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HUMAN CYSTIC ECHINOCOCCOSIS RESEARCH

IN CENTRAL AND EASTERN SOCIETIES (HERACLES)

KICK OFF MEETING, 17-18 DECEMBER 2013

PARTNER 7

PENDIK VETERINARY CONTROL INSTITUTE,

DEPARTMENT OF PARASITOLOGY, TURKEY

WP4. Increasing bioavailability of ABZ and new enantiomeric

drug synthesis

OBJECTIVES

• Determine, in an in vivo model, the protoscolicidal effects of ABZ against

E. Granulosus and whether HP-Βcd improves the oral bioavailability of BZs,

consequently the anthelmintic effectiveness.

• Synthesis of a new enantiomeric pure drug based on ABZ.

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TASK 4.1.Animal sample collection

Five Hundred samples (blood/serum) from CE positive animal and

500 additional blood/serum samples from negative animals will be

collected to support WP2.

Samples will be extracted from different animal (sheep, goats,

cattle, and buffaloes).

Places the samples are collected;

Sheep and cattle: Istanbul, Tekirdag, Kirklareli

Goats: Kırklareli, Antalya, Burdur

Buffaloes: Kirklareli, Samsun

Pig: Tekirdag?

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TASK 4.2. Parasite collection and drug formulations

A. Parasite collection:

• Remove Echinococcus granulosus cyst

• Collect PSC (protoscolices)

• Determine alive PSC (Eosin stain, flame cell activity)

• Examine genotype (Use G1 for infection)

B. Preservation of PSC until inection of mice

There are 2 methods.

B1. Direct usage of PSC for infection (Diker et al 2007; Cucher

et al, 2012)

Infection of mice with 2000 viable protoscolices, IP

B2. Maintain in medium for certain time (Zhang et al, 2005).

After 56 days cultivation yield developed PSC.

Infection with 50 smal cysts, IP

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TASK 4.3. Experimental design in the animal model

Experimental animals (Mice)

Need 190 Balb/c female mice (Ettlin et al.)

Mice will be purchased from licensed private company.

Mice age: 9 weeks; weight : 20-25 g (Kuster et al, 2013)

Experiment 1. Three groups of mice will be tested.

Groups/number of

mice

G1 n:20 G2 n:20 G3 n: 20 Total 60

mice

Drug ABZ ABZ+HP- βCD CONTROL

Experiment 2. Recovered PSCs from Experiment 1 will be used

to re-infect new mice groups

Groups/number of mice G1 n:10 (ABZ) G2 n:10

(ABZ+HP- βCD)

Total 20 mice

Infection trial IP IP

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Experiment 3. Four groups of mice will be tested.

Groups/numb

er of mice

G1 n:20 G2 n:20 G3 n: 20 G4 n:20 Total 80

mice

Drug ABZ ABZSO4(+) ABZSO4(-) CONTROL

Experiment 4. Recovered PSCs from Experiment 3 will be used to re-

infect new mice groups [N=10 for each group, (total 30 mice) excluding

the control group].

Groups/number

of mice

G1 n:10

(ABZ)

G2 n:10

ABZSO4(+)

G3 n: 10

ABZSO4(-)

Total 30 mice

Infection trial IP IP IP

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Applications

•Need 140 infected mice for experiment1 and experiment 3.

•Start giving drug formulations orally with bucco gastric tube 12

months later after intraperitoneal infection.

•Animals will be treated 3 months.

•Administer all formulations daily in 0.2 ml deionized water

TASK 4.4. Necropsy and sample collection

A. Blood samples for HPLC: Blood samples collection will be

started after two months of drug treatment.

Samples will be centrifugated and sera will be stored at -20

degree until HPLC analysis.

B. After drug applications animals will be sacrified by CO2.

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C. Evaluation

Determine cyct type (normal, calsified or caseified)

Fertility: cyst with PSC will be considered fertile.

Count cysts,

Measure cysts

Define location of cyst

for each mouse.

Histopathology

TEM/SEM?

