Malaria intro
Malaria is a parasitic infection with a complex biology, making it hard to tackle. It is transmitted by the Anopheles
mosquito, causing a life-threatening disease. A disease found in tropical regions of the world (high numbers in Africa)
Symptoms: Fever, Headache, Chills, Sweating, Nausea/vomiting, Muscle pain - these are all symptoms of
more common illnesses, like flu. Often malaria is not identified in areas where it is not prevalent.
Species
There are five species of the Anopheles genus that infect humans – only females transmit malaria. It is caused by a
one-celled parasite: plasmodium. Plasmodium falciparum is the major cause of malaria death; other species include
Plasmodium vivax, p. malariae, p. ovale and p. knowelsi (monkey parasite).
Life cycle
Step 1: Human gets bitten by a female mosquito.
Mosquitos inject humans with a cocktail of anti-coagulants,
numbing agents and then parasites too if they are infected
Step 2: Sporozoites migrate to liver
Step 3: Merozoites enter the blood and invade the RBCs
(erythrocytes). The merozoites multiply until the cells burst
(lyse) and then invade more RBCs. Merozoites cause the
symptoms of malaria, so up until now the host will be
asymptomatic. Now the patient will experience fevers each
time the parasite breaks free from a RBC.
Step 4: Some merozoites develop into gametocytes which
are then ingested by the new mosquito that bites and the
cycle continues.
Consequences: Cyclic fevers. Anaemia. Coma.
Death. Cerebral malaria - Spleen cleanses old red blood cells naturally so it will also clear malarial infected cells if it gets the chance. The parasite
decorates the RBC with Knob Associated Histidine Rich Protein (KAHRP) which makes the RBC sticky so that it does not travel to the spleen and is not expelled;
the problem with this is that the sticky RBCs will stick to important capillaries around the body leading to coma and death.
Statistics
In recent years: 2.5x109 people exposed per year, ~2x108 clinical cases per year, ~500,000 child deaths per year.
Plasmodium reproduce rapidly with 1 P. vivax = 250 million merozoites in 14 days.
Anti-malarials: Chloroquine was first used in the 1950s but resistance to it has spread globally.
In pharmaceuticals the development of an antibacterial can make up to $16 billion per year whereas an anti-malarial is only
$200 million per year. This is because the market anti-malarials would be aimed at are poor 3rd world residents who cannot
afford it and therefore they are only sold to richer travellers. New drugs can take up to 12 years to develop and cost up to $868
million. Medicines for Malaria Venture (MMV) is a charity that provides the upfront costs of developing an anti-malarial to
pharma companies. 13 new anti-malarials have been developed saving many lives.
Diagnosis
1. Giemsa stain: Microscopy with visualization of Giemsa-stained parasites in a blood sample. Species determination
is made based on morphological characteristics of the four species of human malaria parasites and the infected red
blood cells. Takes around 5-45 minutes.
2. Rapid Diagnostic Test (RDT): Detects malaria antigens.
- Takes 15 mins and requires no microscopy equipment or expertise.
- Cannot detect low levels and not good at detecting p. malariae and p. ovale
- Must be followed by microscopy to determine level of parasitaemia
3. PCR:
- Good to detect low level infection
- Good to verify which species is involved
- Too slow for acute use for primary diagnosis
Malaria treatment
, Target validation
In vitro enzyme assay – creating method of screening to check compounds are inhibiting a particular target of
interest. Useful in validating the target.
However, in drug design we must properly understand the target being hit because you could design a drug to hit
one enzyme but when placed in a whole organism it may have huge effect on a different enzyme or system.
Target knockout – CRISPR allows us to create knockouts in parasites and confirm that what is being targeted will be
effective. If the knockout causes the parasite to die this validates the target for the production of inhibitors.
Limitations of in vitro
- Host metabolism is important in making the drugs active in the body
- No pharmacokinetics – effect the body has on the drugs e.g. absorption, distribution, metabolism and
secretion
- Toxic compounds – to make sure drug is effective against parasite but not damaging to host
After performing these screens, you would want to see a compound with IC50 inhibitory concentration – kills 50% of
parasites less than micromolar before moving to animals.
Animal testing
Mouse models
Various rodent models available with different plasmodium.
