Efficacy and safety results after >3.5 years of treatment with the Bruton's tyrosine kinase inhibitor evobrutinib in relapsing multiple sclerosis: Long-term follow-up of a Phase II randomised clinical trial with a cerebrospinal fluid sub-study.

BACKGROUND: Evobrutinib - an oral, central nervous system (CNS)-penetrant, and highly selective Bruton's tyrosine kinase inhibitor - has shown efficacy in a 48-week, double-blind, Phase II trial in patients with relapsing MS. OBJECTIVE: Report results of the Phase II open-label extension (OLE; up to week 192 from randomisation) and a cerebrospinal fluid (CSF) sub-study. METHODS: In the 48-week double-blind period (DBP), patients received evobrutinib 25 mg once-daily, 75 mg once-daily, 75 mg twice-daily or placebo (switched to evobrutinib 25 mg once-daily after week 24). Patients could then enter the OLE, receiving evobrutinib 75 mg once-daily (mean (± standard deviation (SD)) duration = 50.6 weeks (±6.0)) before switching to 75 mg twice-daily. RESULTS: Of 164 evobrutinib-treated patients who entered the OLE, 128 (78.0%) completed ⩾192 weeks of treatment. Patients receiving DBP evobrutinib 75 mg twice-daily: annualised relapse rate at week 48 (0.11 (95% confidence interval (CI) = 0.04-0.25)) was maintained with the OLE twice-daily dose up to week 192 (0.11 (0.05-0.22)); Expanded Disability Status Scale score remained stable; serum neurofilament light chain fell to levels like a non-MS population (Z-scores); T1 gadolinium-enhancing lesion numbers remained low. No new safety signals were identified. In the OLE, evobrutinib was detected in the CSF of all sub-study patients. CONCLUSION: Long-term evobrutinib treatment was well tolerated and associated with a sustained low level of disease activity. Evobrutinib was present in CSF at concentrations similar to plasma.

Safety, Tolerability, and Pharmacodynamics of the ADAMTS-5 Nanobody M6495: Two Phase 1, Single-Center, Double-Blind, Randomized, Placebo-Controlled Studies in Healthy Subjects and Patients With Osteoarthritis.

OBJECTIVE: To assess the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of single and multiple injections of M6495, a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5)  nanobody, in healthy volunteers and patients with osteoarthritis. METHODS: Two randomized, placebo-controlled, double-blind studies were performed. Study 1 enrolled 54 healthy male volunteers who received one subcutaneous (s.c.) injection of M6495 (1-300 mg) or placebo (ratio 2:1), evaluating safety, PK, and PD as changes in the serum aggrecan fragment alanine-arginine-glycine-serine (ARGS). Study 2 enrolled 32 patients with osteoarthritis with Kellgren-Lawrence grades 2 to 4 and pain greater than or equal to 40 on the Western Ontario and McMaster Universities Arthritis Index pain subscale at screening and evaluated the safety, PK, and PD of three doses every two weeks (75-300 mg per dose) or six once-weekly M6495 s.c. doses (300 mg) or placebo (ratio 3:1) over 106 days' follow-up. RESULTS: M6495 in single and multiple doses of less than or equal to 300 mg s.c. weekly was well tolerated with no clinically significant changes in any safety parameter. Adverse events more frequently reported in the M6495 groups were mostly mild cases of injection site reactions, myalgia, and nausea, which resolved after treatment cessation. The elimination half-life of single s.c. doses of M6495 ranged from 79 to 267 hours. M6495 administration substantially reduced serum ARGS levels, indicative of target engagement and indicating disease-modifying potential of M6495. CONCLUSION: Treatment with M6495 in single and multiple doses up to and including 300 mg s.c. was found to be well tolerated and adequately safe for further clinical evaluation of potential disease-modifying effects.

A plain language summary of what clinical studies can tell us about the safety of evobrutinib - a potential treatment for multiple sclerosis.

