attenuates (a) systemic and peri-articular inflammation, (b) bone and cartilage degradation, and (c) arthritis-related body weight loss. Treatment with folate-hapten conjugates is comparable to methotrexate, etanercept, anakinra, and celecoxib at alleviating the symptoms of arthritis. We conclude that reduction of activated macrophages by folate-targeted immunotherapy can ameliorate the symptoms of arthritis in two rodent models of the disease.

The protective effects of incadronate on inflammation and joint destruction in established rat adjuvant arthritis

The effects of a new generation bisphosphonate, incadronate, in established adjuvant arthritis rats were evaluated according to the arthritis index, hind paw volume, and radiological and histopathological examinations. Incadronate suppressed the radiological and histopathological changes of hind paws, as well as the joint swelling in a dose-dependent manner. In contrast, the arthritis control rats showed drastic joint inflammation, marked destruction of bone and articular cartilage. The remains of articular cartilage lost Safranin O staining, and were attached with numerous TRAP-positive multinuclear cells. Some of resorption lacunas could be seen at the cartilage matrix nearby the TRAP-positive multinuclear cells. As regards the chondroprotective effects of bisphosphonates, we speculate that it is probably concerned with the inhibition of the chondroclasts. These data indicate that bisphosphonates may be a class of effective agent that can be considered for treatment of various arthritic conditions, including human rheumatoid arthritis.

Anti-CD2 (OX34) MoAb treatment of adjuvant arthritic rats: attenuation of established arthritis, selective depletion of CD4+ T cells, and CD2 down-modulation

SUMMARY Anti-CD2 MoAbs have previously been shown to induce tolerance and to block B cell differentiation, T cell and monocyte activation. Since these immune functions are important in joint inflammation, we asked whether administration of the anti-CD2 MoAb OX34 has a beneficial effect on established rat adjuvant arthritis, a model of human rheumatoid arthritis, and how it affects CD2-bearing leucocyte subsets. Female Lewis rats with established adjuvant arthritis received a total of 5 mg OX34 or isotype-matched control MoAb starting on day 15 after adjuvant injection. Weight and arthritis score (AS) were measured in a blinded fashion. Peripheral blood cells were analysed for numbers of leucocyte subsets at various time points. Animals were killed on day 30 and lymphatic organs were processed for immunohistology. Clinically, OX34 treatment led to increased body weight and reduced AS. Although OX34 binds to CD4+ and CD8+ T cells in a comparable fashion, OX34 treatment reduced CD4+ T cells, but not CD8+ T cells. Among CD4+ T cells CD45RC+ (‘naive’) T cells virtually disappeared; CD45RC− (‘recently activated’) T cells were slightly reduced. A reduction of CD4+ T cells was also found in the lung, liver, bone marrow, spleen and lymph nodes. Down-modulation of the CD2 molecule by OX34, again, affected CD4+ T cells, suggesting a specific signal for CD4+ but not CD8+ T cells. In conclusion, the anti-CD2 MoAb OX34 attenuates established rat adjuvant arthritis. In spite of similar binding to CD4+ and CD8+ T cells, OX34 depletes only CD4+ T cells and down-modulates the CD2 molecule on these cells. These results suggest a therapeutic benefit from CD2-directed therapy for chronic types of arthritis.

Involvement of macrophage migration inhibitory factor in the evolution of rat adjuvant arthritis.

Recent studies have established an essential role for macrophage migration inhibitory factor (MIF) in T cell and macrophage activation, both of which are characteristics of rat adjuvant arthritis. This study investigated the role of MIF in early adjuvant arthritis. MIF was detected in rat synovium by immunohistochemistry and enzyme-linked immunosorbent assay using specific monoclonal antibodies (MAb). Anti-MIF MAb treatment was administered, and the effects on clinical aspects of adjuvant arthritis were assessed. MIF was absent from normal rat synovium prior to adjuvant injection, but was detectable on day 4 after injection (6 days before the onset of clinical disease) and was colocalized with ED-1+ macrophages throughout the evolution of the disease. Levels of MIF were increased in established adjuvant arthritis sera, and adjuvant arthritis synovial macrophages released MIF at a mean +/- SEM concentration of 607.9 +/- 201.5 pg/ml. Anti-MIF treatment led to profound, dose-dependent inhibition of the adjuvant arthritis clinical score, paw swelling, and synovial lavage leukocyte numbers (P < 0.001), and also resulted in reduced synovial macrophage and T cell accumulation. These findings demonstrate an important role for MIF in the evolution of rat adjuvant arthritis.

