B. D. Williams

Treatment of rheumatoid arthritis with recombinant human interleukin‐1 receptor antagonist

OBJECTIVE To evaluate the efficacy and safety of interleukin-1 receptor antagonist (IL-1Ra) in patients with rheumatoid arthritis (RA). METHODS Patients with active and severe RA (disease duration <8 years) were recruited into a 24-week, double-blind, randomized, placebo-controlled, multicenter study. Doses of nonsteroidal antiinflammatory drugs and/or oral corticosteroids (< or =10 mg prednisolone daily) remained constant throughout the study. Any disease-modifying antirheumatic drugs that were being administered were discontinued at least 6 weeks prior to enrollment. Patients were randomized to 1 of 4 treatment groups: placebo or a single, self-administered subcutaneous injection of IL-1Ra at a daily dose of 30 mg, 75 mg, or 150 mg. RESULTS A total of 472 patients were recruited. At enrollment, the mean age, sex ratio, disease duration, and percentage of patients with rheumatoid factor and erosions were similar in the 4 treatment groups. The clinical parameters of disease activity were similar in each treatment group and were consistent with active and severe RA. At 24 weeks, of the patients who received 150 mg/day IL-1Ra, 43% met the American College of Rheumatology criteria for response (the primary efficacy measure), 44% met the Paulus criteria, and statistically significant improvements were seen in the number of swollen joints, number of tender joints, investigators assessment of disease activity, patients assessment of disease activity, pain score on a visual analog scale, duration of morning stiffness, Health Assessment Questionnaire score, C-reactive protein level, and erythrocyte sedimentation rate. In addition, the rate of radiologic progression in the patients receiving IL-1Ra was significantly less than in the placebo group at 24 weeks, as evidenced by the Larsen score and the erosive joint count. IL-1Ra was well tolerated and no serious adverse events were observed. An injection-site reaction was the most frequently observed adverse event, and this resulted in a 5% rate of withdrawal from the study among those receiving IL-1Ra at 150 mg/day. CONCLUSION This study confirmed both the efficacy and the safety of IL-1Ra in a large cohort of patients with active and severe RA. IL-1Ra is the first biologic agent to demonstrate a beneficial effect on the rate of joint erosion.

Therapeutic efficacy of a novel membrane-targeted complement regulator in antigen-induced arthritis in the rat.

OBJECTIVE Complement system activation is strongly implicated as a factor in the pathogenesis of chronic synovitis in human rheumatoid arthritis. The objective of this study was to explore the therapeutic potential and local retention of a novel membrane-targeting complement regulatory protein, derived from human complement receptor 1, in the experimental setting of rat antigen-induced arthritis. METHODS Sensitized animals were treated at the time of arthritis induction with a single intraarticular (IA) dose of the membrane-targeting regulator APT070, a non-membrane-targeting control regulator (APT898), or vehicle control, and disease was assessed clinically and histologically. In addition, immunocytochemical analysis was performed on sections from normal rat knee joints at various time points after IA injection with APT070. RESULTS Animals treated with APT070 showed a dose-dependent therapeutic effect, with significantly milder clinical and histologic disease compared with both other treatment groups (P < 0.008 at the higher dose) and minimal evidence of erosive disease at study end in the active treatment group. Immunoperoxidase and immunofluorescence studies demonstrated local retention of APT070 on cell surface membranes within the normal joint up to 48 hours after IA injection. CONCLUSION These results show that IA complement inhibition represents an effective therapeutic strategy in experimental arthritis, by demonstrating that the exogenous delivery of a membrane-targeting complement regulator can result in prolonged synovial cell surface binding and significant clinical benefit in vivo. Complement inhibitory strategies of this type should be considered as novel therapies in human inflammatory arthritis.

Soluble complement receptor one (sCR1) inhibits the development and progression of rat collagen-induced arthritis

We set out to determine whether inhibition of complement using sCR1 could influence the development and progression of collagen arthritis in the Lewis rat. Collagen arthritis was successfully established in the Lewis rat, using a novel immunization schedule. In separate experiments, cobra venom factor (CVF) and sCR1 were used to achieve systemic complement inhibition. Their respective effects on disease onset and on the progression of established disease compared with saline‐treated control animals was explored. Arthritis was assessed by measurement of clinical score, paw diameter and paw volume. Complement inhibition using either CVF or sCR1, prior to the onset of clinical signs of inflammation, delayed the development of disease. CVF was ineffective in the treatment of established disease, whereas sCR1 delayed the progression of disease in affected joints and prevented the recruitment of further joints while the animals were complement‐depleted. In the control saline‐treated groups the disease continued to progress relentlessly. We conclude that complement activation is important in the initiation and maintenance of inflammation in collagen arthritis. The potent disease‐modulating effect of sCR1 provides persuasive evidence that specific complement inhibiting agents may be an effective approach to the treatment of inflammatory joint diseases

