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Discovery of Anti-Leishmanial Leads from Natural Lead

H. E. J. Research Institute of Chemistry

International Center for Chemical and Biological SciencesUniversity of Karachi, Karachi-75270, Pakistan

Sammer Yousuf and M. Iqbal Choudhary

04/21/23

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One of the greatest public health problems in many developing countries.

Second most prevailing tropical disease, after malaria.

Widely distributed in 88 countries of world including Afghanistan, Pakistan, Bangladesh, India, China and the southern United States and Europe.

Transmitted by the bite of infected sandfly (Phelobotomus).

Leishmaniasis-An Old Disease

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Leishmaniasis

WHO has estimated that more than 350 million people are at risk of infection with Leishmania species with 600,000 new clinical cases reported annually.

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Classification of Leishmaniasis

Caused by Lieshmania species in humans, depending on the ability of the organism to penetrate in the deep tissues.

• Visceral Leishmaniasis: Visceral leishmaniasis, the most severe clinical form, is caused by the Leishmania donovani. It characterized by its affects on internal organs, such as liver, spleen and bone marrow.

•Mucocutaneous Leishmaniasis: Mucocutaneous leishmaniasis can cause facial disfiguration due to erosion in the mucocutaneous sites of the mouth and

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•Cutaneous Diffuse Leishmaniasis: Cutaneous diffuse Leishmaniasis is characterized by the formation of nodules, multiple lumps, specially around the face and

on the external surface of arms and legs.

•Cutaneous Leishmaniasis: Cutaneous leishmaniasis is a least severe form of leishmaniasis. Leishmania maxicana and Leishmania major is responsible for cutaneous leishmaniasis.

Classification of Leishmaniasis

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Geographical Distribution of Cutaneous Leishmaniasis due to Leishmania major

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90% of the cutaneous leishmaniasis cases are reported from Afghanistan, Brazil, Iran, Peru, Saudi Arabia and Syria

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Leishmaniasis in Pakistan

Major outbreaks were observed in NWFP, Jacobabad, Larkana, and Dadu districts of Sindh province.

Prevalent in Pakistan and has been reported from

almost all parts.

Diagnosis and treatment is difficult due to poor access to health services.

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• Leishmaniasis is a poverty-related disease. • It affects the poorest of the poor and is associated

with • Malnutrition • Weakness of the immune system • Lack of resources

According to WHO!

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• Antimonial compounds, amphotericin B, pentamidine, pyrazolopyrimidine, aminosidine, with a combination of antimony salts-pyrazolopyrimidine

• Stiff joints, gastrointestinal problems, cardiotoxicity and in some cases hepatic and renal insufficiency.

Current Treatment

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• Pakistan is a country with a rich tradition of medicinal herbs used by people to treat their health disorders

• Physalis angulata and Physalis purviana has already been reported previously for their leishmanicidal potential

Search Start

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•Physalis minima (Linn. var. indica) is a herb which is bitter in taste and widely used in folk medicines.

•The plant is used in vitiated conditions of gallbladder, burning sensation, bronchitis, for the treatment of inflammation, enlargement of spleen, urinary disorders, abdominal troubles and headache.

•The roots are used as a vermifuge and febrifuge.

•The 50% alcoholic extracts of the plant showed in vivo antimalarial, in vitro antigonorrhoeal activity.

BIOLOGICAL AND PHARMACOLOGICAL IMPORTANCE OF PHYSALIS MINIMA

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The fruits of Physalis minima are used as appetizer and have also been recorded as an ingredient of medicinal oil, which is given for spleen disorders.

• Antimycobacterial

• Immunomodulatory

• Hypoglycemic

BIOLOGICAL AND PHARMACOLOGICAL IMPORTANCE OF PHYSALIS MINIMA

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Methanolic Extract

Extracted with pet. ether (20 L)

(2.5 Kg)

Pet. ether Extract (200 g)

Insoluble part

Extracted with CH2Cl2 (20 L)

Chlorofrom Extract (150 g)

Ethylacetate Extract ( 200 g)

Extracted with Etylacetate (20 L)

Insoluble part

Insoluble part

Methanol water soluble part ( 200 g)

Extracted with MeOH: H2O (1:1)

Insoluble part

(20 L)

EXTRACTION OF EXTRACTION OF PHYSALIS MINIMAPHYSALIS MINIMA

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Medicinal Plant Plant’s Extracts (IC50 µg/mL ± S.D) % Reduction in

Amastigotes

(25 µg/mL ± S.D)

Physalis minima

Linn.

