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Preclinical Screening Methods Kala Azar

Presenter: Dr Pranav SoporyJunior Resident (Academic)

All Indian Institute of Medical SciencesNew Delhi- 110029

Mob: +91-9999491690Email: pranav.sopory@gmail.com

In vivo methods

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Contents

Introduction: Leishmaniasis

Kala Azar

Current treatment

Requirement of screening for new drugs

In vivo methods

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IntroductionLeishmaniasis• Parasitic disease • Spread by the bite of an infected sandfly• Common in tropical countries

• Clinical features range from:-self-resolving cutaneous ulcer-mutilating muco-cutaneous disease-lethal systemic illness

Cont’d(Medscape)

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Etiology

Parasite : Leishmania

• “Kinetoplastida” group

• Obligate Intracellular organism

• > 20 pathogenic species

• Zoonotic disease (except Kala Azar)

Vector: Sandfly

• “Phlebotomous” species

• Thrives in a hot climate

• > 30 species transmit Leishmaniasis

• Single bite egests > 1,000 parasites

Cont’d

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Disease Cutaneous Leishmaniasis(a.k.a. Delhi Boil)

Muco-cutaneous Leishmaniasis(a.k.a. Espundia)

Indian Visceral Leishmaniasis

(a.k.a. Kala Azar)Sandfly P. Sergentii P. Pessoai P. Aregentipes

Protozoa L. Tropica L. Brazilliens L. DonovaniL. infantum

Organ affected Skin Skin + Mucous membrane Inner Organs

Common varieties of Leishmaniasis

Ref: Rajesh Bhatia: Essentials of Microbiology

(NVBDCP website) 6

Kala Azar

• Endemic in poor socio-economic groups of population primarily living in rural areas

Location No. of endemic districts

Cases Deaths

Bihar 33 6517 5

Jharkhand 4 1262 0

West Bengal 11 576 0

Uttar Pradesh 6 131 0

India 54 8500 5

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Kala Azar: Life Cycle

(CDC website)

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Current Treatment Criteria for use Drug Mechanism of action

First Line Rx Sodium Stibogluconate (SSG)20mg/kg B.W. I/M for 20 days

-Inhibits Glutathione metabolism via rapid efflux.

-Oxidative stress in parasite

SSG failure Amphotericin-B1 mg/kg B.W. I/V on alternate days for 15

days

-Irreversibly binds to Ergosterol -Disrupts membrane integrity

First line in areas with SSG resistance

Miltefosine100 mg O.D. for 28 days

-inhibition of cytochrome c oxidase-Disrupts mitochondrial function

SSG and Miltefosine resistance

Liposomal Amphoteracin B3 mg/kg/day on Days 1-5 and

Days 14 and 21

Same as Amphotericin-B

NVBDCP website

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Drug Organ Affected Mechanism ToxicitySodium Stibogluconate Heart • Reduces myocardial

contraction force1. Bradycardia2. Hypotension

Kidney Acute Renal Failure

Amphotericin B Kidney • Causes tubular injury and electrolyte leak

• Renal Vasoconstriction

1. Hypokalemia2. ↓ Urine output

Hypersensitivity Reaction • Microbial origin (Streptomyces Nodosus) –

• Stimulates immune response – Increased Cytokines

1. Fever2. Rigors/Chills3. Hypotension4. Hypoxeia

Miltefosine Gastrointestinal tract 1. Nausea & VomitingTeratogenic in animals 1. Fetal Compromise

(contraindicated in pregnant women)

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Resistance to Sodium Stibogluconate

• 1. Diminished reduction of SSG to its active trivalent form(unknown mechanism)

• 2. Mutation in Aquaglyceroporin (AQP) channel that helps mediate uptake of the drug

• 3. Mutated MRP(Multi-drug Resistance associated Protein) gene – decreased intracellular sequestration of the drug

• 4. Increased levels of RLE synthesizing Glutathione

(Ref: J Glob Infect Dis. 2010 May-Aug 167-176 Drug Resistance in Leishmaniasis, J Chakravarty)

http://www.nvbdcp.gov.in/Doc/IVM-Manual-Draft-2015.pdf

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Guidelines on Vector control• Indoor Residual Spraying:

• Application of insecticide on walls and other surfaces that serve as a resting place for infected sandflies.