Anti-CD2 (OX34) MoAb treatment of adjuvant arthritic rats: attenuation of established arthritis, selective depletion of CD4+ T cells, and CD2 down-modulation.

Anti-CD2 MoAbs have previously been shown to induce tolerance and to block B cell differentiation, T cell and monocyte activation. Since these immune functions are important in joint inflammation, we asked whether administration of the anti-CD2 MoAb OX34 has a beneficial effect on established rat adjuvant arthritis, a model of human rheumatoid arthritis, and how it affects CD2-bearing leucocyte subsets. Female Lewis rats with established adjuvant arthritis received a total of 5 mg OX34 or isotype-matched control MoAb starting on day 15 after adjuvant injection. Weight and arthritis score (AS) were measured in a blinded fashion. Peripheral blood cells were analysed for numbers of leucocyte subsets at various time points. Animals were killed on day 30 and lymphatic organs were processed for immunohistology. Clinically, OX34 treatment led to increased body weight and reduced AS. Although OX34 binds to CD4+ and CD8+ T cells in a comparable fashion, OX34 treatment reduced CD4+ T cells, but not CD8+ T cells. Among CD4+ T cells CD45RC+ ('naive') T cells virtually disappeared; CD45RC- ('recently activated') T cells were slightly reduced. A reduction of CD4+ T cells was also found in the lung, liver, bone marrow, spleen and lymph nodes. Down-modulation of the CD2 molecule by OX34, again, affected CD4+ T cells, suggesting a specific signal for CD4+ but not CD8+ T cells. In conclusion, the anti-CD2 MoAb OX34 attenuates established rat adjuvant arthritis. In spite of similar binding to CD4+ and CD8+ T cells, OX34 depletes only CD4+ T cells and down-modulates the CD2 molecule on these cells. These results suggest a therapeutic benefit from CD2-directed therapy for chronic types of arthritis.

Treatment of established adjuvant arthritis in rats with monoclonal antibody to CD18 and very late activation antigen‐4 integrins suppresses neutrophil and T‐lymphocyte migration to the joints and improves clinical disease

The migration of leucocytes from blood into the joint is a key feature of human and experimental arthritis. Adhesion molecules on leucocytes and vascular endothelium are important in this process and may be therapeutic targets for intervention in arthritis. We investigated whether monoclonal antibody treatment to block the alpha 4 integrin, very late activation antigen-4 (VLA-4), and beta 2 integrins (CD11/CD18) administered to rats during the preclinical (day 5) or clinical phase (day 10+) would modify disease. When treatment was initiated 5 days after induction of disease, development of arthritis was significantly reduced by either anti-alpha 4 (TA-2) or anti-beta 2 (WT.3) monoclonal antibodies (mAb) and the combination of both mAb was even more effective (clinical scores: control 11.4; anti-alpha 4 6.6; anti-beta 2 6.8; anti-alpha 4 + anti-beta 2 3.9). When treatment was delayed until arthritis was apparent (day 10), the anti-alpha 4 + anti-beta 2 mAb combination still significantly diminished the arthritis score on day 14 (control 13; anti-alpha 4 + anti-beta 2 7.9). Treatment with anti-alpha 4 + anti-beta 2 mAb decreased the migration to the joints of blood polymorphonuclear leucocytes (PMNL) by 66-79% and of spleen T lymphocytes by 56-75%, depending on the joint. In contrast, PMNL migration was abolished (> 98%) and T-cell migration markedly (87%) inhibited to dermal inflammatory reactions in the same animals. These findings demonstrate: that blocking mAb to alpha 4 and beta 2 integrins can reduce the severity of adjuvant arthritis, even after joint inflammation has developed; that this treatment can markedly inhibit PMNL and T-lymphocyte migration to the joints; and that yet to be defined mechanisms distinct from alpha 4 (CD49d) and beta 2 (CD11/CD18) integrins, also contribute to leucocyte migration to inflamed joints. Identifying these additional adhesion mechanisms may be required to control joint inflammation further.

Long-term amelioration of rat adjuvant arthritis following systemic elimination of macrophages by clodronate-containing liposomes.

