AbstractNumerous pharmaceuticals and recently an increasing list of monoclonal antibodies undergoing regulatory preclinical trials in nonhuman primates are administered intravenously, often by continuous infusion. In consideration of “Refinement” in the 3 Rs, an existing infusion technique was required to be transferred to a new laboratory setting. During a pre-clinical safety assessment study, one naïve control monkey of each sex (Macaca fasicularis, 2.9-3.9 kg, Mauritius) underwent port catheter surgery. The subcutaneous port (PortHold™ and accompanying needles, Access Technologies, USA) was implanted with the tip being inserted in the vena cava caudalis. Dosing [Model 500 V Pump (Pegasus® Orchesta Infusion)] commenced following a 4-week recovery, system flushing with NaCl [incl. a heparin block (10 IU/mL), 0.69 mL dead space], jacket training, pump precision test and pre-study diagnostics. The animals were free-ranging (max. 20% back pack vs body weight ratio) administered continuously for 24 hours and were recorded with stable body weights throughout the training and dosing period. Housing and dosing was conducted under ETS123 conditions1 and clinical signs during the recovery period were limited to minimal swollen inguinal areas. Post-surgery week 4 clinical pathology proved the absence of inflammatory processes before start of dosing. Port patency was given for the complete duration of the study and great care was implemented to conduct port-needle changes under aseptic conditions.

IntroductionNumerous pharmaceuticals and recently an increasing number of monoclonal antibodies (related to implementation of the ICH S6 guideline “Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals”), as well as antibiotics (Korte 2014) are undergoing regulatory preclinical trials in nonhuman primates (NHP) being intravenously administered, often by continuous 24 hours or longer infusion.

Materials and MethodsSafe Harbor Statement

Prior to conduct of the study, the study protocols were reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and all study tasks were performed in accordance with facility Standard Operating Procedures (SOPs).

The animals were housed in pens that conform to the ‘Code of practice for the housing and care of animals used in scientific procedures’ (Home Office, London, 2014) and in compliance with ETS 123, 2007.

Animals (2.9-3.9 kg and 2-3 years from Mauritius) were surgically prepared (McNamee 1984, Healing 2000) approximately three weeks before the commencement of any pre-treatment procedures. Prior to surgery animals were satisfactorily acclimatised to the jackets and assessed as behaviourally acceptable to use for study.

Animals were fasted overnight prior to anaesthesia and were anaesthetised with ketamine 10 mg/kg intramuscularly followed by medetomidine [Domitor® (Orion Corp.)] 80 µg/kg intravenously. An endotracheal tube was placed in the trachea. Oxygen was provided via a T-piece breathing circuit at a nominal fresh gas flow rate of 2 litres/minute. Anaesthesia was supplemented with isoflurane as necessary. Animals were placed on a Bair Hugger™ (3M Company) warm air heating blanket while anaesthetised. Heart rate and tissue oxygen saturation was monitored by pulse oximetry in accordance with good veterinary anaesthetic practices. Following induction of anaesthesia but prior to surgery the following was administered: carprofen 4 mg/kg intravenously, buprenorphine 10 µg/kg intramuscularly and cefuroxime 15 mg/kg intravenously. Hartmann’s solution was administered at a nominal rate of 10 mL/kg/hr via a cannula placed in a suitable peripheral vein (cephalic or saphenous). Surgery was carried out using rigorous aseptic techniques and good operating theatre practice in accordance with relevant Covance standard operating procedures. A vascular access port (VAP) was implanted subcutaneously in a left paramedian location caudal to the shoulder. The VAP was anchored to underlying musculature using 2/0 Prolene® (Johnson & Johnson, Corp.). A catheter was inserted into the vena cava caudalis, via an incision in the left femoral vein, and tunnelled subcutaneously to the VAP. The femoral vein was ligated and the catheter anchored using 4/0 Ethibond™ (Johnson & Johnson, Corp.). Fascial planes and subcutaneous tissues was closed with continuous 4/0 Vicryl sutures. Skin wounds were closed using a continuous subcuticular suture of 4/0 Vicryl. Following completion of surgery, anaesthesia was reversed with atipamezole [Antisedan® (Orion Corp.)] 400 µg/kg intravenously. Animals were kept on oxygen and warmth until recovery and then they were individually housed for 10 days following surgery. For analgesia meloxicam [Metacam® (Boehringer Ingelheim Vetmedica GmbH)] was administered orally, 0.1 mg/kg once daily for 5 days and for antibiosis amoxicillin and clavulanate [Synulox™ (Beecham, Inc.)] were given orally at 50 mg twice daily for 5 days. Surgery sites (inguinal and dorsal) were inspected daily, whilst the catheter was flushed approximately twice weekly with sterile 0.9% saline containing heparin (10 IU/mL).

