Download - Preclinical Development Handbook || Repeat Dose Toxicity Studies

213

8 REPEAT DOSE TOXICITY STUDIES

Shayne Cox Gad Gad Consulting Services, Cary, North Carolina

Preclinical Development Handbook: Toxicology, edited by Shayne Cox GadCopyright © 2008 John Wiley & Sons, Inc.

Contents

8.1 Introduction 8.2 Objectives 8.3 Regulatory Considerations

8.3.1 Good Laboratory Practices (GLPs) 8.3.2 Animal Welfare Act 8.3.3 Regulatory Requirements for Study Design

8.4 Study Design and Conduct 8.4.1 Animals 8.4.2 Routes and Setting Doses 8.4.3 Parameters to Measure 8.4.4 Other In - Life Endpoints for Evaluation 8.4.5 Histopathology 8.4.6 Study Designs

8.5 Study Interpretation and Reporting References

8.1 INTRODUCTION

In the broadest sense, subchronic and chronic studies for pharmaceutical products can incorporate any of the routes used to administer a therapeutic agent, use any of a number of animal models, and conform to a broad range of experimental designs. They can be two weeks long (what used to be called “ subacute ” studies because they were conducted at dose levels below those employed for single dose or acute studies) or last up to a year. Another name for these studies is repeat dose studies [1, 2] — that is, those studies whereby animals have a therapeutic agent


administered to them on a regular and repeated basis by one or more routes over a period of one year or less. There is great fl exibility and variability in the design of such studies.

The fi rst thing to note about repeat dose studies and their design are the prinici-ples of dose response — a governing general principle of toxicology:

The Three Dimensions of Dose Response

As dose increases:

• Incidence of responders in an exposed population increases. • Severity of response in affected individuals increases. • Time to occurrence of response or of progressive stage of response decreases. • An implication is that as duration of dosing increases, NOEL/NOAEL

decreases.

This chapter seeks to provide a fi rm grasp of the objectives for repeat dose studies, the regulatory requirements governing them, the key factors in their design and conduct, and the interpretation of their results.

8.2 OBJECTIVES

As with any scientifi c study or experiment (but especially for those in safety assess-ment), the essential fi rst step is to defi ne and understand the reason(s) for the conduct of the study — that is, its objectives. There are three major (scientifi c) reasons for conducting repeat dose toxicity studies, but the basic characteristic and objective of all but a few repeat studies needs to be understood. The repeat dose study is (as are most other studies in whole animal toxicology) a broad screen. It is not focused on a specifi c endpoint; rather, it is a broad exploration of the cumulative biological effects of the administered agent over a range of doses: so broad an exploration, in fact, that it can be called a “ shotgun ” study.

The objectives of the typical repeat dose study fall into three categories. The fi rst is to broadly defi ne the toxicity (and, if one is wise, the pharmacology and hyper-pharmacology) of repeated doses of a potential therapeutic agent in an animal model [3, 4, 5] . This defi nition is both qualitative (what are the target organs and the nature of the effects seen) and quantitative (at what dose levels, or, more impor-tantly, at what plasma and tissue levels, are effects defi nitely seen and not seen). It seeks to characterize the cumulative toxicity of the drug under study. Such cumula-tive toxicity is rarely predicted by single dose toxicity.

The second objective (and the one that in the pharmaceutical industry usually compels both timing and compromising of design and execution) is to provide support for the initiation of and/or continued conduct of clinical trials in humans [6, 7] . As such, repeat dose studies should provide not only adequate clearance (therapeutic margin) of initial dose levels and duration of dosing, but also guidance for any special measures to be made or precautions to be taken in initial clinical trials. Setting inappropriate dose levels (either too low or too high) may lead to the failure of a study. A successful study must both defi ne a safe or “ clean ” dose level

(one that is as high as possible, to allow as much fl exibility as possible in the conduct of clinical studies) and demonstrate and/or characterize signs of toxicity at some higher dose. The duration of dosing issue is driven by a compromise between meeting established regulatory guidelines (as set out in Table 8.1 ) and the economic pressure to initiate clinical trials as soon as possible.

The third objective is one of looking forward to later studies. The repeat dose study must provide suffi cient information to allow a prudent setting of doses for later, longer studies (including, ultimately, carcinogenicity studies). At the same time, the repeat dose study must also provide guidance for the other (than dose) design features of longer - term studies (such as what parameters to measure and when to measure them, how many animals to use, and how long to conduct the study).

These objectives are addressed by the usual repeat dose study. Some repeat dose studies, however, are unusual in being conceived, designed, and executed to address specifi c questions raised (or left unanswered) by previous preclinical or early clinical studies. Such a special purpose is addressed separately. Common study durations are 2, 4, and 13 weeks (all species), 26 weeks (rodent chronic toxicity), 39 or 52 weeks (nonrodent chronic toxicity), and 2 years (rodent carcinogenicity).

Chronic studies (those that last six or nine months or a year) may also be con-ducted for the above purposes but are primarily done to fulfi ll registration require-ments for drugs that are intended for continuous long - term (lifetime) use or frequent intermittent use.

8.3 REGULATORY CONSIDERATIONS

Much of what is done (and how it is done) in repeat dose studies is a response to a number of regulations. Three of these have very broad impact. These are the Good Laboratory Practices requirements, Animal Welfare Act requirements, and regula-tory requirements that actually govern study design.

