trans

402 DRUG RESISTANCE
is again assumed to take place in the terminal
food vacuole. Within this pathway host hemoglobin
is enzymatically degraded, liberating
potentially toxic ferriprotoporphyrin IX or
heme. Under normal conditions the heme is
converted into malaria pigment or hemozoin.
Chloroquine (and related drugs) bind with high
affinity to heme and the resulting complex,
which cannot be incorporated into pigment,
retains its cytotoxic potential.
Detailed investigation of this accumulation
process has separated a high affinity, low capacity,
saturable drug accumulation component
from a low affinity, non-saturable process. The
K a of the high affinity process was shown to
correlate with drug susceptibility in a range
of parasite isolates. The kinetics of this uptake
process are similar to those for heme–chloroquine
binding. Furthermore, inhibition of heme
generation using specific inhibitors of the
aspartate protease involved in the initial hemoglobin
cleavage results in a loss of chloroquine
accumulation in parasitized cells. Thus the
accumulation process can be largely explained
in terms of drug binding to heme, and chloroquine
resistance results from a reduced access
to this binding site as measured by the apparent
K a of binding
It was rapidly recognized that this altered
drug accumulation phenotype was similar to
that observed in multidrug-resistant (MDR)
cancer cells. In these cells it had been demonstrated
that resistance was the result of overexpression
of an ATP-dependent P-glycoprotein
membrane transporter, capable of extruding
drug from the cell. In addition a range of unrelated
compounds, as exemplified by the calcium-channel
blocker verapamil, were capable
of reversing resistance in vitro through an
undefined interaction with the MDR pump.
Similar experiments in chloroquine-resistant
parasites revealed a similar, although only partial,
resistance reversal by verapamil. It was
suggested that chloroquine resistance was, by
analogy with MDR cancer cells, a drug efflux
phenomenon, but detailed investigations have
comprehensively failed to show enhanced drug
efflux from resistant parasites.
The currently accepted chloroquineresistance
phenotype is one of reduced drug
accumulation associated with reduced drug
susceptibility (IC 50 100 nM) that can be partially
reversed by a range of ‘reversers’ such as
verapamil. In fact a careful analysis of drug sensitivity
data reported in the literature suggest
that three phenotypes can be identified:
1. Fully chloroquine-sensitive parasites, IC 50
20 nM , no verapamil effect
2. Highly chloroquine-resistant parasites,
IC 50 100 nM, verapamil effect
3. Partially chloroquine-resistant, IC 50 20 nM–
100 nM, no verapamil effect or significantly
reduced verapamil effect
Parasite isolates in the intermediate group are
readily selected for high-level resistance with
a greatly enhanced verapamil effect under
drug pressure in vitro, whereas fully sensitive
parasites cannot be selected for high-level
resistance.
The initial studies aimed at elucidating
the molecular basis of chloroquine resistance
focused on the possible involvement of a multidrug
resistance (MDR) P-glycoprotein-based
mechanism. Two MDR homologs were identified
in P. falciparum, pfmdr1 and pfmdr2,
with pfmdr1 looking the most likely MDR
gene. Pgh1, the protein product of pfmdr1,
shares structural similarity with many members
of the MDR family. The protein has two
nucleotide binding sites, twelve transmembrane
domains and has been localized to the
parasite’s digestive food vacuole membrane. It
was originally argued that resistance resulted
from an over-amplification of pfmdr1, by analogy
with MDR cancer cells, but experimental
MEDICAL APPLICATIONS