The effect of free and liposome-encapsulated clodronate on the hepatic mononuclear phagocyte system in the rat

Clodronate, encapsulated within small unilamellar vesicles (SUVc) will deplete hepatic macrophages after intravenous injection. Functional studies, using probes to evaluate hepatic Fc and C3b uptake, showed a close correlation between the inhibition of receptor‐mediated uptake and the depletion of hepatic macrophages. Twenty milligrams of clodronate encapsulated within SUVc produced · 90% inhibition of uptake and clearance of Fc‐ and C3b‐coated erythrocytes and a comparable reduction of hepatic macrophage numbers. Inhibition of macrophage receptor‐mediated uptake of these erythroctyes was closely related to the reduction in macrophage numbers. Repopulation of macrophages within the liver took place over 2 weeks. At 1 week after depletion, although repopulation was taking place, receptor‐mediated function remained suppressed. In a preliminary experiment, treatment of rats with adjuvant arthritis with 20 mg clodronate encapsulated in SUV suppressed the inflammation and reversed the course of the disease, while treatment with 20 mg free clodronate in saline or 20 mg clodronate in multilamellar vesicles (MLVc) did not.

The suppression of rat collagen-induced arthritis and inhibition of macrophage derived mediator release by liposomal methotrexate formulations

Abstract.Objective and Design: This study was designed to determine whether liposomes are suitable vehicles for the delivery of methotrexate (MTX-γ-DMPE) for arthritis therapy.¶Material or Subjects: Liposomal formulations containing either egg lecithin (EPC), cholesterol (CHOL) and phosphatidic acid (PA) (MTX-EPC) or distearoylphosphatidylcholine (DSPC), CHOL and distearoylphosphatidylethanolamine conjugated to polyethyleneglycol (PEG) (MTX-PEG) were employed. Rat peritoneal macrophages (rPMφ) were used to test the mechanism of action of these liposomes in vitro, whilst, the rat collagen-induced arthritis (CIA) model was used to evaluate the in vivo efficacy of MTX-EPC and MTX-PEG.¶Treatment: In vitro, rPMφ were incubated with liposomal MTX concentrations ranging from 0 to 15 μg/well. In vivo, rats were given 4 daily intravenous injections of liposomal MTX (2.5 mg/Kg).¶Methods: IL-1β and prostaglandin-E2 (PGE2) release from rPMφ were quantified by immunoradiometric assay. Arthritis progression, in vivo, was measured by serial clinical score and hind paw diameter measurements.¶Results: MTX-EPC and MTX-PEG respectively (15 μg of MTX and 0.15 mg of lipid) were powerful inhibitors of both IL-1β (77 ± 2.3%; 79 ± 4.0%) and PGE2 (75.5 ± 4.9%; 68.5 ± 2.3%) release (mean ± SEM % inhibition) from lipopolysaccaride stimulated rPMφ. In vivo, only MTX-EPC exerted an anti-inflammatory effect, clinical score (p < 0.001) and paw diameter (p < 0.001) measurements being significantly lower than in control rats, after 2 days treatment.¶Conclusions: MTX-EPC and MTX-PEG are potent inhibitors of pro-inflammatory mediators in vitro, but liposomes with long circulation times do not appear to have therapeutic potential for treating arthritis in vivo.

Effect of liposome surface charge on the stability of technetium (99mTc) radiolabelled liposomes

Using liposomes radiolabelled by the 99mTechnetium-stannous chloride technique we have investigated the effect of surface charge on the stability of the isotope in vitro and in vivo. Dialysis of 99mTc-labelled positive, negative and neutral liposomes, which had been incubated in either saline or normal rat serum showed no significant loss of the isotope from the liposome surface with only 2 per cent of the isotope dialysed. A comparison of gel chromatography with dialysis confirmed that most of the isotope remained attached to the liposome surface, but it did reveal greater loss of the isotope, between 15 and 23 per cent. The liposome clearance rates obtained from 125I-egg phosphatidylcholine (EPC) and 99mTc dual-labelled positive or neutral liposomes were significantly different. The 99mTc marker was cleared five times faster from the positive liposomes and twice as fast from the neutral liposomes as the 125I-EPC integral membrane marker. The 99mTc attached to liposomes with a negative surface charge was stable in vivo and had the same clearance rate from the circulation as the 125I-EPC marker. These results indicate that the commonly used in vitro techniques for assessing liposome radiolabel stability are unsuitable for predicting the stability of the 99mTc in vivo.

