Low levels of type II collagen formation (PRO-C2) are associated with response to sprifermin: a pre-defined, exploratory biomarker analysis from the FORWARD study.

OBJECTIVE: Osteoarthritis (OA) is characterized by the gradual loss of cartilage. Sprifermin, a recombinant FGF18, is being developed as a cartilage anabolic drug. PRO-C2 is a serum marker of type II collagen formation and low levels have been shown to be prognostic of radiographic progression. The aim of the study was to investigate whether the patient groups with either high or low PRO-C2 levels responded differently to sprifermin. DESIGN: PRO-C2 was measured in synovial fluid (SF) (n = 59) and serum samples (n = 225) from participants of the FORWARD study, a 2-year phase IIb clinical trial testing the efficacy of intra-articular (IA) sprifermin over placebo. The difference between sprifermin and placebo in respect to in change cartilage thickness (measured by quantitative (q) MRI) was analyzed in groups with either high or low (3rd vs 1st-2nd tertiles) baseline serum PRO-C2 levels. RESULTS: SF levels of PRO-C2 increased over time in response to sprifermin, but not to placebo. In the placebo arm, significantly (p = 0.005) more cartilage was lost in the low vs high PRO-C2 group over the 2-year period. The contrast between sprifermin and placebo was significant (p < 0.001), ranging from 0.104 mm at week 26 to 0.229 mm at week 104 in the low PRO-C2 group. This result was not significant in the high PRO-C2 group ranging from -0.034 to 0.142. CONCLUSIONS: Patients with low serum PRO-C2 levels lost more cartilage thickness over time and grew more cartilage in response to sprifermin vs a placebo when compared to patients with high PRO-C2 levels.

Automated MRI assessment confirms cartilage thickness modification in patients with knee osteoarthritis: post-hoc analysis from a phase II sprifermin study.

BACKGROUND: Sprifermin is under investigation as a potential disease-modifying osteoarthritis drug. Previously, 2-year results from the FORWARD study showed significant dose-dependent modification of cartilage thickness in the total femorotibial joint (TFTJ), medial and lateral femorotibial compartments (MFTC, LFTC), and central medial and lateral TFTJ subregions, by quantitative magnetic resonance imaging (qMRI) using manual segmentation. OBJECTIVE: To determine whether qMRI findings from FORWARD could be reproduced by an independent method of automated segmentation using an identical dataset and similar anatomical regions in a post-hoc analysis. METHOD: Cartilage thickness was assessed at baseline and 6, 12, 18 and 24 months, using automated cartilage segmentation with active appearance models, a supervised machine learning method. Images were blinded for treatment and timepoint. Treatment effect was assessed by observed and adjusted changes using a linear mixed model for repeated measures. RESULTS: Based on automated segmentation, statistically significant, dose-dependent structural modification of cartilage thickness was observed over 2 years with sprifermin vs placebo for TFTJ (overall treatment effect and dose response, both P < 0.001), MFTC (P = 0.004 and P = 0.044), and LFTC (both P < 0.001) regions. For highest dose, in the central medial tibial (P = 0.008), central lateral tibial (P < 0.001) and central lateral femoral (P < 0.001) regions. CONCLUSIONS: Cartilage thickness assessed by automated segmentation provided a consistent dose response in structural modification compared with manual segmentation. This is the first time that two independent quantification methods of image analysis have reached the same conclusions in an interventional trial, strengthening the conclusions that sprifermin modifies structural progression in knee osteoarthritis.

Predicting knee replacement in participants eligible for disease-modifying osteoarthritis drug treatment with structural endpoints.

OBJECTIVE: Evaluate associations between 2-year change in radiographic or quantitative magnetic resonance imaging (qMRI) structural measures, and knee replacement (KR), within a subsequent 7-year follow-up period. METHOD: Participants from the Osteoarthritis Initiative were selected based on potential eligibility criteria for a disease-modifying osteoarthritis (OA) drug trial: Kellgren-Lawrence grade 2 or 3; medial minimum joint space width (mJSW) ≥2.5 mm; knee pain at worst 4-9 in the past 30 days on an 11-point scale, or 0-3 if medication was taken for joint pain; and availability of structural measures over 2 years. Mean 2-year change in structural measures was estimated and compared with two-sample independent t-tests for KR and no KR. Area under the receiver operating characteristic curve (AUC) was estimated using 2-year change in structural measures for prediction of future KR outcomes. RESULTS: Among 627 participants, 107 knees underwent KR during a median follow-up of 6.7 years after the 2-year imaging period. Knees that received KR during follow-up had a greater mean loss of cartilage thickness in the total femorotibial joint and medial femorotibial compartment on qMRI, as well as decline in medial fixed joint space width on radiographs, compared with knees that did not receive KR. These imaging measures had similar, although modest discrimination for future KR (AUC 0.62, 0.60, and 0.61, respectively). CONCLUSIONS: 2-year changes in qMRI femorotibial cartilage thickness and radiographic JSW measures had similar ability to discriminate future KR in participants with knee OA, suggesting that these measures are comparable biomarkers/surrogate endpoints of structural progression.

