DESAIN PENELITIAN

BIOMOLEKULER, IMUNOLOGI
DAN
HEWAN COBA

NOOR WIJAYAHADI
 MENGAPA PENELITIAN PERLU
DIRANCANG

Untuk memperoleh data yang relevan

(karena data dapat diperoleh dengan
berbagai macam cara)

Cara mana yang

menjamin diperolehnya Cara mana yang secara
data : - obyektif teknis efisien & efektif
- vallid
- reliabel
RELEVAN
Lima Kriteria : OBJECTIF
VALID
RELIABEL
EKONOMIS
 •
PENELITIAN EKSPERIMENTAL
 Dua keadaan yang identik, sejajar dalam
segala hal, kecuali satu yi
perlakuan/manipulasi
 Perlakuan = prosedur yang efeknya akan
diukur dan dibandingkan
 Mengadakan percobaan untuk melihat
hasil
 Hubungan kausal antara variabel-2 yang
diteliti
 •
TRUE EXPERIMENT
 Penelitian untuk menyelidiki kemungkinan
hubungan sebab-akibat dengan memberikan
1/> kel.eks, 1/> kondisi perlakuan &
bandingkan hasilnya dgn kel.kontrol
 Ciri-ciri :
. Pengaturan variabel
. Gunakan kel.kontrol
. Pusatkan pada kontrol varians
. Perhatian validitas Eks/Int.
 •
QUASI EXPERIMENT
 Penelitian untuk memperoleh informasi
yg merupakan perkiraan yg diperoleh
dengan eks,murni, dalam keadaan yg
t’memungkinkan mengontrol semua
variabel yang relevan
 Sebagai contoh : penelitian sosial
 Ciri-ciri : Tidak mungkin melakukan
kontrol terhadap variabel
(1) RA : Kel. Eks
KONTROL NEG
Kel.kontrol KONTROL POS
Waktu
(2) - Awal Penelitian
Akhir Penelitian
(3) X - Intervensi
• O - Observasi/Pengukuran
O1,2,3 - Pengukuran (1), (2), (3) dst.
• O,x dalam satu garis : Kel.Ind / Subyek sama
• Garis pemisah kel yg tak dilakukan randomisasi
Contoh : RA Kel. Eksp. O1 x O2
The Role of Animal Models
in Drug Discovery
 Definition of Animal Models

A living organism in which normative biology

or behavior can be studied, or in which a
spontaneous or induced pathological process
can be investigated, and in which the
phenomenon in one or more respects
resembles the same phenomenon in humans
or other species of animals.

- By Joe Held on the basis of Wessler’s original definition

 Classification of Animal Models

• Exploratory
to understand a biological mechanism
• Explanatory
to understand a complex biological problem
• Predictive
to discover and quantify the impact of a
treatment
 Animal Models to Humans

• Fidelity
The resemblance of the biological structure in
the animal with the corresponding structure in
humans
• Discriminating ability (predictability)
The similarity between humans and model
species with respect to relevant biological
mechanism is more important than the fidelity
of the model.
 Classification of Disease Models

1. Induced (experimental) disease models

2. Spontaneous (genetic) disease models
3. Transgenic disease models
4. Negative disease models
Mechanism of resistance
5. Orphan disease models
Naturally occurs in a non-human species but
has not yet been identified in humans
 Induced (Experimental) Disease
Models
• Healthy animals initially.
• Condition to be investigated is experimentally
induced. For example, partial hepatectomy
for liver regeneration, streptozotocin-induced
type 1 diabetes
• Unfortunately, few induced models completely
mimic the etiology, course, and pathology of
the target disease in the humans.
 Spontaneous (Genetic) Disease
Models
• Naturally occurring genetic variants
(mutants) in studying human diseases
For example: nude mice, Snell’s dwarf
mice (lacking pituitary), BB rats

• Concern – the compensatory

mechanisms may differ between
humans and the animal model species
 Transgenic Disease Models

• The advance in genetic engineering and

embryo manipulation technology facilitates
the development of transgenic models.

• Mutations induced by mutagens such as

ethyl-nitroso-urea (ENU) represent another
approach to the generation of new mutants.
 Animal Models in Drug Discovery

• Pharmacodynamics
• Pharmacokinetics
• Toxicology
 Pharmacodynamics

Primary Effect

Secondary Effect
 Pharmacodynamic Primary Effect
Animal Models for Type 2 Diabetes

• Genetic models
db/db mice, ob/ob mice, KK mice, fa/fa
Zucker rats
• Oral Glucose Tolerance Test
Pharmacodynamic Seconday Effect

Adverse effects

Safety Pharmacology
 ICH Topic S7A
Safety Pharmacology Studies for Human
Pharmaceuticals
NOTE FOR GUIDANCE ON SAFETY PHARMACOLOGY
STUDIES FOR HUMAN PHARMACEUTICALS

