Pre Clinical Studes in Animals

8/7/2019 Pre Clinical Studes in Animals

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Pre clinical studies in animals

Table of contents

1.1 Introduction....................................................................................................... 2

1.2 Objectives............................................................................................... ............2

1.3 Scope.............................................................................................. ....................2

2 An introduction about nude mice........................................................................ .3

3 Specification of test material................................................................................. 3

4 Pre clinical tests ................................................................. ............................. 3

4.1 Cross reactivity testing of monoclonal

antibodies......................................................................... .................................... .......3

4.2 In vitro and in vivo testing................................................ .................................... 4

4.3 Pharmacology and toxicity testing..................................................... ...................4

4.4 Pre clinical safety testing...................................................................................... 4

4.4.1 General parameters............................................................................................. 4

4.4.2 Animal species selection...................................................... .............................. 5

4.4.3 Dose selection..................................................................... ............................... 6

5.1 Single dose toxicity studies................................................................................... 6

5.2 Repeated dose toxicity studies.............................................................................. 6

6 Pharmacokinetics and toxicokinetics studies........................................................ .6

7 Immunogenicity testing¶s........................................................... .............................7

8 Acute and chronic toxicity testing¶s........................................................................ 7

9 Carcinogenicity testing¶s.......................................................................................... 8

10 Reproductive toxicity testing.......................... .......................................................... 8

11 Genotoxicity testing¶s............................................................................................... 8



1: Introduction

Monoclonal antibodies are genetically engineered immune monospecific antibodies. They arecategorised in immunoglobulins which have some specific molecular interactions with

specific molecular targets. Typically they are formed by the fusion of myeloma cells with

spleen cells. Mouse which is immunized with specific antigen is used in this process. One of

the most important property of monoclonal antibodies is binding with the cancer cell specific

antigens and generate immunological response against cancer cell. Monoclonal antibodies

holds great importance in generation of many biotechnology derived pharmaceuticals .The

origination of Biotechnology derived pharmaceuticals can be found in 1980s; however it took

large time to bring them to market because they were not given authorisation. In order to

show safety of these products various guide lines and points were written and shown by

several regulatory agencies. Honest review of these documents can led us to very good results

if we want to discover or develop new biopharmaceuticals.

1.2: Objectives

As we know day by day world is growing with high advance technologies. In order to

maintain eaquvillance there is need for common understanding, therefore in case of

biotechnology derived pharmaceuticals governing standards¶ are compared between

European Union, United States and Japan. All these there have acquired same parameters and

scientific approach to preclinical safety evaluation, development and market authorisation of

biotechnology derived pharmaceuticals.

The main objective of our study is to identify preclinical safety of monoclonal antibodies in

background of ICH guidelines and how this drug can be led to market. The main points of

preclinical safety evaluation for monoclonal anti bodies according to ICH s6 guidelines are as

fallows.

1: to know initial and final dose in human beings.

2: to discover those organs which are targeted/harmed by toxicity and is there a chance that

this toxicity can be reversible.

3: to evaluate principles for safety supervision.

1.3: Scope

Aim is to propose fundamental framework for safety evaluation of biotechnology derived

pharmaceuticals for human use. We can measure monoclonal antibodies for the following

things listed below.

1: Stability



2: Potency

3: Purity

4: Pharmacology and toxicology

5: and Safety.

2: An introduction about NUDE MOUSE

Nude mouse is a laboratory mouse. Due to genetic mutation occurred in one of its strain

degeneration of it thymus had occurred. Genetic base for this mutation spontaneous deletion

of FOXN1 gene. Because of lack of thymus this mouse cannot produce mature T

lymphocytes. Morphologically it has no hair on its body. As it shows no rejection in response

after receiving many tissues and tumor grafts, that¶s why it is extensively used for research in

various laboratories. In nude mouse Similar to other drugs monoclonal antibodies also shows

very good results for treatment of human¶s colon carcinoma that is why this species was

selected.

