COMPARATIVE TOXICITY STUDY OF PHENYTOIN IN JUVENILE AND ADULT CYNOMOLGUS MONKEYS CYNOMOLGUS

Rosario M. Perez1, Flordeliza P. de Villa1, Lourdes S. Antonio1, Takashi Hayashi1, Norio Muto2, Emako Suzuki2 and Mamoru Nomura2
1

INA RESEARCH PHILIPPINES, INC., P-2 B-7 L1-A Technology Avenue, Laguna Technopark, Bian, Laguna, 4024, Philippines P- B- L1Bi 2 Ina Research Inc., 2148-188 Nishiminowa, Ina-shi, Nagano-ken, 399-4501, Japan 2148InaNagano399-

Body weight changes:

CYP2C9

CYP2C19

250

(nmol/mg protein)

12 0

200

Body w e ight cha nge (%)

11 0

150

100

OBJECTIVES

10 0

90

To validate the experimental procedures for the nonclinical toxicological evaluation of a pediatric drug product using the juvenile cynomolgus monkey as the test system

80 1 3 7 10 13 16 1 4

6 -m o n th o ld tre a te d 1 8 -m o n th o ld tre a te d tre a te d a d u lt 6 -m o n th o ld co n tro l 1 8 -m o n th o ld co n tro l

7 10 13 15

50

Figure 4.

Da y of Me a sure m e nt

Mean CYP2C9, CYP2C19 and CYP3A4 enzyme activities (expressed as % of control) in juvenile and adult cynomolgus monkeys

To determine age-related physical and physiological agechanges in the juvenile compared to the adult monkey

Figure 1.

MATERIALS AND METHODS

Hematology Parameters
160

ANIMALS
Twenty cage-bred juvenile monkeys (10 six- month and 10 cagesixeighteeneighteen -month old males and females) were randomly assigned to the control (n=6 per juvenile age group) and treatment groups (n = 4 per juvenile age group). Additionally, 2 male and 2 female nave, cage-bred adult na cage(>48 months) monkeys were included in the treatment group. All the animals in this study were used in accordance with the ethical procedures approved for the care and use of laboratory animals by the Institutional Animal Care and Use Committee of INA RESEARCH PHILIPPINES, INC. (INARP), an AAALAC-accredited facility. AAALACEXPERIMENTAL DESIGN The monkeys were grouped as shown in Table 1.
Daily Dose Duration (mg/kg/day) (weeks) 0 0 15 30 15 30 15 II C 30 4 4 2 2 2 2 2 2 >48 2* 2* Number of Animals Age Male Female (months) 6 18 6 18 3 3 2 2 3 3 2 2

300

Blood Chemistry Parameters

250 200 150 100 50 0

6-month old treated 18-month old treated treated adult

GLU

CREA

LDH

CPK

ALB

ALP

UN

TP

Ca

GOT

GPT

CHOL

BIL-T

Figure 2.

Group IA (Control) IB (Control) II A II B

Treatment

Distilled water

No treatment- or age-related changes in urinalysis, ECG, treatmentagegross and histopathological findings

TK/PK of PHT:
Cmax (ng/mL)

18000 16000 14000 12000 10000 8000 6000 Day 1 15 mg/kg



PHT

6-mo old treated 18-mo old treated treated adult 2201 2208

The possible influence of other factors such as reduced protein binding and/or delayed developmental expression or paucity of drug transporter proteins (P(Pglycoprotein) that could significantly influence the differential penetration of PHT into the brain were considered for the young monkey. Polymorphism affecting the CYP2C isoforms was also considered for the adult female in addition to the effects of PHT saturation kinetics and CYP450- mediated CYP450metabolism of this drug.

* Liver samples for enzyme activity assay obtained from additional untreated adult males (2) and females (2) at INARP and Ina Research Inc. w ere used as were control in vitro.


