Animal models and the search for anti-rheumatic drugs

Animal models and the search for antirheumatic drugs Objectives To understand the problems with existing therapies for RA The reasoning behind screening cascades and the position of in vivo pharmacology To appreciate the specific problems related to animal experimentation To understand how to manipulate in vivo models (immune/non immune etc) To give an example of a tertiary model of arthritis its similarities and differences to RA Rheumatoid arthritis Diagnosis of rheumatoid arthritis Any 4 of the following must be present to allow diagnosis of RA:Morning stiffness  1h Three or more joints involved Arthritis of hand joints Symmetric arthritis Present for  6wk Rheumatoid nodules Rheumatoid factor (positive < 5% normal subjects) Radiographic changes (must show erosion/decalcification) Pathology of rheumatoid arthritis Traditional pyramid for treatment of RA Assumptions:1) RA relatively benign disease 2) NSAIDs less toxic than DMARDs Exper imental therapy High dose corticosteroids Cyclophosphamide Methotrexate, azathiaprine Hydroxychloroquine Sulphasalazine NSAIDs Patient education Physical therapy Gold salts Low dose prednisone Occupational therapy High dose salicylates Sawtooth strategy for treatment of RA Typical course of disease Sawtooth course of disease 1 2 3 4 5 6 7 DMARD 0 5 10 15 20 25 Years Current therapies: Treatment cascade & limitations NSAIDs Volterol, Vioxx, Celebrex (GI ulceration & Bleeding. Not DMARD) DMARDs Methotrexate, Leflunamide, Hydroxychloroquine Efficacy (refractory), Safety – Myelosuppression, Hepatic toxicity BIOLOGICALS Enbrel, infliximab, Adalimumab, Anakinra (IL-1r) Expensive, inconvenient Admin, Partial/Non-responders?, Safety – TB, Op infection, CHF, Demyelinating Disease, Lymphoma NCEs and NBEs • NCEs generally work well across species – tend to target well-conserved sites eg enzyme active site • NBEs are often species specific – Large molecular interactions are less likely to be well-conserved – May need parallel reagent for animal models with final testing of human reagent in primates – Use human transgenic animal – Use human explant tissue Screening cascade for NCE Isolated protein Functional cell assay Ex vivo assay Counter screens for selectivity In vitro toxicology CYP induction/inhibition Mini Ames Micronucleus test In vitro Metabolism Microsomal stability CaCO-2 studies In vivo PK In vivo efficacy Primary model Secondary model Tertiary model Screening cascade for NBEs: concept of parallel reagents Murine parallel reagent In vitro studies isolated target and murine cell systems Human therapeutic In vitro studies isolated target and human cell systems Furry test tube Furry test tube? Primary model Hu-Mouse transgenic? Single dose PK Human explant studies? Medium term secondary models Primate studies Multidose PK Clinical trial Tertiary disease model Animal models: considerations for use and interpretation of data • Ethical concerns: When to use. Distress scoring. • Legal: Licensing issues. • Practical concerns: Is this a good experiment? • Statistical concerns: Group sizes. Most appropriate analysis. What to do with data from culled animals. Effects of compound on plasma TNF 90 mins after challenge with LPS 30 Plasma TNF ng/ml 20 10 * ** 0 0 3 10 30 mg/kg p.o. Paw oedema •Sub plantar injection of irritant eg carrageenan •Reaction usually maximal by 3 hours •Readily quantified by plethysmometry Disadvantages •Not an arthritis •Not amenable to histological or biochemical assay •End point is crude •Very popular in industry! Measurement of paw oedema using plethysmometry Results expressed as paw volume (ml) or change in paw volume (Δml) Cavity models •Peritonitis •Pleurisy •Air pouches Advantages •Exudate volume •Total and differential cell counts •Amenable to biochemistry Effects of experimental compound on exudate volume recovered from a 4 hour carrageenan pleurisy in the rat Exudate volume(ml) 0.75 Vehicle= 10% DMSO and 90% Labrafil Drugs were administered 30 mins before carrageenan injection 0.50 ** *** 0.25 *** 0.00 Vehicle 0.1 1 10 +1%carrageenan (mg/kg) Means  s.e.m., n=8/9 per treated group, n=4 in untreated group Statistical analysis: One way ANOVA followed by a Bonferroni's multiple comparisons test ** p<0.01, ***p<0.001comparison with vehicle control Effects of experimental compound on total cells recovered from a 4 hour carrageenan pleurisy in the rat 20 Total cells (x10 ) * *** 10 Vehicle= 10% DMSO and 90% Labrafil Drugs were administered 30 mins before carrageenan injection 6 *** *** 0 Vehicle 0.1 1 10 +1%carrageenan (mg/kg) Means  s.e.m., n=8/9 for treated groups, n=4 in untreated group Statistical analysis: One way ANOVA followed by a Bonferroni's multiple comparisons test p<0.05, ***p<0.001comparison with vehicle control Air pouches •No interfering visceral organs •Pouch lining cells have similarities to synovial cells •Prolonged inflammation can be induced Disadvantages •Preformed cavities (6-day-old-pouches) give best results Accumulation of cells in a murine 6-day-old air pouch in response to LPS 10 6 Total cells x10 5 0 0.000 0.025 0.050 0.075 0.100 0.125 LPS mg/air pouch Manipulation of cavity models •Non-immune models -complement dependent -complement independent •Immune models -active (sensitisation and challenge) -passive (adoptive transfer of serum or cells) Models of arthritis •Matrix degradation •Polyarthritis -Adjuvant arthritis -Collagen II arthritis •Monoarticular arthritis -non-immune -Immune Collagen induced arthritis Similarities to RA • Susceptibility linked to MHC • immunologically mediated • Erosions of cartilage & bone • Autoimmunity to collagen seen in some RA patients • Arthritic rather than multisystem Differences to RA • Disease is induced • The model “resolves” • New bone formation more marked • No synovial vasculitis • No cycles of relapse & remission • No RF • No sex predilection Collagen induced arthritis •Animals (rats or mice) sensitised to collagen ll in adjuvant •Booster injection (mice) •Distress scoring •Examine for clinical signs •Measurement of hind paw volume (rats) •Histology Assessment of CIA Clinical assessment Disease score Body weight Analgesia Drug specific Plasma levels Bioactivity markers Joint pathology Histology Radiographs Serum markers of disease progression Acute phase protein COMP Bone sialoprotein Immunology CII Ab Ex vivo lymphocyte proliferation Hypersensitivity to CII Clinical presentation of murine CIA Score 0 1 2 3 Pictures Remi Okoye NORMAL WRIST SWOLLEN WRIST + PAD SWOLLEN WRIST + PAD + DIGITS SWOLLEN Effects of an experimental compound on rat CIA Paw volumes Change in paw volumes AUC Days 14-24 AUC Dpaw volume (Days 14-24) 3 1.4 Paw volume (ml) 1.3 1.2 Vehicle 0.03mg/kg 0.3mg/kg 3mg/kg 10mg/kg 2 Means  s.e.m., n=10/group Statistical analysis ANOVA With Bonferroni post test **p<0.01 vs Vehicle 1.1 1 ** ** 0 1.0 0.9 0 5 10 15 20 25 Vehicle 0.03 0.3 3 10 Days post sensitisation mg/kg u.i.d. Effects of leflunomide on murine CIA 8 Clinical score 6 Vehicle Leflunomide 4 ** 2 0 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Animals dosed day -1 Leflunomide 3mg/kg PO once daily Vehicle = 1% Methyl Cellulose DBA/1 mice N = 15 Day P= <0.05 Day 31/32 * P= <0.01 Day 30/33-36** Dosing strategies in CIA Therapeutic dosing Clinical score Dosing through sensitisation period Sensitisation Boost Time Prophylactic dosing CIA Histopathology Score 3 0 1 2 3 normal inflammatory cell influx, cell influx & focal erosion cartilage & bone loss of joint architecture Score 0 X-ray changes in CIA CLINICAL SCORE = 0 CLINICAL SCORE = 3 Serological markers • Anticollagen II antibodies and their isotypes • Acute phase proteins • Cartilage oligomeric matrix protein (COMP) • Bone sialoprotein Summary • There remains an unmet need in the treatment of RA • In vivo models of inflammation are an essential part of drug discovery • The strategy for NCEs and NBEs is different • Efficacy in models helps define species for toxicology • Models can help with calculation of therapeutic dose in man and with identifying surrogate markers of drug activity for use in the clinic