8.3.1 Good Laboratory Practices ( GLP s )

Since 1978, the design and conduct of preclinical safety assessment studies for phar-maceuticals in the United States (and, indeed, internationally) have been governed and signifi cantly infl uenced by GLPs. Strictly speaking, these regulations cover qualifi cations of staff and facilities, training, record - keeping, documentation, and actions required to ensure compliance with and the effectiveness of these steps. Although the initial regulations were from the U.S. FDA [8] , they have always

TABLE 8.1 Duration of Treatment Supported by Preclinical Studies

Animal Study Length Generally Allowed Human Dosing

2 weeks Up to 3 doses (but could be as much as 12 consecutive doses)

1 month Up to 30 days (though usually 28) 3 months 3 months 6 months (rodent); 9 – 12 months (dog)(U.S.)/6

months (EEC and Japan) Unlimited

REGULATORY CONSIDERATIONS 215


extended to cover studies performed overseas [9] . Most other countries have adopted similar regulations. A discussion of these regulations is beyond the scope of the current chapter, but several aspects are central to this effort. Each technique or methodology to be employed in a study (such as animal identifi cation, weighing and examination, blood collection, and data recording) must be adequately described in a standard operating procedure (SOP) before the study begins. Those who are to perform such procedures must be trained in them beforehand. The actual design of the study, including start date and how it is to be ended and analyzed, plus the principal scientists involved (particularly the study director), must be specifi ed in a protocol that is signed before the study commences. Any changes to these features must be documented in amendments once the study has begun. It is a good idea that the pathologist who is to later perform or oversee histopathology be designated before the start of the study, and that the design be a team effort involving the best efforts of the toxicologist, pathologist, and (usually for subchronic studies) the drug metabolism scientist [10] .

8.3.2 Animal Welfare Act

Gone are the days when the pharmaceutical scientist could conduct whatever pro-cedures or studies that were desired using experimental animals. The Animal Welfare Act [11] (and its analogues in other countries) rightfully requires careful consider-ation of animal usage to ensure that research and testing uses as few animals as possible in as humane a manner as possible. As a start, all protocols must be reviewed by an Institutional Animal Care and Use Committee. Such review takes time but should not serve to hinder good science. When designing a study or devel-oping a new procedure or technique, the following points should be kept in mind:

1. Will the number of animals used be suffi cient to provide the required data yet not constitute excessive use? (It ultimately does not reduce animal use to utilize too few animals to begin with and then have to repeat the study.)

2. Are the procedures employed the least invasive and traumatic available? This practice is not only required by regulations but is also sound scientifi c practice, since any induced stress will produce a range of responses in test animals that can mask or confound the chemically induced effects.

8.3.3 Regulatory Requirements for Study Design

The fi rst consideration in the construction of a study is a clear statement of its objec-tives, which are almost always headed by meeting regulatory requirements to support drug development and registration. Accordingly, the relevant regulatory requirements must be analyzed, which is complicated by the fact that new drugs are no longer developed for registration and sale in a single - market country. The expense is too great, and the potential for broad international sales too appealing. While each major country has its own requirements as to study designs and studies required (with most of the smaller countries adhering to the regulations of one of the major players), harmonization has done much to smooth these differences [12] . Meeting these regulatory requirements is particularly challenging for several reasons. First, the only offi cial delineation of general requirements in the Untied States is dated

STUDY DESIGN AND CONDUCT 217

[13] , and recently special cases have arisen (anti - HIV agents, biotechnologically derived agents, therapeutic agents for neonates and the very elderly, etc.) that try the utility of these requirements. These needs have led to a stream of points - to - consider, which seek to update requirements. Second, the term “ guidelines ” means different things in different countries (in the United States it means “ requirements, ” and in Japan, “ suggestions ” ).

Agents intended to treat or arrest the progress of rapidly spreading life - threaten-ing diseases (such as AIDS) are subject to less stringent safety assessment require-ments prior to initial clinical evaluations than are other drugs. However, even though approval (if clinical effi cacy is established) for marketing can be granted with preclinical testing still under way, all applicable safety assessments (as with any other class of drugs) must still be completed [14] .

Drugs intended for use in either the elderly or the very young have special addi-tional requirements for safety evaluation, in recognition of the special characteris-tics and potential sensitivities of these populations. For the elderly, these requirements call for special consideration of renal and hepatic effects [15] . Likewise, drugs intended for the young require special studies to be performed in neonates and juvenile animals (usually of 2 – 4 weeks ’ duration in rats).

In the last fi ve to six years, a number of potentially important drugs have been produced by recombinant DNA technology. These biomacromolecules, which are primarily endogenously occurring proteins, present a variety of special consider-ations and concerns, including the following:

• Because they are endogenously occurring molecules, assessing their pharmaco-kinetics and metabolism presents special problems.

• One must determine if the externally commercially produced molecule is bio-logically equivalent to the naturally occurring one.

• Since they are proteins, there is the question of whether they are immunogenic or provoke neutralizing antibodies that will limit their usefulness.

• Because they are available only in very small quantities, the use of traditional protocols (such as those that use ever - increasing doses until an adverse effect is achieved) is impractical.

• Agents with such specifi c activity in humans may not be appropriately evalu-ated in rodents or other model species.

Each of these points must be addressed in any safety testing plan [16] . The require-ments set out in this chapter are designed to do this (for repeat dose testing).

8.4 STUDY DESIGN AND CONDUCT

8.4.1 Animals

In all but a few rare cases, for pharmaceutical safety assessment, separate studies in at least two species are required. Regulations require that both species be mam-malian, and one of these must be a nonrodent; practice and economics dictate that the other species will be a rodent. With extremely rare exception, the rodent species employed is the rat (although the mouse also sees signifi cant use). There is consider-


ably more variability in the nonrodent species, with a range of factors determining whether the dog (most common choice), a primate species (typically the rhesus or cynomolgus, although some others are used in particular cases), the pig (particularly in Europe), or some other animal (e.g., the ferret) is selected. The factors that should and do govern species selection are presented in detail in Gad and Chengelis [17] . The use of multiple species is a regulatory requirement arising from experience and the belief (going back to 1944 at least) that it will provide a better chance of detect-ing the full range of biological responses (adverse and otherwise) to the new molec-ular entity being evaluated. This belief has come under fi re in recent years [18] , but is unlikely to be changed soon. Along the same lines, unless an agent is to be used by only one sex or the other of humans, equal numbers of both sexes of an animal species are utilized in the studies, with the sexes being treated as unrelated for pur-poses of statistical analysis. Also except in rare cases, the animals used are young, healthy adults in the logarithmic phase of their growth curve. (The FDA specifi es that rodents be less than 6 weeks of age at the initiation of dosing.)

