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Dr. Alexis N. LaCrue and Dr. Dennis E. Kyle
September 8, 2011
 2.1 billion live in malarious areas
 Affects 300-500 million people
  worldwide; one million deaths
  annually
 Transmitted by the bite of a female
  Anopheles mosquito
 Five species that affect humans:
  Plasmodium falciparum, P. vivax, P.
  malariae, P. ovale, and P. knowlesi
Plasmodium Lifecycle
Principle clinical sign is periodic fever Paroxysm: relapsing or periodic
   fever
 Periodicity corresponds to erythrocytic development (48 or 72 hr)
     benign tertian: P. vivax
     malignant tertian : P. falciparum
     quartan : P. malariae
 The pattern of intermittent chills/fever mirrors the synchronized
   parasite development in an infected person’s blood
 This response is primarily due to toxins released with schizont
   rupture
Plasmodium Lifecycle
Reasons for Malaria
Resurgence
  •   Insecticide resistant vectors
  •   Parasite drug resistance
  •   Demographics
  •   Economics and politics
• Vector control

• Vaccines

• Drugs
Plasmodium Lifecycle
Global distribution of dominant or potentially important malaria vectors. This map does not include all regionally
important vectors or species complexes. (Ex Kiszewski et al. [2004] Am. J. Trop. Med. Hyg. 70[5].)
GAMETOCYTES




                                           GAMETES
                                  14-16                  ZYGOTE           24hr
                                 days PI
                                                                           PI
                                                               OOKINETE
                          SALIVARY                    8-10
                           GLANDS                    days PI




                                                                          PERITROPHIC MATRIX



        SPOROZOITES


                                OOCYST      EPITHELIAL
                                              CELLS


                      BASAL LAMINA
 Distribution
      Over 420 species, most in tropics and subtropics
      Temperate and summer arctic distribution
      ~70 species capable of transmission (40 important)
 Feeding habits
      Female requires blood meals for egg broods
      Males feed on nectar
 Life cycle – 7 to 20 days (egg to adult)
      egg > larva > pupa > adult
      Females mate once and lay 200-1000 eggs in 3-12
       batches over a lifetime
      Find their host by chemical and physical stimuli
      Average life span of mosquito < 3 weeks
 Malaria development – 7 to 12 days
      Each male & female gametocyte produce >10,000
       sporozoites
Eggs are Deposited Singly on Water Surface

                                          Lateral floats function to keep
                                          eggs on water surface




    Drawing of Anopheles eggs




                                          Chorion of egg is sculpted

 Larval embryogenesis (72 hr) and hatching must occur in 4 days
•Four larval stages (instars)
•Feed on microbes
•Breath at the water surface
•Lie horizontally at the water surface
•One week to months, temperature
dependent

                                         Non-wetable spiracle opens at water surface for respiration
The pupa is non-feeding, surface breathing, and is the stage of
transition from aquatic larva to winged adult (24 hr)
 Fully formed adult mosquito emerges from
    pupal stage at water surface.
   Males often emerge first and form swarms,
    can’t copulate until genitalia rotate 180°
   Females emerge, enter swarm, copulate in the
    air
   Females may mate more than once
   Sperm is stored in the spermatheca for lifetime
   Males feed on nectar, females primarily on
    blood
   Aestivation in adult females
     cessation or slowing of activity in winter;
        especially slowing of metabolism
Factors that influence presence, abundance and longevity of mosquitoes.




  1.   Temperature
  2.   Rainfall
  3.   Relative Humidity                          Land cover
  4.   Topography                                 (vegetation)
  5.   Soil Type
Plasmodium Lifecycle
 Entomologic Inoculation
  Rate (EIR)
   EIR = mosquito biting rate
    times the proportion of
    infected mosquitoes
   Sporozoite rates usually 1-20%
Plasmodium Lifecycle
Plasmodium Lifecycle
Plasmodium Lifecycle
Plasmodium Lifecycle
Transgenic mosquitoes to
block transmission of
malaria
Plasmodium Lifecycle
Plasmodium Lifecycle
Currently, there is no vaccine.

3. Pre-erythrocytic/anti-infection vaccines
     • Directed against sporozoites and/or liver
        stages
         • Abundant surface proteins (CSP, TRAP)
         • Attenuated sporozoites
     • Designed to prevent blood-stage infection
        and thereby avoid all manifestations of
        disease.
2. Anti-morbidity/mortality vaccines
     • Vaccines directed against asexual blood
        stages
     • Designed to reduce clinical severity.
3. Transmission blocking vaccines
     • Directed against mosquito stages
     • Designed to halt development in the
        mosquito                                   Protective mechanisms of immunity are shown for each stage.
     • Would be used in combination with other
        vaccines and/or drug therapy
CREDIT: KATHARINE
SUTLIFF/SCIENCE
•Does not treat the human but prevents infection
•Better to have a vaccine or drug that does both.
•We have new drug candidates that may do both.
Plasmodium Lifecycle
 Prophylaxis
     Causal prophylaxis
     Suppressive prophylaxis
     Post-exposure “prophylaxis”
   Treatment of acute, uncomplicated malaria
   Treatment of severe malaria
   Radical cure
   Presumptive Intermittent Therapy (IPT)
Malaria: progress, perils, and prospects for eradication
Brian M. Greenwood, David A. Fidock, Dennis E. Kyle, Stefan H.I. Kappe, Pedro L. Alonso, Frank
H. Collins, Patrick E. Duffy. J Clin Invest. 2008;118(4):1266–1276 doi:10.1172/JCI33996
1. Cinchona alkaloids      7. Sulfonamides
        quinine                    sulfadoxine (with
        quinidine                  pyrimethamine)
2. 4-aminoquinolones       8. Sulfones
        chloroquine                dapsone (with
                                   chlorproguanil)
        amodiaquine
                           9. Antifols
3. 4-quinolinemethanol
                                   pyrimethamine
        mefloquine
                                   proguanil
4. 8-aminoquinolines
                           10. Artemisinins
        primaquine
                           11. Antibiotics
5. Phenanthrenemethanols
                                   Doxycycline
        halofantrine
                                   Tetracycline
6. Biguanides
                                   Azithromycin
        proguanil
ACT resistance-Thai
                                                   cambodia border




