Application of biomarkers in the development of drugs intended for the treatment of osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is a chronic and slowly progressive disease for which biomarkers may be able to provide a more rapid indication of therapeutic responses to therapy than is currently available; this could accelerate and facilitate OA drug discovery and development programs. The goal of this document is to provide a summary and guide to the application of in vitro (biochemical and other soluble) biomarkers in the development of drugs for OA and to outline and stimulate a research agenda that will further this goal. METHODS: The Biomarkers Working Group representing experts in the field of OA biomarker research from both academia and industry developed this consensus document between 2007 and 2009 at the behest of the Osteoarthritis Research Society International Federal Drug Administration initiative (OARSI FDA initiative). RESULTS: This document summarizes definitions and classification systems for biomarkers, the current outcome measures used in OA clinical trials, applications and potential utility of biomarkers for development of OA therapeutics, the current state of qualification of OA-related biomarkers, pathways for biomarker qualification, critical needs to advance the use of biomarkers for drug development, recommendations regarding practices and clinical trials, and a research agenda to advance the science of OA-related biomarkers. CONCLUSIONS: Although many OA-related biomarkers are currently available they exist in various states of qualification and validation. The biomarkers that are likely to have the earliest beneficial impact on clinical trials fall into two general categories, those that will allow targeting of subjects most likely to either respond and/or progress (prognostic value) within a reasonable and manageable time frame for a clinical study (for instance within 1-2 years for an OA trial), and those that provide early feedback for preclinical decision-making and for trial organizers that a drug is having the desired biochemical effect. As in vitro biomarkers are increasingly investigated in the context of specific drug treatments, advances in the field can be expected that will lead to rapid expansion of the list of available biomarkers with increasing understanding of the molecular processes that they represent.

A walking model to assess the onset of analgesia in osteoarthritis knee pain.

OBJECTIVE: To assess a walking model utilizing a set of standardized treadmill walks to measure acute analgesic response in osteoarthritis (OA) of the knee. DESIGN: Randomized, double-blind, placebo-controlled, multiple dose, three-period crossover study. Patients > or =45 years of age (N=22) with symptomatic knee OA were randomized to naproxen 500 mg bid, tramadol/acetaminophen 37.5 mg/325 mg in forced titration, or placebo in each of three periods. Patients performed multiple 20-minute treadmill walks on Day 1 and Day 3 at a consistent self-selected pace predetermined at screening. Pain intensity (PI) during the walks was assessed on an 11-point numerical rating scale at 0, 3, 6, 9, 12, 15, 18, and 20 min. The primary endpoint was the time-weighted average (TWA) change from baseline PI on Day 3 for the two self-paced walks for the active treatments vs placebo. Time to moderate pain (TTMP) was a key secondary endpoint. RESULTS: Compared with placebo, the TWA change from baseline PI on Day 3 was significantly better with tramadol/acetaminophen (P=0.043) but not with naproxen (P=0.089). TWA change from baseline on Day 1 was also significantly better with both tramadol/acetaminophen (P=0.001) and naproxen (P=0.048) compared with placebo. TTMP was significantly better for tramadol/acetaminophen and naproxen than placebo (P<0.001 to P=0.015) for walks on Day 1 after a single dose and on Day 3. CONCLUSIONS: This novel OA pain model was able to discriminate both tramadol/acetaminophen and naproxen from placebo after single and multiple doses. ClinicalTrials.gov identifier: NCT00772967.

OARSI recommendations for the management of hip and knee osteoarthritis: part III: Changes in evidence following systematic cumulative update of research published through January 2009.