WHAT IS THIS SUMMARY ABOUT?: This summary explains the findings from a recent investigation that combined the results of over 1000 people from three clinical studies to understand the safety of evobrutinib. Evobrutinib is an oral medication (taken by mouth), being researched as a potential treatment for multiple sclerosis (MS). This medication was also investigated in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Over 1000 people have taken evobrutinib as part of three separate phase 2 clinical studies. These studies looked at how much of the drug should be taken, how safe the drug is, and how well it might work for treating a certain medical condition. WHAT WERE THE RESULTS?: Evobrutinib was well-tolerated by participants in all three studies. The number of side effects reported by participants taking the medication was very similar to those reported by participants taking the placebo (a 'dummy' treatment without a real drug). The most common side effects in clinical studies were urinary tract infections, headache, swelling of the nose and throat, diarrhoea and blood markers of potential liver damage (these returned to normal once the treatment was stopped). WHAT DO THE RESULTS MEAN?: The safety data from all three clinical studies are encouraging and can be used to inform further research into using evobrutinib in MS. Clinical Trial Registration: NCT02975349 (multiple sclerosis), NCT03233230 (rheumatoid arthritis), NCT02975336 (systemic lupus erythematosus) (ClinicalTrials.gov).

Characterisation of the safety profile of evobrutinib in over 1000 patients from phase II clinical trials in multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus: an integrated safety analysis.

OBJECTIVE: Analyse the integrated safety profile of evobrutinib, a Bruton's tyrosine kinase inhibitor (BTKi), using pooled data from multiple sclerosis (MS), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) trials. METHODS: Phase II, randomised, double-blind, placebo-controlled trial data were analysed (N=1083; MS: n=213, 48 weeks (W); RA: n=390, 12W; SLE: n=480, 52W). The analysis included all patients who received ≥1 dose of evobrutinib (25 mg or 75 mg once daily, or 50 mg or 75 mgtwice daily) or placebo. Descriptive statistics and exposure-adjusted incidence rates (EAIR) were used to report treatment-emergent adverse events (TEAEs). RESULTS: Data from 1083 patients were pooled: evobrutinib, n=861; placebo, n=271 (sum >1083 due to MS trial design: n=49 received both placebo (W0-24) and evobrutinib 25 mg (W25-48)); median follow-up time (pt-years): evobrutinib, 0.501; placebo, 0.463. Across indications, the proportion of patients with TEAEs and the EAIR were similar for evobrutinib and placebo (66.2% (247.6 events/100 pt-years) vs 62.4% (261.4 events/100 pt-years)). By indication, the EAIR (events/100 pt-years) of TEAEs for evobrutinib versus placebo were: MS: 119.7 vs 148.3; RA: 331.8 vs 306.8; SLE: 343.0 vs 302.1. Two fatal events occurred (in SLE). The serious infections EAIR was 2.7 and 2.1 events/100 pt-years for evobrutinib and placebo. For previously reported BTKi-class effects, the EAIR of transient elevated alanine aminotransferase/aspartate aminotransferase TEAEs (events/100 pt-years) with evobrutinib versus placebo was 4.8 vs 2.8/3.5 vs 0.7, respectively. IgG levels were similar in evobrutinib/placebo-treated patients. CONCLUSIONS: This is the first BTKi-integrated safety analysis that includes patients with MS. Overall, evobrutinib treatment (all doses) was generally well tolerated across indications. TRIAL REGISTRATION NUMBERS: NCT02975349, NCT03233230, NCT02975336.

Efficacy and Safety of the Bruton's Tyrosine Kinase Inhibitor Evobrutinib in Systemic Lupus Erythematosus: Results of a Phase II, Randomized, Double-Blind, Placebo-Controlled Dose-Ranging Trial.

