This document discusses various screening methods for testing potential antimalarial compounds, including both in vitro and in vivo models. In vitro methods include culturing Plasmodium falciparum in human erythrocytes and assessing drug efficacy using assays like 3H hypoxanthine uptake, Giemsa stained slide counting, and flow cytometry. Common in vivo models use rodents infected with Plasmodium berghei or P. yoelii to test drug efficacy over 4 days or in prophylaxis and residual activity tests. More advanced models include using immunocompromised mice that can support P. falciparum infection and testing transmission-blocking activity in mosquitoes.

1. From-
DR.HARSH YADAV & DR. GOPAL JHALANI
Resident, SMS Medical College
Jaipur

2. 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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3.  Malaria:one of the oldest recorded disease.
 Protozoal disease caused by plasmodium
 Transmitted to man by infected female Anopheline
mosquito
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4.  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 countries
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5. Screening process of antimalarial
compound
•In vitro screening (3H Hypoxanthine uptake,
Giemsa stained slide method (MIC method ),Flow
cytometry, micro test.
• In vivo screening(plasmodium berghei 4 day
suppression test ,Hill’s test for causal prophylaxis
and residual activity)
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6.  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 dose
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7. 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)
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8.  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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9. 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.
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10. 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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11.  Parasites are cultured in different concentration of test
compounds in media containing reduced
concentration of hypoxanthine
 after which 3H Hypoxanthine is added.
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12.  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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13. shortcomings are :
 Expensive
 Complicated
 Involves usage of radioactive substance.
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14. 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
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15.  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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16.  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).
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17. 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.
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18. Micro-test (Mark III)
 Most commonly used method for the antimalarial testing for resistance
 Provides 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 with
 Chloroquine
 Mefloquine
 Quinine
 Amodiaquine
 Artemisinin
 Sulfadoxine (SDX)/
pyrimethamine (PYR)
 Pyrimethamine (PYR)
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19.  Patient’s blood sample is inoculated in the wells and
incubated with suitable medium.
 The number of schizonts is counted and compared
with control well.
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20.  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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21. 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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22. 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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23. 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 models
 Rodent malaria parasite. P. berghei, P. yoelii, P.
chabaudi, P. vinckei have been used extensively in
drug discovery and early development.
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24. Rodent models:
 a) Plasmodium berghei 4 day suppression test
 Most widely used preliminary test.
 Efficacy assessed by comparison of
blood parasitemia
mouse survival time
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25. Mice maintained at
 220C at 50-70% humidity
 Diet containing p-aminobenzoic acid 45 mg/kg and water
ad libitum.
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26. 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.
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27.  On day 1 to 3, the experimental groups are treated.
 On day 4, blood smears from all animals are prepared
with Giemsa stain.
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28.  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 calculated
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29.  Untreated control mice typically die approximately
one week after infection.
 For treated mice mean survival time is calculated in
comparison with the untreated and standard drug
treated groups.
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30.  Mice without parasitemia on day 30 of post-infection are
considered cured.
 Compounds identified as active in this test are progressed
through several of the following secondary tests.
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31.  In the ‘Dose ranging full four day test’, compounds are
tested at four doses, by different routes of administration,
to determine ED50 value.
 This test also leads to information about relative potency
and bioavailability
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32. Recrudescence test
Mice are administered with single dose of the test compound
on day 3 of postinfection.
 Control mice receive the suspension vehicle alone.
 Blood smears are prepared at intervals of 12 h, 24h and
then daily till day 33
 and assessed for parasitemia.
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33.  Results are expressed in terms of rapidity of onset of
activity, time to onset of recrudescence, increase of
parasitemia and duration of survival (in days).
 Compounds are also tested for prophylactic activity by
administrating them prior to infection, followed by
daily examination of smears.
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34. b) Hill’s test for causal prophylaxis
and residual activity
 In this model, mice (Charles River strain) are inoculated
with P. yoelii (N67 strain) sporozoites
 Each mouse receives approximately 104-105 sporozoites
intravenously in a total volume of 0.2ml.
 For a compound to be considered truly causal
prophylactic it must pass through four different phases
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35. Phase 1
 involves detection of causal prophylactic activity of the
test compound in mice.
 Test compound is administered 3hr after a sporozoite
inoculation.
 During 14 day period, blood films are taken
 If parasitemia is not detected for 14 days the compound is
considered to be fully protective.
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36. Phase 2
 Compound is then tested for residual activity directed
against blood stage parasites
 By administrating a single dose of the test compound
 After 48hr 104 trophozoites are injected intravenously.
 If the time interval to reach 2% parasitemia is similar to
that of the control group, then it is considered that no
residual activity has occurred
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37. Phase 3
• Compounds suspected of prolonged residual activity are
tested by administering sporozoites followed by the drug 3 h
later.