            Chloroquine resistance                 Mefloquine resistance
            Sulfodoxine-pyrimethamine resistance   Malaria-free areas
WHO/UNICEF, 2005
Year of     1st case of
  Antimalarial drug   introduction   resistance

 Quinine               1632          1910
 Chloroquine           1945          1957 12 years
 Proguanil             1948          1949 1 year
 Sulfadoxine-
  pyrimethamine         1967          1967 <1 year
 Mefloquine            1977          1982 5 years
 Atovaquone            1996          1996 <1 year
 Treatment                   Result
   Halofantrine                Recrudesced at 4 wk
   Quinine (iv) plus           MFQ prophylaxis,
    halofantrine                 Recrudesced ~3 wk
   Quinine (iv) followed       Cleared yet
    by Quinine (po) and          recrudesced 7d after Rx
    Doxycycline                  , but asymptomatic
   Halofantrine (po) with      Recrudesced at 12 days
    whipping cream!
 Treatment                                      Result
   Mefloquine plus Doxycycline                      Recrudesced at day 27
    (7 days)
   Artesunate (po) followed by                      Success at last!
    Mefloquine




    “This case of imported multi-drug resistant falciparum
    malaria shows that artemisinin and derivatives will soon
    be needed, in fact are already needed, in the western
    world.”                 Lancet 1994
 Common name: Qinghaosu
 Isolated from: Chinese herb Artemisia annua
 Characteristics:
   1. Rapidly kills asexual stages
   2. Short half-life
   3. Frequent recrudescence when used as
        monotherapy
 WHO recommendation: Use in combination
  w/ other antimalarials which have a longer
  half-life
    Artemisinin Combination Therapy (ACT)
    High cure rate in 3 days
 Severe malaria disease
 High levels of parasites in the blood
 Inability to take oral medications
 Lack of timely access to intravenous quinidine
 Quinidine intolerance or contraindications
 Quinidine failure
 Recrudescence rates
    5 days of treatment < 10%
    3 days of treatment 40% - 70%
    1 day of treatment > 90%
 Recrudescent parasites remain susceptible to drug in in vitro drug
   susceptibility tests
Plasmodium Lifecycle
Survival of erythrocytic forms that leads to renewed manifestation
Plasmodium Lifecycle
In vitro Evidence                                        In vivo Evidence




  Recovery rates ranging from 0.044% to 1.313%   Recovery based on number of dormant parasites present in host
  Teuscher et al. (2010). JID 202: 1362-1368     LaCrue et al. (2011).
Problem:
    Very few in peripheral blood
        Difficult to identify in blood smears
    Want to be able to easily identify in the field


Goal:
Develop a method that will enhance the detection of dormant
parasites in patient samples and thick smears
    1.Faster identification and quantification of dormant parasites
    (i.e. distinguish dormant rings from rings, merozoites, Howell
    Jolly bodies)
    2.Determine if there is a correlation between the number of
    dormant rings in the first 72hr of treatment and recrudescence
    3.Predict optimal dosing regimen of artemisinin combinations
 Not for profit partnership established in Switzerland in
  1999
 Mission: To reduce burden of malaria in endemic
  countries through the development of novel and
  effective anti-malarials
 Vision: To have a malaria-free world


www.mmv.org
MMV- R and D
                 Lead identification
         (in vitro high throughput screens
                 and in vivo studies)


                 Lead optimization
           (in vitro and in vivo studies to
         identify absorption, distribution,
            metabolism, and excretion
                   characteristics)


           Preclinical development and
                 candidate selection
           (in vitro and in vivo studies to
             assess safety in humans)


                  Clinical Phase I
             (volunteers administered
         increasing doses of drug; adverse
                 effects assessed)


                 Clinical Phase II
          (Proof of concept- small group
                    of patients)

                 Clinical Phase III
             (Large group of patients)

             Registration and Launch
MMV- R and D
                 Lead identification
         (in vitro high throughput screens
                 and in vivo studies)


                 Lead optimization
           (in vitro and in vivo studies to
         identify absorption, distribution,
            metabolism, and excretion
                   characteristics)


           Preclinical development and
                 candidate selection
           (in vitro and in vivo studies to
             assess safety in humans)


                  Clinical Phase I
             (volunteers administered
         increasing doses of drug; adverse
                 effects assessed)


                 Clinical Phase II
          (Proof of concept- small group
                    of patients)

                 Clinical Phase III
             (Large group of patients)

             Registration and Launch
 To determine if compounds from the Roman
 Manetsch lab at USF and the Michael Riscoe
 lab at the Portland Veterans Affairs Medical
 Center have anti-malarial activity in vivo and
 in vitro.
O                        O                                            O        O
                                                                                                5                            8     9    1                              5
                                                                                                     4        3 R                                                          4

    Interested in exploring the anti-malarial activity of
                                                                                                                                                                                    3

                                                                                        6                                7                  2                     6
                                                                                                                                                                                            OR
                                                                                   R'                         2     R'                          R
                                                                                                                                                        O                           2
                                                                                        7                N1              6        N10       3                    O 7           N1
    quinolones and tetrahydroacridones                                                          8        H                   5
                                                                                                                                  H     4                              8       H

                                                                                                    4Q                           THA                             PEQ
   Endochin,- an experimental anti-malarial quinolone from                                                   O
                                                                                                                                                                           O        O

    the 1940s                                                                                                                                       O
                                                                                                                                                                                        O

                                                                                                                                                             O             N
                                                                                                                                                                           H
             Recently shown to have poor activity in mammalian
                                                                                            O              N
                                                                                                           H
                                                                                                          endochin                                          ICI56,780

              systems                                                                                    (RMMC103)                                          (RMMC128)