3: Specification of the test material

Removal of impurities is of highly importance because we can come with false results at the

end. To trust on purification processes is necessary then to develop a pre clinical testing

programme. Whatever the aspect is the product should satisfy the criteria of preclinical safety

studies.

In general comparison should be done between the product which is used in pharmacology

and toxicology and the product which is used for initial clinical studies, however during

developmental and constructing stages if some modifications are done in product then thoseare highly marked. Biochemical and Biological properties i.e. stability, purity, identity play a

vital role in comparing a product. In order to judge the product in real manner some extra

studies i.e. pharmacokinetics and pharmacodynamics should be done.

4: Preclinical tests

Before initiation of humans trials a number of tests have to be done for the given drug. the

main purpose to do these test is to know about pharmacological and toxicological effects

before it is subjected to human beings. In order to know in detail we have to do both in vivoand in vitro tests. As mentioned in ICH S6 and M3 guide lines preclinical tests should give

detail about characterization of the drug, route of administration, duration of total exposure

proposed in individuals patients, and use in special population.

Different preclinical tests performed are illustrated under

4.1: cross reactivity testing of monoclonal antibody



Cross reactivity should be done prior to phase1 because we can easily identify non target

tissues which non target tissues can give us serious negative results. ICH S6 guide line clearly

tells us not to satisfy our self only by immunohistochemical examinations for binding of

monoclonal antibody binding for cross reactivity in relevant species but other techniques like

in situ hybridization must be performed. In addition both in vitro and in vivo testing cross

reactivity should be performed.

4.2: In vitro and in vivo testing

For in vitro testing as given in ICH S6 guidelines for monoclonal anti body detail profile for

immunogenic properties, complement binding, antigenic specificity, and in last cytotoxic

reactions should be completely known. However in vivo studies should evaluate sensitivity of

various species to the given drug and also to identify safety and toxicity.

4.3: pharmacology and toxicity testing

The main theme of pharmacology studies is to understand and functional effects on major

systems like cardiovascular, central nervous, renal, and respiratory systems. The purpose of

these tests is to identify adverse events, possible toxicity, and safe initiation of the drug. In

case of monoclonal antibody product testing immunogenicity, difference in species, value of

cross reactivity, are of most importance. In pharmacology and toxicology area normally

pharmacokinetics and pharmacodynamics studies are done by which we can easily recognise

antigen of interest. In nude mice we can easily generate xenograft models by inserting the cell

which is expressing antigen of our interest. Homologous proteins can be used for this purpose

but as given in ICH S6 and M3 guidelines this should be done in relevant species because

they provides very good results.

4.4: Preclinical safety testing

4.4.1: General parameters

The main theme of preclinical safety testing is to know pharmacological and toxicological

out come in all modes not during initiation of human studies, keeping in mind that both in

vivo and in vitro are its features. There is a less chance to test extensive toxicity testing of

biopharmaceuticals as it is having same configuration both in structure and pharmacology as

that of chemically synthesised products.

However these points should be considered:

1: age

2: choice of animal species

3: the way of delivery, type of dose



4: through which route dose is delivered and its way of treatment

5: physiological state

6: and to know stability and conditions of use of test material.

Good laboratory practise (GLP) is of high importance for toxicity studies, because there arehigh chances of false results if it is ignored; however in some cases some specialised tests

needed for biopharmaceuticals are not needed to be followed by good laboratory practise

these should be pointed out in other words these should be denoted. In some cases if data

from GLP is not present neither it means that the study doesn¶t favours clinical trials nor will

not be approved for market.

4.4.2: Animal species selection

Various numbers of techniques gives us information that which specie is relevant for testing

or experiment. In order to understand potential in vivo toxicity we should have sufficient

knowledge about receptor distribution. That¶s why it is often said that a relevant specie is theone in which the test material is pharmacologically active as receptor are expressed for test

material, here in case of monoclonal anti bodies we selected nude mice.