OBJECTIVE: Evobrutinib is a highly selective, orally administered Bruton's tyrosine kinase (BTK) inhibitor. The objective of this phase II, multicenter, randomized, double-blind, placebo-controlled trial was to evaluate the efficacy and safety of evobrutinib in patients with active autoantibody-positive systemic lupus erythematosus (SLE). METHODS: Patients were diagnosed with SLE by either the Systemic Lupus International Collaborating Clinics criteria or at least four American College of Rheumatology criteria 6 months or more prior to screening, had an SLE Disease Activity Index-2000 score of 6 or more, were autoantibody-positive and on standard-of-care therapy. Randomization was 1:1:1:1 to oral evobrutinib 25 mg once daily (QD), 75 mg QD, 50 mg twice daily, or placebo. Primary efficacy endpoints were SLE responder index (SRI)-4 response at week 52 and SRI-6 response at week 52 in the high disease activity subpopulation. Safety endpoints included treatment-emergent adverse events (TEAEs). RESULTS: A total of 469 patients were randomized and received at least one dose of evobrutinib or placebo at the time of primary analysis. Mean (SD) age at baseline was 40.7 (±12.3) years; 94.9% of patients were female. Neither primary efficacy endpoint was met. All doses of evobrutinib were well tolerated, and there was no clear dose effect on the incidence of reported TEAEs, or serious TEAEs, including severe infections. CONCLUSION: This phase II, dose-ranging trial in SLE failed to show a treatment effect of evobrutinib versus placebo at any dose. Evobrutinib was generally well tolerated, with no dose effect observed for TEAEs. These results suggest that BTK inhibition does not appear to be an effective therapeutic intervention for patients with SLE.

99mTc-NTP 15-5 is a companion radiotracer for assessing joint functional response to sprifermin (rhFGF-18) in a murine osteoarthritis model.

With the emergence of disease modifying osteoarthritis drugs (DMOAD), imaging methods to quantitatively demonstrate their efficacy and to monitor osteoarthritis progression at the functional level are urgently needed. Our group showed that articular cartilage can be quantitatively assessed in nuclear medicine imaging by our radiotracer 99mTc-NTP 15-5 targeting cartilage proteoglycans. In this work, surgically induced DMM mice were treated with sprifermin or saline. We investigated cartilage remodelling in the mice knees by 99mTc-NTP 15-5 SPECT-CT imaging over 24 weeks after surgery, as wells as proteoglycan biochemical assays. OA alterations were scored by histology according to OARSI guidelines. A specific accumulation of 99mTc-NTP 15-5 in cartilage joints was evidenced in vivo by SPECT-CT imaging as early as 30 min post-iv injection. In DMM, 99mTc-NTP 15-5 accumulation in cartilage within the operated joints, relative to contralateral ones, was observed to initially increase then decrease as pathology progressed. Under sprifermin, 99mTc-NTP 15-5 uptake in pathological knees was significantly increased compared to controls, at 7-, 12- and 24-weeks, and consistent with proteoglycan increase measured 5 weeks post-surgery, as a sign of cartilage matrix remodelling. Our work highlights the potential of 99mTc-NTP 15-5 as an imaging-based companion to monitor cartilage remodelling in OA and DMOAD response.

Recombinant fibroblast growth factor-18 (sprifermin) enhances microfracture-induced cartilage healing.

Posttraumatic osteoarthritis is a disabling condition impacting the mostly young and active population. In the present study, we investigated the impact of intra-articular sprifermin, a recombinant truncated fibroblast growth factor 18, on the outcome of microfracture treatment, a widely used surgical technique to enhance cartilage healing at the site of injury. For this study, we created a cartilage defect and performed microfracture treatment in fetlock joints of 18 horses, treated joints with one of three doses of sprifermin (10, 30, or 100 µg) or with saline, hyaluronan, and evaluated animals functional and structural outcomes over 24 weeks. For primary outcome measures, we performed histological evaluations and gene expression analysis of aggrecan, collagen types I and II, and cartilage oligomeric matrix protein in three regions of interest. As secondary outcome measures, we examined animals' lameness, performed arthroscopic, radiographic, and computed tomography (CT) scan imaging and gross morphology assessment. We detected the highest treatment benefit following 100 µg sprifermin treatment. The overall histological assessment showed an improvement in the kissing region, and the expression of constitutive genes showed a concentration-dependent enhancement, especially in the peri-lesion area. We detected a significant improvement in lameness scores, arthroscopic evaluations, radiography, and CT scans following sprifermin treatment when results from three dose-treatment groups were combined. Our results demonstrated, for the first time, an enhancement on microfracture outcomes following sprifermin treatment suggesting a cartilage regenerative role and a potential benefit of sprifermin treatment in early cartilage injuries.

Long-term structural and symptomatic effects of intra-articular sprifermin in patients with knee osteoarthritis: 5-year results from the FORWARD study.

