basics of screening

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
4/24/2016 Yadav H, SMS Medical College, Jaipur 2

3.  Malaria:one of the oldest recorded disease.
 Protozoal disease caused by plasmodium
 Transmitted to man by infected female Anopheline
mosquito
4/24/2016 Yadav H, SMS Medical College, Jaipur 3

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
4/24/2016 Yadav H, SMS Medical College, Jaipur 4

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)
4/24/2016 Yadav H, SMS Medical College, Jaipur 5

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
4/24/2016 Yadav H, SMS Medical College, Jaipur 6

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)
4/24/2016 Yadav H, SMS Medical College, Jaipur 7

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 8

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 9

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 10

11.  Parasites are cultured in different concentration of test
compounds in media containing reduced
concentration of hypoxanthine
 after which 3H Hypoxanthine is added.
4/24/2016 Yadav H, SMS Medical College, Jaipur 11

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
4/24/2016 Yadav H, SMS Medical College, Jaipur 12

13. shortcomings are :
 Expensive
 Complicated
 Involves usage of radioactive substance.
4/24/2016 Yadav H, SMS Medical College, Jaipur 13

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
4/24/2016 Yadav H, SMS Medical College, Jaipur 14

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 15

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).
4/24/2016 Yadav H, SMS Medical College, Jaipur 16

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 17

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)
4/24/2016 Yadav H, SMS Medical College, Jaipur 18

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 19

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
4/24/2016 Yadav H, SMS Medical College, Jaipur 20

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 21

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 22

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 23

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
4/24/2016 Yadav H, SMS Medical College, Jaipur 24

25. Mice maintained at
 220C at 50-70% humidity
 Diet containing p-aminobenzoic acid 45 mg/kg and water
ad libitum.
4/24/2016 Yadav H, SMS Medical College, Jaipur 25

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 26

27.  On day 1 to 3, the experimental groups are treated.
 On day 4, blood smears from all animals are prepared
with Giemsa stain.
4/24/2016 Yadav H, SMS Medical College, Jaipur 27

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
4/24/2016 Yadav H, SMS Medical College, Jaipur 28

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 29

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 30

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
4/24/2016 Yadav H, SMS Medical College, Jaipur 31

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 32

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 33

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
4/24/2016 Yadav H, SMS Medical College, Jaipur 34

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 35

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
4/24/2016 Yadav H, SMS Medical College, Jaipur 36

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 37

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 38

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
4/24/2016 Yadav H, SMS Medical College, Jaipur 39

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 40

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 41

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 42

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
4/24/2016 Yadav H, SMS Medical College, Jaipur 43

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 44

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 45

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 46

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 47

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 48

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 49

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 50

51.  Avian models have become unpopular primarily due to
the introduction of the rodent models, as mammalian
models are more relevant to human infections
4/24/2016 Yadav H, SMS Medical College, Jaipur 51

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
4/24/2016 Yadav H, SMS Medical College, Jaipur 53

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 54

55. Plasmodium cynomolgi rhesus
model
 This model runs a close parallel to P. vivax infection in
man.
4/24/2016 Yadav H, SMS Medical College, Jaipur 55

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 56

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 57

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%.
4/24/2016 Yadav H, SMS Medical College, Jaipur 58

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 59

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 60

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 61

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 62

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 63

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 64

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 65

66. THANK YOU
4/24/2016 Yadav H, SMS Medical College, Jaipur 66

67. References
 1.Tracy JW, Webster LT Jr. Drugs used in the chemotherapy of protozoal infections. In: Hardman JG,
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.
 5. Desjardins RE. In vitro techniques for antimalarial development and evaluation. In: W.Peters and W.H.G.
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.
 7. Berenbaun MC. A method for testing synergy with any number of agents. J Infect Dis 1978;137:122-30.
 8. Ohrt C, Willingmyre CD, Lee P, Knirsh C, Milhous W. Assessment of azithromycin in combination with
other antimalarial drugs against plasmo
 dium in vitro. Antimicrob Agents Chemother 2002;46:2518-24.
 9. Carfield CJ, Podney M, Gutteridge WE. Interactions of atovaquone with other antimalarial drugs against
Plasmodium falciparum in vitro. Exp Parasitol 1995;80:373-81.
 10. WHO.Roll back Malaria Department. c2004; [cited 2005 May 6]. Available from:
http://www.who.int/malaria/resistance.
4/24/2016 Yadav H, SMS Medical College, Jaipur 67

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.
4/24/2016 Yadav H, SMS Medical College, Jaipur 68