Auranofin-induced suppression of autoimmune antibody production and inflammation in genetically autoimmune-prone mice.

The gold compound auranofin and lobenzarit (CCA) were compared in regard to effects on development of an autoimmune-like disease in MRL/1 mice, which normally develop elevated levels of serum anti-DNA antibodies and rheumatoid factor as well as joint lesions similar to those seen in patients with rheumatoid arthritis. MRL/1 mice, which are genetically prone to development of autoimmune disease, were given auranofin or lobenzarit by gavage for 15 weeks, starting at 6 weeks of age. Mice were examined periodically for immunological abnormalities as well as histologic changes in articular joints. The auranofin-treated mice showed marked diminution in development of anti-DNA antibodies and serum rheumatoid factor as compared to control animals. Although higher than in the auranofin-treated animals, CCA-treated mice also had lower levels of serum autoantibodies than those seen in the control animals. Examination of limb joints for histopathologic changes indicated that the auranofin-treated animals developed only the slightest evidence of lesions as compared to control animals. CCA-treated mice also had a lessening of lesion development compared to control animals, but lesions were more developed than in auranofin-treated mice. This study indicates that auranofin is more effective than CCA in diminishing development of autoimmunity in MRL/1 mice.

5,6-Diaryl-2,3-dihydroimidazo[2,1-b]thiazoles: a new class of immunoregulatory antiinflammatory agents.

A series of substituted 5,6-diaryl-2,3-dihydroimidazo[2,1-b]thiazoles were synthesized and evaluated in the rat adjuvant-induced arthritis and mouse oxazolone-induced contact sensitivity assays to determine the potential of these compounds for use as immunoregulatory antiinflammatory agents. This class of compounds was derived by combining salient structural features of the antiinflammatory agent flumizole and the immunoregulatory drug levamisole. Unlike the latter two, a number of compounds in the target series were found to possess the desired combination of activities. Exploration of structure-activity relationships in the adjuvant-induced arthritic rat assay revealed that optimal potency was exhibited by symmetrically substituted 5,6-diaryl compounds having one of the following alkyl heteroatom or halogen functions at the para position: methoxy, ethoxy, methylthio, N-ethyl-N-methylamino, fluoro, or chloro. Scrambling of these two substituent classes to yield the asymmetrically substituted 5,6-diaryl compounds resulted in potent activity only with the 5-alkyl heteroatom, 6-halo-substituted regioisomers. However in the oxazolone-induced contact sensitivity assay, no consistent relationship of variation in activity with structural change was apparent. The initial target compound 5,6-bis(4-methoxyphenyl)-2,3-dihydroimidazo[2,1-b]thiazole (1) was compared with its progenitors in additional models of inflammation and immunoregulation.

Antiinflammatory activity of 5,6-diaryl-2,3-dihydroimidazo[2,1-b]thiazoles. Isomeric 4-pyridyl and 4-substituted phenyl derivatives.

Isomeric 5(6)-(4-pyridyl)- and 6(5)-(4-substituted-phenyl)-2,3-dihydroimidazo[2,1-b]thiazoles were prepared by a mixed benzoin-imidazothione route, and their structures were assigned by spectral comparison to compounds of established substitution pattern. The structural assignment was confirmed by X-ray analysis. Examination of the compounds for antiinflammatory activity by an adjuvant arthritic rat assay revealed strikingly higher potencies for one analogous series than for their isomers. This selectivity was paralleled in the ability to stimulate cell-mediated immunity, as reflected in an oxazolone-induced contact sensitivity model. A drug-receptor complex is proposed that requires at least three sites of interactions.

Biologic actions and pharmacokinetic studies of auranofin.

The preclinical profiles of auranofin (Ridaura), an oral chrysotherapeutic agent, parenteral gold sodium thiomalate, gold thioglucose, and their respective ligands were compared. Auranofin was more effective than gold sodium thiomalate in suppressing inflammation and stimulating cell-mediated immunity. In contrast to gold sodium thiomalate and gold thioglucose, auranofin inhibited cellular release of lysosomal enzymes, antibody-dependent cellular cytotoxicity, production of antibodies in adjuvant arthritic rats, and antibodies involved in cytotoxicity reactions. The respective ligands were without significant biologic activity. In rats, a higher fraction of gold was associated with blood cells after auranofin administration than after gold sodium thiomalate. The absorption, distribution, metabolism, and excretion of auranofin are uniquely different from other gold compounds.

Superoxide radical production by human leukocytes exposed to immune complexes: inhibitory action of gold compounds.

Superoxide radicals produced by phagocytic cells are considered to be important mediators in the rheumatoid inflammation. The effect of the gold compounds auranofin (AF) and gold sodium thiomalate (GST) on superoxide production by human leukocytes was investigated in two models of immunologic injury: immune-complex phagocytosis and frustrated phagocytosis. In both systems, AF (0.5-1.0 micrograms Au/ml) showed a potent inhibitory activity on superoxide generation, quantitated by ferricytochrome c and NBT reduction. GST showed only modest inhibition at higher concentrations (100 microM). The thiol protecting agent dithiothreitol, 1 mM, completely blocks the inhibitory effect of AF. The inhibition of the oxy radical generation by AF may play an important role in the control of rheumatoid inflammation; it is suggested that this action might be mediated through sulfhydryl-AF interaction at the cellular membrane level.

