Lecture 4 Bioactivity Testing and Clinical Studies

Bioassays and clinical studies

Oliver Kayser

Types of Assays

• Chemical Assays: Spectrophotometry, Spectrofluorimetry, Chromatography,

• Immunoassays

• Microbiological assays

Definition

– Estimation of the conc / potency of a substance by measuring its biological response in living systems

– i.e.Observation of pharmacological effects on

[1] living tissues, or cells (in vitro)

[2] microorganisms

[3] animals (in vivo)

Indications for Bioassay• Active principle of drug is unknown• Active principale cannot be isolated, e.g. insulin,

posterior pituitary extract etc. • Chemical method is either

– not available – if available, too complex, – insensitive to low doses e.g. Histamine can be bioassayed in

microgram conc. • Unknown Chemical composition, e.g. long acting

thyroid stimulator. • Chemical composition of drug variable but has same

pharmacological action e.g. cardiac glycosides isolated from diff sources, catecholamines etc.

Principles of Bioassay• Active principle to be assayed should show the same

measured response in all animal species• The degree of pharmacological response produced

should be reproducible under identical conditions [Eg Adrenaline shows same rise in BP in the same species under identical conditions: wt, age, sex, strain / breed etc]

• The reference standard must owe its activity to the principle for which the sample is being bioassayed

• Activity assayed should be the activity of interest• Individual variations must be minimised / accounted

for• Bioassay might measure a diff aspect of the same

substance compared to chemical assay [Eg testosterone & metabolites

Types of Bioassays[1] Quantal Assays [ Direct endpoint ]

– Elicits an ‘All or None’ response in different animals

– Eg. • Digitalis induced cardiac arrest in guinea pigs• hypoglycemic convulsions in mice. • Digitalis induced head drop in rabbits• Calculation of LD50 in mice or rats

[2] Graded Response Assays [mostly on tissues]– Graded responses to varying doses– Unknown dose response measured on same tissue

Methods of Bioassay con1[2] Graded Response Assays [ Direct comparison on same tissues]– Interpolation: Conc. of unknown is read from a standard plot of a log dose response curve of at least 4 sub maximal concentrations

– Matching / Bracketing: Const dose bracketed with varying doses of standard till exact match is obtained

• Used when test sample is too small

• Inaccurate & margin of error difficult to estimate• Eg histamine on guinea pig ileum, Posterior pituitary on rat uterus

– Multiple Point Assays• 3 point assay [combines pples of matching with interpolation]

• 4 point assay [combines pples of matching with interpolation]

3 point assay [2+1 dose assay]• Fast & convenient• Procedure [Eg Ach bioassay]

– Log dose response [LDR] curve plotted with varying conc of std Ach solutions and given test solution

– Select two std doses s1& s2 [ in 1:2 dose ratio] from linear part of LDR [ Let the corresponding response be S1, S2]

– Choose a test dose t with a response T between S1 & S2 – Record 4 sets data [Latin square: Randomisation reduces

error] as follows• s1 s2 t• t s1 s2• s2 t s1• s1 s2 t

– Plot mean of S1, S2 and T against dose. Calculate – Log Potency ratio [ M ] = [ (T –S1) / (S2‐S1) ] X log d

[d = dose ratio]

4 point assay [2 +2 dose assay]• Procedure [Eg Ach bioassay]

– Log dose response [LDR] curve plotted with varying conc of std Ach solutions and given test solution

– Select two std doses s1& s2 from linear part of LDR [ Let the corresponding response be S1, S2]

– Choose two test doses t1 & t2 with response T1 &T2 between S1 & S2 ; Also s2/s1 = t2/t1 = 2

– Record 4 data sets [Latin square: Randomisation reduces error]• s1 s2 t1 t2• s2 t1 t2 s1• t1 t2 s1 s2• t2 s1 s2 t1

– Plot mean of S1, S2 and T1, T2 against dose. Calculate – Log Potency ratio [M] = [ (T1 –S1 + T2 –S2) / (S2‐S1 + T2‐T1) ] X

log d [d = dose ratio]

HTPA

• Expensiv e and time consuming

• Example:– 1 plant = 3 years analysis

– Automatisation is needed

• How to screen plants?

College 1/1 ‐ 11

Drug discovery and lead discovery

• Lead or lead compound

• A lead is a compound with pharmacological options for further developments

• Nature is most valuable source for drug leads

Zie ook G. Thomas, Medicinal Chemistry, chapter 5.6

HTPA

College 1/1 ‐ 13

Mega HPLC

Puffer

organischesLösungsmittel

Wasser

... usw.10 SPE-Säulen

Desorption vonden SPE-Säulen

zum Abfall

zum Fraktionen-sammler

AufgabesäuleHaupttrennsäulefür Trennschritt I

... usw.18 SPE-Säulen

Trennschritt IImit 6 Trennsäulen

zur Auswahl

Wasser

College 1/1 ‐ 14

Bioguided fractionation of Retama sphaerocarpa

Bioguided Fractionation


Dereplication

College 1/1 ‐ 16

Dereplication is the use of chemical methods and bioanalytical techniques for the discrimination of already known compunds in extracts ver y importantThese techniques could be:

‐ NMR‐Massa‐ UV

All technical reporting systems are coupled tonatural product libaries Zie ook G. Thomas, Medicinal Chemistry, chapter 5.6

HTPS

• HTPS = How to find a needle in a hairstack

• Principal: To screen the sample completly in time

• Need of bioinformatic suuport necessary

Why perform High Throughput Screening?