• IRS kills sandflies when they come in contact with treated surfaces, preventing disease transmission.

Agent DDT (dichloro-diphenyl-trichloroethane)Concentration 50%

Dose 1gm/sq. meter Timing Twice a year: February & June

Mechanism of action: Rapid opening of Sodium ion channels in neurons, causing them to fire spontaneously, which leads to spasms and eventual death

Cause of resistance Upregulation of genes expressing Cytochrome P450, accelerate DDT’s metabolism.

Alternative Deltamethrin

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Need of Preclinical Screening for new drugs

• Difficulty in administration• Increased length of treatment• Increased cost and toxicity• Lack of effective vaccine• Difficulty in controlling vectors• Resistance to current chemotherapy• Emerging HIV/VL coinfection

Gupta et. Al. Visceral Leishmaniasis: experimental models for drug discovery

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In vivo models

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Hamster model• Meso-cri-cetus Au-ratus reproduces clinicopathological features

similar to human VL such as:• Fever• Pancytopenia• Hypergammaglobulinemia• Absence of T cell response (# of APC to induce specific T Cells)• Parasitemia localized maximally to the liver and spleen

• Currently the most commonly used model

Cont’dRef: Parasite Burden in Hamsters infected with Two Different strains of Leishmania: LD units

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Quantification at month 1, 3, 6 and 9

Inoculation

Reach stationary phase at Day 7

Cultured in McNeal, Novy, Nicolle (NNN) medium

Strain of L. Infantum -PP75-Wild strain

-Mimics sandfly midgut-Promastigotes

107 promastigotes/ ml

Intracardiac, Intraperitoneal, Intradermal

-LD units(No. of Am. /1000 host cells)

Methodology

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Parasite Positivity

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Final Quantification at 9 months

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Novel Agent: Triazine• Analogue of Pentamidine• Inhibitor of enzyme: Pterin Reductase (PTR1)• Essential in Pterin and Folate metabolism for L. Donovani• Developed at Central Drug and Research Institute, Lucknow(2013)

• Preclinical trial of two Triazine derivatives:• Compound 14• Compound 15

Chauhan K et. al., Discovery of triazine mimetics as potent antileishmanial agents

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Day 0

Day 15

Day 17-21

Day 28

Intracardiac injection of107 amastigotes of Dd8 Strain of L. Donovani.

Pre-treatment spleen biopsy done.Hamsters with >10 amastigotes/ spleen cell included in study

Compound 14, Compound 15 administered 50mg/kg B.W./day I.P. for 5 days.Newer agents compared with Pentamidine, Sodium Stibogluconate and Miltefosine.

Post-treatment spleen biopsy done.Efficacy measured via Percent Inhibition (PI).

Methodology

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Percent Inhibition: Percent of parasite reduction in the spleen PI = 100– No. of Amastigotes after treated X 100

No. Amastigotes before treatment

Compound 14: 74.41 ± 10.26% - Good Parasite Inhibition

Compound 15: 62.64 ± 10.74% - Moderate Parasite Inhibition

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BALB/c mouse model• Immune response depends upon the individual genetic makeup of the

animal

• Inbred BALB/c strains have decreased genetic variation making it is possible to collect more biological data over time rarely achieved in other mammalian systems.

• Immunology of this model is very well understood

• Useful in combining standard drugs with Immuno-modulators

Cont’dHenry W. et. al., Immunoenhancement Combined with Amphotericin B as Treatment for Experimental

Visceral Leishmaniasis

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Methodology

• Strain: L. Donovani• Dose: 0.2 ml Normal Saline containing 1.5 X 107 amastigotes• Inoculation Site: Tail Vein • Parasite burden: Giemsa-stained liver imprint (LD Units)• Peak in parasite burden: Day 28 (then declines)

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Cause of Prolonged Infection by Kala Azar

• Leishmania is an obligate intracellular parasite• For such infections, clinical features depend the level of Th1 response• Th1 induces• IL-12• IFN-γ