OBJECTIVE: The FORWARD (FGF-18 Osteoarthritis Randomized Trial with Administration of Repeated Doses) trial assessed efficacy and safety of the potential disease-modifying osteoarthritis drug (DMOAD) sprifermin in patients with knee osteoarthritis. Here, we report 5-year efficacy and safety results. METHODS: Patients were randomised to intra-articular sprifermin 100 µg or 30 µg every 6 months (q6mo) or 12 months, or placebo, for 18 months. The primary analysis was at year 2, with follow-up at years 3, 4 and 5. Additional post hoc exploratory analyses were conducted in patients with baseline minimum radiographic joint space width 1.5-3.5 mm and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain 40-90, a subgroup at risk (SAR) of progression. RESULTS: 378 (69%) patients completed the 5-year follow-up. A significant dose-response in total femorotibial joint cartilage thickness with sprifermin (trend test, p<0.001) and a 0.05 mm mean difference with sprifermin 100 µg q6mo versus placebo (95% CI 0.00 to 0.10; p=0.015) were sustained to year 5. WOMAC pain scores improved ~50% from baseline in all groups. No patient in the 100 µg q6mo group had replacement of the treated knee. 96%-98% of patients receiving sprifermin and 98% placebo reported adverse events, most were mild or moderate and deemed unrelated to treatment. Adverse event-related study withdrawals were <10%. Differentiation in WOMAC pain between sprifermin 100 µg q6mo and placebo in the SAR (n=161) at year 3 was maintained to year 5 (-10.08; 95% CI -25.68 to 5.53). CONCLUSION: In the longest DMOAD trial reported to date, sprifermin maintained long-term structural modification of articular cartilage over 3.5 years post-treatment. Potential translation to clinical benefit was observed in the SAR. TRIAL REGISTRATION NUMBER: NCT01919164.

The effects of sprifermin on symptoms and structure in a subgroup at risk of progression in the FORWARD knee osteoarthritis trial.

OBJECTIVE: To assess pain outcomes and cartilage thickness change in a subgroup at risk (SAR) of further progression in the FORWARD trial of knee osteoarthritis patients treated with sprifermin. METHODS: Patients were randomised 1:1:1:1:1 to: sprifermin 100 µg every 6 months (q6mo), 100 µg q12mo, 30 µg q6mo, 30 µg q12mo, or placebo for 18 months. SAR was defined as baseline medial or lateral minimum joint-space width (mJSW) 1.5-3.5 mm and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain score 40-90 units. Follow-up to 3 years was included in the analysis. Treatment benefit was explored by repeated measures, linear dose-effect trends by timepoint. RESULTS: The SAR comprised 161 (29%) of 549 patients. Mean difference (95% CI) in WOMAC pain at year 3 for sprifermin 100 µg q6mo vs placebo SAR was -8.75 (-22.42, 4.92) for SAR vs 0.97 (-6.22, 8.16) for the intent-to-treat population. SAR placebo patients lost more cartilage over 2 years than the modified ITT (mITT) placebo arm (mean change from baseline, mm [SD]: -0.05 [0.10] vs -0.02 [0.07]). Net total femorotibial joint thickness gain with sprifermin 100 µg q6mo (adjusted mean difference from placebo [95% CI] was similar in the SAR and in the mITT group: 0.06 [0.01, 0.11] vs 0.05 [0.03, 0.07]). CONCLUSIONS: Selection for low mJSW and moderate-to-high pain at baseline resulted in more rapid disease progression and demonstrated translation of structure modification (with maintained net benefit on total cartilage thickness) into symptomatic benefit. This subgroup may represent a target population for future trials. CLINICAL TRIAL REGISTRATION: NCT01919164.

Serum C-reactive protein metabolite (CRPM) is associated with incidence of contralateral knee osteoarthritis.