HTS enables the testing of large numbers of chemicalsubstances for activity in diverse areas of biology ina relatively short time.

The entire chemical space of small organic molecules isestimated to be > 1060. Of those, ~ 2.7 x 107 compoundshave been registered and made. (Nature Insight, 2004)

Responses studied can range from biochemicalsystems of purified proteins or enzymes to signaltransduction pathways to complex cellular networks(Systems Biology).

How Does One Select a Library to Screen?

Random SelectionRandom high throughput screeningLittle is known about the targetFew or no active compounds as guides

Computational Chemistry/Virtual ScreeningCreation of ‘Focused Libraries’Requires prior knowledge about target

Active compounds, 3D‐StructureSequence homology

Prior ExperienceLibrary successfully used for similar

or related targets

Stages of Commercial Drug Development

Drug Development is a game of attrition. The Challenge … Select 1‐2 compounds from the millions of possibilities that will be safe and efficacious in humans

Basic ResearchBasic Research Target ValidationTarget Validation HTS and Lead IdHTS and Lead Id Lead OptLead Opt

Preclinical ToxPreclinical Tox File IND File IND Clinical Phase 0/1Clinical Phase 0/1 Clinical Phase 2Clinical Phase 2

Clinical Phase 3Clinical Phase 3 File NDAFile NDA ApprovalApproval Sales Marketing Phase 4

Sales Marketing Phase 4

Stages of Academic Drug Development

Still a game of attrition. The Challenge …Identify agents that increase the fundamental scientific knowledgefor a particular target with the possibility of providing further validationof the target as a ‘druggable’ target.

Retain the possibility of identifying a lead series of compoundsthat could take our research in new, unexpected directions.

The possibility of establishing intellectual property and the basis for afuture pharmaceutical.

Basic ResearchBasic Research HTS and Lead IdHTS and Lead Id Lead OptLead Opt

The Screening Continuum

Fa. Robocon

Working with samples


Parameter Traditional Assay HTS AssayProtocol Complex, numerous steps

•Multiple Additions•Multiple Aspirations•Multiple Washes

Simple, few steps (3-5)•Additions•Aspirations - discouraged•Washes - discouraged




Assay Container VariedTubes/slides/dishes/cuvettes/animals

Microtiter plates96/384/1536-Wells






Assay Volume 0.1 to 1 mL < 1 μl to 100 μl






Assay Volume 0.1 to 1 mL < 1 μl to 100 μlReagents Varied

•Limited quantity•Various batches•Limited stability

Consistent•QS for complete screen•Single batch - preferred•Prolonged stability









Variables Many•Time/temperature•Ligand/substrate concentration•Cell type

Compound










Compound

Readout Time Milliseconds to months Minutes to hours










Compound

Readout Time Milliseconds to months Minutes to hoursOutput Formats Varied

Readers/scoring/image interpretation

Plate readerAbsorbance/fluorescence/luminescence

Compare Traditional Assays with HTS AssaysParameter Traditional Assay HTS Assay

Critical Issues to be Addressed Prior to Testing the First Compound

Key factors that must be addressed prior to screening:

• Assay protocol (miniaturization/simplification)

• DMSO resistance (test 0.1 ‐ 5%), standard compound vehicle

• Reagent quantity and batch consistency

• Reagent stability for storage and use under assay conditions

• Appropriate positive and negative controls

• Assay reproducibility and signal stability

• Available secondary or counter screen to test target

specificity and selectivity

HTSScreen

Time

CostsQuality

time/wellwells/dayscreens/yearproject time

reagentsconsumablesinstrumentationpersonnel

few false positivesfew false negativesS:N,SW,z’‐FactorValidated ‘Hits’

Key factors for successful HTS

College 1 ‐ 28

Why is High Troughput so important?