   ICI 56, 780- Screened by Walter Reed and shown to have
    activity against liver stages in P. cynomolgi in 1970s
   Tetrahydroacridones (THAs)- anti-malarial activity
    known since 1940s

                 Name                           Type                                                              Activity
         Endochin               4(1H)-quinolone (4Q)                   EE and erythrocytic activity in avians not mammals

         ICI 56,780 (RMMC128)   Phenoxy-ethoxy-4(1H)-quinolone (PEQ)   EE stages in monkeys

         RMMC93                 Tetrahydroacridone (THA)               In vitro erythrocytic activity and gametocyte activity


               Potent blood stage activity and demonstrated potential to kill hypnozoites makes the 4Qs, THAs
                and PEQs ideal for novel drug development.
                   Novel antimalarials were subjected to an in vitro high throughput screen and those with low
                    nanomolar activity were selected for in vivo studies.
Team: USF and
                                     Portland
                                     Manetsch lab
                                     Riscoe lab


                                     Team: USF and
                                     Portland
                                     Manetsch lab
                                     Riscoe lab

                                     Team: USF
                                     Kyle lab


Hit Validation
                 Kyle lab   Manetsch and Riscoe




Hit to Lead
                 Kyle lab      Charman lab



Early Lead
Candidate
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     Must be given access to a server
     Share folders and calendars
     Share microsoft excel, word, powerpoint and adobe acrobat documents
          Great for working from the same files
     Create team sites within a main site
     Give specific permissions to documents, folders, and sites
Plasmodium Lifecycle
Overview




 Compounds with low nM in vitro activity
 Schmatz Scouting protocol to screen for
  compounds to use in the Thompson test
Methods- Thompson Test



          Infect Mice with 1x106 Plasmodium
                 berghei-GFP parasites



         Day 3-5 PE-Treat mice with 10 mg/kg
         and 50 mg/kg of compound diluted in
                      PEG400



           Day 3,6,9,13,21 and 30PE-Check
           parasitemia via flow cytometry




         Euthanize when animals reach >40%     Compounds with ≥90% reduction in
                    parasitemia                    parasitemia go to GSK
Compound 4




                                             C= CURATIVE (100% INHIBITION)
                                             A= ACTIVE (>80% INHIBITION)
                                             S= SUPPRESSIVE (20-80%
                                             INHIBITION)
                                             N= NOT ACTIVE (<20% INHIBITION)
          Drug       Dose per day (3 days)      Vehicle   Route Activity (day 30PE)

       UNTREATED            None                 None     None         N/A
      AMODIAQUINE         30 mg/kg              PEG400    Oral          C
      ATOVAQUONE          50 mg/kg              PEG400    Oral          C
        (0.3MG/KG)        10 mg/kg              PEG400    Oral          N
        (1.0MG/KG)        50 mg/kg              PEG400    Oral          C
        (3.0MG/KG)        50 mg/kg              PEG400    Oral          C
       (10.0MG/KG)        50 mg/kg              PEG400    Oral          C
Summary- Thompson Test


       Group          Drug      Dosages per day   Vehicle     Route        Activity
                                   (3 days):
                                    -1,0, +1
       Control      Untreated                      None        None          N/A

     Experimental       1        10 or 50 mg/kg   PEG 400      PO       Suppressive at
                                                                          50 mg/kg
     Experimental      2         10 or 50 mg/kg   PEG400       PO     Suppressive at both
                                                                        concentrations

     Experimental       3        10 or 50 mg/kg   PEG 400      PO         CURATIVE


     Experimental      4         0.3-10 mg/kg     PEG 400      PO         CURATIVE




    Compound 4 was selected for further modifications to improve the bioavailability
    and produce a lead candidate.
Thompson Test-Results
      Group         Drug       Dosages per day     Vehicle   Route      Activity
                                   (3 days):
                                    -1,0, +1
   Experimental      4           0.3-10 mg/kg      PEG 400    PO       CURATIVE
                                                                         (all)
    Experimental     5           0.3-10mg/kg       PEG 400    PO       CURATIVE
   (October 2010)                                                        (all)
    Experimental     6           0.3-10mg/kg       PEG 400    PO        CURATIVE
   (October 2010)                                                    (3 and 10mg/kg)

    Experimental     7           0.3-10mg/kg       PEG 400    PO       CURATIVE
  (November 2010)                                                    (0.3-10mg/kg)
    Experimental     8           0.3-10mg/kg       PEG400     PO       CURATIVE
  (November 2010)                                                     (1-10mg/kg)
    Experimental     9           0.3-10mg/kg       PEG400     PO       CURATIVE
  (December 2010)                                                     (1-10mg/kg)
    Experimental     10          0.3-10mg/kg       PEG400     PO       CURATIVE
  (December 2010)                                                        (all)
    Experimental     11          0.3-10mg/kg       PEG 400    PO       CURATIVE
   (January 2011)                                                        (all)
    Experimental     12          0.3-10mg/kg       PEG 400    PO       CURATIVE
   (January 2011)                                                     (1-10mg/kg)


   Compound 4 and 11 are now pre-clinical lead candidates.
Plasmodium Lifecycle
Primaquine




               Most of the drugs
               currently in use:
              CQ, QN, AMO, MQ,
                  AS, ATOV
Artemisinin
derivatives
Plasmodium berghei-
              infected mouse

                        6-7 days


               Naïve female
            Anopheles stephensi
            takes a blood meal




                        20-24 days

            Dissect infected Sgs
                 and purify
                sporozoites




Dose mice     Inject 10,000          Dose mice   Follow for
 (Day -1)   spz/mouse + Dose          (Day +1)    30 days
                 (Day 0)
Group          Drug      Dosages per day      Vehicle       Route    Activity
                               (3 days):
                                -1,0, +1
 Infection      Untreated                         None         None      N/A
  control
Drug control       1           50 mg/kg       10% DMSO, 0.5%    SC     CURATIVE
                                                  Tween
Drug control   PRIMAQUINE      50 mg/kg          PEG400         PO     CURATIVE