Numbers of animals to be used in each dose group of a study are presented in Table 8.2 . Although the usual practice is to use three different dose groups and at least one equal - sized control group, this number is not fi xed and should be viewed as a minimum (see Section 8.4.6 ). Use of more groups allows for a reduction in the risk of not clearly defi ning effects and establishing the highest possible safe dose at a modest increase in cost. There must be as many control animals as are in the largest - size test group to optimize statistical power.

Animals are assigned to groups (test and control) by one or another form of statistical randomization. Prior to assignment, animals are evaluated for some period of time after being received in - house (usually at least 1 week for rodents and 2 weeks for nonrodents) to ensure that they are healthy and have no discernible abnormalities. The randomization is never pure; it is always “ blocked ” in some form or another (by initial body weight, at least) so that each group is not (statistically) signifi cantly different from the others in terms of the “ blocked ” parameters.

Proper facilities and care for test animals are not only a matter of regulatory compliance (and a legal requirement), but also essential for a scientifi cally sound and valid study.

Husbandry requires clean cages of suffi cient size and continuous availability of clean water and food (unless the protocol requires some restriction on their avail-ability). Environmental conditions (temperature, humidity, and light – dark cycle) must be kept within specifi ed limits. All of these must, in turn, be detailed in the

TABLE 8.2 Numbers of Animals for Chronic and Subchronic Study per Test Group

Study Length Rats per Sex Dogs per Sex Primates per Sex

2 – 4 weeks 5 – 10 3 – 4 3 3 months a 20 6 5 6/9/12 months 30 8 5 Carcinogenicity (24 months) 50 10 10

a Starting with 1 month studies, one should consider adding animals (particularly to the high dose and control groups) to allow evaluation of reversal (or progression) of effects. The FDA is becoming more invested in this approach — to evaluate both recovery from effects and possible progression after dosing.


protocols of studies. The limits for these conditions are set forth in relevant NIH and USDA publications.

8.4.2 Routes and Setting Doses

Route (how an agent is administered to a test animal) and dose (how much of and how frequently an agent is administered) are inseparable in pharmaceutical safety assessment studies and really cannot be defi ned independently. The selection of both begins with an understanding of the intended use of the drug in humans. The ideal case is to have the test material administered by the same route, at the same fre-quency (once a day, three times a day, etc.), and for the same intervals (continuously, if the drug is an intravenously infused agent, for example) as the drug ’ s eventual use in people. Practical considerations such as the limitations of animal models (i.e., there are some things you can ’ t get a rat to do), limitations on technical support, 1 and the like, and regulatory requirements (discussed below as part of dose setting) frequently act or interact to preclude this straightforward approach.

Almost 30 routes exist for administration of drugs to patients, but only a handful of these are commonly used in preclinical safety studies [19] . The most common deviation from what is to be done in clinical trials is the use of parenteral (injected) routes such as IV (intravenous) and SC (subcutaneous) deliveries. Such injections are loosely characterized as bolus (all at once or over a very short period, such as 5 minutes) and infusion (over a protracted period of hours, days, or even months). The term continuous infusion implies a steady rate over a protracted period, requir-ing some form of setup such as an implanted venous catheter or infusion port.

It is rare that the raw drug itself is suitable (in terms of stability, local tissue toler-ance, and optimum systemic absorption and distribution) for direct use as a dosage form. Either it must be taken into a solution or suspension in a suitable carrier, or a more complex formulation (a prototype of the commercial form) must be devel-oped. Gad and Chengelis [20] should be consulted for a more complete discussion of dose formulation for animals or humans. One formulation or more must be developed (preferably the same one for both animals and humans) based on the specifi c requirements of preclinical dosage formulation. For many therapeutic agents, limitations on volumes that can be administered and concentrations of active ingredient that can be achieved impact heavily on dose setting.

Setting of doses for longer - term toxicity studies is one of the most diffi cult tasks in study design. The doses administered must include one that is devoid of any adverse effect (preferably of any effect) and yet still high enough to “ clear ” the projected clinical dose by the traditional or regulatory safety factors. Such safety or uncertainty factors are currently predictions on the FDA HED guidance [21] . At the same time, if feasible, at least one of the doses should characterize the toxicity profi le associated with the agent (for some biotechnologically derived agents, par-ticularly those derived from endogenous human molecules, it may only be possible to demonstrate biological effects in appropriate disease models, and impossible to

1 Many antiviral agents, particularly some anti - HIV agents, have rather short plasma half - lives, which requires frequent oral administration of the agent. Thirteen week studies have been conducted with tid (3x/day) dosing of rats and monkeys, requiring around - the - clock shift work for technical staff of the laboratory.


demonstrate toxicity). Because of limitations on availability of protodrugs, it is generally undesirable to go too high to achieve this second (toxicity) objective.

Traditionally, studies include three or more dose groups to fulfi ll these two objec-tives. Based on earlier results (generally single dose or two week studies), doses are selected. It is, by the way, generally an excellent idea to observe the “ decade rule ” in extrapolation of results from shorter to longer studies; that is, do not try to project doses for more than an order - of - magnitude - longer study (thus the traditional pro-gression from single dose to 14 day to 90 day studies). Also, one should not allow the traditional use of three dose groups plus a control to limit designs. If there is a great deal of uncertainty, it is much cheaper in every way to use four or fi ve dose groups in a single study than to have to repeat the entire study. Finally, remember that different doses may be appropriate for the different sexes.

It should also be kept in mind that formulating materials may have effects of their own, and a “ vehicle ” control group may be required in addition to a negative control group.

