천 연 물 약 품 화 학 연 구 실

석 사 1 기 장 성 완

From Natural Product to Clinical Trials:

Bevirimat, a Plant-Derived Anti-AIDS Drug

Index0. AIDS

1. Introduction2. Bioactivity-directed Fractionation and Isolation3. Lead Identification4. Lead Optimisation and SAR Study 4.1 Modification of the BA Triterpene Skeletion 4.2 Modification on C-3 Position of BA 4.3 Introduction of C-28 Side Chain into BA 4.4 Bifunctional BA Analouges – Potential for Maturation Inhibitor Development

5. Mechanism of Action Studies of Bevirimat6. Preclinical Studies of Bevirimat7. Clinical Trials and Current Status of Bevirimat8. Conclusions

0. AIDS

ⅰ. What is the AIDS

ⅱ. Classification of HIV

ⅲ. HIV Life Cycle

ⅳ. Current status ; Anti-HIV Drug

ⅴ. Domestic research trend

ⅰ. What is the AIDSAIDS - Acquired Immune Deficiency Syndrome

the human

immune system

caused by infection

with human

immunodeficiency

virus (HIV).

ⅱ. Classification of HIV

Species HIV-1 HIV-2

Virulence High Lower

Infectivity High Low

Prevalence Global West Africa

Inferred Origin

Common chimpanzee

Sooty Mangabey

HIV-1

Group : Group (ssRNA-RT)Ⅵ

Family : Retrociridae

Genus : Lentivirus

ⅲ. HIV Life Cycle

1. Binding

2. Reverse Transcription

3. Integration

4. Transcription

5. Translation

6. Viral Assembly and

Maturation

1

2

3

4

5

6

1. Binding

- HIV binding T4-cell’s CD4+ receptor

- CD4+ receptor be generated HIV binding

- This step can be blocked by entry

inhibitors

2. Reverse Transcription

- Making proviral DNA by RTase

- This step can be blocked by

NRTIs(Nucleoside Reverse

Transcripatase Inhibitors) and NNRTIs

(Non-Nucleoside Reverse Transcriptase

Inhibitors)

3. Integration

- another viral enzyme called integrase

hides the proviral DNA into the cell's DNA

- This step can be blocked by intergrase

inhibitors

4. Transcription

- creating a complementary strand of

genetic material called messenger RNA

or mRNA

- This step can be blocked by antisense

antivirals or transcription inhibitors (TIs),

5. Translation

- The mRNA carries instructions for making

new viral proteins from the nucleus

6. Viral Assembly and Maturation

-Final step virus infection

-Viral Assembly step can be blocked by

Protease Inhibitors (PIs)

-Maturation step can be blocked by

Maturation Inhibitors(Mis)

ⅳ. Current status ; Anti-HIV Drug

ⅳ. Current status ; Anti-HIV Drug

ⅴ. Domestic research trend

Patent

- 태림제약 anti-virus drug

Raw matrial supply

- 유한양행 GWM 사와 계약

Technology Licensing

- 화학硏 - Gilead

1. Introduction 29 approved drugs marketed to treat HIV-1 infection ex) Fuzeon [enfuvirtide,ENF,T-20] – inhibit viral entry

Selzentry[maraviroc,MVC] - CCR5 coreceptor antagonist

HAART(Highly active antiretroviral therapy) combination of drugs with different targets

HAART improved patient’s life quality and length

But HAART made drug-resistant virus

up to 78% if HIV-1-infected individuals harbour drug-resistance virus with a rapidly growing subgroup

Therefore, novel potent antiviral agents need

2. Bioactivity-directed Fractionation and Isolation

QSAR(Qunatitative Structure-activity relationship)ADMET (absorption, distribution, metabolism, excretion, toxicity)

3. Lead Identification

- Triterpene have diverse structure and pharmacological activities

- Several naturally occurring triterpenes have been reported to show anti-HIV activity

- Bevirimat, the first in a new class of compound termed HIV maturation inhibitor(Mis)

- betulinic acid and platanic acid can inhibit HIV-1 BⅢ replication (EC50/bet 1.4 μM /

EC50/pla 6.5 μM)

- betulinic acid contains many plant species

( e.g Tryphyllum peltatum, Ancistrocladus heyneanus, Ziziphi fructus, Tetracera boloviana )- betulin can modify to betulinic acid (betulin in birch bark)

4.Lead Optimisation and SAR Study4.1 Modification of the BA Triterpene Skeletion

- C–3 alcohol

This functional group

can be eliminated or

oxidised

- Betulinic acid have a readily chemical

modification site

C–3 alcohol

C–19 isopropenyl

C–28 carboxyl group - C–19 isopropenyl

This functional group

undergoes typical

reaction with allylic

group

- C–28 carboxyl group

This functional group

can be derivatised

providing ester and

amide

- C–19 isopropenyl

reaction with Pd-C

(Catalytic hydrogenation)

EC50 0.9μM / TI value 14

- C–3 alcohol Oxd

EC50 0.22μM

CC50 0.9μM (cytotoxic)

- C-2 Hydroxylation

EC50 42.3μM(weak

activity)