1. INTRODUCTION

1.1 Objectives Of The Guideline

This guideline was developed to help protect clinical trial
participants and patients receiving marketed products from
potential adverse effects of pharmaceuticals, while avoiding
unnecessary use of animals and other resources. This
guideline provides a definition, general principles and
recommendations for safety pharmacology studies.
www.ich.org
 ICH Guidelines
www.ich.org

• Carcinogenicity Studies
• Genotoxicity Studies
• Toxicokinetics and Pharmacokinetics
• Toxicity Testing
• Reproductive Toxicology
• Pharmacology Studies
• Immunotoxicology Studies
 Genotoxicity
•S2A: Guidance on Specific Aspects of Regulatory
Genotoxicity Tests for Pharmaceuticals

The tripartite harmonised ICH guideline was finalised (Step 4) in July 1995.
This document provides specific guidance and recommendations for
in vitro and in vivo tests and on the evaluation of test results. It  includes
a glossary of terms related to genotoxicity tests to improve consistency in
applications.

• S2B: Genotoxicity: A Standard Battery for

Genotoxicity Testing for Pharmaceuticals

The tripartite harmonised ICH guideline was finalised (Step 4) in July 1997.
This document addresses two fundamental areas of genotoxicity testing:
the identification of a standard set of assays to be conducted for
registration, and the extent of confirmatory experimentation in any
particular genotoxicity assay in the standard battery.
 Concordance of the Toxicity of
Pharmaceuticals in Humans and in Animals
• A multinational pharmaceutical company survey
• Adverse findings of 150 compounds in human clinical data
and data from preclinical tests in animals

HT* Concordance rate – 71% (rodents + non-rodents)

63% (non-rodents)
43% (rodents)
High concordance rate – cardiovascular (80%)
hematological (91%)
gastrointestinal (85%)
Low concordance rate – neurological (ex. headache, dizziness)
the only gastrointestinal (nausea)

* HT : human toxicity Regulatory Toxicology and Pharmacology 32:56 (2000)

Time to First Detection of Relevant
Toxicity in Animals
38%

94% detection within one month

Regulatory Toxicology and Pharmacology 32:56 (2000)

Concordance Rate versus Species

The choice of species used might be subject to more

thoughtful consideration.
Regulatory Toxicology and Pharmacology 32:56 (2000)
 Concerns

Though the predictive value of animal studies may seem high, but

 Penicillin is fatal for guinea pigs, but generally well tolerated by

humans
 Aspirin is teratogenic in mice, rats, cats, dogs, monkeys, but
obviously not in pregnant women
 Thalidomide but not cause birth defects in rats or many other
species, but does so in primates

A close phylogenetical relationship or anatomical similarity is

not a guarantee of identical biochemical mechanisms and
parallel physiological response.
 Further Complication

•To which humans?

Often results are obtained from genetically defined and

uniform animal models, but humans are genetically
highly variable, with cultural, dietary and environmental
difference.
 Pharmacokinetics

• A  Adsoprtion (digestive tract, blood-

brain barrier, bioavailability)
• D  Distribution (effector sites)
• M  Metabolism (cytochrome P450
enzymes, metabolites)
• E  Excretion (remove from the body,
kidney (urine) and feces)
Extrapolation from Animals to Humans

Extrapolation :
how data obtained from animal studies reliably
applies to the human
 Pharmacodynamics
 Adverse effects
 Model body size and scaling
 What are the alternatives?

• Reduction - fewer animals

• Refinement - less painful
procedures
• Replacement - alternative
techniques

30
 The 3 Rs

Reduction: any decrease in the numbers of animals

used to obtain information of a given amount and
precision

Refinement: any decrease in the incidence or severity

of procedures applied to animals necessarily used

Replacement: the substitution of conscious living

vertebrates by non-sentient material

31
The 4th R
The 4th R

• Responsibility

32
 Reduction alternatives
Good planning of studies
• Rational and efficient use of animals
• no wasting
• pilot studies
• screening tests
• Proper statistical design
• Use of inbred starins (for some study types)

33
 Refinement alternatives
• Minimized potential for pain or distress
• Enhanced animal well-being
• Improved housing conditions and
experimental techniques

34
 Replacement alternatives (1)
• Efficient use of existing information
• In silico methods (computer simulations, mathematical
models, QSAR)
• ”Read-across”, grouping of chemicals
• In vitro methods: isolated organs
tissue slices
tissue cultures
cell cultures
subcellular fractions
• Lower organisms
• Early stages of development

35
 Replacement alternatives (2)

In vitro methods are not primarily “replacements” of in

vivo methods. Typically these methods have different
roles in research, and they are complementary for each
others.