To determine whether systemic elimination of macrophages by means of clodronate-containing liposomes counteracts inflammation and joint destruction in rats with established adjuvant arthritis (AA). Rats with AA received a total of 2.7 mg of clodronate encapsulated in liposomes in 3 intravenous doses on days 10, 11, and 12 of arthritis. Phosphate buffered saline (PBS), PBS-laden liposomes, or free clodronate were used as negative controls. Clinical, hematologic, and histopathologic signs of AA were monitored, and depletion of macrophages by clodronate-liposomes was evaluated both in the synovial membrane (SM) and in organs of the mononuclear phagocyte system (MPS). Clodronate-laden liposomes led to significant, long-term amelioration of the clinical signs of AA, a reduction in the erythrocyte sedimentation rate (ESR), and counteraction of joint destruction, not only immediately after treatment, but also for 2 weeks thereafter. Free clodronate induced moderate clinical improvement and a significant decrease in the ESR, but only during the late phase of AA. Drug-free vesicles even aggravated the joint destruction. Clodronate-laden liposomes did not induce significant depletion of resident macrophages in the SM, but rather, in the paracortical region of popliteal lymph nodes, in the liver, and in the marginal zone and periarteriolar lymphatic sheaths of the spleen. Clodronate-laden liposomes induce long-term amelioration of AA, even if administered for a brief period during the florid phase of the disease. The amelioration is paralleled by the elimination of macrophages in immunocompetent areas of the spleen and draining lymph nodes, but not locally in the SM. This suggests an influence of the treatment on the immunoregulatory rather than effector, functions of macrophages.

Treatment of rat arthritides with clodronate-containing liposomes.

Large multilamellar liposomes containing dichloro-methylene-bisphosphonate (clodronate; Clo), a bisphosphonate that becomes toxic when intracellularly concentrated, were used to therapeutically target macrophages (M phi) in rats with established adjuvant arthritis (AA; i.v. on days 10,11,12) or antigen-induced arthritis (AIA; i.v. or i.a. on 3 h, days 1,2). In established AA, i.v. injection of Clo-liposomes led to significant, long-lasting amelioration of clinical parameters, and to reduced destruction of the ankle joint even several weeks after termination of treatment. In the acute phase of established AIA, intravenous treatment induced transient clinical amelioration, but did not counteract joint destruction. I.a. treatment in AIA was ineffective. Systemic treatment with anti-M phi principles induces amelioration of both AA and AIA; the improvement appears more profound in AA, i.e., the model with a more systemic character. Preliminary data indicate that depletion of M phi occurs in the liver rather than in spleen, draining lymph nodes or synovial membrane. In addition, local treatment with the same principle is ineffective in AIA. Therefore, systemic elimination of M phi in different sites may be crucial for effective therapy of arthritis with anti-M phi agents.

Effect of clodronate on established adjuvant arthritis.

The rat adjuvant arthritis model was used to study the effect of disodium clodronate on inflammation and destruction of tarsal bones and joints. Male Lewis rats were given an intradermal injection of mycobacteria. Fourteen days after immunization, rats with similar scores were assigned to the different experimental groups. They were treated subcutaneously either with saline (controls) or with clodronate at doses of 12.5 and 25 mg/kg/day five times a week for 2 weeks. Clinical signs of arthritis including the severity of paw swelling were assessed weekly. At the time of sacrifice, histological features of the non-decalcified tarsus with ankle, intertarsal and tarsometatarsal joints were assessed for inflammatory soft-tissue, articular and bone changes. The total histological score of the hindpaw indicated that 58% of the control rats developed moderate arthritis and 42%, severe arthritis. The treatment with clodronate (25 mg/kg) decreased clinical signs of arthritis and the activity of the collagen-degrading lysosomal enzyme, beta-N-acetylglucosaminidase, in inflamed hindpaw tissue. Histological evaluation indicated moderate arthritis in 83%, but no severe arthritis. The lower dose of clodronate also decreased the severity of the disease; the decrease was, however, statistically insignificant. The results show that clodronate given therapeutically to adjuvant arthritic rats suppresses the intensity of the inflammation and prevents secondary articular and bone lesions in the tibiotarsal region.

Depletion of alpha/beta T cells by a monoclonal antibody against the alpha/beta T cell receptor suppresses established adjuvant arthritis, but not established collagen-induced arthritis in rats.