8.4.3 Parameters to Measure

As stated earlier, repeat dose studies are “ shotgun ” in nature; that is, they are designed to look at a very broad range of endpoints with the intention of screening as broadly as possible for indications of toxicity. Meaningful fi ndings are rarely limited to a single endpoint — rather, what typically emerges is a pattern of fi ndings. This broad search for components of toxicity profi le is not just a response to regula-tory guidelines intended to identify potentially unsafe drugs. An understanding of all the indicators of biological effect can also frequently help one to understand the relevance of fi ndings, to establish some as unrepresentative of a risk to humans, and even to identify new therapeutic uses of an agent.

Parameters of interest in the repeat dose study can be considered as sets of measures, each with its own history, rationale, and requirements. It is critical to remember, however, that the strength of the study design as a scientifi c evaluation lies in the relationships and patterns of effects that are seen in looking at each of these measures (or groups) not as independent fi ndings but rather as integrated profi les of biological effects.

Body Weight Body weight (and the associated calculated parameter of body weight gain) is a nonspecifi c, broad screen for adverse systemic toxicity. Animals are initially assigned to groups based on a randomization scheme that includes each group varying insignifi cantly from one another in terms of body weight. Weights are measured prior to the initial dose, then typically 1, 3, 5, 7, 11, and 14 days thereafter. The frequency of measurement of weights goes down as the study proceeds; after 2 weeks, weighing is typically weekly through 6 weeks, then every other week through 3 months, and monthly thereafter. Because the animals used in these studies are young adults in the early log phase of their growth, decreases in the rate of gain relative to control animals is a very sensitive (albeit nonspecifi c) indicator of sys-temic toxicity [22] .

Food Consumption Food consumption is typically measured with one or two uses in mind. First, it may be explanatory in the interpretation of reductions


(either absolute or relative) in body weight. In cases where administration of the test compound is via diet, it is essential to be able to adjust dietary content so as to accurately maintain dose levels. Additionally, the actual parameter itself is a broad and nonspecifi c indicator of systemic toxicity. Food consumption is usually mea-sured over a period of several days, fi rst weekly and then on a once - a - month basis. Water consumption, which is also sometimes measured, is similar in interpretation and use [23, 24] .

Clinical Signs Clinical signs are generally vastly underrated in value, probably because insuffi cient attention is paid to care in their collection. Two separate levels of data collection are actually involved here. The fi rst is the morbidity and mortality observation, which is made twice a day. This generally consists of a simple cage - side visual assessment of each animal to determine if it is still alive, and, if so, whether it appears in good (or at least stable) health. Historically, this regulatory required observation was intended to ensure that tissues from intoxicated animals were not lost for meaningful histopathologic evaluation due to autolysis [25] .

The second level of clinical observation is the detailed hands - on examination analogous to the human physical examination. It is usually performed against a checklist (e.g., see Ref. 20 ) and evaluation is of the incidence of observations of a particular type in a group of treated animals compared to controls. Observations range from being indicative of nonspecifi c systemic toxicity to fairly specifi c indica-tors of target organ toxicity. These more detailed observations are typically taken after the fi rst week of a study and on a monthly basis thereafter.

Ophthalmologic examinations are typically made immediately prior to initiation of a study (and thus serve to screen out animals with preexisting conditions) and toward the end of a study.

Particularly when the agent under investigation either targets or acts via a mechanism likely to have a primary effect on a certain organ for which functional measures are available, an extra set of measurements of functional performance should be considered. The organs or organ systems that are usually of particular concern are the kidneys, liver, and cardiovascular, nervous, and immune systems. Special measures (such as creatinine clearance as a measure of renal function) are combined with other data already collected (organ weights, histopathology, clinical pathology, etc.) to provide a focused “ special ” investigation or evaluation of adverse effects on the target organ system of concern. In larger animals (dogs and primates) some of these measures (such as ECGs) are made as a matter of course in all studies.

Clinical Pathology Clinical pathology covers a number of biochemical and mor-phological evaluations based on invasive and noninvasive sampling of fl uids from animals that are made periodically during the course of a subchronic study. These evaluations are sometimes labeled as clinical (as opposed to anatomical) pathology determinations. Table 8.3 presents a summary of the parameters measured under the headings of clinical chemistry, hematology, and urinalysis, using samples of blood and urine collected at predetermined intervals during the study. Conventionally, these intervals are typically at three points evenly spaced over the course of the study, with the fi rst being 1 month after study initiation and the last being immedi-ately prior to termination of the test animals. For a 3 month study, this means that


TABLE 8.3 Clinical Pathology Measures

Clinical Chemistry Hematology Urinalysis

Albumin Erythrocyte count (RBC) Chloride Alkaline phosphatase (ALP) Hemoglobin (HGB) Bilirubin Blood urea nitrogen (BUN) Hematocrit (HCT) Glucose Calcium Mean corpuscular hemoglobin (MCH) Occult blood Chloride Mean corpuscular volume (MCV) pH Creatine Platelet count Phosphorus Creatine phosphokinase

(CPK) Prothrombin time Potassium

Direct bilirubin Reticulocyte count Protein γ - Glutamyltransferase (GGT) White cell count (WBC) Sodium Globulin White cell differential count Specifi c gravity Glucose Volume Lactic dehydrogenase (LDH) Phosphorus Potassium Serum glutamic - oxaloacetic

trans - aminase (SGOT)

Serum glutamic - pyruvic transaminase (SGPT)

Sodium Total bilirubin Total cholesterol Total protein Triglycerides

samples of blood and urine would be collected at 1, 2, and 3 months after study ini-tiation (i.e., after the fi rst day of dosing of the animals). There are some implications of these sampling plans that should be considered when the data are being inter-preted. Many of the clinical chemistry (and some of the hematologic) markers are really the result of organ system damage that may be transient in nature (see Table 8.4 for a summary of interpretations of clinical chemistry fi ndings and Table 8.5 for a similar summary for hematologic fi ndings). The samples on which analysis is per-formed are from fi xed points in time, which may miss transient changes (typically increases) in some enzyme levels.

