1. Abstract
Numerous 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.
Introduction
Numerous 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 Methods
Safe 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 Rs
C. Miller1, S. Korte2, A. Salva1, N. Snippe2, F. Runge2 and H. vanWijk1 : 1Covance Laboratories, Harrogate, UK; 2Covance Preclinical Services, GmbH, Münster, Germany
Sponsor: Sven Korte
Presented at Society of Toxicology 2016
Results
The 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).
Conclusions
The 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.
References
ETS 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.