Two compartment pharmacokinetic model describes the intra-articular delivery and retention of rhprg4 following ACL transection in the Yucatan mini pig

Abstract

Treatment of the injured joint with rhPRG4 is based on recent observations that inflammation diminishes expression of native PRG4. Re-establishing lubrication between pressurized and sliding cartilage surfaces during locomotion promotes the nascent expression of PRG4 and thus intra-articular (IA) treatment strategies should be supported by pharmacokinetic evidence establishing the residence time of rhPRG4. A total of 21 Yucatan minipigs weighing ∼55 kg each received 4 mg of ¹³¹ I-rhPRG4 delivered by IA injection 5 days following surgical ACL transection. Animals were sequentially euthanized following IA rhPRG4 at 10 min (time zero), 24, 72 h, 6, 13 and 20 days later. The decay of the ¹³¹ I-rhPRG4 was measured relative to a non-injected aliquot and normalized to the weight of cartilage samples, menisci and synovium, and known cartilage volumes from each compartment surface obtained from representative Yucatan minipig knees. Decay of ¹³¹ I-rhPRG4 from joint tissues best fit a two-compartment model with an α half-life (t_1/2α ) of 11.28 h and β half-life (t_1/2β ) of 4.81 days. The tibial and femoral cartilage, meniscii, and synovium retained 7.7% of dose at 24 h. High concentrations of rhPRG4 were found in synovial fluid (SF) that was non-aspiratable and resided on the articular surfaces, removable by irrigation, at 10 min following ¹³¹ I-rhPRG4 injection. Synovial fluid K21 exceeded K12 and SF t_1/2β was 28 days indicating SF is the reservoir for rhPRG4 following IA injection. Clinical Significance: rhPRG4 following IA delivery in a traumatized joint populates articular surfaces for a considerable period and may promote the native expression of PRG4. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:386-396, 2019.

Keywords: PRG4; cartilage; half-life; lubricin; pharmacokinetics.

Figure 1.

Timeline of Yucatan mini-pig pharmacokinetic (PK) study of intra-articular (IA) I¹³¹-rhPRG4 at Day 0 (D0) following ACL transection 5 days prior (D-5). The remaining number of porcine following each tissue harvest time point is illustrated. The first harvest involving five animals occurred at 10 min following IA (D0 + 10 m) which served as time zero in PK calculations. Sequential tissue harvests following IA were conducted at 24 h (D1), 72 h (D3), 6 days (D6), 13 days (D13), and 20 days (D20). At each time point synovium and cartilage collected from each joint compartment and both condyles were weighed and counted for radioactivity across 21 animals. Samples of SF, blood, liver, and proximal lymph node were also collected and counted. Three animals were removed from the analysis as they did not receive IA injection correctly and demonstrated very high lymph node radioactivity.

Figure 2.

Cartilage surface area and volume analysis by microCT. Four previously frozen unoperated Yucatan mini-pig left knee joints served as controls to determine the surface area and volume of each cartilage surface from the patellofemoral joint and both compartments of the tibiofemoral joint. The product of the tissue density multiplied by the representative cartilage volume from each area was used as the tissue mass denominator in normalizing gamma counts per tissue weight to determine the total amount of I¹³¹rhPRG4 resident in each cartilage surface. Mean ± SD is reported.

Figure 3.

Least squares fitting model and Akaike Information Criterion (AIC) of the percent of total dose of I¹³¹-rhPRG4 in recovered knee tissue counts including lavage and aspirated SF across time points at 10 min, days 1, 3, 6, 13, and 20 for 18 porcine following intra-articular I¹³¹-rhPRG4 delivered 5 days after ACL transection. (A) 98% of the total dose at 10 min (day 0) was accounted for across the 6 time points by least squares elimination profile which did not conform to a predicted AIC two-compartment model. (B) 33% of the total dose at 10 min was accounted for by considering only knee tissue counts (excluding lavage and aspirated SF) which conformed to an AIC two-compartment model. (D) Akaike Information Criterion parameter tables showing AIC t_1/2 estimates in days, and coefficient of variation (CV%). Error bars are mean ± SE; N = 3 at each sampling time.

Figure 4.

Knee joint tissue concentrations of I¹³¹-rhPRG4 at 10 min (day 0), days 1 and 3 (A), and days 6, 13, and 20 (B). Tibial cartilage and synovial membrane surfaces appeared to contain relatively larger amounts of I¹³¹-rhPRG4 at 10 min and remained greater by days 1, 3, and 6 for synovial surfaces. By day 13 and 20, cartilage surfaces showed greater relative amounts of I¹³¹-rhPRG4. Abbreviations: MFC, Medial femoral condyle; MTP, medial tibial plateau; LFC, lateral femoral condyle; LTP, lateral tibial plateau; MM, medial meniscus; LM, lateral meniscus; SMAC, synovial membrane anterior compartment, and SMPC, synovial membrane posterior compartment. Error bars are mean SE; N = 3 at each sampling time.

Figure 5.

Percent of recovered total knee I¹³¹-rhPRG4 across time points at 10 min (day 0), days 1, 3, 6, 13, and 20 in 18 porcine following intra-articular I¹³¹-rhPRG4 delivered 5 days after ACL transection. (A) Comparison of total femoral and tibial cartilage and (B) total medial and lateral cartilage. Initially I¹³¹-rhPRG4 localized more to the medial joint compartment and tibial cartilage surfaces. Toward the end of the concentration-time profiles studied at Day 20, the lateral joint compartment and femoral cartilage retained a greater relative percentage of recoverable I¹³¹-rhPRG4. Error bars are mean SE; N =3 at each sampling time.

Figure 6.

Densitometry of rhPRG4 (MW 300 kDa) and native porcine PRG4 (MW 180 kDa) bands on Western blots of joint surface lavage in 12 of the 18 porcine following intra-articular I¹³¹-rhPRG4 delivered 5 days after ACL transection. (A) Samples at 10 min (day 0) (N = 3),1 (N = 3), 3 (N = 2), 6 (N = 1), 13 (N = 2), and 20 (N = 1) were analyzed on SDS-PAGE and transferred to nitrocellulose 4 months following recovery to allow sufficient radioactive decay. Blots were probed with polyclonal antibody 1752 and densitometry was performed in triplicate and normalized against a 10 mg/ml preparation of rhPRG4. (B) rhPRG4 decreased over time and was detectable up to 13 days later, whereas porcine PRG4 increased over that same interval. Error bars are mean SD.

Figure 7.

Immunohistochemistry of total PRG4 comprising injected rhPRG4 and native PRG4 across representative porcine at 10 min (Day 0), Days 1, 3, 6, 13, and 20 following intra-articular I¹³¹-rhPRG4 delivered 5 days after ACL transection in 18 porcine. Cartilage surface immunoprobing with monoclonal antibody 9G3 was counterstained with DAPI in sections from the two apposing surfaces from medial and lateral joint compartments. Cartilage surface staining was qualitatively most intense at Days 1 and 3, and least intense by Day 20 which is similar to untreated and historical control porcine cartilage. Chondrocyte positivity for PRG4 (white arrows) appeared most intense at Day 6 and may be indicative of renewed native expression of porcine PRG4.