1. Abstract
Nonclinical studies in cynomolgus monkeys involving direct lumbar cerebrospinal fluid (CSF) administration under anaesthesia
are fundamentally different from studies with more conventional routes and several important points should be considered with
their design especially at time of study report generation. Preclinical safety studies on Central Nervous System (CNS)
administered compounds must differentiate between the effects of the compound, the delivery technique, side effects caused
by e.g. dose volume and sedation, and background findings unrelated to treatment. In this context, only a limited amount of
data is available citing the background clinical signs vs treatment-related findings. Conducting long-term (up to 53 weeks)
cynomolgus monkey studies (Macaca fascicularis, n≥500, Asian origin, 10 months to 4 years) under European housing
conditions, animals were dosed intrathecally only in long intervals (dosing in weekly or monthly regimen), whilst physical
examinations were conducted once weekly, with animals in hand. Occurrence and severity from background findings such as
injuries, tail lesions, tooth fracture, genital region findings, swellings, hair loss, diarrhea, reduced food consumption, blue eye
lids, nasal discharge, vomiting, crusted eyes, squamous skin, hair loss, mammillae findings, thin appearance, low body score
and discolored skin were similar to the same duration of oral gavage or subcutaneous dosing. Anaesthesia of the animals
(ketamine and meditomidine with atipamezole as antidote) and lumbar bolus dosing of up to 2 mL (after withdrawal of equal
amount) resulted in transient neurological findings, such as temporarily unilateral or bilateral, reduced or non-detectable
patellar reflex in controls animals. In addition, single cases of ataxia, lameness of legs or/and slight tremor were present (up to
4 hours) and considered to be related to the anaesthesia. In the cited cases, warming lamp and saline as supportive treatment
were the recommended procedures. A diazepam derivate would be applied if severe post dose findings would be observed,
such as spasms.
Introduction
Many diseases affecting the CNS are inadequately treated by systemically administered therapeutics (Cecchelli 2007). This is
primarily due to the presence of the blood brain barrier (BBB), which represents a major obstacle to achieving adequate drug
concentrations at CNS sites of action. Due to the increasing prevalence of neurodegenerative diseases and the inability of
most systemically administered therapies to cross the BBB, direct CNS delivery will likely play an increasing role in the
treatment of these diseases in the future (Felice 2011).
Preclinical safety studies on CNS administered compounds must differentiate between the effects of the therapy, the delivery
device, and/or the vehicle and assess exacerbations of reactions due to any combination of effects (Turner 2003). Animal
models have been developed and characterized for the safety assessment of CNS administered therapeutics and have
enabled first in human trials, but interpretation of these studies can be challenging due to differences between the human CNS
and that of the test species. This is especially true when conducting long-term intrathecal (lumbar) bolus administration studies
in nonhuman primates (Weinbauer 2015), where a differentiation between the effects of the compound, the delivery technique,
side effects caused by e.g. dose volume and sedation and background findings unrelated to treatment is required to ensure a
valid safety assessment. In this context, only a limited amount of data is available citing the background clinical signs vs
treatment-related indications.
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 standard social housing for cynomolgus monkeys at Covance Münster is according to ETS 123, 2007.
The work summarizes the experience of long-term (up to 53 weeks) cynomolgus monkey studies (n≥500, Asian origin,
10 months to 4 years) under European housing conditions (ETS 123).
In preclinical studies with the designs given in Tables 1-4, animals were
dosed intrathecally only in long intervals (weekly or monthly administration),
whilst physical examinations were conducted once weekly, with animals in
hand. In addition the following evaluations were performed: clinical
observations (including postdose observations on dosing days), body weight,
estimated food consumption, physical and neurologic examinations,
neurobehavioral observations, ophthalmic examinations, cardiovascular
investigations (ECG-JET and blood pressure, respiratory rate), as well as
clinical pathology evaluations including urine and blood gas analysis.
Blood and CSF were collected from all animals for toxicokinetic
evaluation. Furthermore, CSF was collected for clinical chemistry, cell
count and biomarker analysis and CSF and blood was processed for
complement analysis.
In accordance to the regulatory requirements in Europe, the cages used are of stainless steel and have a height of 248 cm, a
width of 151.5 cm/151 cm, a floor space of 2.29 m2 and a volume of 5.68 m3 (plus a balcony, Müller, 2008). For dosing start,
animals were separated in smaller groups of 2 or 3 animals/cage and sex. The animal room was maintained at a temperature
of 19 to 25°C and a relative humidity of 40 to 70%. Animals were kept on a 12:12 light schedule (white light illumination
switched on at 5.30 am). The monkeys were fed a standard monkey diet and UV-irradiated and filtered tap water was provided
to the animals ad libitum. The monkeys were provided environmental enrichment (wooden chips, movable stainless steel
mirrors, coloured plastic tools and coloured plastic balls).