Depending on the objective of the study in vitro

methods may be the most appropriate methods for
certain areas of interest, because they can more
accurately provide the information required (e.g.
cellular and molecular events).

36
 Replacement alternatives (3)

• Absolute replacement: no need to use animals (cell

lines, human or invertebrate cells and tissues)
• no need to test for skin irritation if pH <2.0 or >11.5
• no need to test for eye irritation if the chemical is a
skin irritant
• Relative replacement: humane killing of animals to
provide cells or tissues for in vitro studies
• Partial replacement: e.g. use of non-animal methods as
prescreens in toxicity testing

37
 Replacement alternatives (4)

• Direct replacement: e.g. human or guinea pig skin is

used in vitro to replace guinea pig tests in vivo
• Indirect replacement: e.g. Limulus amoebocyte lysate
(LAL) test or a test based on whole human blood is
used to replace rabbit pyrogen tests

38
 Skin corrosion and skin irritation tests

In vitro In vivo
• Skin corrosion: “artificial” • Corrosivity test on rabbit
human skin cultures skin

39
 Eye irritation tests

In vitro In vivo
• Eye irritation: • Draize test in rabbit’s eye
• HET-CAM (hen’s egg chorio-
allantoic membrane) test

HET-CAM = hen’s egg chorio-allantoic membrane

• BCOP (bovine corneal opacity) test
• ICE (isolated chicken eye) test
40
 Production of monoclonal antibodies
In vitro In vivo
• Cell culture • Mouse ascites method

41
 Pyrogen tests
In vitro In vivo
• Limulus test • Rabbit pyrogen test

Horseshoe grab (Limulus Limulus Amebocyte Lysate

polyphemus) Test Kit

• Human whole blood test

42
 Analysis of biologically active compounds

In vitro In vivo
• Pregnancy test (immune assay) • Frog pregnancy bioassay

• Bioassays in chicken, rats and

• Hormones, vitamins (HPLC, mice
immune assays)
• Convulsion test (mouse), blood
• Insulin determination glucose determination (mouse,
rabbit) 43
 Advantages of in vitro tests

• Controlled testing conditions

• Lack of systemic effects
• Reduction of variability between experiments
• Testing is fast (and cheap)
• Small amount of test material is required
• Limited amount of toxic waste is produced
• Human cells and tissues can be used
• Transgenic cells carrying human genes can be used
• Reduction of testing in animals

44
 Limitations of in vitro tests

• General toxicity profile of a chemical cannot be

assessed
• In vivo dose-responses cannot be obtained (for human
risk assessment)
• Systemic effects cannot be evaluated
• Interactions between tissues and organs cannot be
tested
• Pharmacokinetics cannot be evaluated
• Specific organ sensitivity cannot be assessed
• Chronic effects cannot be tested

45
No relevant replacement alternatives

• Pharmacokinetics / toxicokinetics
• Systemic toxicity
• Organ systems toxicity (CNS, respiratory,
cardiovascular, gastrointestinal etc.)
• Immunotoxicity
• Male and female reproduction toxicity (fertility tests,
developmental toxicity tests, peri- and postnatal
toxicity tests)
• Subchronic and chronic toxicity
• Carcinogenicity tests

46
A Typical Safety Evaluation Program
Chemical Identification and Characterization

Literature Review, Structure-Activity Relationships

Local Toxicity Acute Toxicity Sensitization

Pharmacokinetics
Subchronic Toxicity Genotoxicity
Toxicokinetics

Reproductive and Developmental Toxicity Chronic Toxicity

Special Studies Carcinogenicity 47

Predictive ability of a toxicity test

• Specificity
The percentage of negative chemicals correctly identified.

• Sensitivity
The percentage of positive chemicals correctly identified.

• Predictivity
The percentage of predictions for a particular
classification, which were correct.

• Accuracy
The overall percentage of correct classifications.
48
 Animal Species/Model Selection*
 Standard toxicology paradigms
 Use of relevant species
– single relevant species with justification
– limited toxicology in a single “nonrelevant”
species
 Alternative approaches
– transgenic animals
– homologous proteins
– animal models of disease * Sec 3.3
 BPOM, 2000