Pharmacokinetics and Metabolism Pharmaceutical subchronic toxicity studies are always accompanied by a parallel determination of the pharmacokinetics of the material of interest administered by the same route as that used in the safety study. This parallel determination consists of measuring plasma levels of the administered agent and its major metabolites either in animals that are part of the main study or in a separate set of animals (in parallel with the main study) that are dosed and evaluated to determine just these endpoints. The purpose of these determinations is both to allow a better interpretation of the fi ndings of the study and to encourage the most accurate possible extrapolation to humans. The fi rst data of interest are the absorption, distribution, and elimination of the test material, but a number of other types of information can also be collected [26, 27, 28] . For nonparenteral

223

TAB

LE

8.4

A

ssoc

iati

on o

f C

hang

es in

Bio

chem

ical

Par

amet

ers

wit

h A

ctio

ns a

t P

arti

cula

r Ta

rget

Org

ans

Par

amet

er

Org

an S

yste

m

Blo

od

Hea

rt

Lun

g K

idne

y L

iver

B

one

Inte

stin

e P

ancr

eas

Not

es

Alb

umin

↓ ↓

P

rodu

ced

by t

he li

ver.

Ver

y si

gnifi

cant

red

ucti

ons

indi

cate

ex

tens

ive

liver

dam

age.

A

LP

(al

kalin

e ph

osph

atas

e)

↑ ↑

↑

Ele

vati

ons

usua

lly a

ssoc

iate

d w

ith

chol

esta

sis.

Bon

e al

kalin

e ph

osph

atas

e te

nds

to b

e hi

gher

in

you

ng a

nim

als.

Bili

rubi

n (t

otal

) ↑

↑

U

sual

ly e

leva

ted

due

to

chol

esta

sis

eith

er d

ue t

o ob

stru

ctio

n or

hep

atop

athy

. B

UN

(bl

ood

urea

nit

roge

n)

↑

↓

Est

imat

es b

lood

- fi lt

erin

g ca

paci

ty

of t

he k

idne

ys. D

oesn

’ t b

ecom

e si

gnifi

cant

ly e

leva

ted

unti

l ki

dney

fun

ctio

n is

red

uced

60

– 75%

. C

alci

um

↑

Can

be

life

thre

aten

ing

and

resu

lt

in a

cute

dea

th.

Cho

lines

tera

se

↑

↓

Foun

d in

pla

sma,

bra

in, a

nd

RB

Cs.

CP

K (

crea

tine

ph

osph

okin

ase)

↑

M

ost

ofte

n el

evat

ed d

ue t

o sk

elet

al m

uscl

e da

mag

e bu

t ca

n al

so b

e pr

oduc

ed b

y ca

rdia

c m

uscl

e da

mag

e. C

an b

e m

ore

sens

itiv

e th

an h

isto

path

olog

y.

Cre

atin

e

↑

A

lso

esti

mat

es b

lood

- fi lt

erin

g ca

paci

ty o

f ki

dney

as

BU

N

does

. Mor

e sp

ecifi

c th

an B

UN

.

224

Par

amet

er

Org

an S

yste

m

Blo

od

Hea

rt

Lun

g K

idne

y L

iver

B

one

Inte

stin

e P

ancr

eas

Not

es

Glu

cose

↑ A

lter

atio

ns o

ther

tha

n th

ose

asso

ciat

ed w

ith

stre

ss a

re

unco

mm

on a

nd r

efl e

ct a

n ef

fect

on

the

pan

crea

tic

isle

ts o

r an

orex

ia.

GG

T (

γ - gl

utam

yltr

ansf

eras

e)

↑

Ele

vate

d in

cho

lest

asis

. Thi

s is

m

icro

som

al e

nzym

e an

d le

vels

of

ten

incr

ease

in r

espo

nse

to

mic

roso

mal

enz

yme

indu

ctio

n.

HB

DH

(hy

drox

ybut

yric

de

hydr

ogen

ase)

↑

↑

M

ost

prom

inen

t in

car

diac

mus

cle

tiss

ue.

LD

H (

lact

ic d

ehyd

roge

nase

)

↑ ↑

↑ ↑

In

crea

se u

sual

ly d

ue t

o sk

elet

al

mus

cle,

car

diac

mus

cle,

and

liv

er d

amag

e. N

ot v

ery

spec

ifi c

unle

ss is

ozym

es a

re e

valu

ated

. P

rote

in (

tota

l)

↑

↑

Abs

olut

e al

tera

tion

s ar

e us

ually

as

soci

ated

wit

h de

crea

sed

prod

ucti

on (

liver

) or

incr

ease

d lo

ss (

kidn

ey).

SGO

T (

seru

m g

luta

mic

- ox

aloa

ceti

c tr

ans -

amin

ase)

; al

so c

alle

d A

ST (

aspa

rate

am

inot

rans

fera

se)

↑

↑

↑

↑

Pre

sent

in s

kele

tal m

uscl

e an

d he

art

and

mos

t co

mm

only

as

soci

ated

wit

h da

mag

e to

th

ese.

SG

PT

(se

rum

glu

tam

ic -

pyru

vic

tran

sam

inas

e);

also

cal

led

ALT

(al

anin

e am

inot

rans

fera

se)

↑

Eva

luat

ions

usu

ally

ass

ocia

ted

wit

h he

pati

c da

mag

e or

dis

ease

.

SDH

(so

rbit

ol

dehy

drog

enas

e)

↑ or

↓

L

iver

enz

yme

that

can

be

quit

e se

nsit

ive

but

is f

airl

y un

stab

le.

Sam

ples

sho

uld

be p

roce

ssed

as

soon

as

poss

ible

.