The effects of treatment with a monoclonal antibody (R73 mAb) against T cell receptor alpha/beta (TCR-alpha/beta) on both established adjuvant arthritis (EAA) and established collagen-induced arthritis (ECIA) in rats have been investigated. Rats were treated with R73 mAb when arthritis reached a peak. Treatment with the anti-TCR-alpha/beta mAb markedly suppressed EAA, whereas ECIA was not affected by the mAb treatment. Histologically, R73 mAb-treated rats with EAA showed mild hyperplasia of synovial tissues, sparse infiltration of inflammatory cells, and minimal erosion of cartilage, whereas arthritic rats treated with PBS and an irrelevant control mAb against Giardia had marked hyperplasia of synovium with pannus, massive inflammatory cell infiltrate, and severe destruction of cartilage and subchondral bone. R73 mAb-treated rats with ECIA exhibited pronounced formation of pannus containing many inflammatory cells and marked cartilage and subchondral damage similar to those in arthritic rats that received the control treatments. Treatment with R73 mAb depleted markedly alpha/beta+ T cells in both peripheral blood and synovial tissues of rats with EAA and ECIA. R73 mAb treatment was associated with marked reduction in arthritogen-specific delayed-type hypersensitivity responses in both EAA and ECIA. The titers of antibodies against type II collagen produced in rats with ECIA were not affected by the mAb. Thus, alpha/beta+ T cells appear to have a central role in EAA, but not in chronic ECIA.

In vitro inhibition of arachidonic acid metabolism by two novel retinoid analogs

Ro 23-6457, (all-E)-3,7-dimethyl-9-[2-(trifluoromethyl)-6-(nonyloxy)phenyl]-2, 4,6,8- nonatetraenoic acid, and Ro 23-2895, (all-E)-9-[2-(nonyloxy)phenyl]-3,7-dimethyl-2,4,6,8-nonatetraen oic acid, are two novel retinoid analogs which exhibit antiinflammatory activity in both the developing and the established rat adjuvant arthritis models [8]. Here we investigated the effect of these two compounds on the production of arachidonic acid (AA) metabolites in two in vitro test systems [i.e., Ca2+ ionophore A23187 (I)-stimulated resident rat peritoneal macrophages (MO) and cytokine-stimulated human dermal fibroblasts (HDF)]. Both compounds, Ro 23-6457 and Ro 23-2895, significantly inhibited the release of 14C-AA metabolites and the production of LTB4, PGE2, and 6-keto-PGF1 alpha in I-stimulated MO, at concentrations of 1-33 microM. Both compounds also inhibited the production of PGE2 in HDF stimulated by either rhuIL-1 alpha or huTNF alpha at concentrations of 1 x 10(-5) to 1 x 10(-7) M. Ro 23-2895 was also a potent inhibitor of IL-1-induced collagenase production in rheumatoid synovial cells (IC50 approximately 1 to 2.5 x 10(-8) M). The inhibitory profile of these novel compounds in these cell systems is therefore similar to that of other known antiinflammatory retinoids (e.g., all-trans- and 13-cis-retinoic acid). Inhibitory effects such as those described here might in part contribute to the antiinflammatory activity of these compounds in vivo.

Histopathologic evaluation of the effects of etodolac in established adjuvant arthritis in rats: evidence for reversal of joint damage.

Histopathologic evaluation of hindpaws from control rats with established adjuvant arthritis showed severe alterations in soft tissue and bone, as well as progressive, moderate-to-severe articular changes. Following treatment with etodolac for 28 days, soft tissue and articular changes were rated mild, and bone changes were rated moderate, but with remodeling. These findings indicate that etodolac partially reversed the joint damage in these rats.