OBJECTIVE: To update evidence for available therapies in the treatment of hip and knee osteoarthritis (OA) and to examine whether research evidence has changed from 31 January 2006 to 31 January 2009. METHODS: A systematic literature search was undertaken using MEDLINE, EMBASE, CINAHL, AMED, Science Citation Index and the Cochrane Library. The quality of studies was assessed. Effect sizes (ESs) and numbers needed to treat were calculated for efficacy. Relative risks, hazard ratios (HRs) or odds ratios were estimated for side effects. Publication bias and heterogeneity were examined. Sensitivity analysis was undertaken to compare the evidence pooled in different years and different qualities. Cumulative meta-analysis was used to examine the stability of evidence. RESULTS: Sixty-four systematic reviews, 266 randomised controlled trials (RCTs) and 21 new economic evaluations (EEs) were published between 2006 and 2009. Of 51 treatment modalities, new data on efficacy have been published for more than half (26/39, 67%) of those for which research evidence was available in 2006. Among non-pharmacological therapies, ES for pain relief was unchanged for self-management, education, exercise and acupuncture. However, with new evidence the ES for pain relief for weight reduction reached statistical significance, increasing from 0.13 [95% confidence interval (CI) -0.12, 0.36] in 2006 to 0.20 (95% CI 0.00, 0.39) in 2009. By contrast, the ES for electromagnetic therapy which was large in 2006 (ES=0.77, 95% CI 0.36, 1.17) was no longer significant (ES=0.16, 95% CI -0.08, 0.39). Among pharmacological therapies, the cumulative evidence for the benefits and harms of oral and topical non-steroidal anti-inflammatory drugs, diacerhein and intra-articular (IA) corticosteroid was not greatly changed. The ES for pain relief with acetaminophen diminished numerically, but not significantly, from 0.21 (0.02, 0.41) to 0.14 (0.05, 0.22) and was no longer significant when analysis was restricted to high quality trials (ES=0.10, 95% CI -0.0, 0.23). New evidence for increased risks of hospitalisation due to perforation, peptic ulceration and bleeding with acetaminophen >3g/day have been published (HR=1.20, 95% CI 1.03, 1.40). ES for pain relief from IA hyaluronic acid, glucosamine sulphate, chondroitin sulphate and avocado soybean unsponifiables also diminished and there was greater heterogeneity of outcomes and more evidence of publication bias. Among surgical treatments further negative RCTs of lavage/debridement were published and the pooled results demonstrated that benefits from this modality of therapy were no greater than those obtained from placebo. CONCLUSION: Publication of a large amount of new research evidence has resulted in changes in the calculated risk-benefit ratio for some treatments for OA. Regular updating of research evidence can help to guide best clinical practice.

Absence of association of asporin polymorphisms and osteoarthritis susceptibility in US Caucasians.

OBJECTIVE: An association between osteoarthritis (OA) and functional polymorphisms in the aspartic acid (d) repeat of the asporin (ASPN) gene was reported in Japanese and Han Chinese populations. The aim of this study was to assess the association of variants in the ASPN gene with the presence of radiographic hand and/or knee OA in a US Caucasian population. METHODS: Ten single nucleotide polymorphisms (SNPs) within the ASPN gene were genotyped in 775 affected siblings with radiographically confirmed hand and/or knee OA, and the allelic, genotypic and haplotypic association results were examined. RESULTS: One variant (SNP RS7033979) showed nominal evidence of association with both hand OA (P=0.042) and knee OA (P=0.032). Four additional SNPs showed nominal evidence of association with knee OA only. These associations were only observed with genotypic tests; the corresponding allelic and haplotype tests did not corroborate the single-point association results. CONCLUSION: These data suggest that polymorphisms within ASPN are not a major influence in susceptibility to hand or knee OA in US Caucasians.

Adoptive transfer of suppression of arthritis in the mouse model of collagen-induced arthritis. Evidence for a type II collagen-specific suppressor T cell.

This study details the suppressive mechanism involved in the antigen-specific suppression of collagen-induced arthritis. Intravenous injection of 500 micrograms of soluble native type II collagen 3 d before immunization with native type II collagen emulsified in complete Freund's adjuvant resulted in animals with decreased in vitro cellular and humoral immune response to native and denatured type II collagen compared with control groups. Control groups were composed of animals preinoculated with saline and type I collagen and established the antigen-specific nature of the observed suppression. Mice with reduced immune responses to type II collagen also were observed to portray little or no erythema and edema associated with collagen-induced arthritis. Adoptive transfer experiments established the requirement of T cells for the suppression of collagen-induced arthritis. Analysis of the phenotype of responding splenic cells in chronic immunotherapeutically suppressed mice in vitro revealed that responding cells were Ly1-2+ (suppressor/cytotoxic) T cells. On the other hand, the cellular phenotype of T cells responding to type II collagen in nonsuppressed collagen-induced arthritic mice was Ly1+2- (helper/inducer T cells). The data indicate that type II collagen-specific T cells are generated on intravenous inoculation of soluble native type II collagen. These cells are observed in type II collagen-immune animals, which are nonarthritic and portray reduced humoral and in vitro cellular immune response to type II collagen. This study suggests that specific suppression of immune responses to type II collagen by T-suppressor cells can be immunotherapeutic in certain forms of arthritis.