Dosing was conducted with free-ranging animals (max. 20% backpack vs body weight ratio, Figure 1) administered continuously for 24 hours (1 day) with Model 500 V Pump (Pegasus®), fitted into a backpack.

#1618 Emerging Biologics Development Methods: Free-Ranging Cynomolgus Monkeys Undergoing Continuous Intravenous Infusion with an Implanted Port Catheter System–A Method Transfer in Courtesy to the “Refinement” in the 3 RsC. Miller1, S. Korte2, A. Salva1, N. Snippe2, F. Runge2 and H. vanWijk1 : 1Covance Laboratories, Harrogate, UK; 2Covance Preclinical Services, GmbH, Münster, GermanySponsor: Sven Korte

Presented at Society of Toxicology 2016

ResultsThe following problems occur often when free-ranging infusion is implemented without a knowledge transfer: (a) extraction of the catheter from the vein, (b) necrosis at the port area and (c) needle loss. These were primarily resolved by (a) improved animal handling, (b) closer fixation of jacket to the body and (c) advanced taping down of the needle. During study conduct, body weight development was normal. If a non-test-item-related reduction was noted, enriched feeding was offered to support single animals requiring more nutrition when carrying the weight of the backpack system. Clinical signs during the recovery period (post-surgery) were limited to minimal swollen inguinal areas. Post-surgery week 4 clinical pathology proved the absence of inflammatory processes before start of dosing. Port patency was archived for the complete duration of the study and great care was implemented to conduct port-needle changes under aseptic conditions.

Frequent background histopathological lesions observed in the vessels at the tip of catheters (application site) included: hypertrophy/hyperplasia of the intima (Figure 2a), inflammation of the vascular wall (Figure 2b), thrombi (Figure 2c) and partly organized thrombi (Figure 2d). At the port region (entrance of the catheter into the skin) a granulomatous reaction around suture material was a frequent finding (Figure 2e).

ConclusionsThe successfully transferred infusion technique for free-ranging cynomolgus monkeys is an exceptional example of implementing the 3 Rs in regulatory toxicology, now allowing the conduct of further dose regimens in the predominant nonhuman primate species for the preclinical safety assessment of biologics and small molecules. The outcome of this study also (in the future) will support an ethical approval, required when studies of longer duration or using more animals shall be required.

ReferencesETS 123, 2007: European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. Appendix A. Official Journal of the European Communities K, 2525.

Healing G. and Smith D., Editors. 2000. Handbook of pre-clinical continuous intravenous infusion, Taylor & Francis, London, 241–250.

Korte S, Nowak P, Luft J, Niggemann B (2014): The Marmoset. In: O. Green, G. Healing (Eds.) Non-Clinical Vascular Infusion Technology, Volume II, CRC Press, Taylor & Francis Group, Boca Raton, London, New York, 285-306.

McNamee GA Jr, Wannemacher RW Jr, Dintermann RE, Rozmiarek H, Montrey RD. 1984. A surgical procedure and tethering system for chronic blood sampling, infusion, and temperature monitoring in caged nonhuman primates. Lab Anim Sci. 34, 303-307.

Figure 1. Free-ranging cynomolgus monkey undergoing continuous intravenous infusion with a lightweight infusion backpack system.

Figure 2a. Intimal hypertrophy/hyperplasia (arrows) at the application site of a catheter.

Figure 2c. Thrombus (arrows) obliterating the vascular lumen at the application site of a catheter.

Figure 2e. Granulomatous reaction around suture material (arrows) at the port region.

Figure 2b. Inflammation of the vascular wall at the application site of the catheter.

Figure 2d. Partly organized thrombus (arrows) at the application site of a catheter.