ADAM10 and ADAM17 assays. Both assays were performed as published previously by us (19) Briefly, both assays followed the same general protocol. 5 µL of 2x enzyme solution (20 nM) in assay buffer (10 mM Hepes, 0.001% Brij-35, pH 7.5) were added to solid bottom black 384 plates (Nunc, cat# 264705). Next, test compounds and pharmacological controls were added to corresponding wells using a 384-pin tool device (V&P Scientific, San Diego). After 60 min incubation at RT, the reactions were started by addition of 5 µL of 2x solutions of glycosylated substrate DM2 (20 µM). Reactions were incubated at RT for 2 h, after which the fluorescence was measured using Biotek H1 multimode microplate imager (λexcitation = 360 nm, λemission = 460 nm). IC50 values were calculated by fitting normalized data to sigmoidal log vs. response equation utilizing non-linear regression analysis from GraphPad Prizm 6.
Pharmacokinetics of CID 3117694. All animal studies were conducted in accordance with NSU IACUC guidelines. Study protocol # 2021.04.DM2 was approved by NSU IACUC before the commencement of the studies. CID 3117694 was formulated in 10% DMSO, 40% PEG400, 30% PG, 20% H2O at 2 mg/mL and administered by intragastric (ig) gavage to Sprague-Dawley male rats at 10mg/kg. The animals were anesthetized by isoflurane. Blood and synovial fluid were collected at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h after administration of CID 3117694. Synovial fluid (SF) was collected using the 23-gauge needle connected to a mini peristaltic pump via perfusion tubing. Sterile saline was infused at a constant rate of 100 µl/min − 1. After infusion of 100 µl of vehicle (sterile saline), the outflow tubing was connected to the 25-gauge needle, to minimize pressure build-up within the joint space. Fluid was infused and withdrawn at a constant rate until a 250 µl basal sample was collected in a 1.5 ml centrifuge tube. Samples were immediately frozen at -20°C. Blood was collected using 25G needle directly from the hearts. The samples were prepared for LC-MS/MS analysis using acetonitrile protein precipitation technique. Briefly, blank plasma and subject samples (plasma and SF samples) were retrieved from the deep freezer and allowed to thaw. The calibration curve standards (10 to 10,000 ug/mL of CID 3117694) and Quality Control samples were prepared using blank plasma. The thawed samples were vortexed to ensure complete mixing of contents. 50 µL of each sample were transferred to the vials and 200 µL of Internal Standard (IS, telmisartan) mixed with acetonitrile were added to all the samples and vortexed. Samples were kept on the shaker for 5 min to ensure complete mixing of contents. The samples were centrifuged at 4,000 rpm at 20°C for 10 min and the supernatant was transferred into auto injector vials and loaded into auto sampler. 5 µL was injected on to API-4500 Q TRAP LC-MS/MS system. The samples were separated on Phenomenex, Synergi C18 4.6 × 50 mm,4 µm HPLC column using 10mM ammonium acetate + 0.1% Formic acid buffer as solvent A and 100% Acetonitrile as solvent B. PK parameters were quantitated using WinNonLin (Certara, Princeton, NJ).
In vivo efficacy in the CIA mouse model. All animal studies were conducted in accordance with NSU IACUC guidelines. Study protocol # 2021.04.DM5 was approved by NSU IACUC before the commencement of the studies. CID 3117694 and indomethacin were ground with a small amount of the dispersing agent (0.5% HPMC (3 cP)–0.5% Tween 80), using a mortar and a pestle, and mixed repeatedly to form a smooth paste. The paste was moved to a graduated cylinder and filled up to the required volume with the dispersing agent to form a suspension as described elsewhere (20). Test compound (10, 30, & 50 mg/kg) and Indomethacin (2.5mg/kg) were administered with mouse gavage needle (gauge no: 18, BD, catalog number: 653902) through oral route as suspension with 5 mL/kg dose volume (from day 0 to day 28).
6-8-week-old C57 BL/6 mice from Charles River (male and female) were used in the study. Animals were randomly assigned to six groups: (1) normal (non-RA control), (2) RA control untreated, (3) RA control treated with 2.5 mg/kg/day indomethacin, and 3 test groups (CID 3117694 (10mg/kg/day, 30mg/kg/day, and 50mg/kg/day)) to provide an estimate for lead compound’s efficacy range. Each group had three males and three females assigned to control for gender-based differences.