For monoclonal anti bodies relevant species are those where expression of desired epitope

and tissue cross reactivity are same as that of human tissues. If in an animal species desired

epitope is not expressed again it is having chance that it will show toxicity values if their

tissues cross reactivity is established with humans.

Normally two animal species should be selected; in some cases one animal species is

sufficient if biological activity of biopharmaceutical is well understood. In toxicity short term

studies normally two species are required but it is quite enough if only one species is used for long term toxicity studies.

The use of non relevant species for toxicity studies is not favoured and leads to negative

results. If in worst case there is no relevant specie then relevant transgenic animals which

express human receptors or proteins which shows homologosity should be preferred. As

transgenic animal is expressing human receptor the reactivity of product with this receptor

will be having similar physiological conditions as thought in humans. In addition we can

obtain useful information from homologous proteins. It should be noted that different types of

impurities, pharmacokinetics, and pharmacological mechanism will be having differences

between homologous form and product which will be used in clinic. When we can¶t use

transgenic animal models or homologous proteins still it is careful to evaluate some factors in

limited toxicity in single species. This can be repeated dose study which will be less than 14

days but coming with important endpoints.

Due to advancement in technology increase is found in constructing animal¶s models which

are thought to be similar to human disease. They include transgenic animals and is some

cases gene is induced to spontaneously knocked out in these animals. It is hoped that these



models will provide major information not only regarding pharmacological action and

pharmacokinetics but also safety, efficacy and quality of the product.

4.4.3: Dose selection

The channel and frequency of administration should be kept very close as it is proposed for

clinical use in humans. Special attention should be given to pharmacokinetics, bioavailability

and volume of the test product in those species where it is to be used .for example the

frequency of administration in case of laboratory animals should be increased in equivalence

to suggested agenda for human clinical studies in order to adjust quick clearance rates or low

solubility of the active components. Here degrees of exposure of experimental animal

comparative to clinical exposure should be determined. The effects of volume, concentration,

formulation, should be properly mentioned. Routes used other than clinical routes may be

accepted if the route is having no issues with bioavailability.

Dosage levels should be maintained so that we get data about dose response relationship,

which includes toxic dose and no observed adverse effect level (NOAEL). There are someclasses of products which shows very less or no toxicity for which we can¶t mention specific

maximum dose level. Here we can take help from scientific consideration. To object high

dose selection special care should be taken of pharmacological effects, physiological effects,

clinical use and availability of test material.

5.1: Single dose toxicity studies

In order to understand relationship of dose to systemic or local toxicity, single dose studies

gives us very important data and we can choose doses for repeated dose toxicity studies by

the help single dose studies data. After passing through single dose toxicity data its

information is gathered.

5.2: Repeated dose toxicity studies

Repeated dose toxicity studies are of much more importance in animals. One of most

important thing in this aspect is to mention recovery period. For biopharmaceuticals where

recovery is thought to be prolonged due to pharmacological or toxicological effects recovery

group animals should be observed till reversibility is established. Repeated dose duration

should depend upon propose clinical exposure. For most biotechnology derived

pharmaceuticals animal dosing duration is from one to three months. Those

biopharmaceuticals which are thought to be used in short terms repeated dose more than 7

days and for acute life threatening diseases repeated dose of two weeks are recommended for them in order to support clinical studies. 6 months or longer studies are recommended for

those biopharmaceuticals which are used for chronic diseases if they want to get access to

market.