• After an additional 48h , 0.2 ml blood is and injected
intraperitoneally into a clean mouse.
• Blood films are examined for a 14 day .
• Residual activity is noted if less than 50% of the recipients
develop parasitemia . A compound has no residual activity if
75% or more recipient mice develop patent infection.
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38. Phase 4.
 An additional procedure to clarify whether a compound
has residual effect on erythrocytic stages.
 Mice are injected intravenously with 104 trophozoites 48 h
after the compound administration.
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39. After an additional 3-4 h, 0.2ml of blood is removed and
injected to clean recipient mice.
 Blood films are taken and a comparison of the time
interval to reach 2% parasitemia is made with control
mice
 If the time interval is similar no residual activity is present
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40. Sporonoicidal activity testing
 Albino mice are administered with Plasmodium yoelii
nigeriensis N 67 parasite on day 0.
 Female A. stephensi mosquitoes are placed in containers.
 On third day following infection , mosquitoes were
starved for 24 h are allowed to feed on mice.
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41.  Presence of mature gamets is confirmed on blood film
of mice.
 The mice are then anaesthetized with a single dose of
60 mg/kg of sodium pentobarbitone i.p..
 Mice laid on top of mosquito containers
 Mosquitoes feed on them.
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42.  On the seventh day after the blood feed, a sample of
each batch is removed and dissected
 Midguts are examined with the aid of semi-dark
ground illumination and oocyst counts are made.
 The mean count for each batch is calculated.
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43.  Drug is dissolved in 4% sucrose and fed ad libitum to the
insects following the blood meal.
 The level of drug activity can be calculated from a
comparison of mean oocyst counts in treated and control
batches
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44. Immunocompromised mice.
 A new in vivo mice model for antimalarial efficacy is
designed which comprises of use of
immunocompromised mice.
 It can support Plasmodium falciparum infection as it
lacks T and Lymphokine activated killer cells.
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45.  P. falciparum parasitized human red blood cells are grafted
into immunodeficient mice.
 For immunomodulation dichloromethylene diphosphate
(Cl2MDP) is administered for tissue macrophages.
 The neutrophils controlled by NIMP-R 14 monoclonal
antibody.
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46.  This is the first rodent model in which Plasmodium
falciparum infection can be maintained.
 Test drugs are administered when parasitemia
becomes stable and smears show predominance of
ring forms.
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47. Avian models
 At least 447 species of birds have been found infected
with malarial parasite.
 The avian parasite infects nucleated erythrocytes.
 The vectors of avian species are mosquitoes of the
genera Aedes or Culex
 pre erythrocytic stages of the avian parasites are
found in the mesodermal tissues and two generation.
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48.  Baranger and Filer (1953) using rings, bands or spirals of
various metals placed around chicks infected with P.
gallinaceum found some protective effects.
 prolonged survival time
 Asexual blood parasites were retarded.
 The removal of the band was followed by an increase in
parasitemia on the following day.
 Copper, iron or gold were the most effective.
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49.  Method: When only one or two birds need to be
infected, parasitized blood can be obtained from the
leg vein or wing vein of an infected bird.
 When large number of chicks is to be infected from a
single donor, blood is aspirated directly from the neck
vein or from the heart.
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50.  Drugs may be administered to chicks orally, intravenously
or intramuscularly
 the oral route is the method used more frequently.
 Experimental birds are infected intravenously and receive
the first dose of drug on day 1 followed by twice daily for
the next 3 days. Blood films are made on day 4.
Parasitemia achieved on day 4 in treated and untreated
control group is then compared.
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51.  Avian models have become unpopular primarily due to
the introduction of the rodent models, as mammalian
models are more relevant to human infections
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52. 4/24/2016 Yadav H, SMS Medical College, Jaipur 52

53. Primate models
 Owl monkey (Aotus trivirgatus) and the squirrel
monkey (Saimiri sciureus) have served as experimental
models.
Utility of primate models
 Confirmation of rodent efficacy results.
 Provide clear prediction of drug effect in humans.
 Checks vaccine efficacy
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54.  These primate hosts are primarily used for screening
of antimalarial drugs
 . They also serve as faithful models to investigate
various complications associated with malaria.
 Aotus is one of the WHO recommended model for
studies in malaria.
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55. Plasmodium cynomolgi rhesus
model
 This model runs a close parallel to P. vivax infection in
man.
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56.  It can be used to evaluate causal prophylactic, blood
schizonticidal and hypnozoitocidal activity of a test
compound in one model.
 Young, tuberculin negative rhesus monkeys weighing 3.5
to 6 kg are used.