Cartilage expression of a type II collagen mutation in an inherited form of osteoarthritis associated with a mild chondrodysplasia.

In a family who expressed severe dominantly inherited osteoarthritis, the underlying mutation was traced by genomic sequencing to a single base change which predicts an amino acid substitution of cysteine for arginine at residue 519 of the triple-helical domain of the type II collagen molecule (Ala-Kokko, L., C. T. Baldwin, R. W. Moskowitz, and D. J. Prockop. 1990. Proc. Natl. Acad. Sci. USA. 87:6565-6568). In the present study we examined whether this predicted protein phenotype was evident in articular cartilage obtained from an affected family member who underwent hip surgery. The cartilage collagen was solubilized by CNBr digestion. Cysteine residues were labeled by reduction and alkylation with 14C-iodoacetate. Collagen CNBr-peptides were fractionated by ion exchange and reverse phase column chromatography. One peptide from the alpha 1(II) chain, alpha 1(II) CB8, was found to be radiolabeled. Tryptic peptides were prepared from it and identified by microsequence analysis. The results show that approximately one-quarter of the alpha 1(II) chains present in the polymeric extracellular collagen of the patient's cartilage contained the Arg519-to-Cys substitution. The protein exhibited other abnormal properties including disulfide-bonded alpha 1(II)-dimers and signs of posttranslational overmodification. The premature cartilage failure and osteoarthritis are presumably a result of the abnormal type II collagen being expressed in the cartilage matrix.

Prostaglandin biosynthesis by lapine articular chondrocytes in culture.

Secondary monolayer and spinner cultures of rabbit articular chondrocytes released into the culture medium prostaglandins the synthesis of which was inhibited by sodium meclofenamate. The prostaglandins measured by radioimmunoassay were, in order of decreasing abundance, prostaglandin E2, 6-oxo-prostaglandin F1 alpha (the stable metabolite of prostacyclin) and prostaglandin F2 alpha. Several lines of evidence indicated that chondrocytes synthesize little if any thromboxane B2 (the stable metabolite of thromboxane A2). The presence of prostaglandins was confirmed by radiometric thin-layer chromatography of extracts of culture media incubated with [3H]arachidonic acid-labeled cells. In monolayer culture, chondrocytes synthesized immunoreactive prostaglandins in serum-free as well as serum-containing medium. Monolayer chondrocytes produced higher levels of prostaglandin E2 relative to 6-oxo-prostaglandin F1 alpha than did spinner cells, but the latter synthesized more total prostaglandins. The identity of endogenous prostaglandins as well as those synthesized in short-term culture by rabbit cartilage slices was compared to those produced by chondrocytes in long-term culture. Chondrocytes synthesized all of the prostaglandins found in articular cartilage. Minimal quantities of thromboxane B2 were detected in cartilage. A higher percentage of 6-oxo-prostaglandin F1 alpha relative to other prostaglandins was found in cartilage than in either monolayer or spinner chondrocyte cultures. These results demonstrate that articular chondrocytes synthesize prostaglandins and prostacyclin. These prostaglandins may exert significant physiological effects on cartilage, since exogenous prostaglandins depress chondrocyte sulfated-proteoglycan synthesis and may even promote proteoglycan degradation.

Correlation of the biosynthesis of prostaglandin and cyclic AMP in monolayer cultures of rabbit articular chondrocytes.

