Malaria Drug Action And Resistance

Front 1

What are the basics of drug actions?

Back 1

drugs function by interacting with a cellular 'target'
eg, protein, DNA, RNA, lipids

many targets are enzymes or transporters (eg, inhibitors) or receptors (eg, agonists and antagonists)

Front 2

How do drugs have selective toxicicy?

Back 2

unique target in parasite
- chloroquine blocks hemazoin formation

discrimination between host and parasite targets
- pyrimethamine and DHFR

target is more important to parasite than host
- DNA synthesis (antifolates)

drug activation by parasite
- anaerobes and nitroimidazoles

greater drug accumulation by parasite
- food vacuole and chloroquine

Front 3

What are the major drug targets?

Back 3

food vacuole and digestion of hemoglobin.

As the parasite matures, it develops a special organelle, called the cytostome, for the uptake of host cytoplasm. The hemoglobin-containing vesicles fuse to form a food vacuole. The food vacuole is an acidic compartment (pH 5.0–5.4) that contains protease activities, and in this regard resembles a lysosome.

Digestion of hemoglobin also releases heme. Free
heme is toxic due to its ability to destabilize and lyse membranes, as well as inhibit the activity of several enzymes. Some of this free heme can be degraded in oxidative processes. However, the majority of the heme is sequestered into hemozoin or the malarial pigment

Front 4

How and where is hemoglobin digested?
Identify unlabeled parts.

Back 4

Hemoglobinvis taken up by the parasite via pinocytosis and delivered to the food vacuole where proteases break down the globin into peptides and amino acids that are transported to the host erythrocyte. The heme is crystallized into hemozoin resulting in its detoxification.

The malaria parasite takes up the host erythrocyte cytoplasm and breaks down the hemoglobin into amino acids. These amino acids are then used for the synthesis of parasite proteins or possibly as an energy source.

The food vacuole is an acidic compartment (pH 5.0–5.4) that contains protease activities, and in this regard resembles a lysosome.

Aspartic acid proteases designated as plasmepsins make the initial cleavages of the native hemoglobin causing it to dissociate into large globin fragments and free heme. The globin fragments are then further digested by plasmepsins and falcipains (cysteine proteases) into peptides.

Digestion of hemoglobin also releases heme. Free heme is toxic due to its ability to destabilize and lyse membranes, as well as inhibit the activity of several enzymes. Some of this free heme can be degraded in oxidative processes. However, the majority of the heme is sequestered into hemozoin or the malarial pigment

Front 5

How is chloroquine metabolized?

How does this effect plasmodium?

Back 5

Chloroquine and other 4-aminoquinolines inhibit pigment formation, as well as the heme degradative processes, and thereby prevent the detoxification of heme.

The free heme destabilizes the food vacuolar membrane and other membranes and leads to the death of the
parasite.

The fact that the biocrystallization of heme is a unique process to the parasite and not found in the host accounts for the high therapeutic index of such drugs in the absence of drug resistance.

Front 6

Describe antifolates and nucleotide metabolism

Back 6

folate is necessary co-factor for nucleotide (i.e., DNA) synthesis and other biosynthesis

pyrimethamine preferentially inhibits parasite DHFR, dihydrofolate reductase

sulfadoxine inhibits folate synthesis dihydropteroate synthetase(DHPS)
- parasite cannot salvage folates
- host only salvages

Front 7

What are the drug resistance levels?

Back 7

Traditionally these levels of drug resistance have been defined as sensitive (no recrudescence), RI (delayed recrudescence), RII (early recrudescence), and RIII (minimal or no antiparasite effect). Drug resistance by this protocol is determined by monitoring patients for parasitemia for 28 days following standard drug treatment.

Because of the difficulties in using this protocol in endemic areas a modified protocol based on clinical outcome was introduced by the World Health Organization

Front 8

What is the definition of drug resistance?

Back 8

Drug resistance is defined by a treatment failure and can be graded into different levels depending on the timing of the recrudescence following treatment.

parasite tolerance of higher drug levels

Front 9

What factors do you have to rule out before saying a drug is resistant?

Back 9

non-compliance
bad quality
wrong dose
vomiting

Front 10

What is the modified drug resistance protocol?

Back 10

introduced by WHO in 1996

based on clinical outcome:
- adequate clinical response (ACR)
- late treatment failure (LTF)
- early treatment failure (ETF)

more practical in areas of intense transmission
- difficult to distinguish re-infection from recrudescense
- parasitemia in the absence of clinical symptoms is common

Front 11

What is the definition of adequate clinical response (ACR)?

Back 11

no recrudescence by day 14

ACR is defined as the absence of parasitemia
(irrespective of fever) or absence of clinical symptoms (irrespective
of parasitemia) during 14 days following treatment.

Front 12

What is the definition of late treatment failure (LTF)?