   Test            2        10 and 50 mg/kg      PEG 400        PO      NONE


   Test            3        10 or 50 mg/kg       PEG 400        PO     CURATIVE


   Test            4          3-10 mg/kg         PEG 400        PO     CURATIVE
   In-direct method- Assessment of pre-patent period (i.e. time from injection of
    sporozoites to peripheral blood infection)
      Pros:
         All or none effect of drug or vaccine
      Cons:
         Complicated if drug effects liver and erythrocytic forms
   Direct methods- Study of parasites in vivo (i.e. examination of liver sections, QRT-
    PCR, flow cytometry, intra-vital imaging)
     Pros:
        Significant advances for detecting liver stages
     Cons:
        Mice must be sacrificed. Prevents long term study of infection.
        Labor intensive (requires lots of mice to achieve statistical power)
        Expensive
 LET’S TRY BIOLUMINESCENCE!!
“production and emission of light by a living organism
            as a result of a chemical reaction”

Sea pansy produces GFP protein

                                                                               Firefly

                                      Non-invasive study of on-going biological
                                       processes
                                           No surgery needed
Squid with bioluminescent bacteria
                                      Captures light emitted by the reactions
                                       of luciferase and its substrate D-luciferan
                                           Firefly – attract a mate
                                           Sea pansy- repel predators
                                           Bacteria- repel predators
Bioluminescent marine bacteria
                                      Common applications of BLI:
                                           Studies of infection using bioluminescent
                                            pathogens
                                           Studies of cancer progression using
                                            bioluminescent cell line
                                           Stem cell research
Plasmodium berghei-
  infect donor mice

               6-7 days

     Naïve female
  Anopheles stephensi
  takes a blood meal




              20-24 days

 Dissect infected Sgs
      and purify
     sporozoites




Seed into 96 well plate,
 1,500 sporozoites per
  well & HepG2 cells
Drug concentrations decrease from right to left
Mice infected with parasite   Injected with D-luciferin   Mice anesthetized and placed
containing luciferase gene    which is oxidized by        into Xenogen IVIS Spectrum     ATP only present in living cells so the
                              luciferase + ATP            (Imaged for 5-60 seconds)      reaction allows for measurement of
                                                                                         energy or life




                                                            Imaged for 5-60 seconds
   Parasite used: P. berghei ANKA 1052 cl1: GFP and luciferase under the
    control of AMA-1 promoter (Leiden)
             Compound                          Dose (mg/kg)                        Route
              Untreated                             N/A                     N/A
                  1                                  50                     Sub-q
                  1                                  50                     Oral
                  2                                  10                     Oral
                  2                                  50                     Oral and Sub-q
                  2                                 100                     Oral
                  3                                   3                     Oral
                  3                                  10                     Oral
Plasmodium berghei-
              infected mouse

                       6-7 days


               Naïve female
            Anopheles stephensi
            takes a blood meal




                        20-24 days

            Dissect infected Sgs
                 and purify
                sporozoites




Dose mice     Inject 10,000          Dose mice             BLI
 (Day -1)   spz/mouse + Dose          (Day +1)   (44hr and Days 6,9,13 PE)
                 (Day 0)
44hr post-infection




                   Inject sporozoite-infected mice with D-luciferin
                                      (100mg/kg)




                       Anesthetize for 5 min with isofluorane




                    Image mice using the IVIS spectrum system




 Liver collection group:                                       Blood-stage group:
Euthanize and image livers                               Follow days 6, 9, 13, 21, and 30PE
BLI-LIVER STAGE ACTIVITY

                              Drug 1                         Drug 2
Untreated       SQ (50 mg/kg)          (50 mg/kg)   (10 mg/kg)        (50 mg/kg)   PQ-50




 1          2     1       2            1       2    1       2         1       2    1       2


1                                                   1



2                                                   2
Drug 1
       Untreated   SQ (50 mg/kg)            ORAL (50 mg/kg)   PQ-50




44HR
 PE




DAY
6PE




DAY
9PE




DAY
13PE
Compound        Dose (mg/kg)    Route      Activity (n=5)

Untreated      N/A                 N/A      N/A
1              50                  Sub-q    Curative (all)
1              50                  Oral     Curative (all)
2              10                  Oral     Curative (3)
2              50                  Oral     Curative (4)
2              50                  Sub-q    Curative (4)
2              100                 Oral     Curative (4)
3              3                   Oral     Curative (all)
3              10                  Oral     Curative (all)
 We have screened more than 100 compounds
  since April 2009
 Found compounds with blood, liver, gametocyte,
  and mosquito stage activity
 Best 2 analogs have moved forward as pre-
  clinical leads
Acknowledgments

    Kyle lab
         Dennis E. Kyle (PI)
         Tina Mutka (research assistant)
         Ken Udenze (graduate student)
         Steven Stein (graduate student)
    Manetsch lab
         Roman Manetsch
         Matt Cross
         Andrii Monastyrskyi
         Jordany Maignan
    Riscoe Lab
         Portland, Oregon
    PK studies
         Monash University, Sue Charman
    Parasites
         Leiden University
    Funding
         Medicines for Malaria Ventures (MMV)

