7/31/2019 Comparative Toxicity Study of Phenytoin in Juvenile and Adult Cynomolgus Monkeys

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COMPARATIVE TOXICITY STUDY OF PHENYTOIN IN JUVENILE AND ADULT CYCOMPARATIVE TOXICITY STUDY OF PHENYTOIN IN JUVENILE AND ADULT CYNOMOLGUS MONKEYSNOMOLGUS MONKEYSRosario M.Rosario M. PerezPerez11, Flordeliza P. de, Flordeliza P. de VillaVilla11, Lourdes S. Antonio, Lourdes S. Antonio11, Takashi, Takashi HayashiHayashi11, Norio Muto, Norio Muto22,, EmakoEmako SuzukiSuzuki22 and Mamoru Nomuraand Mamoru Nomura22

11INA RESEARCH PHILIPPINES, INC., PINA RESEARCH PHILIPPINES, INC., P--2 B2 B--7 L17 L1--A Technology Avenue, La guna Technopark, BiA Technology Avenue, La guna Technopark, Bian, Laguna, 40 24, Philippi nesan, Laguna, 40 24, Philippi nes22Ina Research Inc.,Ina Research Inc.,21482148--188188Nishiminowa, InaNishiminowa, Ina--shi, Naganoshi, Nagano--ken, 399ken, 399 --4501, Japa n4501, Japa n

CONCLUSIONCONCLUSION

INTRODUCTIONINTRODUCTION

MATERIALS AND METHODSMATERIALS AND METHODS

RESULTSRESULTS

ANIMALSANIMALS

Twenty cageTwenty cage--bred juvenile monkeys (10 sixbred juvenile monkeys (10 six--month and 10month and 10eighteeneighteen--month old males and females) were randomlymonth old males and females) were randomlyassigned to the control (n=6 per juvenile age group) andassigned to the control (n=6 per juvenile age group) and

treatment groups (n = 4 per juvenile age group).treatment groups (n = 4 per juvenile age group).Additionally, 2 male and 2 female naAdditionally, 2 male and 2 female nave, cageve, cage--bred adultbred adult

(>48 months) monkeys were included in the treatment(>48 months) monkeys were included in the treatmentgroup.group.

OBJECTIVESOBJECTIVES

All the animals in this study were used in accordance withAll the animals in this study were used in accordance withthe ethical procedures approved for the care and use ofthe ethical procedures approved for the care and use of

laboratory animals by the Institutional Animal Care andlaboratory animals by the Institutional Animal Care andUse Committee of INA RESEARCH PHILIPPINES, INC.Use Committee of INA RESEARCH PHILIPPINES, INC.(INARP), an AAALAC(INARP), an AAALAC--accredited facility.accredited facility.

EXPERIMENTAL DESIGNEXPERIMENTAL DESIGN

The monkeys were grouped as shown in Table 1.The monkeys were grouped as shown in Table 1.

** Liver samples forLiver samples for enzyme activity assayenzyme activity assayobtained from additional untreatedobtained from additional untreated

adult males (2) and females (2) at INARP and Ina Research Inc. wadult males (2) and females (2) at INARP and Ina Research Inc. were used asere used as

control in vitro.control in vitro.

2230302*2*2*2*>48>48

221515II CII C

22303022221818

221515II BII B

223030222266

221515

PHTPHT

II AII A

333318184400I BI B

(Control)(Control)

3333664400DistilledDistilled

waterwater

I AI A(Control)(Control)

FemaleFemaleMaleMaleAgeAge

(months)(months)

Number of AnimalsNumber of AnimalsDurationDuration

(weeks)(weeks)

Daily DoseDaily Dose

(mg/kg/day)(mg/kg/day)TreatmentTreatmentGroupGroup

2.2. Induction of CYP3A4 by PHT was markedly observed inInduction of CYP3A4 by PHT was markedly observed in

the 6the 6--month old monkeys.month old monkeys.

1.1. The highest mean % change in total CYP450 contentThe highest mean % change in total CYP450 contentwas recorded for the 6was recorded for the 6--month old monkeys.month old monkeys.

2.2. In terms of CYP2C9, PHT treatment increased VmaxIn terms of CYP2C9, PHT treatment increased Vmax

values except in the adult monkeys.values except in the adult monkeys.

3.3. For CYP2C19, the Vmax values in all groups wereFor CYP2C19, the Vmax values in all groups were

decreased by PHT treatment; Lowest change in Vmaxdecreased by PHT treatment; Lowest change in Vmaxvalues were seen in the treated adult monkeys.values were seen in the treated adult monkeys.