(Sun berry)

Methanol 26.00 ± 0.59 ND

Pet. Ether 31.62 ± 0.30 65

Chloroform 2.05 ± 0.02 90

Ethyl acetate 35.36 ± 0.08 ND

Standard Drugs Amphotericin B 0.12 ± 0.10 73.25 at 10µM

Pentamidine 5.13 ± 0.02 52.05 at 12.5µM

In Vitro leishmanicidal activity of Physalis minima extracts Against Promastigotes and Amstigotes Stages of

Leishmania major Parasite

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In vitro Cytotoxic Activity Against 3T3 Cell Lines (Normal Fibroblast cell).

Extracts of Physalis minima IC50 (µg/ mL) ± S.D.

3T3 cell lines

Methanol 18.78 ± 0.21

Pet. ether > 100

Dichloromethane 13.31 ± 0.70

Ethyl acetate >100

Standard

(Cyclohexamide)

0.26 ± 0.04

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Animal model Doses (mg/Kg) LD50 or Lethality

Wister Rats

(Male 200-250g)

10 Non Lethal

100 Non Lethal

500 Non Lethal

1000 Non Lethal

Animal Toxicity Determination (LD50) Against the

all Extracts of Physalis minima.

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In Vivo Leishmanicidal Studies on BALB/c Mice Model of Cutaneous Leishmanisis by

Topical Application of Gel having Extracts of Physalis minima Linn.

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Based on in vitro antileishmanial studies

Most active and least toxic mathanol and chloroform extracts were selected for

In vivo studiesLimited clinic trials

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In Vivo Leishmanicidal Studies

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Development of Cutaneous Leishmania Lesion

First day of inoculation 5-days of infection inflammation showed

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Inoculated in the base of the tail with 1x 106 to 5x 106

infective promastigotes

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Treatment of Infected Mice by Using White Soft Paraffin Based Gel Containing 25% Plant Extract

2-weeks of treatment Complete recovery after 4 weeks of

treatment.

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1-weeks of treatment

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Clinical Trials For Cutaneous Leishmaniasis

By Using 25% Chloroform Extract of Ariel Parts of Physalis minima

at

Shaheed Mohtarma Benazir Bhutto Medical University, LARKANA

Conducted and Supervised by

PROF. Dr. Farooq Rahim Somroo04/21/23

Before treatment After treatment

Topical Application of 25% Chloroform Extract of Ariel Parts of Physalis minima (Twice in a day)

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Before treatment After treatment

Topical Application of 25% Chloroform Extract of Ariel Parts of Physalis minima (Twice in a day)

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Before treatment After treatment

Topical Application of 25% Chloroform Extract of Ariel Parts of Physalis minima (Twice in a day)

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Topical Application of 25% Chloroform Extract of Ariel Parts of Physalis minima (Twice in a day)

54 Patients : Follow up for 2-4 weeks

39 Patients responded to treatment

39 Patients: Showed Recovery Response After Two Weeks of Treatment

39 Patients: Complete Recovery in 4 weeks

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Clinical Trials For Cutaneous Leishmaniasis

By Using 25% Methanolic Extract of Ariel Parts of Physalis minima

at

Shaheed Mohtarma Benazir Bhutto Medical University, LARKANA

Conducted and supervised by

PROF. Dr. Farooq Rahim Somroo04/21/23

Before treatment After treatment

Topical Application of 25% Mathanolic Extract of Ariel Parts of Physalis minima (Twice in a day)

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Before treatment After treatment

Topical Application of 25% Mathanolic Extract of Ariel Parts of Physalis minima (Twice in a day)

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Before treatment After treatment

Topical Application of 25% Mathanolic Extract of Ariel Parts of Physalis minima (Twice in a day)

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Topical Application of 25% Methanolic Extract of Ariel Parts of Physalis minima (Twice in a day)

37 Patients : Follow up for 2-4 weeks

19 Patients responded to treatment

12 Patients: Showed Recovery Response After Two Weeks of Treatment

19 Patients: Complete Recovery in 4 weeks

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Chemical Constituents of Methanol and Chloroform Extracts of Physalis minima