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Plasmodium Lifecycle

  • 1. Dr. Alexis N. LaCrue and Dr. Dennis E. Kyle September 8, 2011
  • 2.  2.1 billion live in malarious areas  Affects 300-500 million people worldwide; one million deaths annually  Transmitted by the bite of a female Anopheles mosquito  Five species that affect humans: Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi
  • 4. Principle clinical sign is periodic fever Paroxysm: relapsing or periodic fever  Periodicity corresponds to erythrocytic development (48 or 72 hr)  benign tertian: P. vivax  malignant tertian : P. falciparum  quartan : P. malariae  The pattern of intermittent chills/fever mirrors the synchronized parasite development in an infected person’s blood  This response is primarily due to toxins released with schizont rupture
  • 6. Reasons for Malaria Resurgence • Insecticide resistant vectors • Parasite drug resistance • Demographics • Economics and politics
  • 7. • Vector control • Vaccines • Drugs
  • 9. Global distribution of dominant or potentially important malaria vectors. This map does not include all regionally important vectors or species complexes. (Ex Kiszewski et al. [2004] Am. J. Trop. Med. Hyg. 70[5].)
  • 10. GAMETOCYTES GAMETES 14-16 ZYGOTE 24hr days PI PI OOKINETE SALIVARY 8-10 GLANDS days PI PERITROPHIC MATRIX SPOROZOITES OOCYST EPITHELIAL CELLS BASAL LAMINA
  • 11.  Distribution  Over 420 species, most in tropics and subtropics  Temperate and summer arctic distribution  ~70 species capable of transmission (40 important)  Feeding habits  Female requires blood meals for egg broods  Males feed on nectar  Life cycle – 7 to 20 days (egg to adult)  egg > larva > pupa > adult  Females mate once and lay 200-1000 eggs in 3-12 batches over a lifetime  Find their host by chemical and physical stimuli  Average life span of mosquito < 3 weeks  Malaria development – 7 to 12 days  Each male & female gametocyte produce >10,000 sporozoites
  • 12. Eggs are Deposited Singly on Water Surface Lateral floats function to keep eggs on water surface Drawing of Anopheles eggs Chorion of egg is sculpted  Larval embryogenesis (72 hr) and hatching must occur in 4 days
  • 13. •Four larval stages (instars) •Feed on microbes •Breath at the water surface •Lie horizontally at the water surface •One week to months, temperature dependent Non-wetable spiracle opens at water surface for respiration
  • 14. The pupa is non-feeding, surface breathing, and is the stage of transition from aquatic larva to winged adult (24 hr)
  • 15.  Fully formed adult mosquito emerges from pupal stage at water surface.  Males often emerge first and form swarms, can’t copulate until genitalia rotate 180°  Females emerge, enter swarm, copulate in the air  Females may mate more than once  Sperm is stored in the spermatheca for lifetime  Males feed on nectar, females primarily on blood  Aestivation in adult females  cessation or slowing of activity in winter; especially slowing of metabolism
  • 16. Factors that influence presence, abundance and longevity of mosquitoes. 1. Temperature 2. Rainfall 3. Relative Humidity Land cover 4. Topography (vegetation) 5. Soil Type
  • 18.  Entomologic Inoculation Rate (EIR)  EIR = mosquito biting rate times the proportion of infected mosquitoes  Sporozoite rates usually 1-20%
  • 23. Transgenic mosquitoes to block transmission of malaria
  • 26. Currently, there is no vaccine. 3. Pre-erythrocytic/anti-infection vaccines • Directed against sporozoites and/or liver stages • Abundant surface proteins (CSP, TRAP) • Attenuated sporozoites • Designed to prevent blood-stage infection and thereby avoid all manifestations of disease. 2. Anti-morbidity/mortality vaccines • Vaccines directed against asexual blood stages • Designed to reduce clinical severity. 3. Transmission blocking vaccines • Directed against mosquito stages • Designed to halt development in the mosquito Protective mechanisms of immunity are shown for each stage. • Would be used in combination with other vaccines and/or drug therapy
  • 28. •Does not treat the human but prevents infection •Better to have a vaccine or drug that does both. •We have new drug candidates that may do both.
  • 30.  Prophylaxis  Causal prophylaxis  Suppressive prophylaxis  Post-exposure “prophylaxis”  Treatment of acute, uncomplicated malaria  Treatment of severe malaria  Radical cure  Presumptive Intermittent Therapy (IPT)
  • 31. Malaria: progress, perils, and prospects for eradication Brian M. Greenwood, David A. Fidock, Dennis E. Kyle, Stefan H.I. Kappe, Pedro L. Alonso, Frank H. Collins, Patrick E. Duffy. J Clin Invest. 2008;118(4):1266–1276 doi:10.1172/JCI33996
  • 32. 1. Cinchona alkaloids 7. Sulfonamides quinine sulfadoxine (with quinidine pyrimethamine) 2. 4-aminoquinolones 8. Sulfones chloroquine dapsone (with chlorproguanil) amodiaquine 9. Antifols 3. 4-quinolinemethanol pyrimethamine mefloquine proguanil 4. 8-aminoquinolines 10. Artemisinins primaquine 11. Antibiotics 5. Phenanthrenemethanols Doxycycline halofantrine Tetracycline 6. Biguanides Azithromycin proguanil
  • 33. ACT resistance-Thai cambodia border Chloroquine resistance Mefloquine resistance Sulfodoxine-pyrimethamine resistance Malaria-free areas WHO/UNICEF, 2005
  • 34. Year of 1st case of Antimalarial drug introduction resistance  Quinine 1632 1910  Chloroquine 1945 1957 12 years  Proguanil 1948 1949 1 year  Sulfadoxine- pyrimethamine 1967 1967 <1 year  Mefloquine 1977 1982 5 years  Atovaquone 1996 1996 <1 year
  • 35.  Treatment  Result  Halofantrine  Recrudesced at 4 wk  Quinine (iv) plus  MFQ prophylaxis, halofantrine Recrudesced ~3 wk  Quinine (iv) followed  Cleared yet by Quinine (po) and recrudesced 7d after Rx Doxycycline , but asymptomatic  Halofantrine (po) with  Recrudesced at 12 days whipping cream!