Juvenile monkeys exhibit developmental characteristicsJuvenile monkeys exhibit developmental characteristicssimilar to pediatric patients making them potentiallysimilar to pediatric patients making them potentially

excellent test systems for assessing toxicity in theexcellent test systems for assessing toxicity in thepediatric population.pediatric population. Studies have shown that theStudies have shown that thenucleotide and deduced amino acid sequences ofnucleotide and deduced amino acid sequences of

cytochrome P450 (cytochrome P450 (CYP450) enzymes of the cynomolgusCYP450) enzymes of the cynomolgusmonkey were highly homologous (92monkey were highly homologous (92--96%) to those of96%) to those of

humans and that theirhumans and that their 2C enzymes are known to be2C enzymes are known to becomparable to their human counterpart.comparable to their human counterpart. Phenytoin (PHT)Phenytoin (PHT)is largely metabolized by the hepatic CYP450 enzymeis largely metabolized by the hepatic CYP450 enzyme

system beingsystem being mainly oxidized to 5mainly oxidized to 5--(4(4--hydroxyphenyl)hydroxyphenyl)--55--phenylhydantoin by CYP2C9 and to a minor extent byphenylhydantoin by CYP2C9 and to a minor extent by

CYP2C19 in humans.CYP2C19 in humans.

To validate the experimental procedures for theTo validate the experimental procedures for thenonclinical toxicological evaluation of a pediatric drugnonclinical toxicological evaluation of a pediatric drug

product using the juvenile cynomolgus monkey as theproduct using the juvenile cynomolgus monkey as thetest systemtest system

To determine ageTo determine age--related physical and physiologicalrelated physical and physiologicalchanges in the juvenile compared to the adult monkeychanges in the juvenile compared to the adult monkey

To provide some insights into theTo provide some insights into the metabolism and toxicmetabolism and toxiceffects of PHT in the cynomolgus monkey while allowingeffects of PHT in the cynomolgus monkey while allowingcomparisons to be done between juvenile and adultcomparisons to be done between juvenile and adult

monkey age groups and to the corresponding agemonkey age groups and to the corresponding agegroups in humans.groups in humans.

PARAMETERSPARAMETERS

Clinical ObservationClinical Observation

-- IncludingIncludingNeurobehavioralNeurobehavioral

AssessmentAssessmentBatteryBattery

Body weightBody weight

Food ConsumptionFood Consumption

HematologyHematology

Blood ChemistryBlood Chemistry

UrinalysisUrinalysis

Electrocardiography (ECG)Electrocardiography (ECG)

ElectroencephalographyElectroencephalography(EEG)(EEG)

Plasma Phenytoin AssayPlasma Phenytoin Assay

CYP450 Enzyme ActivityCYP450 Enzyme ActivityAssayAssay

Gross and HistopathologyGross and Histopathology

EEG changes: Spindle or high voltage slow wavesEEG changes: Spindle or high voltage slow waveswere observed at 30were observed at 30 mg/kg/day in these 2 PHTmg/kg/day in these 2 PHT--

treated monkeys.treated monkeys.

The possible influence of other factors such as reducedThe possible influence of other factors such as reduced

protein binding and/or delayed developmentalprotein binding and/or delayed developmental

expression or paucity of drug transporter proteins (Pexpression or paucity of drug transporter proteins (P--

glycoprotein) that could significantly influence theglycoprotein) that could significantly influence the

differential penetration of PHT into the brain weredifferential penetration of PHT into the brain were

considered for the young monkey.considered for the young monkey.

Polymorphism affecting the CYP2C isoforms was alsoPolymorphism affecting the CYP2C isoforms was also

considered for the adult female in addition to the effectsconsidered for the adult female in addition to the effects

of PHT saturation kinetics and CYP450of PHT saturation kinetics and CYP450--mediatedmediated

metabolism of this drug.metabolism of this drug.

Due to the small number of animals used, definite ageDue to the small number of animals used, definite age --

related associations between the development of toxicityrelated associations between the development of toxicity

and the TK and enzyme kinetic properties of PHT wereand the TK and enzyme kinetic properties of PHT were

not clearly seen in the young and adult PHTnot clearly seen in the young and adult PHT--treatedtreated

monkeys that manifested toxicity. Othermonkeys that manifested toxicity. Other possiblepossible

interdependent factors that could influence theinterdependent factors that could influence the

evaluation of the experimental endpoints should also beevaluation of the experimental endpoints should also be

considered.considered.