6: Pharmacokinetics and toxicokinetics studies



Relevant animal species should be used for toxicity testing in case of biotechnology derived

pharmaceuticals because they are species specific. Although Single and multiple dose

pharmacokinetic, toxicokinetics, and tissue distribution studies are very useful in applicable

species yet to demonstrate consistent guidelines is very hard task in case of biotechnology

derived pharmaceuticals. It is difference in pharmacokinetics which has a very high effect on

Judgement of dose response relationship in toxicity studies and predictiveness of animalstudies. In case of some products there may be delay in expression of pharmacokinetics as

seen in cytokines or there may be lengthy expression of pharmacodynamics effects as seen in

plasma levels. Care of absorption level should be taken because it can be forced by

concentration rate, volume and its site. During toxicity studies systemic exposure should be

observed. In case of radiolabeled proteins, materials used should be shown. There lies a

strong possibility that we can come with difficulty while interpreting data for tissue

concentration or autoradiography due to rapid in vivo metabolism.

7: Immunogenicity

According to ICH S6 and M3 guile lines immunogenicity testing¶s are neither relevant nor required, because we don¶t find possible adverse reactions and any change in the

pharmacokinetics and toxicokinetics of the given drug. If some immune mediated reactions

like hypersensitivity and anaphylaxis are generated then proper reasons and results of thee

pathways due to which these are emerged should be given. If neutralization reaction with

antibodies occurred then previous bioactivity or direct neutralization assay should be

performed. These studies are very better to performed during pharmacokinetics and tissue

distribution when they are compared to unconjugated antibody and at this stage their stability

data should be submitted. While doing repeated dose toxicity studies proper evaluation of

antibodies affiliated with administration of these types of products should be done. Response

should be categorised and well correlation between their morphology and pharmacological,toxicological changes should be made. Those pathological changes which are concerned with

immune complexion and deposition should be highly noticed. While translating data special

features like inauspicious or adverse effects, activation, outgrowth of new toxic material, and

effects generated from pharmacokinetic and pharmacodynamics should be highlighted.

Results coming up with antibodies should not be fundamental criteria for early ending of

preclinical safety study or its advancement unless and until the immunological response

doesn¶t equalise pharmacological or toxicological effects of biopharmaceutical in high ratio

in animals. It is observed that immune response is having fluctuation to biopharmaceutical as

it is observed in human beings. There is a possibility that human beings will form serum

antibodies versus humanised proteins and their response remains or is found in their presence; however life threatening response to recombinant proteins is very low.

8: Acute chronic toxicity testing¶s

For monoclonal antibodies these normally these tests are not done however six months data

should be provided for those products which are chronically used. on site of administration if



any reaction occurred additional tests for example local tolerance test should be provided in

order to support them.

9: Carcinogenicity testing¶s

Standard carcinogenicity testing¶s are considered inappropriate for biotechnology derived

pharmaceuticals; however product judgement depending upon length of clinical dosing,

patient population, and biological activity of the product may be needed. Those products

which are truly understood to support or accelerate development of transformed cells and

clonal expansion may be leading to neoplasia should be judged with respect to receptor

expression in different malignant and human cells. Here the power of the product should be

defined which enhance growth of the normal or malignant cells. Further studies should be

done in appropriate animal if in vivo data is having concern about carcinogenic potential.

Cellular development may provide useful information if repeated dose studies are done for

long terms.

10:Reproductive toxicity testing¶s

Product, clinical indication and patient population are the most important factors on which

these types of studies are dependent. Along reproduction developmental studies should be

done in relevant species. As mentioned in ICH S6 guidelines proper evaluations from toxicity

to reproduction should be performed in relevant species and proper results should be

submitted. It should be well understood that in case of embryo and fetal development if

proper justification is given then only one species is sufficient as it is given in ICH E5

guidelines. In case of any adverse reactions proper explanation should be given and its results

should be submitted.

11: Genotoxicity studies

Normally Genotoxicity studies often done for pharmaceuticals are not relevant to

biotechnology derived pharmaceuticals therefore, they are not needed. In addition

establishment of immense no of proteins or peptides can generate unexplainable results. It is

thought that these substances will play no role directly with DNA or other chromosomal

material. Studies in new developed systems if are available should be done when their cause

about product is known. In order to calculate the genotoxic potential of process contaminants¶

use of Genotoxicity studies are not considered suitable.

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Pre Clinical Studes in Animals