  • 36.  Treatment  Result  Mefloquine plus Doxycycline  Recrudesced at day 27 (7 days)  Artesunate (po) followed by  Success at last! Mefloquine “This case of imported multi-drug resistant falciparum malaria shows that artemisinin and derivatives will soon be needed, in fact are already needed, in the western world.” Lancet 1994
  • 37.  Common name: Qinghaosu  Isolated from: Chinese herb Artemisia annua  Characteristics: 1. Rapidly kills asexual stages 2. Short half-life 3. Frequent recrudescence when used as monotherapy  WHO recommendation: Use in combination w/ other antimalarials which have a longer half-life  Artemisinin Combination Therapy (ACT)  High cure rate in 3 days
  • 38.  Severe malaria disease  High levels of parasites in the blood  Inability to take oral medications  Lack of timely access to intravenous quinidine  Quinidine intolerance or contraindications  Quinidine failure
  • 39.  Recrudescence rates  5 days of treatment < 10%  3 days of treatment 40% - 70%  1 day of treatment > 90%  Recrudescent parasites remain susceptible to drug in in vitro drug susceptibility tests
  • 41. Survival of erythrocytic forms that leads to renewed manifestation
  • 43. In vitro Evidence In vivo Evidence Recovery rates ranging from 0.044% to 1.313% Recovery based on number of dormant parasites present in host Teuscher et al. (2010). JID 202: 1362-1368 LaCrue et al. (2011).
  • 44. Problem: Very few in peripheral blood Difficult to identify in blood smears Want to be able to easily identify in the field Goal: Develop a method that will enhance the detection of dormant parasites in patient samples and thick smears 1.Faster identification and quantification of dormant parasites (i.e. distinguish dormant rings from rings, merozoites, Howell Jolly bodies) 2.Determine if there is a correlation between the number of dormant rings in the first 72hr of treatment and recrudescence 3.Predict optimal dosing regimen of artemisinin combinations
  • 45.  Not for profit partnership established in Switzerland in 1999  Mission: To reduce burden of malaria in endemic countries through the development of novel and effective anti-malarials  Vision: To have a malaria-free world www.mmv.org
  • 46. MMV- R and D Lead identification (in vitro high throughput screens and in vivo studies) Lead optimization (in vitro and in vivo studies to identify absorption, distribution, metabolism, and excretion characteristics) Preclinical development and candidate selection (in vitro and in vivo studies to assess safety in humans) Clinical Phase I (volunteers administered increasing doses of drug; adverse effects assessed) Clinical Phase II (Proof of concept- small group of patients) Clinical Phase III (Large group of patients) Registration and Launch
  • 47. MMV- R and D Lead identification (in vitro high throughput screens and in vivo studies) Lead optimization (in vitro and in vivo studies to identify absorption, distribution, metabolism, and excretion characteristics) Preclinical development and candidate selection (in vitro and in vivo studies to assess safety in humans) Clinical Phase I (volunteers administered increasing doses of drug; adverse effects assessed) Clinical Phase II (Proof of concept- small group of patients) Clinical Phase III (Large group of patients) Registration and Launch
  • 48.  To determine if compounds from the Roman Manetsch lab at USF and the Michael Riscoe lab at the Portland Veterans Affairs Medical Center have anti-malarial activity in vivo and in vitro.
  • 49. O O O O 5 8 9 1 5 4 3 R 4 Interested in exploring the anti-malarial activity of 3  6 7 2 6 OR R' 2 R' R O 2 7 N1 6 N10 3 O 7 N1 quinolones and tetrahydroacridones 8 H 5 H 4 8 H 4Q THA PEQ  Endochin,- an experimental anti-malarial quinolone from O O O the 1940s O O O N H  Recently shown to have poor activity in mammalian O N H endochin ICI56,780 systems (RMMC103) (RMMC128)  ICI 56, 780- Screened by Walter Reed and shown to have activity against liver stages in P. cynomolgi in 1970s  Tetrahydroacridones (THAs)- anti-malarial activity known since 1940s Name Type Activity Endochin 4(1H)-quinolone (4Q) EE and erythrocytic activity in avians not mammals ICI 56,780 (RMMC128) Phenoxy-ethoxy-4(1H)-quinolone (PEQ) EE stages in monkeys RMMC93 Tetrahydroacridone (THA) In vitro erythrocytic activity and gametocyte activity  Potent blood stage activity and demonstrated potential to kill hypnozoites makes the 4Qs, THAs and PEQs ideal for novel drug development.  Novel antimalarials were subjected to an in vitro high throughput screen and those with low nanomolar activity were selected for in vivo studies.
  • 50. Team: USF and Portland Manetsch lab Riscoe lab Team: USF and Portland Manetsch lab Riscoe lab Team: USF Kyle lab Hit Validation Kyle lab Manetsch and Riscoe Hit to Lead Kyle lab Charman lab Early Lead Candidate
  • 51. SKYPE  Google documents (free)  Everyone must have a google account to access  Share folders and calendars  Share excel, word, powerpoint, and pdf-like documents  export to microsoft excel, microsoft word, or adobe acrobat  work on documents at the same time
  • 52. Microsoft Sharepoint (paid) http://sharepoint.microsoft.com/en-us/Pages/default.aspx  Must be given access to a server  Share folders and calendars  Share microsoft excel, word, powerpoint and adobe acrobat documents  Great for working from the same files  Create team sites within a main site  Give specific permissions to documents, folders, and sites
  • 54. Overview  Compounds with low nM in vitro activity  Schmatz Scouting protocol to screen for compounds to use in the Thompson test
  • 55. Methods- Thompson Test Infect Mice with 1x106 Plasmodium berghei-GFP parasites Day 3-5 PE-Treat mice with 10 mg/kg and 50 mg/kg of compound diluted in PEG400 Day 3,6,9,13,21 and 30PE-Check parasitemia via flow cytometry Euthanize when animals reach >40% Compounds with ≥90% reduction in parasitemia parasitemia go to GSK