The heterogeneous nature of osteoarthritis (OA) and the need to subtype patients is widely accepted in the field. The biomarker CRPM, a metabolite of C-reactive protein (CRP), is released to the circulation during inflammation. Blood CRPM levels have shown to be associated with disease activity and response to treatment in rheumatoid arthritis (RA). We investigated the level of blood CRPM in OA compared to RA using data from two phase III knee OA and two RA studies (N = 1591). Moreover, the association between CRPM levels and radiographic progression was investigated. The mean CRPM levels were significantly lower in OA (8.5 [95% CI 8.3-8.8] ng/mL, n = 781) compared to the RA patients (12.8 [9.5-16.0] ng/mL, n = 60); however, a significant subset of OA patients (31%) had CRPM levels (≥ 9 ng/mL) comparable to RA. Furthermore, OA patients (n = 152) with CRPM levels ≥ 9 ng/mL were more likely to develop contra-lateral knee OA assessed by X-ray over a two-year follow-up period with an odds ratio of 2.2 [1.0-4.7]. These data suggest that CRPM is a blood-based biochemical marker for early identification OA patients with an inflammatory phenotype.

Relationship between motion, using the GaitSmartTM system, and radiographic knee osteoarthritis: an explorative analysis in the IMI-APPROACH cohort.

OBJECTIVES: To assess underlying domains measured by GaitSmartTMparameters and whether these are additional to established OA markers including patient reported outcome measures (PROMs) and radiographic parameters, and to evaluate if GaitSmart analysis is related to the presence and severity of radiographic knee OA. METHODS: GaitSmart analysis was performed during baseline visits of participants of the APPROACH cohort (n = 297). Principal component analyses (PCA) were performed to explore structure in relationships between GaitSmart parameters alone and in addition to radiographic parameters and PROMs. Logistic and linear regression analyses were performed to analyse the relationship of GaitSmart with the presence (Kellgren and Lawrence grade ≥2 in at least one knee) and severity of radiographic OA (ROA). RESULTS: Two hundred and eighty-four successful GaitSmart analyses were performed. The PCA identified five underlying GaitSmart domains. Radiographic parameters and PROMs formed additional domains indicating that GaitSmart largely measures separate concepts. Several GaitSmart domains were related to the presence of ROA as well as the severity of joint damage in addition to demographics and PROMs with an area under the receiver operating characteristic curve of 0.724 and explained variances (adjusted R2) of 0.107, 0.132 and 0.147 for minimum joint space width, osteophyte area and mean subchondral bone density, respectively. CONCLUSIONS: GaitSmart analysis provides additional information over established OA outcomes. GaitSmart parameters are also associated with the presence of ROA and extent of radiographic severity over demographics and PROMS. These results indicate that GaitsmartTM may be an additional outcome measure for the evaluation of OA.

Effect of Intra-Articular Sprifermin vs Placebo on Femorotibial Joint Cartilage Thickness in Patients With Osteoarthritis: The FORWARD Randomized Clinical Trial.

Importance: Sprifermin is under investigation as a disease-modifying osteoarthritis drug. Objective: To evaluate the effects of sprifermin on changes in total femorotibial joint cartilage thickness in the more symptomatic knee of patients with osteoarthritis. Design, Setting, and Participants: FORWARD (FGF-18 Osteoarthritis Randomized Trial with Administration of Repeated Doses) was a 5-year, dose-finding, multicenter randomized clinical trial conducted at 10 sites. Eligible participants were aged 40 to 85 years with symptomatic, radiographic knee osteoarthritis and Kellgren-Lawrence grade 2 or 3. Enrollment began in July 2013 and ended in May 2014; the last participant visit occurred on May 8, 2017. The primary outcome at 2 years and a follow-up analysis at 3 years are reported. Interventions: Participants were randomized to 1 of 5 groups: intra-articular injections of 100 µg of sprifermin administered every 6 months (n = 110) or every 12 months (n = 110), 30 µg of sprifermin every 6 months (n = 111) or every 12 months (n = 110), or placebo every 6 months (n = 108). Each treatment consisted of weekly injections over 3 weeks. Main Outcomes and Measures: The primary end point was change in total femorotibial joint cartilage thickness measured by quantitative magnetic resonance imaging at 2 years. The secondary end points (of 15 total) included 2-year change from baseline in total Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores. The minimal clinically important difference (MCID) is unknown for the primary outcome; for total WOMAC score in patients with hip and knee osteoarthritis, the absolute MCID is 7 U (95% CI, 4 to 10 U) and the percentage MCID is 14% (95% CI, 9% to 18%). Results: Among 549 participants (median age, 65.0 years; 379 female [69.0%]), 474 (86.3%) completed 2-year follow-up. Compared with placebo, the changes from baseline to 2 years in total femorotibial joint cartilage thickness were 0.05 mm (95% CI, 0.03 to 0.07 mm) for 100 µg of sprifermin administered every 6 months; 0.04 mm (95% CI, 0.02 to 0.06 mm) for 100 µg of sprifermin every 12 months; 0.02 mm (95% CI, -0.01 to 0.04 mm) for 30 µg of sprifermin every 6 months; and 0.01 mm (95% CI, -0.01 to 0.03 mm) for 30 µg of sprifermin every 12 months. Compared with placebo, there were no statistically significant differences in mean absolute change from baseline in total WOMAC scores for 100 µg of sprifermin administered every 6 months or every 12 months, or for 30 µg of sprifermin every 6 months or every 12 months. The most frequently reported treatment-emergent adverse event was arthralgia (placebo: n = 46 [43.0%]; 100 µg of sprifermin administered every 6 months: n = 45 [41.3%]; 100 µg of sprifermin every 12 months: n = 50 [45.0%]; 30 µg of sprifermin every 6 months: n = 40 [36.0%]; and 30 µg of sprifermin every 12 months: n = 48 [44.0%]). Conclusions and Relevance: Among participants with symptomatic radiographic knee osteoarthritis, the intra-articular administration of 100 µg of sprifermin every 6 or 12 months vs placebo resulted in an improvement in total femorotibial joint cartilage thickness after 2 years that was statistically significant, but of uncertain clinical importance; there was no significant difference for 30 µg of sprifermin every 6 or 12 months vs placebo. Durability of response also was uncertain. Trial Registration: ClinicalTrials.gov Identifier: NCT01919164.

