Cocaine and amphetamine-regulated transcript mRNA regulation in the hypothalamus in lean and obese rodents.

Cocaine and amphetamine-regulated transcript (CART) mRNA and immunoreactivity are expressed abundantly in the hypothalamus. Central administration of various fragments of this neuropeptide decreases food intake in rodents. To find out whether CART might play a role in the physiological regulation of energy balance, we used in situ hybridization to investigate whether CART mRNA abundance changed in two chronic obese/fat versus lean states and after acute dietary restriction. In the first study, mice were treated with goldthioglucose to destroy glucose-responsive neurones in the ventromedial hypothalamus. This produced hyperphagia and obesity: 7 weeks after treatment, those receiving goldthioglucose weighed 70% more than the controls. CART mRNA abundance in the arcuate nucleus of goldthioglucose-treated mice was decreased by 71% compared to levels in the control mice, but CART expression was unaffected in the dorsolateral hypothalamus. In the second study, male Siberian hamsters were exposed to short days to induce a physiological winter response in which body weight decreases as fat reserves are catabolized, and food intake correspondingly declines. After 8 weeks in short days, body weight had declined by 18% relative to controls maintained in long days in a summer fat state. CART mRNA levels did not differ significantly between the two groups in any hypothalamic areas. In the third study, male Siberian hamsters, either in long days or after 12 weeks exposure to short days to induce weight loss, were subject to a 48-h period of fasting. Although photoperiod per se did not affect CART expression, fasting produced a significant decrease in CART mRNA in the arcuate nucleus of hamsters in both the long- and short-day state. We conclude that CART-producing cells are involved in energy homeostasis: the marked decrease in CART expression in the arcuate nucleus in goldthioglucose-lesioned mice may contribute to the development of obesity, and the decrease following acute dietary restriction in hamsters may reflect a compensatory mechanism to reduce caloric expenditure, but our results do not indicate that CART is involved in long-term seasonal regulation of body weight.

Apoprotein C-III deficiency markedly stimulates triglyceride secretion in vivo: comparison with apoprotein E.

Apoprotein (apo) C-III plays an important role in the development of hypertriglyceridemia by inhibiting triglyceride (TG) removal. However, the effect of apo C-III on TG production remains unclear. We measured TG secretion rate (TGSR) in apo C-III gene-disrupted (apo C-III-null) mice to investigate the influence of this protein on TG turnover. TGSR measured by the Triton WR-1339 method was increased twofold in these mice compared with wild-type (WT) mice. Obesity was induced by the injection of gold-thioglucose (GTG), which made the WT mice hypertriglyceridemic due to a threefold increase of TGSR. However, GTG-induced obesity failed to increase TG in apo C-III-null mice, although TGSR was increased 10-fold, suggesting substantial stimulation of TG removal. Apo E-null mice were severely hypercholesterolemic but were not hypertriglyceridemic, and TGSR was rather decreased. GTG-induced obesity made these mice hypertriglyceridemic because of TG overproduction to an extent similar to that seen in WT mice. These results suggest that apo C-III deficiency potently enhances TG turnover, especially when TG production is stimulated, and that apo E deficiency is not always rate limiting for TG production.

Auranofin or D-penicillamine in the treatment of rheumatoid arthritis.

Ninety patients were entered into a randomized, controlled, double-blind trial lasting 12 months to compare auranofin (6 mg/d), and D-penicillamine (250 mg/d for 4 weeks, 500 mg/d for 4 weeks, then 750 mg/d thereafter) in the treatment of rheumatoid arthritis. Most patients in both groups completed the trial with significant improvement in all quantitative measures of efficacy. Patients treated with D-penicillamine were more likely to have "important improvement" in physician global assessment, swollen joint count, and score and grip strength. The overall frequency of side effects was similar between the two groups; however, more patients were withdrawn for adverse effects from the D-penicillamine group, and proteinuria (greater than or equal to 2+) and thrombocytopenia (less than 100 000 mm3) occurred significantly more frequently with D-penicillamine than auranofin (p = 0.028). These results suggest that in the dosage regimen used, auranofin is safer than D-penicillamine but that D-penicillamine tends to show greater clinical effectiveness in patients with rheumatoid arthritis.