  • 56. Compound 4 C= CURATIVE (100% INHIBITION) A= ACTIVE (>80% INHIBITION) S= SUPPRESSIVE (20-80% INHIBITION) N= NOT ACTIVE (<20% INHIBITION) Drug Dose per day (3 days) Vehicle Route Activity (day 30PE) UNTREATED None None None N/A AMODIAQUINE 30 mg/kg PEG400 Oral C ATOVAQUONE 50 mg/kg PEG400 Oral C (0.3MG/KG) 10 mg/kg PEG400 Oral N (1.0MG/KG) 50 mg/kg PEG400 Oral C (3.0MG/KG) 50 mg/kg PEG400 Oral C (10.0MG/KG) 50 mg/kg PEG400 Oral C
  • 57. Summary- Thompson Test Group Drug Dosages per day Vehicle Route Activity (3 days): -1,0, +1 Control Untreated None None N/A Experimental 1 10 or 50 mg/kg PEG 400 PO Suppressive at 50 mg/kg Experimental 2 10 or 50 mg/kg PEG400 PO Suppressive at both concentrations Experimental 3 10 or 50 mg/kg PEG 400 PO CURATIVE Experimental 4 0.3-10 mg/kg PEG 400 PO CURATIVE Compound 4 was selected for further modifications to improve the bioavailability and produce a lead candidate.
  • 58. Thompson Test-Results Group Drug Dosages per day Vehicle Route Activity (3 days): -1,0, +1 Experimental 4 0.3-10 mg/kg PEG 400 PO CURATIVE (all) Experimental 5 0.3-10mg/kg PEG 400 PO CURATIVE (October 2010) (all) Experimental 6 0.3-10mg/kg PEG 400 PO CURATIVE (October 2010) (3 and 10mg/kg) Experimental 7 0.3-10mg/kg PEG 400 PO CURATIVE (November 2010) (0.3-10mg/kg) Experimental 8 0.3-10mg/kg PEG400 PO CURATIVE (November 2010) (1-10mg/kg) Experimental 9 0.3-10mg/kg PEG400 PO CURATIVE (December 2010) (1-10mg/kg) Experimental 10 0.3-10mg/kg PEG400 PO CURATIVE (December 2010) (all) Experimental 11 0.3-10mg/kg PEG 400 PO CURATIVE (January 2011) (all) Experimental 12 0.3-10mg/kg PEG 400 PO CURATIVE (January 2011) (1-10mg/kg) Compound 4 and 11 are now pre-clinical lead candidates.
  • 60. Primaquine Most of the drugs currently in use: CQ, QN, AMO, MQ, AS, ATOV Artemisinin derivatives
  • 61. Plasmodium berghei- infected mouse 6-7 days Naïve female Anopheles stephensi takes a blood meal 20-24 days Dissect infected Sgs and purify sporozoites Dose mice Inject 10,000 Dose mice Follow for (Day -1) spz/mouse + Dose (Day +1) 30 days (Day 0)
  • 62. Group Drug Dosages per day Vehicle Route Activity (3 days): -1,0, +1 Infection Untreated None None N/A control Drug control 1 50 mg/kg 10% DMSO, 0.5% SC CURATIVE Tween Drug control PRIMAQUINE 50 mg/kg PEG400 PO CURATIVE Test 2 10 and 50 mg/kg PEG 400 PO NONE Test 3 10 or 50 mg/kg PEG 400 PO CURATIVE Test 4 3-10 mg/kg PEG 400 PO CURATIVE
  • 63. In-direct method- Assessment of pre-patent period (i.e. time from injection of sporozoites to peripheral blood infection)  Pros:  All or none effect of drug or vaccine  Cons:  Complicated if drug effects liver and erythrocytic forms  Direct methods- Study of parasites in vivo (i.e. examination of liver sections, QRT- PCR, flow cytometry, intra-vital imaging)  Pros:  Significant advances for detecting liver stages  Cons:  Mice must be sacrificed. Prevents long term study of infection.  Labor intensive (requires lots of mice to achieve statistical power)  Expensive  LET’S TRY BIOLUMINESCENCE!!
  • 64. “production and emission of light by a living organism as a result of a chemical reaction” Sea pansy produces GFP protein Firefly  Non-invasive study of on-going biological processes  No surgery needed Squid with bioluminescent bacteria  Captures light emitted by the reactions of luciferase and its substrate D-luciferan  Firefly – attract a mate  Sea pansy- repel predators  Bacteria- repel predators Bioluminescent marine bacteria  Common applications of BLI:  Studies of infection using bioluminescent pathogens  Studies of cancer progression using bioluminescent cell line  Stem cell research
  • 65. Plasmodium berghei- infect donor mice 6-7 days Naïve female Anopheles stephensi takes a blood meal 20-24 days Dissect infected Sgs and purify sporozoites Seed into 96 well plate, 1,500 sporozoites per well & HepG2 cells
  • 66. Drug concentrations decrease from right to left
  • 67. Mice infected with parasite Injected with D-luciferin Mice anesthetized and placed containing luciferase gene which is oxidized by into Xenogen IVIS Spectrum ATP only present in living cells so the luciferase + ATP (Imaged for 5-60 seconds) reaction allows for measurement of energy or life Imaged for 5-60 seconds
  • 68. Parasite used: P. berghei ANKA 1052 cl1: GFP and luciferase under the control of AMA-1 promoter (Leiden) Compound Dose (mg/kg) Route Untreated N/A N/A 1 50 Sub-q 1 50 Oral 2 10 Oral 2 50 Oral and Sub-q 2 100 Oral 3 3 Oral 3 10 Oral
  • 69. Plasmodium berghei- infected mouse 6-7 days Naïve female Anopheles stephensi takes a blood meal 20-24 days Dissect infected Sgs and purify sporozoites Dose mice Inject 10,000 Dose mice BLI (Day -1) spz/mouse + Dose (Day +1) (44hr and Days 6,9,13 PE) (Day 0)
  • 70. 44hr post-infection Inject sporozoite-infected mice with D-luciferin (100mg/kg) Anesthetize for 5 min with isofluorane Image mice using the IVIS spectrum system Liver collection group: Blood-stage group: Euthanize and image livers Follow days 6, 9, 13, 21, and 30PE
  • 71. BLI-LIVER STAGE ACTIVITY Drug 1 Drug 2 Untreated SQ (50 mg/kg) (50 mg/kg) (10 mg/kg) (50 mg/kg) PQ-50 1 2 1 2 1 2 1 2 1 2 1 2 1 1 2 2
  • 72. Drug 1 Untreated SQ (50 mg/kg) ORAL (50 mg/kg) PQ-50 44HR PE DAY 6PE DAY 9PE DAY 13PE
  • 73. Compound Dose (mg/kg) Route Activity (n=5) Untreated N/A N/A N/A 1 50 Sub-q Curative (all) 1 50 Oral Curative (all) 2 10 Oral Curative (3) 2 50 Oral Curative (4) 2 50 Sub-q Curative (4) 2 100 Oral Curative (4) 3 3 Oral Curative (all) 3 10 Oral Curative (all)
  • 74.  We have screened more than 100 compounds since April 2009  Found compounds with blood, liver, gametocyte, and mosquito stage activity  Best 2 analogs have moved forward as pre- clinical leads
  • 75. Acknowledgments  Kyle lab  Dennis E. Kyle (PI)  Tina Mutka (research assistant)  Ken Udenze (graduate student)  Steven Stein (graduate student)  Manetsch lab  Roman Manetsch  Matt Cross  Andrii Monastyrskyi  Jordany Maignan  Riscoe Lab  Portland, Oregon  PK studies  Monash University, Sue Charman  Parasites  Leiden University  Funding  Medicines for Malaria Ventures (MMV)