Distribution of gold in rat blood after intravenous administration of auranofin, gold sodium thiomalate and aurothioglucose to rats.

Serum, blood and cell-associated gold were determined at various time periods after intravenous administration of 1 mg Au/kg of auranofin (AF), gold sodium thiomalate (GSTM) and aurothioglucose (GTG). AF gold exhibited an early phase of decay with high levels of cell-association; whereas, after 72 h cell-associated gold was not detectable. In contrast, GSTM and GTG serum gold values were generally higher than blood values since cell-associated gold rarely occurred. These observations suggest that, in contrast to GSTM and GTG, intravenous administration of AF results in a dynamic equilibrium of gold between the cellular and serum compartments of blood.

The pharmacological profile of auranofin, an orally active gold compound.

Auranofin (AF; ' Ridaura '), an oral chrysotherapeutic agent, parenteral gold sodium thiomalate (GST) and gold thioglucose (GTG) were evaluated in order to compare their preclinical profiles. AF was found to be more effective than GST and GTG in suppressing inflammation and stimulating cell-mediated immunity. In contrast to GST, AF inhibited cellular release of lysosomal enzymes, antibody-dependent cellular cytotoxicity, production of antibodies in adjuvant arthritic rats, and antibodies involved in cytotoxicity reactions. In pharmacokinetic studies, plasma gold in rats following AF administration, exhibited greater cell association than after GST administration. In conclusion, the pharmacological profile of AF is markedly different from those of GST and GTG and this suggests potential for improvements in chrysotherapy.

Mechanisms of action of auranofin: effects on humoral immune response.

Auranofin (AF) and gold sodium thiomalate (GSTM) were evaluated in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent antibody responses. AF decreased the ability of immune sera to participate in ADCC, whereas GSTM did not. Immune serum from AF-treated rats also exhibited a decreased antibody-dependent complement lysis (ADCL) reactivity. In contrast, immune sera from GSTM-treated rats enhanced ADCL. AF also suppressed '7S' hemagglutinin antibody response to sheep red blood cells in adjuvant arthritic rats, whereas neither GSTM nor gold sodium thioglucose significantly suppressed hemagglutinin antibody titers at doses which produced antiinflammatory activity.

Comparative pharmacology and biological effects of different gold compounds.

Auranofin's (AF) physical, chemical, pharmacological, and pharmacokinetic properties differ from those of gold sodium thiomalate (GSTM). AF is lipid soluble, monomeric, nonconductive and is not a potent sulfhydryl reagent. In further contrast to GSTM, AF gold is orally absorbed, exhibits protracted blood levels, is bound to cellular elements of the blood, excreted mainly in the feces, and exhibits less tissue retention. AF is more effective in acute inflammatory models and is a potent inhibitor of lysosomal enzyme release, antibody-dependent cellular cytotoxicity, and superoxide production. AF can suppress antibodies produced in adjuvant arthritic rats and those involved in cytotoxicity reactions; whereas, GSTM is ineffective or immunoenhancing. AF is more effective in stimulating abnormalized cell-mediated immunity. In conclusion, AF is a unique oral chrysotherapeutic agent which can affect cellular and immunopathological events involved in the perpetuation of inflammation and tissue damage.

Pharmacokinetics of auranofin in animals.

Gold from orally administered auranofin (AF) was absorbed 17-23% in rats and 15-38% in dogs. Gold was highly bound to blood cells and plasma proteins. Gold terminal half life was 1.2-1.8 days in rat blood and plasma (measured for 7 days post dose) and 19.5 days in the dog (measured for 42 days). Excretion of gold (rat and dog) was via feces (84 and 81%) urine (10 and 16%) and bile (3% of dose). Rat tissue levels of gold were highest in the kidney. Evidence indicated that AF was rapidly degraded to triethylphosphine oxide with the remaining molecular fragments postulated to be a protein-gold complex and acetylthioglucose.

Augmentation of concanavalin A-induced immunosuppression by indomethacin.