Tissue distribution of sprifermin (recombinant human fibroblast growth factor 18) in the rat following intravenous and intra-articular injection.

Objective: Fibroblast growth factor 18 (FGF18) is involved in chondrogenesis and articular cartilage repair. We investigated tissue distribution and pharmacokinetics of radioactive [3H]sprifermin, a recombinant human FGF18, in rats after a single intravenous (i.v.) or intra-articular (i.a.) injection. Design: In two studies (48-96-h [n = 23] and 28-day [n = 12]), 35 male albino (Sprague Dawley) rats received single i.v. or i.a. dose [3H]sprifermin (0.24 mg/kg). Radioactivity was measured in blood, serum, and (in animals receiving i.a. administration) in the knee joint by liquid scintillation counting. Radioactivity in organs, tissues, and distribution in the whole body were measured with whole-body autoradiography. Results: After i.v. injection, radioactivity peaked in serum and whole blood after 4 and 24 h, respectively, with greater total radioactivity in serum. After i.a. injection, radioactivity peaked in serum and whole blood after 24 and 48 h, respectively; intact [3H]sprifermin was not detected in vena caval serum and systemic exposure was low, approximately 20% of that with i.v. injection. Following i.v. injection, radioactivity was mainly found in the liver, adrenal glands, kidney, and spleen; following i.a. injection, radioactivity was preferentially concentrated in articular cartilage after initial distribution in the joint capsule, and still evident in the joint after 28 days. Conclusions: After i.a. injection of [3H]sprifermin in rats, radioactivity was concentrated in the knee joint, particularly articular cartilage, with low levels in other investigated tissues. Systemic exposure to sprifermin was greater with i.v. than i.a. injection. Subsequent clinical investigation in patients with osteoarthritis has reported consistent results.

Recombinant human FGF18 preserves depth-dependent mechanical inhomogeneity in articular cartilage.

Articular cartilage is a specialised tissue that has a relatively homogenous endogenous cell population but a diverse extracellular matrix (ECM), with depth-dependent mechanical properties. Repair of this tissue remains an elusive clinical goal, with biological interventions preferred to arthroplasty in younger patients. Osteochondral transplantation (OCT) has emerged for the treatment of cartilage defects and osteoarthritis. Fresh allografts stored at 4 °C have been utilised, though matrix and cell viability loss remains an issue. To address this, several studies have developed media formulations to maintain cartilage explants in vitro. One promising factor for these applications is sprifermin, a human-recombinant fibroblast growth factor-18, which stimulates chondrocyte proliferation and matrix synthesis and is in clinical trials for the treatment of osteoarthritis. The study hypothesis was that addition of sprifermin during storage would maintain the unique depth-dependent mechanical profile of articular cartilage explants, a feature not often evaluated. Explants were maintained for up to 6 weeks with or without a weekly 24 h exposure to sprifermin (100 ng/mL) and the compressive modulus was assessed. Results showed that sprifermin-treated samples maintained their depth-dependent mechanical profile through 3 weeks, whereas untreated samples lost their mechanical integrity over 1 week of culture. Sprifermin also affected ECM balance by maintaining the levels of extracellular collagen and suppressing matrix metalloproteinase production. These findings support the use of sprifermin as a medium additive for OCT allografts during in vitro storage and present a potential mechanism where sprifermin may impact a functional characteristic of articular cartilage in repair strategies.

Sprifermin (rhFGF18) enables proliferation of chondrocytes producing a hyaline cartilage matrix.

OBJECTIVE: Fibroblast growth factor (FGF) 18 has been shown to increase cartilage volume when injected intra-articularly in animal models of osteoarthritis (OA) and in patients with knee OA (during clinical development of the recombinant human FGF18, sprifermin). However, the exact nature of this effect is still unknown. In this study, we aimed to investigate the effects of sprifermin at the cellular level. DESIGN: A combination of different chondrocyte culture systems was used and the effects of sprifermin on proliferation, the phenotype and matrix production were evaluated. The involvement of MAPKs in sprifermin signalling was also studied. RESULTS: In monolayer, we observed that sprifermin promoted a round cell morphology and stimulated both cellular proliferation and Sox9 expression while strongly decreasing type I collagen expression. In 3D culture, sprifermin increased the number of matrix-producing chondrocytes, improved the type II:I collagen ratio and enabled human OA chondrocytes to produce a hyaline extracellular matrix (ECM). Furthermore, we found that sprifermin displayed a 'hit and run' mode of action, with intermittent exposure required for the compound to fully exert its anabolic effect. Finally, sprifermin appeared to signal through activation of ERK. CONCLUSIONS: Our results indicate that intermittent exposure to sprifermin leads to expansion of hyaline cartilage-producing chondrocytes. These in vitro findings are consistent with the increased cartilage volume observed in the knees of OA patients after intra-articular injection with sprifermin in clinical studies.

