SCREENING OF ANTIMALARIAL DRUGS

SCREENING OF ANTIMALARIAL DRUGS From-DR.HARSH YADAV & DR. GOPAL JHALANIResident, SMS Medical CollegeJaipur

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Screening Preclinical testing of drugs in experimental animals or in vitro for their biological and toxic effects and potential clinical applications

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Malaria:one of the oldest recorded disease. Protozoal disease caused by plasmodiumTransmitted to man by infected female Anopheline mosquito 1/15/2014Yadav H, SMS Medical College, Jaipur3

More sensitive and economical screening models are needed because:

Resistance to existing antimalarial drugs

Growing need for newer and more efficacious antimalarial drugs especially in tropical countries1/15/2014Yadav H, SMS Medical College, Jaipur4

Screening process of antimalarial compound1/15/2014Yadav H, SMS Medical College, Jaipur5

Dose range, ED50, Prophylactic activity and residual activity evaluation in rodents

Confirmation of antimalarial efficacy in Primate models e.g Aotus monkey

PK/PD/Toxicity data evaluation in Primates

Initiate Phase 1 or human studies with1/12th of ED50 dose in mice or 1/7th of rat dose1/15/2014Yadav H, SMS Medical College, Jaipur6

IN VITRO methods

Culture Plasmodium falciparum in human erythrocytes .

Plasmodium falciparum maintained human erythrocytes incubated at 380C in RMPI 1640 medium with human serum or albumax (a lipid rich bovine serum albumin) 1/15/2014Yadav H, SMS Medical College, Jaipur7

Human AB group erythrocytes is inoculated with falciparum infected Aotus monkey blood.Parasites are most suitable for drug assays when there is 2-5% parasitemia. All stages of the erythrocytic cycle of parasite must present in the culture.

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Culture of Plasmodium falciparum is used to :- study the mode of entry of parasite into erythrocytes screening of new drugs to isolate and characterize strains and clones to identify immunogenic antigens and genome of parasite. 1/15/2014Yadav H, SMS Medical College, Jaipur9

Materials and Methods 3H Hypoxanthine uptake

Hypoxanthine used by parasite for purine salvage and DNA synthesis.

Radiolabelled hypoxanthine uptake by parasite is an indicator of its growth and multiplication.

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Parasites are cultured in different concentration of test compounds in media containing reduced concentration of hypoxanthine after which 3H Hypoxanthine is added.1/15/2014Yadav H, SMS Medical College, Jaipur11

measurement of radioactivity by a 1205 Betaplate reader. Percent reductions are measured. most commonly used method for assessing antimalarial efficacy of a compound in vitro

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shortcomings are : Expensive Complicated Involves usage of radioactive substance.

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Giemsa stained slide method (MIC method ) Low cost alternative for testing small number of compounds. Parasites are incubated with test compound Parasitemia of control and treated groups are compared by counting Giemsa stained parasites by light microscopy 1/15/2014Yadav H, SMS Medical College, Jaipur14

Parasites incubated in 5% suspension of erythrocytes at 370C.Change in the proportion of infected RBCs is assessed at end of 72 hr. at various concentrations of each drug.

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This method reports a single concentration as the end point i.e. concentration of a drug in the first sample showing complete inhibition of growth. This measurement is classically known as the Minimum Inhibitory Concentration (MIC).1/15/2014Yadav H, SMS Medical College, Jaipur16

Flow cytometry Parasites are fixed after appropriate period of incubation with test compounds The parasite nuclei are stained with DAPI (42, 6-diamidino-2- phenylindole). Counts of treated and control cultures are then obtained by flow cytometry.1/15/2014Yadav H, SMS Medical College, Jaipur17

Micro-test (Mark III) Most commonly used method for the antimalarial testing for resistanceProvides information on the quantitative drug response of P. falciparum . test can be carried out with several drugs, in a Micro test kit with 12 X 8 wells, predosed withChloroquineMefloquine Quinine AmodiaquineArtemisinin Sulfadoxine (SDX)/ pyrimethamine (PYR)Pyrimethamine (PYR)

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Patients blood sample is inoculated in the wells and incubated with suitable medium. The number of schizonts is counted and compared with control well. 1/15/2014Yadav H, SMS Medical College, Jaipur19

For monitoring the level and spread of resistance, molecular diagnostic methods for detecting resistant parasite have been proposed.These molecular tools are based on the detection by PCR of point mutation in the parasite genome responsible for in vitro resistance

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These methods are suitable for use on a large number of samples in malaria endemic areas and have major advantage over in vitro tests that require parasite cultivation which take days to perform.

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Advantages of in vitro methods Precise and efficient Rapid Large number of compounds can be evaluated at the same time Synergism or antagonism with drug combinations can be studied Better assessment of intrinsic activity of a drug.

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Limitations of in vitro methods

Drugs acting through active metabolite cannot be studied. Non reproducibility of pharmacokinetic effects. Toxic compounds also get selected. Expertise and infrastructure needed Lack of clinical correlation.

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In vivo methods Compounds effective in in-vitro screening tests are taken up for in vivo evaluation.Plasmodium species that cause human disease are unable to infect non primate animal modelsRodent malaria parasite. P. berghei, P. yoelii, P. chabaudi, P. vinckei have been used extensively in drug discovery and early development.1/15/2014Yadav H, SMS Medical College, Jaipur23

Rodent models:a) Plasmodium berghei 4 day suppression test

Most widely used preliminary test. Efficacy assessed by comparison of blood parasitemia mouse survival time1/15/2014Yadav H, SMS Medical College, Jaipur24

Mice maintained at 220C at 50-70% humidityDiet containing p-aminobenzoic acid 45 mg/kg and water ad libitum.1/15/2014Yadav H, SMS Medical College, Jaipur25

On day 0, mice are injected with 0.2 ml of aliquot (2X107 parasitized erythrocytes by Plasmodium berghei) Experimental groups are(five each) Vehicle treated (control group) Test drug treated group. Positive control group (chloroquine,reference drug) is administered.1/15/2014Yadav H, SMS Medical College, Jaipur26

On day 1 to 3, the experimental groups are treated. On day 4, blood smears from all animals are prepared with Giemsa stain.1/15/2014Yadav H, SMS Medical College, Jaipur27

Parasitemia is determined microscopically by counting 4 fields of approximately 100 erythrocytes per field.

The difference between the mean value of the control group and those of the experimental groups is calculated1/15/2014Yadav H, SMS Medical College, Jaipur28

For low parasitemias (