Prostaglandin and tumor necrosis factor secretion by peritoneal macrophages isolated from normal and arthritic rats treated with liposomal methotrexate

The effect of a novel liposomal preparation containing a phospholipid conjugate of methotrexate (MTX-LIPO) upon macrophage mediator release was investigated in normal and arthritic rats ex vivo. Peritoneal macrophages isolated from MTX-LIPO-treated arthritic rats and stimulated with lipopolysaccharide produced significantly less tumor necrosis factor (TNF) and prostaglandin (PGE2) than did macrophages isolated from saline-treated controls. In the same experimental system, free methotrexate only inhibited prostaglandin release, but it was more potent than MTX-LIPO in this respect. Additional studies are presently underway to investigate the effect of MTX-LIPO and MTX treatment upon the lipopolysaccharide-induced rise in plasma levels of various proinflammatory mediators in vivo. Haematopoietic toxicity was demonstrated in blood isolated from rats treated with free MTX, and this was as characterized by a significant reduction in reticulocyte count compared with MTX-LIPO and saline-treated rats.

Differential effects of methotrexate and liposomally conjugated methotrexate in rat adjuvant-induced arthritis

In this study we evaluated the comparative efficacy of free and liposomally conjugated methotrexate on both disease indtiction and suppression of acute inflammation in rat adjuvant‐induced arthritis. Rats were given either empty liposomes (E‐LIPO), free methotrexate (MTX) or the liposomally conjugated methotrexate (MTX‐LIPO) at a dose of 100/μg/day for 7 consecutive days by the intravenous route. When MTX treatment was initiated on the day of arthritis induction the drug suppressed bul did not abolish the development of joint inflammation. Free MTX had no significant anti‐inliammatory effect upon an established arthritis when dosing was commenced on day 11 post‐adjuvant induction. Conversely, MTX‐LIPO did not affect the progression of the arthritis when dosing was started on day 0, but exerted a significant anti‐inflammatory effect on an established arthritis. MTX‐LIPO treatment was significantly less haematotoxic than free MTX.

High performance liquid chromatographic analysis of liposome stability

Two techniques have been studied for their suitability for the analysis of the stability of liposomes: (1) High Performance Gel Permeation Liquid Chromatography (HPGPLC), a TSK G5000PW Ultrogel column; (2) Gel Permeation Chromatography (GPC), a Sepharose 4B column. The stability of dual radio-labelled, cholesterol-poor and cholesterol-rich, negatively charged liposomes in vitro (in saline and in serum), and in vivo, have been investigated using these two techniques. The HPGPLC TSK G5000PW column proved to be the superior technique for the analysis of liposome stability with the advantages of rapid run times, increased sample recovery, and smaller sample volumes were required. The results obtained confirm that inclusion of a high ratio of cholesterol into the liposome structure prevents phospholipid loss when exposed to serum, and that the cholesterol-poor liposome structure is dramatically altered under the same conditions. In conclusion, the TSK G5000PW column is ideal for monitoring movement of phospholipid between liposomes and serum proteins and for detecting changes in liposome size.

Interleukin‐1β (IL‐1β) inhibition: a possible mechanism for the anti‐inflammatory potency of liposomally conjugated methotrexate formulations in arthritis

Liposomes with conventional and long‐circulation times were employed as carriers for the methotrexate derivative MTX‐γ‐DMPE (MTX‐EPC and MTX‐PEG respectively), their mechanism of action was investigated in vitro and in vivo and their therapeutic efficacy assessed using the rat collagen‐induced arthritis (CIA) model. At non‐toxic dose, both MTX‐EPC and MTX‐PEG inhibited the lipopolysaccharide (LPS) induced release of IL‐1β from activated rat peritoneal macrophages (rPMΦ) in a dose and time dependent manner. Free methotrexate (MTX) was not active in this respect. After a single intravenous injection (i.v.), and at equivalent doses, both free MTX (500 μg) and MTX‐EPC inhibited the LPS induced rise in plasma IL‐1β levels observed in MTX‐PEG and saline treated rats. When used to treat established CIA, MTX‐EPC resulted in significantly lower clinical score (CS) (1.0±0.42 (P<0.001)) and hind paw diameter (HPD) (6.5±0.34 mm (P<0.001)) measurements than controls (3.0±0.26; 7.33±0.41 mm), after only two i.v. doses, and remained significantly lower for the entire experimental period. By day 24 both CS (2±0.61 (P<0.001)) and HPD (6.97±0.25 mm (P<0.002)) measurements had also become significantly lower in MTX‐PEG treated rats than in saline treated controls (3.62±0.17, 7.92±0.38 mm) and remained lower until day 30. Joint inflammation in MTX treated rats was completely ameliorated by day 20 but the health and well being of the animals was compromised and the experiment terminated at this time‐point. Our results clearly demonstrate that both MTX‐EPC and MTX‐PEG liposomes have potential for development into therapeutic modalities for the treatment of inflammatory joint disease in man.