1. P. berghei – standard but not necessarily the most ideal, it is just the oldest. Plasmodium berghei infect
reticular sites in the late stages of RBC reproduction that still have nuclei in them – therefore more similar to
P. vivax in humans than P. falciparum which is the major cause of malaria.
2. P. chadbaudi
3. P. vinckei – high parasitaemia and synchronous
Parasites can have different sensitivity.
4-day suppression test
1. Infect mice via intra-venous or intra-peritoneal injection with 10 7 parasitised RBC
2. 2-4 hours later you treat you 3 groups:
- Treat one group with just the vehicle – the buffer the drugs would be suspended in (negative
control).
- Second group treated with chloroquine (positive control) – these are chloroquine-sensitive parasites
so this test is the response you would expect if the drug were effective.
- Then the final group you treat with the test compound
3. You then repeat the dose on days 1 to 3
4. On day 4 you look at blood smears and count parasitaemia – what percentage of RBC are infected
5. Stop treatment and measure time till death – work out the ED50 (effective dose to kill 50% of parasites)
Monkey models
Monkey models can help to confirm rodent results and are more predictive for humans. A primate host is used –
usually squirrel monkeys. P. falciparum is used. This step is a compulsory requirement for entry in phase 1 trials.
Drug discovery
ADrugs are either from plants or have been chemically derived from plant compounds. This is reflective of drugs
from throughout human history. E.g. Meadowsweet (Filipendula ulmara) – in 1653, Nicholas Culpeper claimed it
cured malaria. Actually, it is rich in salicylic acid which is similar to aspirin, so it likely eased symptoms rather than
being curative.
Quinghao (artemisia annua)
Historic medicinal use: 168 BCE – Good for treating haemorrhoids.
4th century BCE – fevers/ malaria.
Artemisinin is the active anti-malarial compound of this plant that
was isolated in 1972 and its structure determined in 1979.
Artemisinin structure
Pink part = Oxygen bridge/Endoperoxide bond
Very rare
Highly unstable
In artemisinin it is a more stable bond that can survive and be
found more readily. The bond is not entirely stable and can be broken – the break down is mediated by the presence
Malaria is a parasitic infection with a complex biology, making it hard to tackle. It is transmitted by the Anopheles
mosquito, causing a life-threatening disease. A disease found in tropical regions of the world (high numbers in Africa)
Symptoms: Fever, Headache, Chills, Sweating, Nausea/vomiting, Muscle pain - these are all symptoms of
more common illnesses, like flu. Often malaria is not identified in areas where it is not prevalent.
Species
There are five species of the Anopheles genus that infect humans – only females transmit malaria. It is caused by a
one-celled parasite: plasmodium. Plasmodium falciparum is the major cause of malaria death; other species include
Plasmodium vivax, p. malariae, p. ovale and p. knowelsi (monkey parasite).
Life cycle
Step 1: Human gets bitten by a female mosquito.
Mosquitos inject humans with a cocktail of anti-coagulants,
numbing agents and then parasites too if they are infected
Step 2: Sporozoites migrate to liver
Step 3: Merozoites enter the blood and invade the RBCs
(erythrocytes). The merozoites multiply until the cells burst
(lyse) and then invade more RBCs. Merozoites cause the
symptoms of malaria, so up until now the host will be
asymptomatic. Now the patient will experience fevers each
time the parasite breaks free from a RBC.
Step 4: Some merozoites develop into gametocytes which
are then ingested by the new mosquito that bites and the
cycle continues.
Consequences: Cyclic fevers. Anaemia. Coma.
Death. Cerebral malaria - Spleen cleanses old red blood cells naturally so it will also clear malarial infected cells if it gets the chance. The parasite
decorates the RBC with Knob Associated Histidine Rich Protein (KAHRP) which makes the RBC sticky so that it does not travel to the spleen and is not expelled;
the problem with this is that the sticky RBCs will stick to important capillaries around the body leading to coma and death.
Statistics
In recent years: 2.5x109 people exposed per year, ~2x108 clinical cases per year, ~500,000 child deaths per year.
Plasmodium reproduce rapidly with 1 P. vivax = 250 million merozoites in 14 days.
Anti-malarials: Chloroquine was first used in the 1950s but resistance to it has spread globally.