Sprifermin treatment enhances cartilage integration in an in vitro repair model.

Cartilage integration remains a clinical challenge for treatment of focal articular defects. Cartilage exhibits limited healing capacity that declines with tissue maturation. Many approaches have been investigated for their ability to stimulate healing of mature cartilage or integration of repair tissue or tissue-engineered constructs with native cartilage. Growth factors present in immature tissue may enhance chondrogenesis and promote integrative repair of cartilage defects. In this study, we assessed the role of one such factor, fibroblast growth factor 18 (FGF18). Studies using FGF18 have shown a variety of positive effects on cartilage, including stimulation of chondrocyte proliferation, matrix biosynthesis, and suppression of proteinase activity. To explore the role of FGF18 on cartilage defect repair, we hypothesized that treatment with recombinant human FGF18 (sprifermin) would increase matrix synthesis in a defect model, thus improving integration strength. To test this hypothesis, 6 mm cartilage cylinders were harvested from juvenile bovine knees. A central 3 mm defect was created in each explant, and this core was removed and replaced. Resulting constructs were cultured in control or sprifermin-containing medium (weekly 24-h exposure of 100 ng/ml sprifermin) for 4 weeks. Mechanical testing, biochemical analysis, micro-CT, scanning electron microscopy, and histology were used to assess matrix production, adhesive strength, and structural properties of the cartilage-cartilage interface. Results showed greater adhesive strength, increased collagen content, and larger contact areas between core and annular cartilage in the sprifermin-treated group. These findings present a novel treatment for cartilage injuries that have potential to enhance defect healing and lateral cartilage-cartilage integration. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2648-2656, 2018.

Automated Tracking of Motion and Body Weight for Objective Monitoring of Rats in Colony Housing.

Living together in large social communities within an enriched environment stimulates self-motivated activity in rats. We developed a modular housing system in which a single unit can accommodate as many as 48 rats and contains multiple functional areas. This rat colony cage further allowed us to remotely measure body weight and to continuously measure movement, including jumping and stair walking between areas. Compared with pair-housed, age-, strain-, and weight-matched rats in conventional cages, the colony-housed rats exhibited higher body mass indices, had more exploratory behavior, and were more cooperative during handling. Continuous activity tracking revealed that the amount of spontaneous locomotion, such as jumping between levels and running through the staircase, fell after surgery, blood sampling, injections, and behavioral tests to a similar extent regardless of the specific intervention. Data from the automated system allowed us to identify individual rats with significant differences (>2 SD) from other cohoused rats; these rats showed potential health problems, as verified using conventional health scoring. Thus, our rat colony cage permits social interaction and provides a variety of functional areas, thereby perhaps improving animal wellbeing. Furthermore, automated online tracking enabled continuous quantification of spontaneous motion, potentially providing objective measures of animal behavior in various disease models and reducing the need for experimental manipulation. Finally, health monitoring of individual rats was facilitated in an objective manner.