Back 12

recrudescence during days 4-14

LTF is the reappearance
of symptoms or the presence of parasitemia during days four to 14 of
follow-up.

Front 13

What is the definition of early treatment failure (ETF)?

Back 13

persistence during days 1-3

ETF is the persistence of clinical symptoms or the presence of parasitemia during the first three days of follow-up.

Front 14

What are the drug resistance mechanisms?

Back 14

mutations in target gene
increase production of target (gene amplification)

drug inactivation

decrease drug accumulation (includes increase efflux)

Front 15

What is Multiple Drug Resistance (MDR)?

Back 15

originally identified in drug resistant bacteria and cancer
ubiquitous family of ABC transporters (aka P-glycoprotein)

Chloroquine resistance is associated with a decrease in the amount of chloroquine that accumulates in the
food vacuole. The mechanism for this decreased accumulation
is controversial. However, some studies have shown that the decrease in drug accumulation is due to an increase in drug efflux. The observation that verapamil and related drugs can reverse the chloroquine resistant phenotype has led to speculation that an ATP-dependent transporter plays a role in drug efflux and chloroquine resistance, similar to the multidrug resistance (MDR) in cancer. An MDR-like transporter, designated PfMDR1, has been identified on the food vacuole membrane.

However, no definitive correlations between PfMDR1 and chloroquine resistance could be demonstrated. An ancillary role for PfMDR1 in chloroquine resistance cannot be ruled out though.

Front 16

what is chloroquine resistance trasnporter (CRT)?

Back 16

originally identified in cross between sensitive and resistance strains (saw and increase in polymorphisms)

A genetic cross and mapping studies between a chloroquine resistant clone and a chloroquine sensitive clone resulted in the identification of a 36 kb region on chromosome 7 associated with chloroquine resistance.

The gene has been designated as PfCRT (for P. falciparum chloroquine resistance transporter) and the protein is localized to the food vacuole membrane. Several mutations in the PfCRT gene show correlations with the chloroquine resistance phenotype and one mutation, a substitution of a threonine (T) for a lysine (K) at residue 76 (K76T) shows good correlation with chloroquine resistance.

Presumably these mutations affect the accumulation of chloroquine in the food vacuole, but the exact mechanism of chloroquine resistance is not known.

Front 17

What is the importance of point mutations in drug resistance?

Back 17

point mutations seem to predominate as mechanism of drug resistance

selectable trait under drug pressure

not necessarily a biological advantage

molecular markers for evaluating drug resistance in populations

rapid in vitro test for resistance (eg, PCR, gene chips)

Front 18

What is the major polymorphism for CRT, chloroquine resistance transporter?
- Name its subcellular location, primary function, and major drugs affected.

Back 18

Major Polymorphism: K76T
Subcellular location: food vacuole
Primary function: transporter
Major Drugs Affected: chloroquine

Front 19

What is the major polymorphism forMDR1, multidrug resistance (P-glycoprotein homolog)?
- Name its subcellular location, primary function, and major drugs affected.

Back 19

Major Polymorphism: Amplification, D86Y
Subcellular location: food vacuole
Primary function: putative transporter
Major Drugs Affected: Mefloquine, quinine (?)

Front 20

For protein, DHFR, dihydrofolate
reductase -
- Name its subcellular location, primary function, and major drugs affected.

Back 20

Subcellular location:cytoplasm
Primary function: Folate metabolism
Major Drugs Affected: Pyrimethamine, proguanil

Front 21

For protein, DHPS, dihydropteroate synthetase -
- Name its subcellular location, primary function, and major drugs affected.

Back 21

Subcellular location:cytoplasm
Primary function: Folate metabolism
Major Drugs Affected: Sulfadoxine, dapsone

Front 22

How does drug resistance deveolop?

Back 22

Drug resistance develops when parasites with decreased sensitivities
to antimalarial drugs are selected under drug pressure. Decreased drug
sensitivity can be conferred by several mechanisms and reflects genetic mutations or polymorphisms in the parasite population. The drug-resistant parasites will have a selective advantage over the drug-sensitive parasites in the presence of drug and will be preferentially transmitted. Major factors in the development of drug resistance are the use of subtherapeutic doses of drugs or not completing the treatment regimen.

Front 23

What are the factors that contribute to developing and spread of drug resistance?

Back 23

Self-treatment - Individuals may only take the drug until symptoms clear or will take lower doses to save money

Poor compliance - Individuals may not complete the full course of treatment because of drug side effects

Mass administration - The widespread use of a drug in an area of intense transmission increases drug pressure by
exposing a larger parasite population to the drug

Long drug half-life - Drugs that are slowly eliminated will lead to a longer exposure of the parasite to
subtherapeutic drug concentrations

Transmission intensity - High levels of transmission may allow reinfection while drugs are at subtherapeutic levels