PEDOMAN PELAKSANAAN UJI

KLINIK OBAT TRADISIONAL
UJI PRA-KLINIK OT

Terdiri dari:
1. Uji toksikologik, untuk menilai keamanan OT
yang diuji dan menetapkan spektrum efek
toksik.
2. Uji Farmakodinamik, untuk memberikan
informasi tentang khasiat.
Merupakan penelitian eksperimental dengan
binatang coba (in Vivo maupun in Vitro)
 OT YANG DIUJI
Identitas OT perlu diungkap sebelum Uji pra-
klinik:
• Simplisia yang digunakan diuraikan dalam nama
latin baik genus maupun spesiesnya.
• Ukuran berat / volume
• Langkah-langkah proses pembuatan
(simplisiabentuk siap diujikan)
• Dosis dan cara penggunaan (pemberian,
frekuensi, interval, lama pemberian)
UJI TOKSISITAS OT
TOKSISITAS AKUT
OT dipakai secara singkat
Tujuan:
a. Menetapkan potensi toksisitas akut (LD50)
b. Menilai berbagai gejala klinik
c. Mengetahui spektrum efek toksik
d. Mengetahui mekanisme kematian
Hewan coba species pengerat
Dosis OT bertingkat, terendah sesuai empirik
Pengamatan 7-14 hari
Hewan mati  otopsi : makroskopik dan mikroskopik
Hewan hidup  otopsi : makroskopik dan mikroskopik
 diamati terjadinya pemulihan
 2. Toksisitas jangka panjang

 Tujuan untuk mengetahui spektrum efek toksik serta hubungan dosis

dan toksisitas pada pemberian berulang pada jangka waktu lama
 Uji toksisitas subakut sekurang-kurangnya 1-3 bulan
 Uji toksisitas kronik sekurang-kurangnya 3-6 bulan
 Hewan coba: hewan pengerat
 Dosis 3 tingkat, terendah dosis efektif sesuai hewan coba, dosis
tertinggi diharapkan terjadi perubahan hematologik, biokimia,
anatomik-histologik namun mayoritas masih hidup.
 Dapat mengungkap batas keamanan (margin of safety)
 3. Uji toksisitas khusus
 Termasuk uji mutagenik, teratogenik, karsinogenik.
 Bukan syarat mutlak untuk masuk uji klinik
 Dilakukan secara selektif bila:
1. Formula OT mengandung bahan kimia yang
potensial memberikan efek khusus.
2. Formula OT yang potensial digunakan wanita
usia subur, perlu pertimbangkan efek
teratogenik
3. Formula OT yang secara epidemiologik terkait
dengan penyakit tertentu.
 Uji Farmakodinamik OT
• Tujuan membuktikan kasiat dan menelusuri mekanisme efek dari OT
teruji.
• Eksperimental pada hewan sehat dan dibuat berpenyakit tertentu.
Dapat juga secara in vitro.
• Pemberian obat disesuaikan dengan penggunaan pada manusia
mencakup dosis dan cara penggunaan (pemberian, interval, lama)
• Kelompok pembanding placebo atau obat standard
• Respons yang diamati secara kualitatif maupun kuantitatif sesuai efek
terapi yang diharapkan maupun respons pada sistem-sistem lain.
Methods for testing immunological factors

I.2.1 In vitro methods

I.2.1.1 Inhibition of histamine release from mast cells

I.2.1.2 Mitogen induced lymphocyte proliferation
I.2.1.3 Inhibition of T cell proliferation
I.2.1.4 Chemiluminescence in macrophages
I.2.1.5 PFC (plaque forming colony) test in vitro
I.2.1.6 Inhibition of dihydro-orotate dehydrogenase
Methods for testing immunological factors

I.2.2 In vivo methods for testing immunological factors

I.2.2.1 Spontaneous autoimmune diseases in animals

I.2.2.2 Acute systemic anaphylaxis in rats
I.2.2.3 Anti-anaphylactic activity (Schultz-Dale reaction)
I.2.2.4 Passive cutaneous anaphylaxis
I.2.2.5 Arthus type immediate hypersensitivity
I.2.2.6 Delayed type hypersensitivity
I.2.2.7 Reversed passive Arthus reaction
I.2.2.8 Adjuvant arthritis in rats
I.2.2.9 Collagen type II induced arthritis in rats
Methods for testing immunological factors

I.2.2 In vivo methods for testing immunological factors

I.2.2.10 Proteoglycan-induced progressive polyarthritis in

mice
I.2.2.11 Experimental autoimmune thyroiditis
I.2.2.12 Coxsackievirus B3-induced myocarditis
I.2.2.13 Porcine cardiac myosin-induced autoimmune
myocarditis in rats
I.2.2.14 Experimental allergic encephalomyelitis
I.2.2.15 Acute graft versus host disease (GVHD) in rats
Methods for testing immunological factors

I.2.2 In vivo methods for testing immunological factors

I.2.2.16 Influence on SLE-like disorder in MRL/lpr mice

I.2.2.17 Prevention of experimentally induced myasthenia
gravis in rats
I.2.2.18 Glomerulonephritis induced by antibasement
membrane antibody in rats
I.2.2.19 Auto-immune uveitis in rats
I.2.2.20 Inhibition of allogenic transplant rejection