Groups/number of mice G1 n:10 (ABZ) G2 n:10

(ABZ+HP- βCD)

Total 20 mice

Infection trial IP IP

Experiment 2 Recovered PSCs from Experiment 1 will be used to re-

infect new mice groups

Experiment 4. Recovered PSCs from Experiment 3 will be used to re-

infect new mice groups [N=10 for each group, (total 30 mice) excluding

the control group].

Groups/number

of mice

G1 n:10

(ABZ)

G2 n:10

ABZSO4(+)

G3 n: 10

ABZSO4(-)

Total 30 mice

Infection trial IP IP IP

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Ethic approvals and certificates of people who work with

experimental animals are completed.

TASK 4.5. Pharmacokinetics and Toxicology

TASK 4.6. Statistical analysis and new drug synthesis

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PHARMACOKINETICS

Adriano Casulli EURLP, ISS (Rome, Italy)

WP4

BIOAVAILABILITY of ABZ and ENANTIOMERIC DRUG SYNTHESIS – WP4

Task 4.5: Pharmacokinetics

Infection Experiments Drug bioavailability Parasite viability Toxicology

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OXIDATIVE PATHWAY OF ABZ

CYCLODEXTRINS are cyclic oligosaccharides that tend to form

inclusion complexes. Are used for:

- increasing aqueous solubility of drugs;

- increasing chemical stability of drugs;

- enhancing drug delivery to and through biological membranes.

Increase the absorption of ABZ by preparing a suspension with

Hydroxypropyl-β-Cyclodextrin (HP-βCD).

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ABZ-SO ENANTIOMERS

It is well known the importance of chirality on clinical

pharmacology of chiral drugs: the use of one of them alone instead of

the racemic form could reduce adverse effects or improve therapeutic

effectiveness.

(+)-(R)-ABZ-SO

(-)-(S)-ABZ-SO

*

*

N

HN

NH

O

O

S

O

N

HN

NH

O

O

S

O

a) Monitoring the plasma concentration of the three forms of the

metabolic pathway of ABZ (with or without HP-βCD) by HPLC on

polysaccharide-based chiral stationary phases through in vivo

experiments;

b) Checking the biological activity and efficacy of the ABZ-SO

enantiomers separately by enantioselective through in vivo

experiments and in human patients;

c) Setting HPLC methods capable to isolate the single enantiomers on

a semipreparative scale to evaluate the efficacy of each one in vivo

experiments and in future clinical trials.

THE STUDY WILL FOCUS ON:

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PHARMACOKINETICS

The BIOAVAILABILITY of the metabolic pathway of ABZ will be

evaluated by:

1) maximum plasma concentration (Cmax, μg/mL);

2) time to achieve the maximum plasma concentration (Tmax, h);

3) the area under the plasma concentration–time curve between 0 and

6 h (AUC0–6, μg • h/mL).

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Toxicological endpoints in mouse model - HERACLES project

Francesca Maranghi - Roberta Tassinari – Laura Narciso Dpt. SPVSA – Food and Veterinary Toxicology Unit

francesca.maranghi@iss.it, roberta.tassinari@iss.it, laura.narciso@iss.it

WP4 – Task 4.5 Toxicological endpoints

In the frame of the evaluation of potential side effects of drug formulations administered to BALB/c female mice, the following endpoints have been selected on the basis of the known mode of action - tubulin interaction in target species – and effects - hepatotoxicity, (testicular toxicity), toxicity for immune system.

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…toxicological endpoints Hepatotoxicity Histopathological analysis will be performed on slides of liver of treated and control mice. Quali-quantitative: critical evaluation from Experiments 1, 2, 3, 4 (5 mice/group). Parameters of oxidative stress on hepatocytes: • mitochondrial toxicity • ROS formation • superoxide radical production • lipid peroxidation.

….toxicological endpoints Toxicity for immune system • Cytokines in blood of mice • interleukin (IL)-8 • tumour necrosis factor (TNF) α Tubulin interaction Genotoxicity (e.g.) • chromosomal aberration • abnormal mitosis • micronuclei formation • comet assay