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57.  Each animal receives i.v. inoculum of approximately
500,000 sporozoites in 2 ml of fluid.
 Drugs administered through a stomach tube once daily,
commencing the day before infection and continuing up to
day 8.
 Blood films are made daily till 6 weeks.
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58.  At the end of 6 weeks if the animals are still negetive,
they are rechallenged with a similar inoculum to prove
their susceptibility to infection.
 All animals becoming infected during 4-6 weeks
observation period and also infected animals from
rechallenged group are further treated as soon as the
parasitemia level reaches 0.1 to 0.5%.
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59.  The animals receive 7 daily oral doses of the drug and
blood films are examined daily during and after therapy
up to a total of 30 days.
 If still negative, the animals are then examined twice
weekly until they relapse.
 If the parasites are not cleared by the drug during the
primary attack, the animals are given 7 daily doses of
5mg/kg of chloroquine. If no relapse follows chloroquine
administration, it indicates that the test drug has destroyed
the hypnozoites.
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60.  When no relapse occurs within 8-12 weeks, the animals
are splenectomized.
 Failure to develop further parasitemia within 4 weeks
indicates that the animals are radically cured.
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61. Malarial vaccine
 More than 40 distinct antigens in various stages of the
parasite have been proposed as potential vaccine
candidates.
 Owl and squirrel monkeys are excellent models for
testing vaccines.
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62.  RTS,S is one of several potential vaccines under
development that target the pre-erythrocytic stage of
the disease.
 The RTS,S vaccine was engineered using genes from
the outer protein of Plasmodium falciparum malaria
parasite and a portion of a hepatitis B virus and a
chemical adjuvant to boost the immune system
response.
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63. W.H.O MALARIA VACCINE
 RTS,S/AS01 is the most advanced vaccine candidate against
Plasmodium falciparum.
 A Phase III trial began in May 2009 and has completed
enrolment in 2011 with 15,460 children in the seven countries
in sub-Saharan Africa.
 Two age groups in the trial:
1) children aged 5-17 months receiving only the RTS,S/AS01
vaccine;
• 2) children aged 6-14 weeks, receive the same malaria vaccine
co-administered with pentavalent vaccines in the routine
immunization schedule.
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64.  According to the current trial schedule, the Phase III trial
data expected to become available to WHO in 2014-2015.
 Based on currently available data the vaccine will be
evaluated as an addition to, not a replacement for, existing
preventive, diagnostic and treatment measures.
 The need for long-lasting insecticidal nets, rapid
diagnostic tests and artemisinin-based combination
therapies will continue, if RTS,S/AS01 becomes available
and is used.
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65. Clinical trials
 In October 2013, GlaxoSmithKline (GSK) reported that
the RTS,S vaccine reduced the amount of cases amongst
young children by almost 50 percent and among infants by
around 25 percent.
 GlaxoSmithKline is set to submit an application for a
marketing license with the European Medicines Agency
(EMA) in 2014.
 WHO which states that it will recommend the use of
RTS,S for use starting in 2015, providing it gets approval.
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66. THANK YOU
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67. References
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Limbird LE, editors. The Pharmacological basis of Therapeutics. New York: McGraw-Hill; 2001.
 2. Trager W, Jensen JB. Human malaria parasites in continuous culture. Science 1976;193:673-5.
 3. Fidock DA, Rosenthal PJ, Croft SL, Nwaka S. Antimalarial drug discovery: Efficacy models for
compound screening (supplementary document). Switzerland. Available from:
http://www.mmv.org/filesUpld/164.pdf
 4. Fidock DA, Rosenthal PJ, Croft SL, Nwaka S. Antimalarial drug discovery: Efficacy models for
compound screening. Nat Rev Drug Discov 2004;3:509-20.
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Richards, editors. Handbook of Experimental Pharmacology. Germany: Springer-Verlag;1984.p179-200
 6. Makler MT, Hinrichs DJ. Measurement of the lactate dehydrogenase activity of plasmodium falciparum
as an assessment of parasitemia. Am J Trop Med Hyg 1993;48:205-10.
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 8. Ohrt C, Willingmyre CD, Lee P, Knirsh C, Milhous W. Assessment of azithromycin in combination with
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http://www.who.int/malaria/resistance.
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68. Flow cytometry
 is a technique for counting and examining microscopic
particles, such as cells and chromosomes, by
suspending them in a stream of fluid and passing
them by an electronic detection apparatus.
 It allows simultaneous multiparametric analysis of the
physical and/or chemical characteristics of up to
thousands of particles per second.
 Flow cytometry is routinely used in the diagnosis of
health disorders, especially blood cancers, but has
many other applications in both research and clinical
practice.
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