Lucas A. Mutch

Successful implementation of cooperative handling eliminates the need for restraint in a complex non-human primate disease model.

Background  Streptozotocin‐induced diabetic non‐human primates are used to study efficacy and safety of innovative immunosuppression after islet transplantation. We implemented a training program for medical management of a chronic disease state.

Refining the high-dose streptozotocin-induced diabetic non-human primate model: an evaluation of risk factors and outcomes.

In preparation for islet transplantation, diabetes was induced using streptozotocin (STZ) in non-human primates ranging from juveniles to adults with diverse body types: we studied the process with respect to the diabetic state and emergence of adverse events (AEs) and their severity, and identified risk factors for clinical and laboratory AEs. Pharmaceutical-grade STZ was given based on body surface area (BSA) (1050–1250 mg/m2, equivalent to 80–108 mg/kg) or on body weight (BW) (100 mg/kg) to 54 cynomolgus and 24 rhesus macaques. AEs were related to risk factors, i.e. obesity parameters, BW and BSA, age and STZ dose in mg/m2. Clinical AEs during the first days after infusion prompted euthanasia of three animals. Except for those three animals, diabetes was successfully induced as shown by circulating C-peptide levels, the intravenous glucose tolerance test and/or arginine stimulation test. C-peptide after infusion weakly correlated (P = 0.048) with STZ dose in mg/m2. Grade ≥3 nephrotoxicity or hepatotoxicity (serum markers >3× baseline or >5 × baseline, respectively) occurred in about 10% of cases and were generally mild and reversible. Grade ≥2 clinical AEs occurred in seven of 78 animals, reversed in four cases and significantly correlated with obesity parameters. Taking girth-to-height ratio (GHtR) as an indicator of obesity, with threshold value 0.92–0.95, the positive predictive value of obesity for AEs was 92% and the specificity 94%. We conclude that diabetes is successfully induced in non-obese animals using a 100 mg/kg pharmaceutical grade STZ dose. Obesity is a significant risk factor, and animals with a higher than normal GHtR should preferably receive a lower dose. The incidence of relevant clinical or laboratory AEs is low. Careful monitoring and supportive medical intervention can result in recovery of AEs.

A novel alternative placement site and technique for totally implantable vascular access ports in non-human primates

Background  Two novel approaches to implanting a central venous catheter port in non‐human primates (NHPs) using peripheral insertion are presented and compared.

Evaluation of commercial ELISA and RIA for measuring porcine C-peptide: implications for research.

Pre‐clinical demonstration of porcine islet graft function is necessary to support the clinical transplantation of pig islets. C‐peptide concentration is an especially useful marker of insulin secretion, because its measurement is not confounded by the presence of exogenous insulin. To measure porcine C‐peptide (PCP), researchers in the field exclusively used the Millipore (previously Linco Research) radioimmunoassay (RIA) until 2011, when Mercodia released an alternative enzyme‐linked immunosorbent assay (ELISA). (At the end of 2013, the Millipore RIA was withdrawn from the market for commercial reasons.) In our current study, to directly compare these two assays, we performed validation studies on each. We also performed interlaboratory comparisons. Then, to determine the level of agreement between the assays, we analyzed the porcine serum C‐peptide concentration measurement results obtained from each assay.

Management of adverse side-effects after chemotherapy in macaques as exemplified by streptozotocin: case studies and recommendations

The chemotherapeutic streptozotocin is used for induction of diabetes in animal models including non-human primates. Being a cytotoxic nitrosourea compound, it can be associated with adverse events (AEs), mainly nausea and emesis, nephrotoxicity, elevated liver transaminase levels, pulmonary oedema and, most prominently, metabolic acidosis: these can be severe in some cases. The incidence and gravity are to some extent related to the characteristics of the individual animal, diagnostic tools, prompt recognition of symptoms and supportive measures. Careful animal selection, dose adaptation and supportive actions such as renal protective hydration are the main tools in managing AEs, but do not fully eliminate unavoidable and sometimes life-threatening conditions. In our centre we have built experience in a cohort of 78 cynomolgus and rhesus macaques in which six cases manifested severe AEs (8%). This experience has prompted implementation of strategies for early detection and management of adverse effects, together with an animal refinement programme. We present here specific pretreatment regimens, post-infusion laboratory evaluations, and flow charts to assess/treat metabolic acidosis and precipitating factors. Case reports of the six animals with severe AEs are presented to illustrate management of AEs, especially metabolic acidosis, and criteria for early euthanasia where appropriate. We conclude that improved monitoring and validated tools allow for optimal management of adverse effects in an early stage of their manifestation. Reduced morbidity and mortality not only improve individual animal wellbeing but also avoid model-induced confounding that diminishes the translational value of the experimental protocol.

Long-Term Hepatic Vascular Access in the Nonhuman Primate for Recurrent Portal Vein Infusion

ABSTRACT Islet cell transplantation in nonhuman primates is generally performed in the liver, by infusion of the transplant into the portal vein. We introduced a vascular access port with the catheter tip located in the splenic vein to avoid multiple major survival surgeries. This procedure was conducted in 16 cynomolgus and 9 rhesus macaques. A subset underwent islet cell transplantation. A historic control group (n = 17) received the transplant via open midline laparotomy. The groups did not differ in operation time (median about 60 min): however, animals undergoing midline laparotomy required significantly more opioid pain relief postoperatively than animals implanted with a hepatic vascular access port. Animals after port placement and transplantation had significantly higher blood hemoglobin values than those in the control group, but these values were still in the normal range. In addition to transplantation, the port could be used for administration of biologics and for blood sampling. In all cases, the port remained patent for infusion purposes (median follow-up 336 days, range 62–485 days). Patency for blood sampling was maintained in about half of the animals: the 50% survival of patency for sampling was 255 days. This difference between infusion and sampling patency is most likely due to the location of the catheter tip in the splenic vein, with occlusion caused by the small vessel-to-catheter ratio. We conclude that hepatic vascular access enables long-term frequent administration of cells, medication, or other products and also serves to sample blood: hence, this procedure contributes to a higher level of animals well-being.

Cross-validation of commercial enzyme-linked immunosorbent assay and radioimmunoassay for porcine C-peptide concentration measurements in non-human primate serum

C‐peptide concentration is widely used as a marker of insulin secretion and is especially relevant in evaluating islet graft function following transplantation, because its measurement is not confounded by the presence of exogenous insulin. To address the shortage of human islet donors, the use of porcine islets has been proposed as a possible solution and the stringent pig‐to‐non‐human primate (NHP) model is often the most relevant for pre‐clinical evaluation of the potential for diabetes reversal resulting from an islet xenograft. The Millipore radioimmunoassay (RIA) was exclusively used to measure porcine C‐peptide (PCP) until 2013 when the assay was discontinued and subsequently a commercially available enzyme‐linked immunosorbent assay (ELISA) from Mercodia has been widely adopted. Both assays have been used in pre‐clinical trials evaluating the therapeutic potential of xenograft products in reversing diabetes in the pig‐to‐NHP model, to interpret data in a comparable way it may be useful to perform a harmonization of C‐peptide measurements.