- C–28 carboxyl group

EC50 23μM / TI value

1.9

4.2 Modification on C-3 Position of BA

- C-3 hydroxyl group readily

acylated with variety of anhydrides

and acid chlorides

1 2

Com 8, 9 ) Extremely potent anti-HIV

activity

Com 15, 16) similar potencties compare

com 8,9

Com 10, 17 ) reduction in antiviral

activity

Com 13, 20~22 ) lack or no antiviral

activity

Com 7, 11, 12, 14, 18 ) partial antiviral

activity

- C-3 modification’s result indicates that the two C-3’ methyl group

in the bevirimat side chain contribute anti-HIV activity and terminal

carboxyl acid substitution has antiviral activity potent

Similar activity

Moderate

activity

- C-3 position of BA and betulin,

geminal dimethyl substitution of

the side chain C-3’ position

coupled with a terminal

carboxylic acid are important to

the enhanced antiviral activity for

both of these triterpene

templates

C-3 stereo convertC-3 stereo

eliminate

Two compound anti-viral activity dramatically reduced, indicating the importance of the 3-C stereo

R S

- C-3 oxygen exchange with

nitrogen

Only com 42, 45 weak antiviral

activity

- com 46 have similar activity compare buletin(com 4)

but have cytotoxic (EC50 0.22μM / CC50 0.9μM)

- Similary com 47 has antiviral activity and cytotoxic

(EC50 0.22μM / CC50 0.9μM)

- com 48~50 has similar antiviral activity but decrease

cytotoxic (EC50 0.57~4.57μM / CC50 ＞ 15μM)

4.3 Introduction of C-28 Side Chain into BA

- These type functions by blocking the viral entry into the host cells

- Increasing chain length influence the anti-HIV potency

- compound with amide side chains between aminooctanoic acid and aminododecanoic acid

tend to increase antiviral potency

- com 51 inhibits the infectivity of several HIV-1

strains (EC50 0.05~2μM)

- com 52 equipotent antiviral activity

- com 53 have equipotent against

HIV-1 virus when compare with

com 51, com 52

- com 51 show potent antiviral activity

in vitro but its clinical development

was stopped due to poor

‘pharmacodynamic properties’

4.4 Bifunctional BA Analouges – Potential for Maturation Inhibitor Development

- Two functionalities are on the opposite position of the BA skeleton

- a design combining both modifications led to the development of bi-functional BA

analogues

- EC50 0.0026μM higher potent than C-3 and C-28 parent compound

- This category preserves both the anti HIV-1 entry and maturation inhibit ability

5. Mechanism of Action Studies of Bevirimat

- Bevirimat affect virus replication at a

time point after the completion of viral

DNA integration and Tat expression

- inhibited the processing of the viral

Gag polyprotein at a specific step

- Bevirimat does not affect viral RT,

reverse protease, virus particle budding

- Bevirimat blocks processing of CA-

SP1(p25) to CA (p24) which

necessary for final capsid

condensation and formation of

infectious, bevirimat cause abnormal

morphology to virus

- consequently virus become non-

infectious

- Bevirimat has no effect on

processing at other site of Gag

- Bevirimat-resistant isolates were selected by serial passage of HIV-1

- resistant virus found several point mutations proximal to the cleavage site of

CA-SP1

CA H226Y, L231M, L231F, SP1 A1V and A3V

- no mutation observed in other region of Gag or in the PR coding region

- Bevirimat does not affect SIV / HIV-2

Gag region relate virion

structural proteins protase

6. Preclinical Studies of Bevirimat- EC50 10.3nM against subtype B virus

(similar activity compare with AZT,nevirapine)

-Bevirimat retains nanomolar inhibitory activity

against drug resistant HIV strains(NRTIs, NNRTIs,

PIs resistant)

- no activity against HIV-2, SIV and other similar

envelop virus type

Mouse type : SCID-hu Thy/Liv

Does : Twice daily / Oral

Virus : HIV-1 infected

Dose-dependent manner

>2log10 in HIV-1 RNA

≥ 90% p24 conc.

Gag-p24+ thymocytes at 100mg/kg per day

Observe in the mice at plasma conc.

7. Clinical Trials and Current Status of Bevirimat

- Phase (In 2004 completed)Ⅰ

PanocosPharm dose single and multiple phase clinical trials of bevirimat in

healthy volunteers / Oral solution

It well tolreated half-life of ~ 2.5-3 days / supporting daily dosing regimen

- Phase a (In August 2005, completed)Ⅱ

random double-blind, bevirimat monotherapy for ten days resulted in statistically

significant reduction in viral load compare with placebo (individual decrease up to

1.7log10, 100 and 200mg dose)

No evidence develop drug resistant virus

7. Clinical Trials and Current Status of Bevirimat

- Phase b (In 2008 completed)Ⅱ

patients failing HIV therapy due to drug resistance

Good activity in 369, 370, 371 on Gag amino acid sequence mutant, less activity

in polymorphism(variants)

20μg/mL bevirimat is required for a robust response

the mean viral load reduction was -1.18log10copies/mL after 14days in the patients

who free of key baseline Gag polymorphism and bevirimat conc. Minimum target

of 20μg/mL

91% of patients with these two response predictors han at least a 0.5log10 viral

load reduction by week 2 with a maximum treatment response of 2.03log10

7. Clinical Trials and Current Status of Bevirimat

- Phase b (In 2008 completed)Ⅱ

bevirimat well tolerated, with a safety profile comparable to placebo through the

14days of treatment

Bevirimat tablet formulation dosed twice daily achieved target plasma levels

After 14 days of bevirimat treatment given twice daily at doses of 200mg or

300mg(using the 50 mg tablet)

32% patient (treatment naïve and experienced) had plasma concentrations

well above the previously identified minimum target of 20μg/mL

Phase are being plannedⅢ

8. Conclusions

- HAART has led to a significant improvement in the health and life span

HIV-1-infected patients

- But, major problem is the increasing prevalence of virus strains

that are resistant to approved drug

- Plants are a major source of biologically active compounds and can provide

good leads that are structurally unique and/or have new mechanism of action

Thank You for Your Attention