TAB

LE

8.4

C

onti

nued


TABLE 8.5 Some Probable Conditions Affecting Hematological Changes

Parameter Elevation Depression

Red blood cells 1. Vascular shock2. Excessive diuresis3. Chronic hypoxia4. Hyperadrenocorticism

1. Anemias(a) Blood loss(b) Hemolysis(c) Low RBC production

Hematocrit 1. Increased RBCs2. Stress3. Shock

(a) Trauma(b) Surgery

4. Polycythemia

1. Anemias2. Pregnancy3. Excessive hydration

Hemoglobin 1. Polycythemia (increase in production of RBCs)

1. Anemias2. Lead poisoning

Mean cell volume 1. Amemias2. Vitamin B 12 defi ciency

1. Iron defi ciency

Mean corpuscular hemoglobin

1. Reticulocytosis 1. Iron defi ciency

White blood cells 1. Bacterial infections2. Bone marrow stimulation

1. Bone marrow depression2. Cancer chemotherapy3. Chemical intoxication4. Splenic disorders

Platelets 1. Bone marrow depression2. Immune disorder

Neutrophilis 1. Acute bacterial infections2. Tissue necrosis3. Strenuous exercise4. Convulsions5. Tachycardia6. Acute hemorrhage

1. Viral infections

Lymphocytes 1. Leukemia2. Malnutrition3. Viral infections

Monocytes 1. Protozoal infections Eosinophils 1. Allergy

2. Irradiation3. Pernicious anemia4. Parasitism

Basophils 1. Lead poisoning

routes it is essential to demonstrate that systemic absorption and distribution of the test material did occur; otherwise, it is open to question whether the potential safety of the agent in humans has been adequately addressed (not to mention the implica-tion for potential human therapeutic effi cacy).

Both clinical chemistry, pathology, and pharmacokinetics have in common the requirement to collect blood. There are species - specifi c limitations on how this may be done and how much can be collected without compromising the test animals. These are summarized in Tables 8.6 and 8.7 .


TABLE 8.6 Blood Sample Requirements

Species Body Weight (kg) Blood Volume (mL) Sample / Time Point (mL)

Mouse 0.03 2 0.075 Rat 0.3 20 1 Dog 12 1000 50 Cynomolgus

monkey 3 200 10

Rabbit 3 200 10

TABLE 8.7 Blood Sampling Techniques

Species Techniques

Mouse Orbital sinus, cardiac puncture Rat Sublingual, orbital sinus, jugular vein, cardiac puncture, tail vein Dog Jugular vein, cephalic vein Cynomolgus monkey Femoral vein/artery, cephalic vein Rabbit Jugular vein, marginal ear vein, cardiac puncture

8.4.4 Other In - Life Endpoints for Evaluation

Ophthalmology Ophthalmological examination of all animals in study (particu-larly nonrodents) should be performed both before study initiation and at the completion of the period for which the drug is administered. This should be per-formed by an experienced veterinary ophthalmologist.

Cardiovascular Function Particularly in light of recent concerns with drug - induced arrhythmias, careful consideration must be given to incorporating adequate evaluation of drug - induced alterations on cardiovascular function. This is usually achieved by measuring blood pressure, heart rate, and an EKG prestudy and peri-odically during the course of the study (usually at least one intermediate period and at the end of the study) in the nonrodent species being employed.

Neurotoxicology Table 8.8 presents the FDA ’ s current draft criteria [3, 4] for endpoints to be incorporated in studies as a screen for neurotoxicity. In practice, a functional observation battery is employed at several endpoints (usually 1 and 3 months into the study) to fi ll these requirements.

Immunotoxicology In response to concerns about potential effects of drugs on the immune system, the FDA [4] has proposed that a basic set of criteria (Table 8.9 ) be evaluated and considered in standard subchronic and chronic studies. It should be noted that most of these endpoints are already collected in traditional subchronic designs.

Pharmacokinetics All subchronic and chronic toxicity studies now incoprorate (either in the study itself or in a parallel study) evaluation of the basic pharmaco-kinetics of a compound [29] .


TABLE 8.8 FDA Draft Criteria for a Neurotoxicity Screen as a Component of Short - Term and Subchronic Studies

• Histopathological examination of tissues representative of the nervous system, including the brain, spinal cord, and peripheral nervous system.

• Quantitative observations and manipulative test to detect neurological, behavioral, and physiological dysfuntions. These may include:

General appearance Body posture Incidence and severity of seizure Incidence and severity of tremor, paralysis, and other dysfunction Level of motor activity and arousal Level of reactivity to stimuli Motor coordination Strength Gait Sensorimotor response to primary sensory stimuli Excessive lacrimation or salivation Piloerection Diarrhea Ptosis Other signs of neurotoxicity deemed appropriate

TABLE 8.9 FDA Draft Recommendations for Type I Immunotoxicity Test that Can Be Included in Repeat Dose Toxicity Studies

Hematology • White blood cell counts • Differential white blood cell counts • Lymphocytosis • Lymphopenia • Eosinophilia

Histopathology • Lymphoid tissues • Spleen Lymph nodes Thymus Peyer ’ s patches in gut Bone marrow • Cytology (if needed) a Prevalence of activated macrophages Tissue prevalence and location of lymphocytes Evidence of B - cell germinal centers Evidence of T - cell germinal centers • Necrotic or proliferative changes in lymphoid tissues

Clinical chemistry • Total serum production • Albumin • Albumin - to - globulin ratio • Serum transaminases

a More comprehensive cytological evaluation of the tissues would not be done unless there is evidence of potential immunotoxicity from the preceding evaluations.


8.4.5 Histopathology

Histopathology is generally considered the single most signifi cant portion of data to come out of a repeat dose toxicity study. It actually consists of three related sets of data (gross pathology observations, organ weights, and microscopic pathology) that are collected during the termination of the study animals. At the end of the study, a number of tissues are collected during termination of all surviving animals (test and control). Organ weight and terminal body weights are recorded at study termination, so that absolute and relative (to body weight) values can be statistically evaluated.