Editor's Notes

  1. There has been a resurgence of malaria and some of the reasons include: Drug Resistance In parts of South East Asia strains of malaria have developed resistance to anti-malarial drugs such as mefloquine and chloroquine. Insecticide resistance Mosquitoes are becoming resistant to commonly used insecticides such as DDT. Demographics can lead to a resurgence because, People traveling to and from places where malaria is endemic can be potential carriers of the disease. Politics and Economics Malaria-endemic countries are among the world ’ s most impoverished. A malaria-stricken family spends an average of over one quarter of its income on malaria treatment, as well as paying prevention costs and suffering loss of income because they cannot work when they are ill . The cost of malaria control and treatment can drain an economy. According to the WHO, it costs Arica $10-12 billion every year in gross domestic product.
  2. With the increase in parasite resistance to drug treatments and insecticide resistant mosquitoes, novel control methods, are being looked at as a means of controlling malaria. Currently there is no vaccine against malaria. Drug resistance new drugs need to be developed………….. Insecticide treated bed nets to control the vector. Note: Nets can cut malaria transmission by at least 50% and child deaths by 20%; however, very few people have them due to the cost.
  3. The transmission characteristics of regionally dominant malaria vector species were compared in the recent publication cited on this slide and used to explain regional differences in the force of transmission. This world map shows the tremendous diversity of vector species, both current and historic. Some colors represent only the potential for transmission, because malaria has been eradicated from most temperate zones of former endemicity, such as Europe and North America since ca WW II. Although this map differentiates between some members of species complexes (e.g. gambiae and arabiensis of the Gambiae Complex) others are not broken into their constituent species. I will be showing habitats of some of the South American vector shortly.
  4. Box 1 Parasite stages under attack Current approaches to malaria vaccine development can be classified according to the parasite stages that are targeted: 1. Vaccines directed against sporozoites and/or liver stages (collectively termed pre-erythrocytic stages) are designed to prevent blood-stage infection and thereby avoid all manifestations of disease (anti-infection vaccines). 2. Vaccines directed against asexual blood stages are designed to reduce clinical severity (anti-morbidity/mortality vaccines). 3. Vaccines directed against mosquito stages are designed to halt development in the mosquito (transmission-blocking vaccines). Protective mechanisms of immunity are shown for each stage. In reality, the effects that can be anticipated for each type of vaccine overlap broadly: a pre-erythrocytic-stage vaccine, even if not 100% effective, could reduce transmission and morbidity — the latter is predicted by the reductions in morbidity and mortality associated with the use of insecticide-impregnated bednets; a highly effective blood-stage vaccine could eliminate blood stages as soon as they emerge from the liver, thereby curtailing both infection and transmission; and a transmission-blocking vaccine could reduce population-wide malaria infection rates and malaria-associated morbidity.
  5. The fate of normal and attenuated malaria sporozoites in the host liver. A malaria infection is initiated by injection into the host blood of sporozoites by a female anopheline mosquito as she takes a blood meal. The sporozoites must migrate to the liver and colonize hepatocytes in order for the infection to progress. This involves traversal of resident macrophages (Kupffer cells) lining the liver&apos;s blood vessels and passage through a number of hepatocytes before invading a hepatocyte and beginning to develop. The sporozoite differentiates, grows, and multiplies within a vacuole in the host hepatocyte, giving rise to thousands of merozoites. These are released into the bloodstream where they invade red blood cells, initiating the erythrocytic stage of the disease. Sporozoites attenuated by irradiation (RAS) or by genetic manipulation (GAS) also transit through Kupffer cells and hepatocytes before invading liver cells. RAS undergo growth arrest but the infected hepatocytes remain intact, leading to the generation of a protective immune response involving B cells that attack free sporozoites and T cells that recognize infected hepatocytes. The intracellular development of GAS is different from that of RAS, as GAS-infected hepatocytes in culture disappear 24 hours after infection; the GAS-induced immune response may also be different. CREDIT: KATHARINE SUTLIFF/SCIENCE
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  8. Artemisinin is effective against multi-drug resistant p. fal Qinghaosu used for the treatment of fever in Chinese traditional medicine for more than 2,000 years Front line treatment With ACTs, the duration of treatment is only 3 days (Maude et al) If used alone, the artemisinins will cure falciparum malaria in 7 days, but studies have shown that in combination with certain synthetic drugs they produce high cure rates in 3 days with higher adherence to treatment. (WHO Facts on ACTs January 2006 update)
  9. Teuscher et al, tested different P. falciparum strains and found that only 0.04-1.313% of DHA treated parasites recover to resume growth. In an in vivo study, LaCrue et al found that recovery was based on the number of dormant parasites present in the host. So we know that dormancy occurs in vivo; however…
  10. Following treatment with artemisinin, there are very few dormant parasites circulating in the peripheral blood possibly due to clearance by the spleen. This makes identification of dormant parasites difficult. Our goal is to..