These studies show that, in BALB/C mice, when antibody synthesis against sheep red blood cells is suppressed by concanavalin A, treatment with indomethacin (4-8 mg/kg per os) will augment this suppression. Two other nonsteroidal anti-inflammatory drugs, flufenamic acid and meclofenamic acid (50 mg/kg), also have this effect, whereas phenylbutazone was inactive at this dose. The augmentation of concanavalin A-induced immunosuppression by indomethacin could not be demonstrated on the response to the T-independent antigen polyvinypyrrolidone. In contrast to indomethacin, which inhibits cyclooxygenase, neither nordihydroguaiaretic acid, an inhibitor of the lipoxygenase pathway, nor eicosa-5,8,11,14-tetraynoic acid, an inhibitor of both the cyclooxygenase and the lipoxygenase pathways, had this augmenting effect. Therefore, we do not have strong evidence that the absence of a prostaglandin is responsible for the effect of indomethacin. However, inhibition of the pathway leading to prostaglandin synthesis causes an increase in arachidonic acid metabolism via the lipoxygenase pathway. A product of this pathway, such as a leukotriene, may have immunosuppressive effects in this model. Evidence for the enhancement of a suppressor cell population is provided by an in vitro coculture assay. Cells treated with concanavalin A and indomethacin had more suppressive activity than cells treated with concanavalin A or indomethacin alone.

Effect of selected immunoregulatory agents on low-grade contact sensitivity.

The effect of selected compounds with known immunoregulatory activity was examined in a 45-h sensitization period oxazolone contact-sensitivity reaction. Oxazolone sensitivity was induced by applying 0.1 ml of 5% oxazolone in absolute ethanol to the shaved abdomen of C57Bl/6 mice on day 0. Challenge with oxazolone followed 45 h later and was accomplished by painting a 5% solution of oxazolone in absolute ethanol on the left hindpaw. The response at 24 h was determined plethysmographically. Histamine (0.062-1.0 mg/kg, subcutaneously, twice a day), concanavalin A (0.31-5.0 mg/kg intravenously), penicillamine (6.25-25 mg/kg, subcutaneously), chloroquine (6.25-25 mg/kg, subcutaneously), and thymosin fraction 5 (0.125-1.25 mg/kg subcutaneously) all stimulated the oxazolone reaction when administered on day 0. These data suggest that the low-grade oxazolone response may be a useful assay to detect immunostimulatory activity.

Studies of the effect of auranofin, a new antiarthritic agent, on platelet aggregation.

Auranofin (AF), at a concentration of 10 micrograms/ml, was found to be a potent inhibitor of ADP-, epinephrine-, or collagen-induced platelet aggregation utilizing platelet-rich plasma obtained from human blood. In contrast, aurothioglucose was less effective than AF in inhibiting epinephrine- or collagen- induced platelet aggregation. The inhibitory effect of AF was more evident on the second phase of aggregation. The inhibitory effect of AF was more evident on the second phase of aggregation and was a function of drug preincubation time. Compared to platelet-rich plasma, washed platelets were superior for detecting the inhibitory action of AF (greater than or equal to 0.1 microgram/ml) on ADP-induced platelet aggregation. This potent inhibitory action of AF on ADP-induced platelet aggregation was antagonized by dithioerythriol, a potent reducing agent. These results suggest that AF can inhibit both platelet release and aggregation mechanisms which may be relevant to its antiarthritic activity. Further studies are required to elucidate the cellular mechanism by which AF inhibits platelet aggregation.

Stimulation of contact sensitivity to oxazolone by disodium cromoglycate.

The subcutaneous administration of 0.01-10 mg/kg of the antiallergic agent disodium cromoglycate on day 0 30 min prior to sensitization of C57Bl/6 male mice with 5% 2-phenyl-4-ethoxy-methylene oxazolone proved to cause a significant stimulation of the low-grade volume response as measured plethysmographically in 24 h after challenge. The investigation of the mechanism favors the conclusion that histamine release is involved in the action of disodium cromoglycate as judged by the ability of the antihistaminics chlorpheniramine and metiamide to inhibit the disodium cromoglycate action and by the inhibitory effect of polymyxin B induced depletion of histamine.

Effect of auranofin dose regimen change upon cell-associated gold in rheumatoid arthritic patients.

We examined whether changes in auranofin dose regimen in rheumatoid arthritis (RA) patients affect cell-associated and/or serum gold levels. In 7 RA patients, a reduction in daily dosage of auranofin from 3 mg bid to 3 mg qd was correlated with a marked reduction (81%-95%) in cell-associated gold, whereas a return to the 3 mg bid dose level resulted in a reinstatement of cell-associated gold. These changes in cell-associated gold were not reflected in alterations of serum gold levels. This apparent lack of change in serum gold level as opposed to cell-associated gold suggests that this phenomenon warrants further investigation.

Pharmacokinetics of gold following administration of auranofin (SK+FD-39162) and myochrysine to rats.

Auranofin orally administered to rats resulted in delayed and protracted peak blood and serum gold levels occurring 24 to 48 h post administration. During this period, the gold concentration in blood was higher than in serum indicating that a major portion of gold was associated with the cellular components. During 1st order elimination (greater than 48 h), the blood/serum gold ratio decreased which suggested dissociation of cellular gold. Biliary cannulation experiments demonstrated that the protracted gold levels (24 to 48 h) could not be due to hepatic recirculation. In contrast to auranofin, gold sodium thiomalate produced blood gold levels which peaked within 3 h, rapidly declined and were consistently lower than serum gold levels.