Effects of gold compounds on function of phagocytic cells. Comparative inhibition of activated polymorphonuclear leukocytes and monocytes from rheumatoid arthritis and control subjects.

The effect of sodium aurothiomalate and auranofin on the generation of superoxide anions (O2-) by polymorphonuclear leukocytes (PMNLs) and adherent mononuclear phagocytic cells (AMNCs) has been investigated. Sodium aurothiomalate at final concentrations of 1, 10, and 100 micrograms Au/ml and auranofin ranging from 0.1 to 2.0 micrograms Au/ml were used in the reactions involving all cell types. Results have been compared between cells drawn from normal controls and patients with active rheumatoid disease. The effect of gold compounds on both cell types was assessed following activation by phorbol myristate acetate (1 X 10(-8) M) and N-formyl-methionyl-leucyl-phenylalanine (1 X 10(-4) M) using a cytochrome c reduction method. Sodium aurothiomalate at the maximum concentration modestly inhibited O2- generation by PMNLs but not AMNCs. Auranofin inhibits O2- generation by both cell types. Inhibition of cells from patients with rheumatoid arthritis was greater than that seen with cells from normal controls.

Effect of gold compounds on NADPH oxidase system of human neutrophils.

The inhibitory effects of gold compounds on the NADPH oxidase system of human polymorphonuclear leukocytes (PMNs) has been investigated. Auranofin (0.5-4.0 micrograms Au/ml) suppressed the rate of superoxide anion generation as well as the total yield in cells stimulated with phorbol myristate acetate and f-Met-Leu-Phe. This implies that drug action may be occurring at the level of protein kinase C or steps subsequent to this in the signal transduction sequence. Sodium aurothiomalate (1-100 micrograms Au/ml) lacked such activity. Neither gold compound altered the ability of the granule-rich fraction of PMNs to produce oxy radicals whether this fraction was obtained from drug-treated cells or was treated after its isolation. Therefore, in order for auranofin to exhibit its inhibitory effects on the NADPH oxidase system, an intact cell membrane is necessary.

Effects of the chrysotherapeutic agents auranofin and gold sodium thiomalate on hepatic and renal drug metabolism and heme metabolism.

These studies were designed to investigate the effects of the chrysotherapeutic agents auranofin and myochrysine (GST) on hepatic and renal drug-metabolizing enzymes and heme metabolism. Male Sprague-Dawley rats were either administered a single dose of auranofin (17, 34, or 68 mg/kg, p.o.) or administered daily doses of auranofin (0.2, 0.6, 2, 9, or 40 mg/kg/day, p.o.) or GST (1.2 or 5.8 mg/kg/day, i.p.) for 3 or 14 days. Rats were killed 24 h after the final treatment, and subcellular fractions of liver and kidney were prepared. Cytochrome P-450 (P-450) content and ethoxycoumarin-O-deethylase (ECOD), benzphetamine-N-demethylase (BPND), delta-aminolevulinic acid (ALA) synthetase, and heme oxygenase activities were determined. Twenty-four hours following single doses of auranofin, no effects on hepatic P-450, ECOD, or BPND were observed. Treatment with the positive control compounds, CoCl2 (60 mg/kg) and Co-protophorphyrin IX (33 mg/kg), produced decreases in all three variables at 24 hr. Auranofin, at 2 mg/kg, and GST treatment, at both doses, reduced hepatic P-450 and ECOD activity at 3 days. This effect was reversed with continued treatment for 14 days. BPND activity was unaffected at 3 days but was decreased at 14 days. Heme oxygenase activity was enhanced at 3 days and had returned to control activity at 14 days, while ALA synthetase was unaffected. With the exception of heme oxygenase, which was increased, renal variables were unaltered at 3 days. At 14 days, renal P-450 content was decreased in the high-dose auranofin group, heme oxygenase activity was increased in all groups, and ALA synthetase activity was elevated in high-dose auranofin animals. These data indicate that, at doses twenty times the human dose, auranofin and GST administration produced reversible decreases in hepatic and renal P-450 which may be the result of altered heme metabolism.