The juvenile cynomolgus monkey is a viable and reliableThe juvenile cynomolgus monkey is a viable and reliable

species that can be studied to characterize the ADMEspecies that can be studied to characterize the ADME

processes and assess toxicological effectsprocesses and assess toxicological effects in specificin specific

periods of human development based on:periods of human development based on:

The observed ageThe observed age--related physical and physiologicrelated physical and physiologic

changeschanges

The exhibited pattern of expression of the CYP450The exhibited pattern of expression of the CYP450

enzymes andenzymes and developmental acquisition of CYP2C9developmental acquisition of CYP2C9

activityactivity that were generally similar to humansthat were generally similar to humans..

In this study, the experimental procedures for theIn this study, the experimental procedures for the

nonclinical toxicological evaluation of a pediatric drugnonclinical toxicological evaluation of a pediatric drug

product were demonstrated using juvenile cynomolgusproduct were demonstrated using juvenile cynomolgus

monkeys as test systems.monkeys as test systems.

Body weight changes:Body weight changes:

Figure 1.Figure 1. Mean body weight changes (expressed as %Mean body weight changes (expressed as %change relative to baseline values) of the juvenilechange relative to baseline values) of the juvenile

and adult cynomolgus monkeysand adult cynomolgus monkeys

80

90

100

110

120

1 3 7 10 1 3 1 6 1 4 7 1 0 1 3 1 5

Day of Measurement

Bodyweightchange(%)

6 - m o n t h o ld t r e a t e d1 8 - m o n t h o ld tr e a t e dt r e a t e d a d u l t6 - m o n t h o l d c o n t ro l1 8 - m o n t h o l d c o n t ro l

Clinical Observation:Clinical Observation:

One 6One 6--month old female (No. 2201) and one adultmonth old female (No. 2201) and one adult

female (No. 2208) treated with PHT had lowerfemale (No. 2208) treated with PHT had lowerneurobehavioral assessment battery scores forneurobehavioral assessment battery scores forauditory orientation and coordination at 30auditory orientation and coordination at 30

mg/kg/day.mg/kg/day.

Food Consumption:Food Consumption:

No treatmentNo treatment--related changes were recordedrelated changes were recorded

Hematology and Blood Chemistry Data:Hematology and Blood Chemistry Data:

0

50

100

150

200

250

300

GOT

GPT

CPK

LDH

ALP

GLU

BIL-T

UN

CHOL

CREA

PHOS

Ca

TP

ALB

A/G T

G

Blood Chemistry Parameters 6-month old treated18-month old treated

treated adult

0

20

40

60

80

100

120

140

160

RBC HGB HCT MCV MCH MCHC PLT WBC

Hematology Parameters

6-month old treated

18-montholdt reated

treated adult

Figure 2.Figure 2. Mean hematology and blood chemistry parameterMean hematology and blood chemistry parameterpercent values (expressed as % change relative topercent values (expressed as % change relative tobaseline values) of the juvenile and adultbaseline values) of the juvenile and adultcynomolgus monkeyscynomolgus monkeys

No treatmentNo treatment-- or ageor age--related changes in urinalysis, ECG,related changes in urinalysis, ECG,gross and histopathological findingsgross and histopathological findings

Hepatic cytochrome P450 enzyme activities:Hepatic cytochrome P450 enzyme activities:

TK/PK of PHT:TK/PK of PHT:

Figure 4.Figure 4. Mean CYP2C9, CYP2C19 and CYP3A4 enzymeMean CYP2C9, CYP2C19 and CYP3A4 enzymeactivities (expressed as % of control) in juvenile andactivities (expressed as % of control) in juvenile andadult cynomolgus monkeysadult cynomolgus monkeys

0

50

100

150

200

250

300

6 mo-old treated

18mo-old treated

treated adult

CytochromeP450

content(nmol/mg protein)

Vmax (nmol/mg protein/min)

CYP3A4CYP2C19CYP2C9CYP3A4C YP 2C 9 C YP 2C 19

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

6000

8000

10000

12000

14000

16000

18000

Day 1 Day 14 Day 1 Day 14

15 mg/kg 30 mg/kg

Cmax(ng/mL)

6-mo old treated18-mo old treatedtreated adult22012208

80000

120000

160000

200000

240000

280000

Day 1 Day 14 Day 1 Day 14

15 mg/kg 30 mg/kg

AUC

(ng

6-mo old treated18-mo old treated

treated adult22012208