• Economic reasons– Patent time (20 years)

– Long time for drug development (10‐12 years)

– Increased productivity

Test system Manual monster preparation/d robotic monster preparation/d

Enzyme inhibitor 60-80 3000Receptor binding 300 1600Celcultures 1000 4800

Samle Costs (in €)

96er Mikrotiterplateper samplefor 500 000 monster

0,83415,000

384er Mikrotiterplaatper samplefor 500 000 monster

0,1680,000

1536er Mikrotiterplaatper sample for 500 000 monster

0,0315,000

Consider: Reagent QuantityCompare traditional assays with HTS assays

Traditional Assay 96-Well HTS 384-Well HTS

Plates/Day 1 40 40

Total Plates 5 375 94

Cells/Day 1 x 106 40 x 106 40 x 106

Total Cells 5 x 106 375 x 106 94 x 106

Total Assay Volume (μL)

100 100 25

μL/Well User purifiedreagent

10 10 2.5

mL Purified rgt/plate 1 1 1

mL Total purified rgt 5 375 94

30,000 Compounds Traditional Assay 96-Well HTS

Plates/Day 1 40

Total Plates 5 375

Cells/Day 1 x 106 40 x 106

Total Cells 5 x 106 375 x 106


100 100


10 10

mL Purified rgt/plate 1 1

mL Total purified rgt 5 375

Traditional Assay

Plates/Day 1

Total Plates 5

Cells/Day 1 x 106

Total Cells 5 x 106


100


10

mL Purified rgt/plate 1

mL Total purified rgt 5

HTS: An Iterative Process

HTS GroupSecondary ScreenPurpose: Validate initial ‘Hits’Method: Selection of compoundsor medicinal chemistry

Chemistry groupsAnalysis and interpretation of Data for

Structure Activity RelationshipsRefine and improve identified ‘Hits’

Modeling and medicinal chemistrySelection of compounds for screening via

virtual screening, focused libraries

HTS GroupPerform Primary ScreenPurpose: Identify a starting placeMethod: Interrogate libraries of

compounds/genes

Research groupsTarget Id and ValidationDevelop Primary and Secondary assaysDefine criteria for active compoundsDirect ‘Hit’ improvement process

Natural drugs ?

• More and more important:– Fytotherapy

– Homeopathy

– Alternative treatment

• More than 120 therapies are known!

Use of medicinal plants

• Ca. 80% of world population are dependned on plants as drugs

• Synthetic drugs mostly too expensive

• Deeply depended on cultural background

• WHO consideres plant based therapy as essential part in public health systems and acess to therapies

Pharmacolocical active constitutens

• Some plants contain known pharmacolocigal active constitutens, most plant constituents are unknown

• If possible use synthetic compounds where actions and side effects are known

• Mostly distinct parts of the plants are used (roots, leaves, seeds)

• Biosynthesis should be nown to characterise the plant of use

Ethnobotanie ‐ Ethnofarmacie

• Research to understand traditional use of plants

• Artemisinine ‐ Anti‐malaria

• Podofyllotoxine ‐ Anti‐cancer

• Paclitaxel ‐ Anti‐cancer

College 1/1 ‐ 37

From Artemisinin to Artemether

R = OH dihydroartemisinR = CH3 artemetherR = CH2CH3 arteetherR = COCH2CH2COONa artesunate

O

O

HOO

HH

H

OR

HO

O

HOO

O

HH

H

H

From Podophyllotoxin to Wartec

Podophyllum hexandrum

H3CO OCH3

OCH3

O

O

OH

O

O

Podophyllotoxin

Chemotaxonomy

• Each step in the biosynthetic rout is catalysed by one enzyme

• One enzyme = one gene

• Closly related species can produce similar or the same constitutent

ca. 35.000 genes known =

35.000 natural products in one organism !?

Chemotaxonomie

O

MeO

MeO

OH

OMe

O

O

MeO

MeO

OMe

OMe

O

Arctigenin

Dimethylmatairesinol

O

MeO

HO

OH

OMe

O

O

OH

OMe

OO

O

O

OH

OMe

OO

O

OH

OMe

OHO

MeO

OO

MeO

HO

OH

OMe

O

OH

O

OH

OMe

OO

O

OH

O

OO

O

OO

O

OO

O

OMe

OMe

O

OO

O

OH

OMeMeO

Matairesinol

Hinokinin7'-Hydroxymatairesinol

4'Demethylyatein

Chemotaxonomie

DifferentMentha varieties

Ecology

Why produces...

Taxus brevifolia paclitaxel?

Penicillium penicillin?

Cannabis sativa THC?

Streptomyces doxorubicin?

OO

NH OH

HO

O

O

OH

O

O

O

O

O

O

O

OH

COH2

A plant does it not for us !

Chemical ecology

‐ Communication

‐ attracting insects

‐ Against prediators

‐ Defending against other microorganisms (chemisch oorlog)

‐ Protecting against radiation (e.g. UV)

Chemical ecology

Juglans regens

Inhibition, allelopathic effekt

Hydrojuglon‐β‐glucosid Hydrojuglon Juglon(niot active ) (actiive)

Chemical ecology

‐ Tropisch regenwouden

‐ Coral reef

‐ South Africa

‐ Ecological niches

Where can we benefit from?

Biodiversity

Lecture 4 Bioactivity Testing and Clinical Studies

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