We have utilized ionophores to test whether stimulation of chondrocyte prostaglandin biosynthesis is accompanied by an increase in cyclic nucleotide levels in these cells. Radioimmunoassay of prostaglandin E2, 6-oxo-prostaglandin F1 alpha (the stable metabolite of prostaglandin I2) and prostaglandin F2 alpha showed that synthesis of each was stimulated by the divalent-cation ionophore, A23187 after short-term incubation (1-7 min) in serum-free medium. No stimulation of thromboxane B2 was detected. Two monovalent ionophores, lasalocid and monensin failed to stimulate prostaglandin biosynthesis after short-term incubation. Ionophore A23187-stimulated prostaglandin biosynthesis was variably and partially inhibited by sodium meclofenamate, indomethacin and aspirin, but not by sodium salicylate. Ionophore A23187-stimulated prostaglandin biosynthesis was accompanied by a 7.5-fold increase in cyclic AMP levels after 15 min. Sodium meclofenamate, indomethacin and aspirin which inhibited prostaglandin E2 biosynthesis also reduced cyclic AMP levels. Exogenous prostaglandin E2 (1 microgram/ml) stimulated cyclic AMP biosynthesis, which was not inhibited by aspirin. These results indicated that prostaglandins can be considered as one of the local effectors controlling cyclic AMP production in articular cartilage.

Arachidonic acid metabolism by rabbit synovial cells in culture. Studies of non-cyclooxygenase pathways.

We have investigated arachidonic acid (20:4) metabolism by rabbit synovial cells in culture. The lipoxygenase products 5-HETE, 12-HETE and 15-HETE were not detected, despite the presence of a cyclooxygenase inhibitor sodium meclofenamate (20 microM), nor after incubation with ionophore A23187 (1 microM), 20:4 (10 microM), prostaglandin E2, (1 microM), N-formylmethionylleucylphenylalanine (0.01 microM), or murine spleen cell-conditioned medium. [3H]20:4 (10 microM) was incorporated into phospholipids, triacylglycerols and diacylglycerols. A majority of the 3H content of phosphatidylinositol/phosphatidylserine and of diacylglycerols was already present at 1 min, in contrast to the slower accumulation of 3H in triacylglycerols, phosphatidylcholine and phosphatidylethanolamine. The diacylglycerol fraction contained sn-glycerol-1-acyl-2-20:4. These observations are consistent with phospholipase C activity in synovial cells under those culture conditions. The products generated by these enzymes may play important roles in the physiological processes of synovium.

Neutral proteinases from articular chondrocytes in culture. 2. Metal-dependent latent neutral proteoglycanase, and inhibitory activity.

Monolayer and spinner cultured rabbit articular chondrocytes released into the medium latent metal-dependent enzyme with activity against bovine proteoglycan. Pretreatment of medium with p-aminophenylmercuric acetate or trypsin followed by soybean trypsin inhibitor significantly increased enzyme activity. The monolayer-cultured chondrocytes released more of this activity than spinner cultures. The neutral proteoglycanase activity increased with medium concentration and incubation time. Like the human cartilage proteoglycanase, its pH optimum on proteoglycan subunit was 7.25. Gel filtration on BioGel P-30 indicated that the proteoglycanase occurred in two molecular weight forms: 20 000--30 000 and 13 000. The latent enzyme was about 30 000--40 000. The metal-chelators, o-phenanthroline (5 mM) and EDTA (10 mM) inhibited the activated proteoglycanase almost completely, but trypsin and chymotrypsin inhibitors had little effect. The cultured chondrocytes also released into the media a heat-labile inhibitor against the proteoglycanase. The inhibitory activity was present in the nonactivated media and eluted on Sephadex G-100 chiefly at a position corresponding to molecular weights of 10 000--13 000.

Neutral proteinases from articular chondrocytes in culture. I. A latent collagenase that degrades human cartilage type II collagen.

