Indian Visceral Leishmaniasis: Preclinical (in-vivo) screening methods for newer drug discovery.

1. Preclinical Screening Methods
Kala Azar
Presenter: Dr Pranav Sopory
Junior Resident (Academic)
All Indian Institute of Medical Sciences
New Delhi- 110029
Mob: +91-9999491690
Email: pranav.sopory@gmail.com
In vivo methods
1

2. Contents
Introduction: Leishmaniasis
Kala Azar
Current treatment
Requirement of screening for new drugs
In vivo methods
2

3. Introduction
Leishmaniasis
• 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
3
(Medscape)

4. 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
4

5. 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. Donovani
L. infantum
Organ affected Skin Skin + Mucous membrane Inner Organs
Common varieties of Leishmaniasis
5Ref: Rajesh Bhatia: Essentials of Microbiology

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
6(NVBDCP website)

7. Kala Azar: Life Cycle
7(CDC website)

8. 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-B
1 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
Miltefosine
100 mg O.D. for 28 days
-inhibition of cytochrome c oxidase
-Disrupts mitochondrial function
SSG and Miltefosine
resistance
Liposomal Amphoteracin B
3 mg/kg/day on Days 1-5 and
Days 14 and 21
Same as Amphotericin-B
8NVBDCP website

9. Drug Organ Affected Mechanism Toxicity
Sodium Stibogluconate Heart • Reduces myocardial
contraction force
1. Bradycardia
2. Hypotension
Kidney Acute Renal Failure
Amphotericin B Kidney • Causes tubular injury
and electrolyte leak
• Renal Vasoconstriction
1. Hypokalemia
2. ↓ Urine output
Hypersensitivity Reaction • Microbial origin
(Streptomyces
Nodosus) –
• Stimulates immune
response – Increased
Cytokines
1. Fever
2. Rigors/Chills
3. Hypotension
4. Hypoxeia
Miltefosine Gastrointestinal tract 1. Nausea & Vomiting
Teratogenic in animals 1. Fetal Compromise
(contraindicated in
pregnant women)
9

10. 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
10
(Ref: J Glob Infect Dis. 2010 May-Aug 167-176 Drug Resistance in Leishmaniasis, J Chakravarty)

11. 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.
11http://www.nvbdcp.gov.in/Doc/IVM-Manual-Draft-2015.pdf
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

12. 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
12Gupta et. Al. Visceral Leishmaniasis: experimental models for drug discovery

13. 13
In vivo models

14. 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’d
14Ref: Parasite Burden in Hamsters infected with Two Different strains of Leishmania: LD units

15. 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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16. Parasite Positivity
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17. Final Quantification at 9 months
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18. 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
18Chauhan K et. al., Discovery of triazine mimetics as potent antileishmanial agents

19. Day 0
Day 15
Day 17-21
Day 28
Intracardiac injection of
107 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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20. 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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21. 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’d
21
Henry W. et. al., Immunoenhancement Combined with Amphotericin B as Treatment for Experimental
Visceral Leishmaniasis

22. 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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23. 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
23

24. 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-Gamma
2. Anti-CD 40 MAb: maintains IL-12 and IFN-γ
3. Anti-IL 10 Receptor MAb: Inhibits suppression of IL-12 and IFN-γ
• Sacrifice on Day 21 to check parasite burden
Cont’d
24

25. 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
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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)
26Barbieri et. al., Canine model for leishmaniasis

27. 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 DNA
1. 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)
27
Vaccination against Hemoglobin Receptor-Encoding DNA, Rajan
Guha et. al. (2013)

28. 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)
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Methodology

29. Decrease in Parasite Burden (compared with control group)
29
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

30. 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
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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

31. Summary of In vivo models
Animal Species Examples
Mice BALB/c
C57BL/6
Hamster Syrian Golden Hamster
Dogs Different breeds
Non Human Primates Rhesus Monkey
Owl Monkey
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32. 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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33. Thank You
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