These tissues, along with the organs for which weights are determined, are listed in Table 8.10 . All tissues collected are typically processed for microscopic observa-tion, but only those from the high dose and control groups are necessarily evaluated microscopically. If a target organ is discovered in the high dose group, then succes-sively lower dose groups are examined until a “ clean ” (devoid of effect) level is discovered [30] .

In theory, all microscopic evaluations should be performed blind (without the pathologist knowing from which dose group a particular animal came), but this is diffi cult to do in practice and such an approach frequently degrades the quality of the evaluation. Like all the other portions of data in the study, proper evaluation benefi ts from having access to all data that addresses the relevance, severity, timing, and potential mechanisms of a specifi c toxicity. Blind examination is best applied in peer review or consultations on specifi c fi ndings.

In addition to the “ standard ” set of tissues specifi ed in Table 8.10 , observations during the course of the study or in other previous studies may dictate that addi-tional tissues be collected or special examinations (e.g., special stains, polarized light or electron microscopy, immunocytochemistry, or quantitative morphometry) be

TABLE 8.10 Tissues for Histopathology

Adrenals a Mainstream bronchi Body and cervix Major salivary glands Brain, all three levels a Mesenteric lymph nodes Cervical lymph nodes Ovaries and tubes Cervical spinal cord Pancreas Duodenum Pituitary Esophagogastric junction Prostate Esophagus Skeletal muscle from proximal hind limb Eyes with optic nerves Spleen a Femur with marrow Sternebrae with marrow Heart Stomach Ileum Testes with epididymides a Kidneys a Thymus and mediastinal contents a Large bowel Thyroid with parathyroid a Larynx with thyroid and parathyroid Trachea Liver a Urinary bladder Lungs a Uterus including horns

a Organs to be weighed.


undertaken to evaluate the relevance of, or to understand the mechanisms underly-ing, certain observations.

Histopathology testing is a terminal procedure, and, therefore, sampling of any single animal is a one - time event (except in the case of a tissue collected by biopsy). Because it is a regulatory requirement that the tissues from a basic number of animals be examined at the stated end of the study, an assessment of effects at any other time course (most commonly, to investigate recovery from an effect found at study termination) requires that satellite groups of animals be incorporated into the study at start - up. Such animals are randomly assigned at the beginning of the study, and otherwise treated exactly the same as the equivalent treatment (or control) animals.

8.4.6 Study Designs

The traditional design for a repeat dose toxicity study is very straightforward. The appropriate number of animals of each sex are assigned to each of the designated dose and control groups. Unfortunately, this basic design is taken by many to be dogma, even when it does not suit the purposes of the investigator. There are many possible variations to study design, but four basic factors should be considered: controls, the use of interval and satellite groups, balanced and unbalanced designs, and staggered starts.

Classically, a single control group of the same size as each of the dose groups is incorporated into each study. Some studies incorporate two control groups (each the same size as the experimental groups) to guard against having a statistically signifi cant effect due to one control group being abnormal for one or more param-eters (a much more likely event when laboratory animals were less genetically homogeneous than they are now). The belief is that a “ signifi cant ” fi nding that differs from one (but not both) of the concurrent control groups, and does not differ from historical control data, can be considered as not biologically signifi cant. This is, however, an indefensible approach. Historical controls have value, but it is the concurrent control group(s) in a study that is of concern.

Interval or satellite groups have been discussed at two earlier points in this chapter. They allow measurement of termination parameters at intervals other than at termination of the study. They are also useful when the manipulation involved in making a measurement (such as the collection of an extensive blood sample), while not terminal, may compromise (relative to other animals) the subject animals. Another common use of such groups is to evaluate recovery from some observed effect at study termination.

Usually, each of the groups in a study is the same size, with each of the sexes being equally represented. The result is called a balanced design, with statistical power for detection of effects optimized for each of the treatment groups. If one knows little about the dose – toxicity profi le, this is an entirely sound and rational approach. However, there are situations when one may wish to utilize an unbal-anced design — that is, to have one or more dose groups larger than the others. This is usually the case when either greater sensitivity is desired (typically in a low dose group), or an unusual degree of attrition of test animals is expected (usually due to mortality in a high dose group), or as a guard against a single animal ’ s idiopathic response being suffi cient to cause “ statistical signifi cance. ”


As it is normal practice to have a balanced design, it is also traditional to initiate treatment of all animals at the same time. This may lead to problems at study ter-mination, however. It is a very uncommon toxicology laboratory that can “ bring a study down ” on a single day. In fact, there are no labs that can collect blood and perform necropsies in a single day on even the 48 – 80 dogs involved in a study, much less the 160 – 400+ rats in the rodent version. Starting all animals in a study on the same day presents a number of less than desirable options. The fi rst is to terminate as many animals as can be done each day, continuing to dose (and therefore further affect) the remaining test animals. Assuming that the animals are being terminated in a random, balanced manner, this means that the last animals terminated will have received from 3 to 10 additional days of treatment. At the least, this is likely to cause some variance infl ation (and therefore both decrease the power of the study design and possibly confound interpretation). If the difference in the length of treatment of test animals is greater than 3% of the intended length of the study, one should consider alternative designs.

An alternative approach to study design that addresses this problem employs one of several forms of staggered starts. In these, distinct groups of animals have their dosing initiated at different times. The most meaningful form recognizes that the two sexes are in effect separate studies anyway (they are never compared sta-tistically, with the treatment groups being compared only against the same - sex control group). Thus, if the termination procedure for one sex takes 3 – 5 days, then one sex should be initiated on dosing one week and the other on the following week. This maximizes the benefi ts of common logistical support (such as dose formula-tion) and reduces the impact of differential length of dosing on study outcome.

Another variation recognizes that any repeat dose study using both males and females is really two studies. The different sexes are considered separately and, except for ending up in a single report, are treated as separate studies. Accordingly, each sex can be started on a different day without increasing variability.

A further variation on this is to stagger the start - up either of different dose groups, or of the satellite and main study portions of dose groups. The former is to be avoided (it will completely confound study outcome), while the latter makes sense in some cases (pharmacokinetics and special measures) but not others (recov-ery and interval sacrifi ce).