Culture media collected from secondary monolayer and spinner cultures of rabbit articular chondrocytes showed evidence of collagenolytic activity by the following criteria: (1) Amicon PM-10 concentrates of culture medium released [14C] glycine from reconstituted rabbit skin collagen fibrils at 37 degrees C; (2) medium concentrated by lyophilization decreased the relative viscosity of human cartilage collagen in solution. The loss in viscosity was partially inhibited if medium was preincubated with o-phenanthroline, and (3) degradation of human cartilage collagen after 60 h incubation at 24 degrees C was characterized primarily by the appearance of 75 000 dalton (TCA) and 25 000 dalton ((TCB) products. The majority of the collagenase (EC 3.4.24.3) from cultured chondrocytes was secreted in latent form, since preincubation with either trypsin or p-aminophenylmercuric acetate significantly increased activity against human cartilage collagen. Chondrocyte collagenase may be important in mediating the normal slow turnover of cartilage collagen and may be particularly active in collagen destruction associated with early stages of synovial joint arthritides, before attack by non-cartilage cells or extra-articular soft tissues.

'Gelatinase-like' activity from articular chondrocytes in monolayer culture.

In addition to releasing collagenase and proteoglycanase activity, rabbit articular chondrocytes in monolayer culture released into the culture medium, latent, neutral enzyme activity which when activated by p-aminophenylmercuric acetate degraded fluorescein-labeled polymeric rat tail tendon Type I collagen and the tropocollagen TCA and TCB fragments of human Type II collagen into smaller peptides at 37 degrees C. Enzyme activity was abolished if p-aminophenylmercuric acetate-activated culture medium was preincubated with 1.10-phenanthroline, a metal chelator. Thus, articular chondrocytes in monolayer culture are capable of producing neutral proteinases which acting together can result in complete degradation of tendon and cartilage collagen to small peptides.

Spontaneous termination of prolonged ventricular fibrillation after acute myocardial infarction.

We describe a case of ventricular fibrillation occurring during acute myocardial infarction converting to sinus rhythm without specific additional drug therapy or direct current cardioversion. Ventricular fibrillation occurred during therapy with a temporary transvenous bipolar pacemaker and prophylactic intravenous lidocaine. Reversion of ventricular fibrillation without direct current cardioversion is unusual. Direct current shock remains the first form of treatment for witnessed ventricular fibrillation.

Effect of 13-cis-retinoic acid on the hamster flank organ.

Administration of 13-cis-retinoic acid subcutaneously to mature male hamsters produced a marked decrease in the size of the sebaceous glands of the flank organ, without diminution of other hormonally dependent structures of the flank organs. Subcutaneous administration of 13-cis-retinoic acid to female hamsters treated simultaneously with injections of testosterone enanthate prevented the androgen-induced growth of the flank organ sebaceous glands but did not prevent the growth of other hormonally dependent structures such as the dermal pigment cells and large pigmented hair follicles. The sebaceous gland progressively decreased during 3 weeks of treatment and the effect persisted at least 3 weeks after cessation of treatment but was completely reversed by 6 mos after treatment. In vitro studies of testosterone metabolism by hamster flank organ indicated the lack of inhibition of 5 alpha-reduction by 13-cis-retinoic acid. It seems likely that systemically administered 13-cis-retinoic acid, unlike antiandrogens, exerts a specific extrahormonal effect on the sebaceous glands of the hamster flank organ without affecting other androgen dependent cells.

Hemodynamic effects of oxdralazine and hydralazine in hypertension.

Oxdralazine, a pyridazine shown to induce prolonged arteriolar dilation in animals, was given orally in doses of 15 to 30 mg to 7 subjects with hypertension. Arterial pressure fell in 2 hr (average mean of 16 mm Hg), peaking in 3 to 4 hr, and was sustained for more than 6 hr. Cardiac output and heart rate rose in 2 hr (2.1 l/min and 17 bpm) and were elevated at 6 hr (3.1 l/min and 22 bpm). Pulmonary arterial pressure and pulmonary arteriolar resistance did not change. In 6 subjects receiving oxdralazine with hydralazine 50 to 75 mg at 1-wk intervals, hydralazine induced earlier, less sustained decreases in arterial pressure and systemic vascular resistance and less of a rise in heart rate than oxdralazine alone. Circulating norepinephrine levels (radioenzymatic method) 3 hr after oxdralazine rose from a mean of 159 to 294 pg/ml, a greater (p less than 0.05) effect than after hydralazine. At the doses tested, oxdralazine is a potent systemic arteriolar dilator with longer-sustained action and more prominent reflex sympathetic stimulation than hydralazine.