Induction of mammalian stress proteins by a triethylphosphine gold compound used in the therapy of rheumatoid arthritis.

In vitro exposure of cultured human, murine and rat cells to pharmacologic concentrations (10(-8) to 10(-6) M) of auranofin, 2,3,4,6,-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S- triethylphosphine gold(I) (Ridaura), a gold containing compound approved for the treatment of rheumatoid arthritis, results in the induction of several stress proteins. The enhanced synthesis of two polypeptides, p32 and p34, was particularly prominent. A similar response was observed in freshly collected human monocytes challenged with auranofin. In addition, oral administration of auranofin to rats induced enhanced synthesis of a 32-kDa protein in peritoneal exudate cells analyzed ex vivo at various times following drug treatment. These data suggest that increased synthesis of p32 and p34 might participate in mediating certain aspects of auranofin pharmacology.

Treatment of chronic discoid lupus erythematosus with an oral gold compound (auranofin).

Twenty-three patients with severe longstanding discoid lupus erythematosus, unresponsive to conventional treatments, were treated with oral gold in a multicentre open study. Nineteen patients showed clinical improvement and in four of these there was complete resolution of lesions. Adverse reactions were generally mild and self limiting.

Effect of auranofin on absorptive processes in the rat small bowel.

Mucosal auranofin (AF) caused a concentration dependent inhibition of fluid, Na+, glycine and galactose transport by everted sacs of rat small intestine (IC50 = 2 X 10(-4) M). Inhibition of nutrient absorption was not due to reduced fluid uptake since a similar reduction in fluid uptake induced by a mucosal osmotic load failed to alter glycine transport. Inhibitory effects of AF were not observed when metabolism was reduced, suggesting that carrier mediated entry processes were unaltered. AF inhibited Na+, K+-ATPase activity in isolated enterocytes (IC50 = 2 X 10(-4) M), without affecting Mg2+-dependent ATPase activity. Our studies suggest that the actions of AF on small intestinal absorption may result from inhibition of the Na+ pump.

Studies on type II collagen induced arthritis in mice.

A consistent and reproducible polyarthritis was induced in mice by immunizing them with type II collagen in Complete Freunds adjuvant (CFA) and Bacillus Calmette-Guerin (BCG) vaccine. Several inbred strains of mice were investigated for the ability to develop collagen induced arthritis (CIA). DBA/1 mice (H-2q) produced the highest incidence and the most severe arthritis of all the strains examined. Viable BCG vaccine was essential for the induction of a reproducible disease in this strain. The effects of some anti-inflammatory and anti-rheumatic compounds were examined on the developing and established lesions of CIA. These effects were determined by assessing the paw inflammation using a subjective scoring system and measuring foot weight. Furthermore, levels of serum amyloid P component (SAP) were also determined. Benoxaprofen, cyclophosphamide, indomethacin and prednisolone inhibited the paw inflammation in the developing disease whilst the anti-rheumatic compounds auranofin and D-penicillamine exacerbate the paw inflammation. Cyclophosphamide and prednisolone inhibited the established lesions but only prednisolone prevented the development of further lesions in the established disease. The SAP levels in the prednisolone treated group were also reduced. Auranofin treatment exacerbated the inflammation of both the established and the developing lesions in the same animal. D-penicillamine was inactive in the established disease.

Enhanced interleukin 1 and depressed interleukin 2 production in juvenile arthritis.