γ-glutamyl transpeptidase: A novel biochemical marker in inflammation

γ-Glutamyl transpeptidase (γ-GT) plays an important role in the turnover of glutathione and protein biosynthesis. Because in inflammation both catabolic and anabolic steps are activated together with migration of cells, an alteration in γ-GT activity was postulated to occur at the site of inflammation with the development of the inflammatory process. We discovered that γ-GT activity was increased markedly at sites of inflammation produced in several ways in rats. A significant increase in enzyme activity appeared soon after the induction of inflammation. In carrageenin-induced acute inflammation, the paw tissue attained a 3- to 4-fold increase in enzyme activity within 4 hr; in established adjuvant arthritis, a 20- to 24-fold increase over its basal activity occured in the rat paw tissue. The specific enzyme activity was 20–22 nmoles/min/mg protein in the cellular sediment of carrageenin-induced pleural exudate. In cotton granulomatous tissue it was 5- to 6-fold higher compared to the enzyme activity of the skeletal muscles. The in vivo in γ-GT activity was prevented from occuring in proportion to the anti-inflammatory potencies of the test drugs given orally. The prevention of enzyme activity was observed with indomethacin in carrageenin-induced edematous paw tissue and with phenylbutazone in both adjuvant arthritis and carrageenin-induced pleural exudate. Prednisolone was observed to be the most potent drug against cotton granuloma. Nonsteroidal anti-inflammatory drugs (NSAIDs) were not found to affect enzyme activity in vitro when incubated with cellular infiltrate from a cotton pellet granuloma. Differences in certain physico-chemical characteristics, viz. stability at 50°, pH dependency and effects of solvents, were not discernible in between the enzyme activities of the untreated and edematous paw tissues. The studies thus suggest that measurement of γ-GT in inflammation may prove to be a valuable biochemical marker for the assessment of anti-inflammatory activity of drugs in vivo.

Effect of α-(3,5-Di-Tert-Butyl-4-Hydroxybenzylidene)-γ-Butyrolactone (KME-4), a New Anti-Inflammatory Drug, on the Established Adjuvant Arthritis in Rats

The effect of α-(3,5-di-tert-butyl-4-hydroxybenzylidene)-γ-butyrolactone (KME-4), a new anti-inflammatory drug, on established adjuvant arthritis in rats was compared to that of indomethacin. When administered orally daily from days 14 to 27 starting on day 14 after the adjuvant (day 0), KME-4 (2 to 10 mg/kg) produced a dose-related reduction of the swelling of both injected and uninjected hindpaws, and it retarded body weight loss. The initiation of paw swelling after the cessation of therapy was not observed at either 5 mg/kg or 10 mg/kg of KME-4. On day 42 (15 days after discontinuation of-dosing), KME-4 caused the recovery of organ weight, erythrocyte sedimentation rate (ESR) and serum albumin/globulin (A/G) ratio towards normal levels, and it also decreased radiographic bone damage scores in a dose-dependent manner The results indicate that KME-4 produces the improvement of systemic symptoms in the established adjuvant arthritis. The results obtained with indomethacin were si miliar to those with KME-4. However, the degree of the efficacy of indomethacin (2 mg/kg) was lower than that of KME-4 (10 mg/kg) as judged by the measured parameters (ESR, serum A/G ratio and bone damage).

The absence of antibodies to type II collagen in established adjuvant arthritis in rats.

Utilizing an adjuvant arthritis model in rats, we examined humoral immunity to collagen and inflammation in animals with active disease and during drug therapy. Humoral immunity to types I or II collagen was not detected in the sera of rats with advanced adjuvant arthritis; this was in marked contrast to rats with type II collagen-induced arthritis which possessed serum antibodies to native and denatured type II collagen. Hind paw edema and bone pathology were monitored as parameters of inflammation. A new investigational drug, Wy-41,770, was most effective in reducing all of these aspects of inflammatory disease while indomethacin, methylprednisolone, and D-penicillamine caused a less significant diminution of only some of these parameters of inflammation. Antibodies to collagen were not detected in the sera of rats treated with the drugs under study. These data demonstrate that adjuvant arthritis can occur in rats in the absence of antibodies to types I or II collagen.

Lipophilic copper(II) formulations: some correlations between their composition and anti-inflammatory/anti-arthritic activity when applied to the skin of rats.

Copper complexes of phenols related to salicylic acid were prepared in DMSO and applied to the shaved dorsal skin of rats. The following activities were assayed: (i) suppression of the carrageenan or hydroxylapatite paw oedemas; (ii) reduction of chronic inflammation in established adjuvant arthritis; (iii) local skin toxicity. Cu(II) was an essential component. Some limited structure-activity correlations were made among alternative cupriphores. DMSO solutions of copper complexes were more potent than their solutions in ethanol. Glycerol was a beneficial additive. Reducing the acidity of some copper salicylate formulations also reduced their potency. Niflumic acid and phenylbutazone were effective non-salicylate transcutaneous cupriphores.