Delivering rhFGF-18 via a bilayer collagen membrane to enhance microfracture treatment of chondral defects in a large animal model.

Augmented microfracture techniques use growth factors, cells, and/or scaffolds to enhance the healing of microfracture-treated cartilage defects. This study investigates the effect of delivering recombinant human fibroblastic growth factor 18 (rhFHF18, Sprifermin) via a collagen membrane on the healing of a chondral defect treated with microfracture in an ovine model. Eight millimeter diameter chondral defects were created in the medial femoral condyle of 40 sheep (n = 5/treatment group). Defects were treated with microfracture alone, microfracture + intra-articular rhFGF-18 or microfracture + rhFGF-18 delivered on a membrane. Outcome measures included mechanical testing, weight bearing, International Cartilage Repair Society repair score, modified O'Driscoll score, qualitative histology, and immunohistochemistry for types I and II collagen. In animals treated with 32 µg rhFGF-18 + membrane and intra-articularly, there was a statistically significant improvement in weight bearing at 2 and 4 weeks post surgery and in the modified O'Driscoll score compared to controls. In addition, repair tissue stained was more strongly stained for type II collagen than for type I collagen. rhFGF-18 delivered via a collagen membrane at the point of surgery potentiates the healing of a microfracture treated cartilage defect.

The effect of recombinant human fibroblast growth factor-18 on articular cartilage following single impact load.

The aim of this in vitro study was to ascertain the effect of recombinant human Fibroblast Growth Factor-18 (rhFGF18) on the repair response of mechanically damaged articular cartilage. Articular cartilage discs were harvested from healthy mature horses (n = 4) and subjected to single impact load (SIL). The impacted explants, together with unimpacted controls were cultured in modified DMEM ± 200 ng/ml rhFGF18 for up to 30 days. Glycosaminoglycan (GAG) release into the media was measured using the dimethylmethylene blue (DMMB) assay. Aggrecan neopepitope CS846, collagen type II synthesis (CPII) and cleavage (C2C) were measured by ELISA. Histological analysis and TUNEL staining were used to assess repair cell number and cell death. Impacted explants treated with rhFGF18 showed significantly more GAG and CS846 release into the media (p < 0.05), there was also a significant decrease in C2C levels at Day 20. Loaded sections treated with rhFGF18 had more repair cells and significantly less cell death (p < 0.001) at Day 30 in culture. In an in vitro damage/repair model, rhFGF18 increases the proteoglycan synthesis, the repair cell number and prevents apoptosis at Day 30. This suggests that rhFGF18 may be a good candidate for enhancement of cartilage repair following mechanical damage.

Identification of fibroblast growth factor-18 as a molecule to protect adult articular cartilage by gene expression profiling.

To identify genes that maintain the homeostasis of adult articular cartilage and regenerate its lesions, we initially compared four types of chondrocytes: articular (AA) versus growth plate (AG) cartilage chondrocytes in adult rats, and superficial layer (IS) versus deep layer (ID) chondrocytes of epiphyseal cartilage in infant rats. Microarray analyses revealed that 40 and 186 genes had ≥10-fold higher expression ratios of AA/AG and IS/ID, respectively, and 16 genes showed ≥10-fold of both AA/AG and IS/ID ratios. The results were validated by real-time RT-PCR analysis. Among them, Hoxd1, Fgf18, and Esm1 were expressed more strongly in AA than in IS. Fgf18 was the extracellular and secreted factor that decreased glycosaminoglycan release and depletion from the cartilage, and enhanced proliferation of articular chondrocytes. Fgf18 was strongly expressed in the articular cartilage chondrocytes of adult rats. In a surgical rat osteoarthritis model, a once-weekly injection of recombinant human FGF18 (rhFGF18) given 3 weeks after surgery prevented cartilage degeneration in a dose-dependent manner at 6 and 9 weeks after surgery, with significant effect at 10 µg/week of rhFGF18. As the underlying mechanism, rhFGF18 strongly up-regulated Timp1 expression in the cell and organ cultures, and inhibition of aggrecan release by rhFGF18 was restored by addition of an antibody to Timp1. In conclusion, we have identified Fgf18 as a molecule that protects articular cartilage by gene expression profiling, and the anticatabolic effects may at least partially be mediated by the Timp1 expression.