Day 14

Day 1 30 mg/kg

Day 14

PARAMETERS

280000 240000
6-m o old treated 18-m o old treated treate d adult 2201 2208


AUC

Clinical Observation - Including Neurobehavioral Assessment Battery Body weight Food Consumption Hematology Blood Chemistry

(ng

Urinalysis Electrocardiography (ECG) Electroencephalography (EEG) Plasma Phenytoin Assay CYP450 Enzyme Activity Assay Gross and Histopathology

200000 160000 120000 80000

Day 1

Day 14 15 mg/kg

Day 1 30 mg/kg

Day 14

Figure 3. Cmax and AUC(0-24h) (15 and 30 mg/kg/day) of (0juvenile and adult cynomolgus monkeys at different time points

Due to the small number of animals used, definite ageagerelated associations between the development of toxicity and the TK and enzyme kinetic properties of PHT were not clearly seen in the young and adult PHT -treated PHTmonkeys that manifested toxicity. Other possible interdependent factors that could influence the evaluation of the experimental endpoints should also be considered.

Mean hematology and blood chemistry parameter percent values (expressed as % change relative to baseline values) of the juvenile and adult cynomolgus monkeys

PHOS

A/G

TG



To provide some insights into the metabolism and toxic effects of PHT in the cynomolgus monkey while allowing comparisons to be done between juvenile and adult monkey age groups and to the corresponding age groups in humans.

Mean body weight changes (expressed as % change relative to baseline values) of the juvenile and adult cynomolgus monkeys

2. Induction of CYP3A4 by PHT was markedly observed in the 6-month old monkeys. 61. The highest mean % change in total CYP450 content was recorded for the 6- month old monkeys. 62. In terms of CYP2C9, PHT treatment increased Vmax values except in the adult monkeys. 3. For CYP2C19, the Vmax values in all groups were decreased by PHT treatment; Lowest change in Vmax values were seen in the treated adult monkeys.

Food Consumption: No treatment-related changes were recorded treatmentHematology and Blood Chemistry Data:

140 120 100 80 60 40 20 0

6-month old treated 18-month old treated treated adult

CONCLUSION
RBC HGB HCT MCV MCH MCHC PLT WBC

In this study, the experimental procedures for the nonclinical toxicological evaluation of a pediatric drug product were demonstrated using juvenile cynomolgus monkeys as test systems. The juvenile cynomolgus monkey is a viable and reliable species that can be studied to characterize the ADME processes and assess toxicological effects in specific periods of human development based on: The observed age-related physical and physiologic agechanges The exhibited pattern of expression of the CYP450 enzymes and developmental acquisition of CYP2C9 activity that were generally similar to humans. humans.

Juvenile monkeys exhibit developmental characteristics similar to pediatric patients making them potentially excellent test systems for assessing toxicity in the pediatric population. Studies have shown that the nucleotide and deduced amino acid sequences of cytochrome P450 (CYP450) enzymes of the cynomolgus (CYP450) monkey were highly homologous (92-96%) to those of (92humans and that their 2C enzymes are known to be comparable to their human counterpart. Phenytoin (PHT) is largely metabolized by the hepatic CYP450 enzyme system being mainly oxidized to 5-(4-hydroxyphenyl)-55-(4 hydroxyphenyl)phenylhydantoin by CYP2C9 and to a minor extent by CYP2C19 in humans.

Clinical Observation:

One 6-month old female (No. 2201) and one adult 6female (No. 2208) treated with PHT had lower neurobehavioral assessment battery scores for auditory orientation and coordination at 30 mg/kg/day.

INTRODUCTION

RESULTS

EEG changes: Spindle or high voltage slow waves were observed at 30 mg/kg/day in these 2 PHTPHTtreated monkeys. Hepatic cytochrome P450 enzyme activities:
300
Cytochrome P450 content

Vmax (nmol/mg protein/min) CYP3A4 CYP2C9 CYP2C19 CYP3A4

6 mo-old treated 18 mo-old treated treated adult