Sustained-release indomethacin in the comprehensive management of osteoarthritis.

Osteoarthritis, although primarily a degenerative joint disease, may be associated with significant secondary inflammation. Hydrolytic enzymes result in primary cartilage degradation; secondary inflammation occurs in response to degenerative cartilage breakdown products and crystal deposition. Humoral and cell-mediated immune responses have been described. Analgesic and anti-inflammatory agents play a major role in symptomatic relief. Anti-inflammatory activity of the nonsteroidal anti-inflammatory drugs has been ascribed to prostaglandin synthesis inhibition; recent studies suggest additional effects based on inhibition of neutrophil aggregation, superoxide radical generation, and lysosomal enzyme release. Indomethacin, the first of the newer nonsteroidal anti-inflammatory drugs, has a long history of use and patient acceptance. Sustained-release indomethacin (Indocin SR), a 75 mg formulation equivalent to three consecutive doses of conventional indomethacin, adds ease of administration and potential for improved compliance. Nonsteroidal anti-inflammatory drugs, used in conjunction with other therapeutic approaches, provide the opportunity for response that can be gratifying for both patients and physicians.

Primary osteoarthritis: epidemiology, clinical aspects, and general management.

The etiology of osteoarthritis, the most common articular disorder, is still unknown. Epidemiologic studies demonstrate a relationship to aging and to certain occupations; relationships to obesity as an etiologic factor remain controversial. An inverse relationship of osteoarthritis and bony density has been observed. Medical management is primarily symptomatic; surgery, particularly of the hip or knee, restores function and relieves pain at late stages of the disease. Investigational trials of specific therapeutic agents suggest possible forthcoming interventions to prevent, retard, or reverse the disease process.

Steroid myopathy in connective tissue disease.

In eight women with polymyositis (three patients), systemic lupus erythematosus (SLE) (three patients), rheumatoid arthritis (one patient) and shoulder-hand syndrome (one patient), weakness developed during high dose prednisone therapy. These women were studied using serial functional and manual muscle tests, determination of serum glutamic oxaloacetic transminase (SGOT), creatine phosphokinase (CPK) and serum aldolase levels, and urinary excretion of creatine. Insidious onset of weakness was characteristic. Myalgias were seen in five patients and unusual sudden weakness in two. Weakness was always most severe in the pelvic girdle muscles; there was a lesser involvement of shoulder girdle and distal muscles. Serum muscle enzyme levels were normal in all cases, but urinary creatine excretion was invariably increased and proved to be the most sensitive laboratory indicator for clinical diagnosis and for monitoring patient improvement. Serial urinary creatine excretion and serum enzyme studies were of value in differenting steroid myopathy from a flare of myositis in patients with connective tissue disease. Diagnosis and effective management were achieved by the use of readily available laboratory and clinical procedures without resorting to muscle biopsy.

Arthritis of hemochromatosis. Clinical spectrum, relation to histocompatibility antigens, and effectiveness of early phlebotomy.

Five patients who presented with arthritis as the sole manifestation of hereditary hemochromatosis and 51 family members were studied. Studies included clinical evaluation for the presence of arthritis and hemochromatosis, roentgenography of hands, knees, and pelvis, serum iron and serum ferritin measurements, complete HLA typing for 50 of the A and B loci, and, when indicated, liver biopsy. Arthritis occurred in 45 percent of persons with hemochromatosis. Although typical involvement of second and third metacarpophalangeal joints was observed in all five patients and some family members, two with typical arthritis did not have characteristic radiographic changes, two had constitutional symptoms without arthropathy, and one had unilateral hand changes. A specific HLA haplotype (A2/B17 in Family 1 and A29/B15 in Family 2) correlated with hereditary hemochromatosis but not with the arthropathy. Phlebotomy alleviated the early constitutional symptoms but did not help advanced arthritis. Anti-inflammatory drugs, intraarticular injections of glucocorticoids, and resection osteotomies of metacarpal heads were other treatment modalities.