Intra-articular injection of rhFGF-18 improves the healing in microfracture treated chondral defects in an ovine model.

Microfracture is a common cartilage repair procedure. Strategies to improve healing post-microfracture include the use of growth factors to enhance hyaline cartilage production. This study investigates the effect of intra-articular recombinant human fibroblastic growth factor 18 (rhFHF18) on the healing of a chondral defect treated with microfracture in an ovine model. Chondral defects (8 mm diameter) were created in the medial femoral condyle of 80 sheep (n = 16/treatment group). Defects were treated with microfracture alone or microfracture + intra-articular rhFGF-18 (administered either as one or two cycles of 3× weekly injections). Outcome measures included mechanical testing, macroscopic International Cartilage Repair Society repair score, modified O'Driscoll histology score, qualitative histology, and immunohistochemistry for types I, II, and VI collagen. In treated animals, there was a statistically significant improvement in ICRS tissue repair score and tissue infill score, in the modified O'Driscoll score between control and 1 cycle of rhFGF-18 at 6 m, and in the cartilage repair score and structural characteristic score between the control and both rhFGF-18 groups at 6 m. There was no evidence of degeneration of adjacent cartilage in the rhFGF-18 treated cartilage. The increase in hyaline cartilage-like tissue formed in the microfracture + rhFGF-18 treated groups indicates that rhFGF-18 potentiates the formation of hyaline cartilage repair following microfracture.

Osteochondral tissue engineering using a biphasic collagen/GAG scaffold containing rhFGF18 or BMP-7 in an ovine model.

BACKGROUND: The aim of this study was to investigate the effect of combining rhFGF18 or BMP-7 with a biphasic collagen/GAG osteochondral scaffold (Chondromimetic) on the repair of osteochondral defects in sheep. METHODS: Osteochondral defects (5.8x6mm) were created in the medial femoral condyle (MFC) and the lateral trochlea sulcus (LTS) of the stifle joint of 24 female sheep. Sheep were randomly assigned to four groups (n = 6); 1) empty defect, 2) scaffold only, 3) scaffold + rhFGF-18 (30 µg) and 4) scaffold + BMP-7 (100 µg). At 6 months the defects underwent non-destructive mechanical testing, gross assessment of repair tissue (ICRS score) and histological analysis (Modified O'Driscoll score). RESULTS: ICRS repair score: Defects treated with scaffold + rhFGF18 (mean 9.83, 95% CI 8.43-11.23) and scaffold + BMP-7 (10, 9.06-10.94) in the MFC had significantly improved ICRS scores compared to empty defects (4.2, 0-8.80) (p = 0.002). Mechanical properties: BMP-7 treated defects (mean 64.35, 95% CI 56.88-71.82) were significantly less stiff than both the rhFGF18 (mean 84.1, 95% CI 76.8-91.4) and empty defects in the LTS, compared to both contralateral limb (p = 0.003), and the perilesional articular cartilage (p < 0.001). HISTOLOGY: A statistically significant improvement in the modified O'Driscoll score was observed in the rhFGF18 treated group (mean 16.83, 95% CI 13.65-20.61) compared to the empty defects (mean 9, 95% CI 4.88-13.12) (p = 0.039) in the MFC. Excellent tissue fill, lateral integration and proteoglycan staining was observed. Only the rhFGF18 defects showed pericellular type VI collagen staining with positive type II collagen and reduced positive type I collagen staining. The majority of defects in the control and BMP-7 groups demonstrated fibrocartilagenous repair tissue. CONCLUSION: Statistically significant improvements in gross repair, mechanical properties and histological score were found over empty defects when Chondromimetic was combined with rhFGF18. These results suggest that rhFGF18 may play a significant role in articular cartilage repair applications.