John A. Beutler, Ph.D.

Natural Product Drug Discovery and Development - an NCI Perspective

The funnel of drug discovery and development

• The task: find the “needle in the haystack”

• Estimated 10,000 compounds tested to find one which has clinical activity in humans

• Cost of process increases as one nears the goal

• Estimated $300-600 million average development cost for a single drug

Number of Compounds

Number of Compounds

1e-1 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5

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Total Cost

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1e-1 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5 1e+6 1e+7 1e+8

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Discovery

Pharmacology

ADME

Toxicology

Phase I Clinical

Phase II Clinical

Phase III Clinical

Primary screening

• This is the filter that is supposed to separate the “wheat” from the “chaff”

• Cost, speed, relevance all important

• Whole animals precluded due to cost

• Tissue assays too slow

• Cellular or biochemical assays best – using a validated molecular target

But….

What does one lose by throwing out the context in which the target operates?

• Penetration to target inside cell

• Metabolism, excretion

• Toxicity through other mechanism

Secondary screening

• Attempts to put back some of the factors which the primary screen ignored

• Biochemical mechanisms, tissue assay, or whole animal

• Reduce the number of active samples to be considered by eliminating the least desirable ones

Animal testing

• Toxicology – what dose kills or injures animal, in what organ system? (e.g.,liver, brain)

• Can therapeutic blood levels of drug be obtained?• Extrapolation from model animal to human

imprecise but necessary• Cost requires small animals: mouse, then dog• Computer models limited usefulness, but cellular

toxicology increasingly used

Clinical trials

• Phase 0 – sub-pharmacologic doses

• Phase I – first time in humans - safety

• Phase II – efficacy in small numbers

• Phase III – larger populations

Postmarket processes

• Newly introduced drugs are closely monitored to detect lower frequency adverse effects

• Unacceptable toxicity may lead to drug withdrawal

• Broader indications may be explored without FDA approval (“off-label uses”)

Plants for medicines

• Direct application of ethnomedical use

• Source of chemical diversity for screening

• Herbal products under DSHEA

The ethnomedical approach

• Problem: It may be hard to correlate folk medical concepts to Western medicine

• Shaman Pharmaceuticals: antifungals, antidiabetics

• Property rights asserted by countries over ethnomedical knowledge, genetic heritage

Herbal products under DSHEA

• Dietary Supplement Health and Education Act of 1994

• No premarket evaluation for efficacy or toxicity

• Requires correct labeling, GMPs

• Established Office of Dietary Supplements within NIH

Chemical diversity of plants

• Attractive for screening in bioassays – each extract can contain many novel compounds [x number of plant species]

• Plant defensive compounds may be tailored to interfere with mechanisms common to human diseases

• Antibiotic/antifungal compounds may be useful both to the plant and to human medicine

• Many drugs originally obtained from nature

A Case History

NCI collectors collect tree Calophyllum lanigerum from Malaysian rainforest

Screening for HIV inhibitors

• Extracts of many plants screened for the ability to protect host cells against the cell-killing action of HIV-1

• Protection vs. cellular toxicity (TI)

Extract Compound

Bioassay-guided fractionation

• Chromatographic separation of crude extract into fractions• Test each fraction, further separate most active fractions until pure

compound is obtained• NCI chemists isolate calanolide A as anti-HIV principle of Calophyllum

lanigerum extract cf. J.Med.Chem.35: 2735, 1992.

Calanolide A

Structure determined by NMR: O

O O

OH

O

Other related compounds isolated with lesser activity

Mechanism of action• Calanolide A found to inhibit HIV reverse

transcriptase in unique fashion (NNRTI)• Binds to enzyme at site distinct from TIBO and

other NNRTIs• Development of drug resistance – mutant enzyme

resistant to AZT is more sensitive to calanolide A cf. J.Virol. 67: 2412, 1993.

• T139I mutant resistant to calanolide

Supply of compound problematic

• Recollection attempt – tree had been logged

• Other collections had only <0.1 % calanolide A

• Taxonomy of Calophyllum difficult – new species & varieties recognized

• Related compounds considered as drug development leads

Lead compound synthesized

• SmithKline scientists isolate inophyllums from giant African snail as HIV RT inhibitors cf. J.Med.Chem. 36: 4131, 1993.

• traced to Calophyllum in diet• Calanolide superior to inophyllums• Total synthesis of calanolide A

achieved by SmithKline

O

O

O O

OH

Plant sources identified

• Latex of Calophyllum species contains large quantities of calanolides ~5%

• Tap like rubber tree – renewable resource

• Different species of Calophyllum surveyed for calanolide content

cf. J.Nat.Prod.61: 1252, 1998.

Calanolide licensed

• Medichem Co.

• Partner with Sarawak (Malaysia) gov’t. “Sarawak Medichem Pharmaceuticals”

• access to Malaysian resources

Preclinical development

• formulation – how to administer orally

• pharmacokinetics – how fast do blood levels rise and fall in body (HPLC: rat, dog)

• distribution – to what organs – crosses blood-brain barrier

• toxicology – negative effects

Hollow fiber HIV model

• Hollow fibers containing HIV-infected cells implanted i.p. or s.c. in mice

• Calanolide A administered oral or parenteral • Fibers removed and cells assayed for HIV

cytopathicity• Calanolide blocked HIV cytopathicity and was

synergistic with AZT cf. Bioorg.Med.Chem.Lett. 9: 133, 1999.

Calanolide A Phase I trialssafety in humans

Phase IA (1998):

47 healthy adults, US

single oral doses up to 600 mg

higher blood levels than animal studies predicted

side effects – dizziness, oily aftertaste, headache, belching, nausea

Phase I trials, cont’d.

Phase IB (1999):

43 patients, HIV-infected asymptomatic

randomized, placebo-controlled, double- blind

adverse effects – mild to moderate, transient

nausea, dyspepsia and headache

no drug resistance

modest viral load reduction –0.8 log

Asian patients had drug-related toxicity – fever & rash

Further trials

Clinical trial evaluated the therapy's safety and pharmacokinetics (n=48)

Complete March 2003Calanolide A in combination therapy for HIV

Evaluated the therapy's effect on pharmacokinetic enhancement and safety. Results confirmed that the combination therapy was effective in increasing the blood levels of calanolide in human volunteers. Additionally, no serious adverse events were noted in any subjects and the small number of adverse events observed were similar to those previously associated with the drug.

A new wrinkle• Calanolide A also possesses activity against

Mycobacterium tuberculosis. • No other anti-HIV agent, either in development or

approved, possesses this dual therapeutic capability. • Since patients with HIV/AIDS have weakened

immune systems and are more susceptible to contracting TB compared to healthy individuals, the dual therapeutic properties of (+)-Calanolide A make it a valuable therapeutic agent to this patient group and will help to distinguish it from other agents in its class.

Timeline• Discovery - 1991• Patenting & publishing - 1992• Resupply compound – synthesis 1993

– Natural source 1994

• Mechanism of action - 1993• Licensing - 1995• Phase I - 1997• Phase II – 2002 ??