8.5 STUDY INTERPRETATION AND REPORTING

For a successful repeat dose study, the bottom line is the clear demonstration of a no - effect level, characterization of a toxicity profi le (providing guidance for any clinical studies), enough information on pharmacokinetics and metabolism to scale dosages to human applications, and at least a basic understanding of the mechanisms involved in any identifi ed pathogenesis. The report that is produced as a result of the study should clearly communicate these points — along with the study design and experimental procedures, summarized data, and their statistical analysis — and it should be GLP compliant, suitable for FDA submission format.

Interpretation of the results of a study should be truly scientifi c and integrative. It is elementary to have the report state only each statistically and biologically sig-nifi cant fi nding in an orderly manner. The meaning and signifi cance of each in rela-

tion to other fi ndings, as well as the relevance to potential human effects, must be evaluated and addressed.

The author of the report should ensure that it is accurate and complete, but also that it clearly tells a story and concludes with the relevant (to clinical development) fi ndings.

REFERENCES

1. Ballantyne B. Repeated exposure toxicity . In Ballantyne B , Marrs T , Syversen T , Eds. General and Applied Toxicology . London : MacMillan ; 2000 , pp 55 – 66 .

2. Wilson NH , Hardisty JF , Hayes JR. Short - term, subchronic and chronic toxicity studies . In Hayes AW , Ed. Principles and Methods of Toxicicology , 4th ed. Philadelphia : Taylor & Francis ; 2001 , pp 917 – 958 .

3. FDA . Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food , Redbook II, p 86. Washington, DC : Center for Food Safety and Applied Nutrition, FDA ; 1993 .

4. FDA . Toxicological Principles for the Safety of Food Ingredients , Redbook 2000. Washington, DC : Center for Food Safety and Applied Nutrition, FDA ; 2000 .

5. OECD . Guidance Notes for Analysis and Evaluation of Chronic Toxicity and Carcinoge-nicity Studies 1998.

6. O ’ Grady J , Linet OI. Early Phase Drug Evaluation In Man . Boca Raton, FL : CRC Press ; 1990 .

7. Smith CG. The Process of New Drug discovery and Development . Boca Raton, FL : CRC Press ; 1992 .

8. Food and Drug Administration (FDA) . Good Laboratory Practices for Nonclinical Labo-ratory Studies. CFR 21 Part 58, March 1983 .

9. Food and Drug Administration (FDA) . Good Laboratory Practices. CFR 21 Part 58, April 1988 .

10. OECD . Principles of Good Laboratory Practice 1997. 11. Animal and Plant Health Inspection Service (APHIS) . United States Department of

Agriculture (31 August 1989) . Fed Reg 1989 ; 54 ( 168 ): 36112 – 36163 . 12. Alder S , Zbinden G. National and International Drug Safety Guidelines . Zollikon,

Switzerland : M.T.C. Verlag ; 1988 . 13. Food and Drug Administration (FDA) . FDA Introduction to Total Drug Quality .

Washington, DC : US Government Printing Offi ce ; 1971 . 14. Food and Drug Administration (FDA) . Investigational new drug, antibiotic, and biologi-

cal drug product regulations, procedures for drugs intended to treat life - threatening and severely debilitating illness . Fed Reg 1988 ; 53 ( 204 ): 41516 – 41524 .

15. Center for Drug Evaluation and Research (CDER) . Guideline for the Study of Drugs Likely to Be Used in the Elderly . FDA ; November 1989 .

16. Weissinger J. Nonclinical pharmacologic and toxicologic considerations for evaluating biologic products . Regul Toxicol Pharmacol 1989 ; 10 : 255 – 263 .

17. Gad SC , Chengelis CP. Animal Models in Toxicology . New York : Marcel Dekker ; 1992 . 18. Zbinden G. The concept of multispecies testing in industrial toxicology . Regul Toxicol

Pharmacol 1993 ; 17 : 84 – 94 . 19. Gad SC. Routes in safety assessment studies . J Am Coll Toxicol 1994 ; 13 : 1 – 17 .

REFERENCES 231


20. Gad SC , Chengelis CP. Acute Toxicology , 2nd ed. San Diego, CA : Academic Press ; 1999 .

21. CBER . Estimating the Safe Starting Dose in Clinical Trials for Therpeutics in Adult Healthy Volunteers . 2005 . http://www.fda.gov/cber/gdlns/dose.htm .

22. Allaben WT , Hart RW. Nutrition and toxicity modulation: the impact of animal body weight on study outcome . Int J Toxicol 1998 ; 17 (Suppl 2) : 1 – 3 .

23. CPMS/SWP/2877/00 Note for Guidance on Carcinogenic Potential . 24. Ellaben WT , Hart RW. Nutrition and toxicity modulation: the impact of animal body

weight on study outcome . Int J Toxicol 1998 ; 17 (Suppl 2) : 1 – 3 . 25. Arnold DL , Grice HC , Krawksi DR. Handbook of In Vivo Toxicity Testing . San Diego,

CA : Academic Press ; 1990 . 26. Yacobi A , Skelly JP , Batra VK. Toxicokinetics and New Drug Development . New York :

Pergamon Press ; 1989 . 27. Tse FLS , Jaffe JM. Preclinical Drug Disposition . New York : Marcel Dekker ; 1991 . 28. EMEA . Guideline on the Evaluation of Control, Samples for Toxicokinetics Parameters

in Toxicology Studies: Checking for Contamination with the Test Substance . 2004 . 29. CPMP/ICH/384/95; ICH S3A. Toxicokinetics: A Guidance Assessing Systematic Exposure

in Toxicology Studies . 30. Haschek WM , Rousseaup CG. Handbook of Toxicology Pathology . San Diego, CA : Aca-

demic Press ; 1991 .

Download - Preclinical Development Handbook || Repeat Dose Toxicity Studies

Top Related