In pharmaceuticals the development of an antibacterial can make up to $16 billion per year whereas an anti-malarial is only
$200 million per year. This is because the market anti-malarials would be aimed at are poor 3rd world residents who cannot
afford it and therefore they are only sold to richer travellers. New drugs can take up to 12 years to develop and cost up to $868
million. Medicines for Malaria Venture (MMV) is a charity that provides the upfront costs of developing an anti-malarial to
pharma companies. 13 new anti-malarials have been developed saving many lives.
Diagnosis
1. Giemsa stain: Microscopy with visualization of Giemsa-stained parasites in a blood sample. Species determination
is made based on morphological characteristics of the four species of human malaria parasites and the infected red
blood cells. Takes around 5-45 minutes.
2. Rapid Diagnostic Test (RDT): Detects malaria antigens.
- Takes 15 mins and requires no microscopy equipment or expertise.
- Cannot detect low levels and not good at detecting p. malariae and p. ovale
- Must be followed by microscopy to determine level of parasitaemia
3. PCR:
- Good to detect low level infection
- Good to verify which species is involved
- Too slow for acute use for primary diagnosis
Malaria treatment
, Target validation
In vitro enzyme assay – creating method of screening to check compounds are inhibiting a particular target of
interest. Useful in validating the target.
However, in drug design we must properly understand the target being hit because you could design a drug to hit
one enzyme but when placed in a whole organism it may have huge effect on a different enzyme or system.
Target knockout – CRISPR allows us to create knockouts in parasites and confirm that what is being targeted will be
effective. If the knockout causes the parasite to die this validates the target for the production of inhibitors.
Limitations of in vitro
- Host metabolism is important in making the drugs active in the body
- No pharmacokinetics – effect the body has on the drugs e.g. absorption, distribution, metabolism and
secretion
- Toxic compounds – to make sure drug is effective against parasite but not damaging to host
After performing these screens, you would want to see a compound with IC50 inhibitory concentration – kills 50% of
parasites less than micromolar before moving to animals.
Animal testing
Mouse models
Various rodent models available with different plasmodium.
1. P. berghei – standard but not necessarily the most ideal, it is just the oldest. Plasmodium berghei infect
reticular sites in the late stages of RBC reproduction that still have nuclei in them – therefore more similar to
P. vivax in humans than P. falciparum which is the major cause of malaria.
2. P. chadbaudi
3. P. vinckei – high parasitaemia and synchronous
Parasites can have different sensitivity.
4-day suppression test
1. Infect mice via intra-venous or intra-peritoneal injection with 10 7 parasitised RBC
2. 2-4 hours later you treat you 3 groups:
- Treat one group with just the vehicle – the buffer the drugs would be suspended in (negative
control).
- Second group treated with chloroquine (positive control) – these are chloroquine-sensitive parasites
so this test is the response you would expect if the drug were effective.
- Then the final group you treat with the test compound
3. You then repeat the dose on days 1 to 3
4. On day 4 you look at blood smears and count parasitaemia – what percentage of RBC are infected
5. Stop treatment and measure time till death – work out the ED50 (effective dose to kill 50% of parasites)
Monkey models
Monkey models can help to confirm rodent results and are more predictive for humans. A primate host is used –
usually squirrel monkeys. P. falciparum is used. This step is a compulsory requirement for entry in phase 1 trials.
Drug discovery
ADrugs are either from plants or have been chemically derived from plant compounds. This is reflective of drugs
from throughout human history. E.g. Meadowsweet (Filipendula ulmara) – in 1653, Nicholas Culpeper claimed it
cured malaria. Actually, it is rich in salicylic acid which is similar to aspirin, so it likely eased symptoms rather than
being curative.
Quinghao (artemisia annua)
Historic medicinal use: 168 BCE – Good for treating haemorrhoids.
4th century BCE – fevers/ malaria.
Artemisinin is the active anti-malarial compound of this plant that
was isolated in 1972 and its structure determined in 1979.
Artemisinin structure
Pink part = Oxygen bridge/Endoperoxide bond
Very rare
Highly unstable
In artemisinin it is a more stable bond that can survive and be
found more readily. The bond is not entirely stable and can be broken – the break down is mediated by the presence
