recrudescence of parasitaemia 18 days later (6). In a later study conducted during 1991, the in vitro parasite susceptibility to HAL was determined in 76 isolates ...
'
INFORMAL CONSUL'TATION ON AhlTlMALARlAL DRUG POLICIES: DATA REQUIREMENTS, TREATMENT OF UNCOMPLICATED MALARIA, A N D PROPHYLAXIS IN PREGNANCY '14-18 M A R C H 1994
R O O M M 505 WHO HEADQUARTERS GENEVA .
W O R K I N G PAPER 1 0
I N VlTRO ASSESSMENT OF THE SEJSCEYT15II-ITY OF P. FAPCIPARUM T O FiALOFANTRINE, ARTEMIS! N IN A N D DERIVATIVES, AND THE TETRACYCLINES
by li.t i . I16ng/ml).
A recent study conducted in the Congo i s of special interest because RIIIRIII levels of resistance were reported in 221128 patients treated with HAL (10). Plasma HAL concentrations were in the range of 70-350 n d m l on day 3. In vitro studies in a distinct group of 1 1 patients from the same area showed that 6 of them had IC50 levels between 10-23ng/ml. All these studies, considered together with other studies in Nigeria (1 1) and South Africa (12), suggest that the IC50 threshold for HAL resistance, using the semi-microtest, i s higher than the cut-off point of 2-4ng/ml referred to by some authors (10,13). In fact, i t may be more of the order of 10-2Ong/ml. The threshold for MIC values using the W H O microtest will probably be of the order of 20-30ng/ml. However, this can only be determined following the completion of further comparative in vitro-in vivo studies. .
.
In addition to determining the parasite susceptibility to HAL, i t may be appropriate to determine the parasite susceptibility to desbutylhalofantrine (BHAL), i t s principal metabolite. BHAL tends to persist in the blood of treated Melanesian and Thai patients at higher concentrations than i t s parent compound (14). Although its antimalarial activity i s at present comparable to that of HAL, this may change under the influence of differential drug pressures: by HAL and BHAL.
'Conclusions and recommendations 1.
The WHO microtest, using inhibition of schizont maturation as the end point, can be used to determine the parasite susceptibility to HAL.
2.
If culture medium can be supplemented with 5-10% non-immune human serum, incubation can be extended from 24/30 hours to 48 hours and inhibition of parasite re-invasion of erythrocytes can be used as the endpoint.
3.
During further clinical and field trials with HAL, the parasite susceptibility to HAL and, possibly, BHAL could be determined before treatment and during any recrudescence of parasitaemia. In addition, whole blood and serum concentrations of HAL and BHAL should be determined 6 or 12 h. after the last dose and whenever a recrudescence occurs within 21 days of treatment.
Parasite susceptibility to arternisinin and its derivatives
Parasites show varying degrees of susceptibility to artemisinin and its derivatives. Studies with a limited number of well-defined strains show IC50 values for artemisinin varying between 1-30ng/ml (3-85nM). In general, parasites are more susceptible to dihydroartemisinin, the putative metabolite of artemisinin and many of its derivatives. They are also more susceptible to 2 of the main artemisinin derivatives, artemether and artesunate. As an example, the comparative susceptibility of the CQ-resistant K1 and the CQ-sensitive FC27 strains to a number of drugs are shown below.
Susceptibility of the K 1 and FC27 strains of P. falciparurn to arternisinin, dihydroarternisinin and artelinic acid (ng/rnl)
K1 strain
Drug
FC27 strain
lC50
MIC
IC50
MIC
Artemisinin
1.9
4.3
4.9
8.1
Artesunate
0.8
1.7
1.7
2.9
Artemether
0.5
1.2
1.3
2.5
Artejinic acid Dihydroartemisinin
3.7
7.5
16.0
0.4
0.8
0.7
27.7 1.2
10% serum, 2% red cells and 0.5% parasitized cells
In assessing parasite susceptibility to artemisinin and its derivatives, it is important to take into consideration the fact that erythrocytes and, especially parasitized erythrocytes, concentrate these drugs from the medium. This means that there is a progressive increase in M I C and IC50 values with rising erythrocyte concentrations. For example, an MIC value (K1 strain) for artemisinin of 4.3ng/ml at 2% haematocri: rises to 12.0 ng/ml at 8%. The rise in MIC values w i t h increasing erythrocyte concentrations i s even more pronounced with dihydroartemisinin but is less marked with artelinic acid.
MIC values are also affected by serum concentrations and parasite densities, although to a lesser extent than by varying erythrocyte concentrations. For example, the MIC of the K1 strain for dihydroartemisinin increases from 0.8 to 1.3ng/ml when the serum concentration i s increased from 10% to 50% and i s about doubled when the percentage of parasitized erythrocytes is increased from 0.2% to 2.0%. In the first larger-scale study on the in vitro susceptibility of African isolates of
P. falciparum to artemisinin, Doury et a1 (8) reported IC50 values ranging from 0.3-24 ng/ml (1.1-85nM). The lC5O value was below 7.3 ng/ml (26nM) in 68 isolates and above 9.0 ng/ml (32nM) in 11 isolates. Based on the reference provided by the authors, it is presumed that the tests were performed using 5% erythrocyte suspensions. As far as I am aware, similar studies to assess parasite susceptibilities to di hydroartemisinin, artemether and artesunate have not yet been