Dosing Procedure
A dose volume of 0.75 mL-2.0 mL, in most cases 1 mL, of vehicle or test item formulation, followed by 0.25 mL of an artificial
CSF was used for each injection, and the calculated dosing volume of CSF (max. 1.5 mL) was withdrawn in forefront. For best
practice and to apply aseptic techniques, a micro incision of the skin was conducted using a 20G needle, before introduction of
the spinal needle. Dosing was elaborated with a Pencan Paed® pencil-point needle (25 G, B. Braun Melsungen AG, Germany)
for paediatric use to minimize the impact on dura mater spinalis. Intrathecal administration was performed via lumbar puncture
between L3-L5 by slow manual bolus infusion over 1 min to fasted, slightly anesthetized animals [s-ketamine
(5 mg/kg i.m.) and medetomidine (0.06 mg/kg i.m.); atipamezole (0.3 mg/kg i.m.) was used as antidote].
#1633 Long-Term Intrathecal Bolus Administration Studies in Cynomolgus Monkeys:
Expected Background Findings, Procedure-Related Observations and Recommended Procedures
M.O. Niehoff, S. Korte and F.T. Ludwig
Covance Preclinical Services GmbH, Münster, Germany
Presented at Society of Toxicology 2016
Results
Occurrence and severity from background findings such as injuries, tail lesions, tooth fracture,
genital region findings, swellings, hair loss, diarrhea, reduced food consumption, blue eye lids,
nasal discharge, vomiting, crusted eyes, squamous skin, hair loss, mammillae findings, thin
appearance, low body score and discolored skin were recorded as detailed below (Table 5).
Anaesthesia of the animals and lumbar bolus dosing of up to 2 mL (after withdrawal of equal CSF amount (if possible)
resulted in transient neurological findings, such as temporarily unilateral or bilateral, reduced or non-detectable patellar
reflex in control animals as detailed below:
Pre-Dose Findings without Intrathecal Delivery
In healthy animals, the only background finding on multiple (n=17 animals of 240) occasions pre-dose (before the dosing
phase of the study) was a decreased patellar tendon reflex present on several days for the same animal. These findings
were not transient and remain in the vast majority of animals throughout the study until necropsy.
Control Group Findings after Intrathecal Bolus Delivery
Effects that were related to the intrathecal administration (procedure-related, as also occurring in control animals and not
being present before the dosing phase), were either a temporarily absent or reduced patellar tendon reflex (4, 8 and 24
hours after dosing) or the absence of the anal reflex (4 and 8 hours). These findings were all of transient nature.
In addition, single cases of ataxia, lameness of legs and/or slight tremor were present (up to 4 hours) and considered to be
related to the anaesthesia. In the cited cases, warming lamp and saline as supportive treatment were the recommended
procedures. A diacepam derivate would be applied if severe post dose findings would be observed, such as spasms.
Conclusion
The provided work now gives reference for severity and number of clinical signs observed during the conduct of intrathecal
studies using nonhuman primates. For this type of studies, transient neurological findings considered procedure related were
as follows: absent or reduced patellar tendon reflex, absence of the anal reflex and single cases of ataxia, lameness of legs
and/or slight tremor for up to 4 hours post dose administration. Predose, only a decreased patellar tendon reflex was observed.
References
Cecchelli R, Berezowski V, Lundquist S, Culot M, Renftel M, Dehouck MP, Fenart L (2007): Modelling of the blood–brain barrier in drug discovery and development, Nature Rev. Drug Discov, 6:650-661.
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.
Felice BR, Wright TL, Boyd RB, Butt MT, Pfeifer RW, Pan J, Ruiz JA, Heartlein MW, Calias P. (2011): Safety evaluation of chronic intrathecal administration of idursulfase-IT in cynomolgus monkeys. Toxicol Pathol, 39(5):879-92.
Müller, W. (2008): The new European housing regulations for nonhuman primates: opportunities and challenges. In: Weinbauer, G.F., Vogel, F., (Eds.), Critical contributions of primate models for biopharmaceutical drug development. Waxmann Verlag GmbH,
Münster, pp. 53-74.
Turner, MS, 2003: intrathecal drug delivery 2002, Acta Neurochir, 87:29-35.
Weinbauer GF, Korte SH (2015): Juvenile Toxicity Testing: experience Using Nonhuman Primate Models. In: Gerhard F. Weinbauer,
Friedhelm Vogel (Eds.) Primate Biologics Research at a Crossroads; 2015, 174 Pages, E-Book (PDF), ISBN 978-3-8309-7961-6.
Figure 1. Number of intrathecal
oligonucleotide nonhuman primate
studies (2011-2015) at Covance Preclinical
Services GmbH, Münster, Germany (MUE).