Blood mononuclear cells from a total of 23 children with juvenile arthritis were stimulated in vitro to produce interleukin 1 (IL-1) and interleukin 2 (IL-2) and compared with age matched healthy controls. Peripheral blood monocytes from patients with juvenile arthritis produced a higher amount of IL-1 than controls, whereas peripheral blood lymphocytes from the same patients produced lower amount of IL-2 than controls. These findings could not be explained by concurrent therapy. The increase of IL-1 production was more marked in patients with active disease and therefore may have been secondary to the pathological process. However, the decrease of IL-2 production did not depend on disease activity, thus suggesting an immunoregulatory abnormality.

The effect of auranofin on the colonic transport of Na+ and fluid in the rat.

Auranofin in the mucosal fluid caused a dose-dependent inhibition of fluid and Na+ absorption by everted sacs of rat colon. Serosal auranofin was without effect. (Na+ + K+)ATPase activity of homogenates of mucosal scrapes of rat colon was inhibited by auranofin in a dose-related manner, while Mg2+-ATPase activity was little affected. These actions of the drug on colonic transport mechanisms could contribute to the diarrhoea associated with auranofin therapy.

Metallothionein in cultured human epithelial cells and synovial rheumatoid fibroblasts after in vitro treatment with auranofin.

Radioimmunoassay (RIA) and reversed-phase high-pressure liquid chromatography (HPLC) were used to investigate gold-binding proteins of possible metallothionein (MT) nature occurring upon auranofin exposure of cultured human cells. An epithelial cell line (HE) and two sub-strains were examined. The HEAF sub-strain had been made resistant to 2 mumole auranofin/l culture medium. The resistance was associated with the appearance of gold-binding substances with gel filtration characteristics like MT. The HE100 sub-strain had been made resistant to 100 mumole CdCl2/l and contained high amounts of cytosolic Cd-induced MT. In addition, cultured synovial fibroblasts, derived from normal (SN) and rheumatoid (SRA) synovial tissues, were investigated. Evidence was obtained by RIA that the low molecular weight (mol.wt. 6000-7000) gold-binding proteins occurring in the HEAF cells and SRA cells following auranofin exposure, were of MT nature. The relative amounts of MT in the epithelial cell lines were: HE:HEAF:HE100 = 1:18:100. The relative amounts in the synovial fibroblasts were: SN:SRA:SRA treated with auranofin = 1:3:10. The HPLC methods used were found suitable for isolation of Cd-MT in the HE100 cells, but not for the Au-MT in the HEAF cells. By HPLC, the Cd-MT in the HE100 cells was resolved into 3 MT-1 and 1 MT-2 iso-proteins exhibiting the amino acid composition typical of MT. Judged by HPLC, the MT in these cells constituted 0.4% of the cytosolic proteins.

Effect of auranofin (SK&F 39162) on water and electrolyte flux in canine small bowel: a possible diarrheogenic mechanism.

Auranofin (AF) a new antiarthritic gold compound effective when administered orally, frequently causes diarrhea with abnormal stool electrolyte content. Studies were designed to determine the mechanism of the diarrhea caused by AF. In perfused canine Thiry-Vella loops, AF caused significant elevations in effluent volume, osmolarity, and sodium concentration and a significant decrease in potassium concentration. In mucosal homogenates of rat small bowel, AF inhibited sodium, potassium ATPase in a concentration dependent manner. AF did not alter canine colonic smooth muscle activity in vitro. We suggest that AF induced diarrhea results from interruption of normal water and electrolyte absorption by inhibition of enterocyte sodium, potassium ATPase activity.

Bronchiolitis in a rheumatoid arthritis patient receiving auranofin.

A patient with severe rheumatoid arthritis and sicca symptoms was treated with auranofin. During auranofin therapy, she developed irreversible airways obstruction due to bronchiolitis. Whereas this complication could have been due to her underlying disease, we discuss here the possibility of its being related to the auranofin therapy.