Mathanol and Chloroform extracts are rich in

secondary metabolites commonly known as

steroidal lactones

Physalins

Withanolides

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PHYSALINS

CH3

O

CH3

O

HO

OO

O

OH

O

CH3

O1

3 5 7

9

19 11 13

14

16

1820

21

23 25

26

27

28

H

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CH2Cl2: MeOH 95: 5

CH2Cl2: MeOH 98: 2

CH2Cl2: MeOH 96: 4

CH2Cl2: MeOH 95: 5

CH2Cl2: MeOH 93: 7

CH2Cl2: MeOH 91: 9

Pet. ether: Acetone 80: 20

Pet. ether: Acetone 90: 10

Pet. ether: Acetone 84: 16

Pet. ether: Acetone 85: 15

CH2Cl2: MeOH 95: 6

Pet. ether: Acetone 80: 20

Dichloromethane Extract

Column Chroromatography (CC)

Fr. 1 (1.4 g)

Fr. A (120.5 mg)

Fr. B(98.8 mg)

Fr. E(88.5 mg)

5,6-Epoxy physalin B (3)Physalin H (1)

2,3-Dihydro-5,6-epoxy-3-methoxy-physalin B (6)

(25.2 mg)

(20.0 mg)

(12.2 mg)

(15.4 mg)

Isophysalin B(2)(20.2 mg)

27-Hydroxy-1-oxo-witha-24-enolide (8)

(20.0mg)

6-Deoxy physalin D (4)

(10.0 mg)

6-Oxo-11-hydroxyphysalin D (5)

(20.0 mg)

11-Hydroxyphysalin D (3)

Fr. C(50.5 mg)

6-Deoxy-11-hydroxyphysalin I (7)

(10.0mg)

Fr. 2 (2.5 g)

Physalin H(505 mg)

Fr. D(40.5 mg)

PURIFICATION OF PHYSALINS FROM PURIFICATION OF PHYSALINS FROM PHYSALIS MINIMA EXTRACTPHYSALIS MINIMA EXTRACT

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Antileishmanial Activity of Physalins

IC50 (M)= 0.92 0.001

O

O O

H

O

HCH3

OO

OHO

HO

CH3

H

CH3

OHOH

HO H

O

O O

H

O

HCH3

OO

OHO

HO

CH3

H

CH3

OH

H

IC50 (M)= 5.00 0.01

O

O O

H

O

HCH3

OO

OHO

HO

CH3

H

CH3

OH

H

O

HO

IC50 (M)= 3.65 0.070 11 -Hydroxy physalin D 6-Oxo-11 -Hydroxy physalin D

6-Deoxy physalin D

Amphotericine B0.12 0.105

O

O O

H

O

HCH3

OO

OHO

HO

CH3

H

CH3

Cl

H

OH Physalin H

IC50 (M)= 3.39 0.005

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O

O O

H

O

HCH3

OO

OHO

HO

CH3

H

CH3

OCH3

HHO

IC50 (M)= 4.86 0.0562

O

O O

H

O

HCH3

OO

OHO

HO

CH3

H

CH3

H

O

IC50 (M)= 3.39 0.005

O

O O

H

O

HCH3

OO

OHO

HO

CH3

H

CH3

O

H

H3CO

IC50 (M)= 19.4 0.18 2,3-Dihydro-5,6b-epoxy-3-methoxy-physalin B (4)

6-Dehydroxy-11-hydroxy-physalin I (7)

Amphotericine B0.12 0.105

O

O O

H

O

HCH3

OO

OHO

HO

CH3

H

CH3

H

Isophysalin B

IC50 (M)= 7.05 0.05 5,6-Epoxyphysalin B

The encouraging results of this study

are suggestive that we are on the way

for developing cost effective/efficient

local therapy (Ointment) for the

treatment of cutaneous leishmaniasis

in the near future.

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CONCLUSION

Physalis minima was identified as potent antileishmanial agent against cutaneous leishmaniasis by employing in vitro, in vivo and limited clinical trails.

Methanol and Chloroform extracts of the plant were found to be the most active one therefore selected for the limited clinical trails

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CONCLUSION

Chloroform, the most active extract was further subjected to detailed phytochemical investigation to isolate many steroidal lactones

Discovery is patented

“Formulations Against Cutaneous Leishmaniasis”Atta-ur-Rahman, M. Iqbal Choudhary, Sammer Yousuf, Samreen, Farooq Rahman Soomro, Shahida Perveen(US patent 8,287,921 B1), Date of Patent Oct. 16, 2012

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ACKNOWLEDGEMENT

Prof. Atta-ur-Rahman N.I., H.I., S.I., T.I.

Miss Samreen

Higher Education Commission, Islamabad, Pakistan its financial support

4004/21/23

Thank you