0
2
4
6
8
10
12
14
2008 2009 2010 2011 2012 2013 2014 2015
Total Number of Studies by Year - Covance Preclinical Services
GmbH, Münster, Germany (MUE)
Primate (Intrathecal)
Table 1. Example of a Pharmacokinetic Study with Subsequent
CSF Sampling on Multiple Intervals after Dosing
Group
Number
Dosing
Days
Dose Volume*
(mL/kg)
Dose
Route
Animals/Group Necropsy After ...
One Sex 16 Days 37/38 Days
1 to 4 Groups 1, 5, 9 1 IT Bolus 6 3 M 3 M
* 1.0 mL, followed by 0.25 mL aCSF flush. The approximate dosing volume was removed in forefront.
Table 2. Example of a Screening Study
Group
Number
Dosing Days Dose Volume*
(mL)
Dose
Route
Animals/Group
One Sex
Necropsy
On Day
1 to 10 Groups 1, 8 1.0 IT Bolus 4 36
* 1.0 mL, followed by 0.25 mL aCSF flush. The approximate dosing volume was removed in forefront.
Table 3. Example of 3 Months with a 6-Month Recovery Phase Study
Design with a Subsequent Toxicokinetic Group
Group
Number
Dosing
Days
Dose
Volume*
(mL)
Dose
Route
Animals/Group Necropsy on Study Day ... (Male/Female)
Male Female 8 35 42 70 91 98 155 175
1
1, 14,
28, 56,
84
2.0 IT Bolus 6 6 - - - - 4/4 - - 2/2
2
1, 14,
28, 56,
84
2.0 IT Bolus 3 3 - - - - 3/3 - - -
3
1, 14,
28, 56,
84
2.0 IT Bolus 3 3 - - - - 3/3 - - -
4
1, 14,
28, 56,
84
2.0 IT Bolus 7 7 - - - - 5/5 - - 2/2
5 1, 14, 28 2.0 IT Bolus 8 8 1M/1F 1M/1F 2M/1F 1M/2F - 2M/1F 1M/2F -
* 2.0 mL, followed by 0.25 mL aCSF flush. The approximate dosing volume was removed in forefront.
Table 4. Example of 9 Months with a 6-Month Recovery Phase Study Design
Group
Number
Dosing Days
Dose
Volume*
(mL)
Dose
Route
Animals/Group Necropsy in Week
Male Female 37 Weeks 65 Weeks
1
1, 29, 57, 85, 113, 141,
169,197, 225, 253
0.75 IT Bolus 5 5 3 M/3 F 2 M/2 F
2
1, 29, 57, 85, 113, 141,
169,197, 225, 253
0.75 IT Bolus 3 3 3 M/3 F – / –
3
1, 29, 57, 85, 113, 141,
169,197, 225, 253
0.75 IT Bolus 3 3 3 M/3 F – / –
4
1, 29, 57, 85, 113, 141,
169,197, 225, 253
0.75 IT Bolus 7 7 5 M/5 F 2 M/2 F
* 0.75 mL, followed by 0.25 mL aCSF flush. The approximate dosing volume was removed in forefront.
Table 5. Summary of Clinical Signs from Control Group Monkeys
over the Course of Three 13-Week Studies (N=28)
Observation
(Study 1, Study 2
and Study 3)
Males
(n=6)* 1.
Females
(n=6)* 1.
Males
(n=5)* 2.
Females
(n=5)* 2.
Males
(n=3)* 3.
Females
(n=3)* 3.
Tail distal, bloody/crusted 0/0 9/1 0/0 7/2 0/0 0/0
Trunk, reddened skin; lower
part of back, slight
0/0 1/1 0/0 0/0 0/0 0/0
Trunk, spots, wheals, right
inguen, red
0/0 1/1 4/1 0/0 0/0 0/0
Trunk, upper part of back,
crusted
0/0 0/0 0/0 0/0 5/1 0/0
Surgery, clamp removed 0/0 0/0 0/0 1/1 0/0 0/0
Left finger crusted 0/0 0/0 0/0 1/1 0/0 0/0
Right eye, bloody 0/0 0/0 0/0 1/1 0/0 0/0
Lower lip, bloody 0/0 0/0 0/0 1/1 0/0 0/0
Left side of head, crusted 0/0 0/0 8/1 0/0 0/0 0/0
Inflammation, back, bloody/
purulent
0/0 0/0 0/0 0/0 1/1 0/0
Small suture 0/0 0/0 0/0 0/0 1/1 0/0
Fur abnormalities: hair loss,
large area
0/0 0/0 0/0 0/0 0/0 5/1
Right eye, crusted 0/0 0/0 0/0 0/0 0/0 1/1
Discolored skin, left side of
head, blue
0/0 0/0 0/0 0/0 0/0 1/1
Supplement food 0/0 0/0 0/0 0/0 22/1 0/0
Vomiting, excessive salivation,
before dosing
0/0 0/0 0/0 0/0 2/1 0/0
* observations/affected animals.