In Vivo Animal Studies

The single- and repeat-dose studies were conducted in Sprague–Dawley (Crl:CD^®[SD]) rats (Charles River Laboratories). All rats were acclimated to the laboratory environment for a minimum of 14 (single-dose study) or 13 (repeat-dose study) days prior to initiation of dosing. The IV route was chosen because it is consistent with the intended clinical route of administration and was used during the nonclinical program of infliximab. Toxicokinetic (TK) parameters were calculated from individual animal data using noncompartmental analysis (Watson LIMS, version 7.4.1; Thermo Inc). TK parameters included C _max, time to C _max, and AUC. Samples tested below the limit of quantification (<0.1 μg/mL in 100% serum) were assigned a value of 0 μg/mL for the TK calculations.

The objective of this study was to determine the TK and tolerability of PF-06438179 compared with infliximab-EU when administered as a single dose to male rats (aged 9–10 weeks and weight at dose initiation, 337.9–392.6 g). PF-06438179 or infliximab-EU was administered by single IV bolus injection (volume of 5 mL/kg) to male rats (n = 5/group) at doses of 10 or 50 mg/kg. A separate group of five male rats received vehicle #1, and another group of five male rats received vehicle #2. Vehicles #1 and #2 consisted of excipients and buffers identical to those used with PF-06438179 and infliximab-EU, respectively, but with no active protein.

Following dose administration, the animals were retained for an 8-week observation and TK and anti-drug antibodies (ADA) sampling period. Clinical signs were assessed daily, and body weights and quantitative assessment of food consumption were recorded weekly through to day 57. For the TK comparison of PF-06438179 (vehicle #1 and PF-06438179 groups) versus infliximab-EU (vehicle #2 and infliximab-EU groups), approximately 0.25–0.55 mL of blood was collected from the jugular vein of each rat prior to dosing, and at 0.5, 4, 8, 24, 48, 96, 168, 336, 672, 1008, and 1344 h following the first and only dose (administered on day 1). The samples collected prior to dosing and at 1008 and 1344 h after dosing were also used for detection of serum ADA.

Serum PF-06438179 or infliximab-EU concentrations were determined using a validated enzyme-linked immunosorbent assay (ELISA). PF-06438179 or infliximab-EU was captured using immobilized recombinant human TNF adsorbed on a microtiter sample plate. Bound PF-06438179 or infliximab-EU was detected using a donkey anti-human IgG antibody conjugated with horseradish peroxidase and a 3,3′,5,5′-tetramethylbenzidine peroxidase substrate solution to generate a colorimetric readout. Sample concentrations were determined by interpolation from a calibration curve generated using a 4PL curve-fitting program. Area under the concentration–time curve, AUC at 1344 h and extrapolated to infinity (AUC₁₃₄₄ and AUC_inf, respectively), was estimated using the linear trapezoidal rule.

Serum samples were analyzed for anti-PF-06438179 or anti-infliximab-EU antibodies using electro-chemiluminescent assays validated on the Meso Scale Discovery (MSD^®) assay platform. Affinity-purified rabbit anti-infliximab-EU IgG was the positive control, and pooled normal SD rat serum was the negative control. Test samples were first incubated with biotinylated and ruthenylated PF-06438179 or infliximab-EU. Aliquots were added to the wells of a streptavidin-coated microtiter plate and, following a brief wash, tripropylamine-containing detection buffer was added; the resulting chemiluminescent signal that was proportional to the amount of ADA present was measured. Samples were tested using a tiered approach of screening and titer. The final assessment of induction of an ADA response for each animal was based on the comparison of the pre-dose and post-dose sample results.

The objective of this study was to determine the toxicity and TK of PF-06438179 following administration of 10 or 50 mg/kg (with a volume of 5 mL/kg) by IV injection once weekly (days 1, 8, and 15) for 2 weeks to male and female rats (aged 9 weeks and weight at dose initiation: male, 289.3–341.7 g; female, 203.7–252.4 g). In the toxicity arm of the study (n = 10 per sex/group), doses were administered on days 1, 8, and 15; in the TK arm (test article-dosed group, n = 4 per sex; control group, n = 3 per sex), doses were administered on days 1 and 8. Vehicle consisted of excipients and buffer, but no active protein.

Rats were monitored for clinical signs daily, and for changes in body weight, and food consumption, weekly, through day 15. Ophthalmic examinations were performed on the animals in the toxicity arm prior to the initiation of dosing (baseline) and on day 13. Hematology, coagulation, clinical chemistry parameters, and urinalysis were evaluated on day 16, just prior to necropsy on this day, which included full tissue collection, evaluations of organ weights, and macroscopic and microscopic observations. Clinical and anatomic pathology findings were peer reviewed. For the TK analysis in satellite animals, approximately 0.30–0.55 mL of blood was collected from each rat 5 days prior to the initiation of dosing (day −5) and at 1, 7, 24, 72, 120, and 168 h post-dose on days 1 and 8. In the vehicle control group, only the 1-, 7-, and 24-h post-dose samples were analyzed. Serum samples were analyzed for PF-06438179 using a validated ELISA assay and analysis methods as described above. AUC₁₆₈ was estimated using the linear trapezoidal rule. For calculations of day 8 AUC₁₆₈, the serum concentration of PF-06438179 at time 0 was set to the day 1, 168-h post-dose concentration.