Arthritis was induced to right paw of the mice under anaesthesia. Left paw of the mice was used as a control. A total of 10 µl collagen from bovine (type II collagen) nasal septum emulsion (2 mg/ml) was injected into the right knee joint with glass Hamilton syringe and 25G needles for intraarticular (i.a.) injection, followed by a total of 100 µl Complete Freund’s adjuvant (CFA) emulsion injected at the back of mice using the syringe with 25G needles subcutaneously (s.c.). On day 14 after the first injection, the mice were injected i. a. with 10 µl of collagen from the bovine nasal septum (2 mg/kg) and s.c. with 100 µl CFA emulsion for boost injection.
Arthritis progression was monitored using a clinical arthritis scoring system (0–4) and paw swelling was measured with help of a plethysmograph on day 0, 14, 21 and 28 after induction of arthritis. To assign a clinical score, interphalangeal, metacarpophalangeal, and carpal and tarsal joints were observed for swelling and redness. Normal joints were assigned score 0, in case when only one joint type was affected the score was 1, in case when two joint types were affected the score was 2, in case when three joint types were affected the score was 3, maximal redness and swelling of the entire paw with the loss of anatomic definition resulted in a score of 4.
After administration of primary dose of collagen from the bovine nasal septum and CFA scoring was assessed. Measurements were taken on day 0, 14, 21 and 28 after induction of arthritis in induced paws (right paw) and non-arthritis paws (left paw). CID 3117694 (10 mg/kg/day, 30 mg/kg/day, and 50 mg/kg/day) and pharmacological control (indomethacin 2.5 mg/kg/day) treatment was given every day for 28 days beginning from day 1 by oral gavage in hydroxy-propyl-methyl cellulose (HPMC) suspension.
Mice were weighed and observed every day and notes were made on the signs of distress. More specifically, mice were observed for posture, vocalization, ease of handling, lacrimation, chromodacryorrhea, salivation, coat condition, unsupported rearing, arousal, piloerection, motor movements, diarrhea, tail pinch reaction, constipation, and death.
The sample size for therapeutic evaluation was calculated by power analysis based on experimental data from similar studies. In a two-sided test and setting α value at 0.05 and desired power at 0.95, the sample size turned out to be 5 per group. We also expected deaths during experiments; therefore, we used the formula, Nt = N/1-q, where Nt is the number of mice initially used, N is the number required at the end of the study and q is the proportion of attrition which is generally 10%. Thus, at the end of the study period we expected to have data from at least 6 mice per group which is enough to attain statistical significance, if exists. Data was analyzed using a one-way ANOVA followed by Dunnett's test compared with Vehicle Control. At the end of the study, the mice were anesthetized and sacrificed by CO2 euthanasia. The hind paws of the mice were subjected to histopathological examination and the serum was analyzed for CRP, TNF-α, IL-6, and IL-10 as described in below sections.
All the data was expressed as mean ± SEM and statistically analyzed by IBM-SPSS version 20 software using one-way ANOVA followed by post hoc Dunnett-t test at different variance levels. A p-value of 0.05 or less was considered statistically significant.
RA serum marker analysis.
Blood was collected from the tail vein, 100 µL was used for the assays. All assays were conducted using manufacturer’s instructions for the respective assay kits. The following assay kits were purchased: Mouse TNF-α GENLISA™ ELISA (Krishgen BioSystems cat# KB2145), Mouse IL-10 GENLISA™ ELISA (Krishgen BioSystems cat# KB2072), Mouse hsC-Reactive Protein, hs-CRP GENLISA™ ELISA (Krishgen BioSystems cat# KLM0318), Mouse IL-6 GENLISA™ ELISA (Krishgen BioSystems cat# KB2068).
Histology.
Limbs from euthanized animals were preserved in buffered formalin, decalcified, embedded, sectioned, and stained with hematoxylin and eosin (H & E). Microscopic images were acquired with Lx 400 microscope (Labomed, Fremont, CA).