• In Kala Azar APC fail to produce sufficient Th1 response• Instead Th2 response is stimulated producing Antibodies unable to

penetrate cells resulting in• Hypergammaglobulinemia• Reversal of A/G ratio

Cont’d

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Immunoenhancement of AMB with Immunomodulators• Low dose AMB (1mg/kg B.W.) given on Day 14• At this stage, BALB/c mice show both Th1 and Th2 response

• Comparators:1. IL-12: induces IFN-Gamma2. Anti-CD 40 MAb: maintains IL-12 and IFN-γ3. Anti-IL 10 Receptor MAb: Inhibits suppression of IL-12 and IFN-γ

• Sacrifice on Day 21 to check parasite burden

Cont’d

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Treatment Low-dose AMB LDU on Day +21

None - 1,514

None + 1,086

IL-12 - 893

IL-12 + 242

Treatment Low-dose AMB LDU on Day +21

None - 1,589

None + 1,060

Anti-CD40 MAb - 1,165

Anti-CD40 MAb + 236

Treatment Low-dose AMB LDU on Day +21

None - 1,236

None + 1,078

Anti-IL-10R MAb - 1,058

Anti-IL-10R MAb + 302

Barbieri et. al., Canine model for leishmaniasis 26

Canine model for vaccine• Dogs are a domestic reservoir for Visceral Leishmaniasis in Middle

Eastern and South American countries.• Humans: accidental hosts• Vaccination of dogs constitutes a major step towards controlling

transmission

• In India, Kala Azar is Anthroponotic (Human-Sandfly-Human)

Vaccination against Hemoglobin Receptor-Encoding DNA, Rajan Guha et. al. (2013)

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Vaccination with Hemoglobin Receptor-Encoding DNA• Leishmania: • Lacks a Heme synthesis pathway• Therefore, it scavenges hosts heme via Hb Receptor(HbR) on its cell surface

• Aim: To generate Th1 response

• Probable vaccine candidate: HbR DNA1. HbR-FL (Full length)2. HbR-N (Amino terminal)

• Tested on • BALB/c mice (useful model in immunological studies)• Syrian Golden Hamster (mimics clinical features of Kala Azar)

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Intramuscular administration of HbR-N and HbR-FL

Intracardiac Challenge with L. Donovani strain AG83

Day 21: -Th1 response measured

-Parasite load from spleen and liver (sacrifice)

Day 60:-Parasite load measured from spleen and liver (sacrifice)

Methodology

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Decrease in Parasite Burden (compared with control group)

HbR-N (Mice) Day 21 Day 60

Spleen 91 % 99%

Liver 95% 99%

HbR-FL (Mice) Day 21 Day 60

Spleen 86% 99%

Liver 92% 99%

HbR-N (Hamster)

Day 21 Day 60

Spleen 96% 99%

Liver 95% 99%

HbR-FL (Hamster)

Day 21 Day 60

Spleen 96% 99%

Liver 88% 99%

Measured via calculating LDU

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Measuring Th1 response• Measured via Cytometric Bead Array – • enables simultaneous measurement of cytokines too small for traditional

immunoassays.• uses fluorescence detection and antibody-coated beads to efficiently capture

analytes

• Tissue sample:• BALB/c: Day 21 Splenocytes• Compared with control group

IFN-γ IL-2 TNF-α IL-10

HbR-FL ↑ 2.27 ↑ 2 ↑ 2.37 ↓ 2.15

HbR-N ↑ 1.88 ↑ 2 ↑ 2.69 ↓ 10.0

Summary: HbR is a promising candidate for studies in humans

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Summary of In vivo models

Animal Species Examples

Mice BALB/cC57BL/6

Hamster Syrian Golden Hamster

Dogs Different breeds

Non Human Primates Rhesus MonkeyOwl Monkey

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Conclusion• Animal models are expected to mimic the pathological features and

immunological responses observed in humans when exposed to a variety of Leishmania species. • Many experimental models have been developed, each with specific

features, but none accurately reproduces what happens in humans.• The use of a natural model of transmission, using sand fly saliva plus low

doses of parasites has shown that components present in sand fly saliva can modify the course of infection. • The aim of using the animal model is to find a drug that can be

administered orally, be effective in a short course (< 10 days) and have no indication of toxicity at the highest doses tested (100 mg/kg).• Kala Azar, which is fatal to the majority of its victims, still remains a

neglected tropical disease.

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Thank You