Disease-modifying treatments for osteoarthritis (DMOADs) of the knee and hip: lessons learned from failures and opportunities for the future.

Osteoarthritis (OA) is the biggest unmet medical need among the many musculoskeletal conditions and the most common form of arthritis. It is a major cause of disability and impaired quality of life in the elderly. We review several ambitious but failed attempts to develop joint structure-modifying treatments for OA. Insights gleaned from these attempts suggest that these failures arose from unrealistic hypotheses, sub-optimal selection of patient populations or drug dose, and/or inadequate sensitivity of the trial endpoints. The long list of failures has prompted a paradigm shift in OA drug development with redirection of attention to: (1) consideration of the benefits of localized vs systemic pharmacological agents, as indicated by the increasing number of intra-articularly administered compounds entering clinical development; (2) recognition of OA as a complex disease with multiple phenotypes, that may each require somewhat different approaches for optimizing treatment; and (3) trial enhancements based on guidance regarding biomarkers provided by regulatory agencies, such as the Food and Drug Administration (FDA), that could be harnessed to help turn failures into successes.

An in vivo rat model to study calcitonin gene related peptide release following activation of the trigeminal vascular system.

Calcitonin gene related peptide (CGRP) released from the C-fibers projecting from the trigeminal ganglion to the meninges has been suggested to play a crucial role in the pathophysiology of headache, particularly migraine. In humans it has been shown that CGRP is released during migraine-attacks, and this is attenuated by the administration of typical anti-migraine drugs such as dihydroergotamine or sumatriptan. We describe a new rat model which allows the study of CGRP release from the meninges into venous blood following activation of the trigeminal vascular system. The effects of classical and new anti-migraine drugs such as acetylsalicylic acid (ASA), sumatriptan and the new high efficacy 5-HT1B/1D agonist donitriptan (4-[4-[2-(2-aminoethyl)-1H-indol-5-yloxyl]acetyl]piperazinyl-1-yl]benzonitrile) were evaluated in comparison with the established model of neurogenic inflammation in the meninges. Sumatriptan and donitriptan inhibited CGRP release as well as neurogenic inflammation. ASA, however, attenuated neurogenic inflammation, but not CGRP release, confirming the concept of prejunctional inhibition of CGRP release by 5-HT1B/1D receptors. This new model allows the further study of prejunctional pharmacology and mechanisms of neuropeptide release in the trigeminal vascular system, which might be crucial for the further development of potent, more effective anti-migraine drugs.

Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice.

Concanavalin A (Con A) causes severe TNF-alpha-mediated and IFN-gamma-mediated liver injury in mice. In addition to their other functions, TNF-alpha and IFN-gamma both induce the inducible nitric oxide (NO) synthase (iNOS). Using different models of liver injury, NO was found to either mediate or prevent liver damage. To further elucidate the relevance of NO for liver damage we investigated the role of iNOS-derived NO in the Con A model. We report that iNOS mRNA was induced in livers of Con A-treated mice within 2 hours, with iNOS protein becoming detectable in hepatocytes as well as in Kupffer cells within 4 hours. iNOS-/- mice were protected from liver damage after Con A treatment, as well as in another TNF-alpha-mediated model that is inducible by LPS in D-galactosamine-sensitized (GalN-sensitized) mice. iNOS-deficient mice were not protected after direct administration of recombinant TNF-alpha to GalN-treated mice. Accordingly, pretreatment of wild-type mice with a potent and specific inhibitor of iNOS significantly reduced transaminase release after Con A or GalN/LPS, but not after GalN/TNF-alpha treatment. Furthermore, the amount of plasma TNF-alpha and of intrahepatic TNF-alpha mRNA and protein was significantly reduced in iNOS-/- mice. Our results demonstrate that iNOS-derived NO regulates proinflammatory genes in vivo, thereby contributing to inflammatory liver injury in mice by stimulation of TNF-alpha production.

Determination of L-iminoethyl-L-lysine in serum by liquid chromatography.

A selective and sensitive method is presented for the determination of L-iminoethyl-L-lysine (L-NIL) in rat serum. L-NIL is a selective inhibitor of the inducible nitric oxide synthase. The analytical technique is based upon a two-buffer reversed-phase HPLC system with fluorescence detection of pre-column derivatized amino acid analogue with o-phtaldialdehyde. The retention time of L-NIL was 19.4 min. The limit of quantification was 0.5 mg/l. After validation, the method was used to study the pharmacokinetic profile of L-NIL in rats after intravenous as well as oral administration of a single dose.