A bioassay based on disk diffusion technique was applied for determination of fosmidomycin in plasma and urine as the drug is the only bioactive compound. The analysis of plasma concentrations of clindamycin was performed by HPLC since bioassay lacks ability to discriminate between clindamycin and its active metabolites (
N-desmethyl clindamycin and clindamycin sulphoxide) [
18‐
20]. Fosmidomycin and clindamycin were well absorbed orally, but bioavailability of fosmidomycin may not be complete which may be due to its high water solubility property [
21]. The bioavailability of fosmidomycin and clindamycin have been reported to be 30 and 72%, respectively [
18,
19,
22]. Pharmacokinetics of fosmidomycin and clindamycin following multiple oral dosing either as mono- or combination therapy in patients with acute uncomplicated falciparum malaria observed in this study are comparable with those reported in healthy subjects following a single dose of 500 mg or multiple doses of 250 or 500 mg given every 6 hours [
21,
23,
24]. In single dose studies, mean peak plasma concentrations of 2.45 μg/ml were attained following doses of 500 mg. In repeated dose studies in which fosmidomycin was administered every 6 hours for periods of 5 to 7 days in doses of 250, 500 mg and 1 g, steady-state concentrations of 2.45, 4.01 and 5.0 μg/ml, respectively were achieved after 24 hr. Plasma concentration-time profiles of both drugs were best fitted with a one-compartment open model with first-order absorption and elimination with absorption lag time. It is noted for a marked variability in fluctuation of C
max-ss and C
min-ss of fosmidomycin and clindamycin following multiple dosing regimens. The trough/peak plasma concentrations after both dosage regimens increased up to the second or third dose and remained at steady-state thereafter. Fosmidomycin was not metabolised and excreted unchanged in urine as a bioactive substance. The urinary recovery expressed as percent of the administered doses were relatively low (18 and 20% following mono- and combination therapy) comparing to those reported in healthy subjects (26%) [
24,
25]. This extent of urinary excretion supports the low bioavailability of the drug after oral dose administration. Elimination of both fosmidomycin and clindamycin were not affected by dosing, which suggests no accumulation and dose linearity. This was supported by the consistency of C
max-ss and C
av-ss values, the unchanged elimination half-lives after the first and last dose, and the constant urinary recovery during 0–7 days of dosing. In a previous study, saturated absorption of fosmidomycin after multiple dosing was suggested as there were a progressive decline in total urinary recovery from 35.9 to 22.4% during multiple dosing [
21].
The non-compartment pharmacokinetic analysis of fosmidomycin monotherapy was investigated during the acute phase of malaria infection on the first day whereas its kinetics following combination therapy with clindamycin was investigated following the last dose on day 7. Data suggest that malaria infection may not have significant influence on the pharmacokinetics of both fosmidomycin and clindamycin. Although amount of fosmidomycin excreted in urine appears to be relatively low in patients with malaria compared with healthy subjects, overall pharmacokinetic profiles were similar. The pharmacokinetics of fosmidomycin when given alone or with clindamycin were generally comparable except for the significant paralleled reduction of V
z/F and CL/F when clindamycin doses were added, while t
1/2z remained unchanged. It is not clear whether these changes are contributed mainly by malaria infection or pharmacokinetic interaction with clindamycin or both. Reduction of renal clearance of fosmidomycin when clindamycin was given concurrently may account partly for this pharmacokinetic difference. Fosmidomycin is only eliminated by renal clearance while clindamycin is metabolised by CYP3A in liver to clindamycin sulphoxide and
N-desmethyl clindamycin [
25]. Plasma protein binding of fosmidomycin in human is about 1% but clindamycin binds extensively about 90% to plasma protein, mainly to α
1-acid glycoprotein [
19‐
23]. Therefore, the interaction if actually occurred, cannot be explained at the level of hepatic metabolism or plasma protein binding displacement.
Fosmidomycin monotherapy was shown safe and effective blood schizonticide in the initial clearance of asexual parasitaemia. However, the high rate of recrudescence precludes its use in monotherapy [
6,
10] Monotherapy with fosmidomycin was investigated in a pilot study for 1,200 mg every eight hours for seven days with cure rate of 100% (10/10). When the duration was reduced to five, four and three days, cure rates decreased to 89, 88 and 60%, respectively. These observations signify the importance of optimal fosmidomycin dose regimens in the treatment of malaria. In the present study, the cure rate of 22% was observed with monotherapy with fosmidomycin at a dose of 1,200 mg administered orally every eight hours for seven days in a total of 15 Thai patients [
10]. The cure rate with the same dose regimen was, however, 100% in Gambian patients [
10]. MIC (minimum inhibitory concentration) of fosmidomycin for the treatment of multidrug resistant falciparum malaria remains to be determined but is expected to be higher than that in Gambian isolates. The IC
50 value for recombinant
P. falciparum DOXP reductoisomerase is approximately 40 nM and varied from 300 to 1,200 nM in
P. falciparum in vitro without obvious cross-resistance with other antimalarials [
7,
26]. Plasma concentrations in two patients with successful treatment following monotherapy were as high as 1.24 and 2.1 μg/ml, which are considered adequate for radical cure. It was noted that some patients among the 10 cases had recrudescence despite plasma concentrations markedly greater than these levels (4.52 μg/ml). This suggests intrinsically low sensitivity of some parasite strains in Thailand to fosmidomycin and that monotherapy of the drug may not be effective for treatment of multidrug resistant falciparum malaria. When fosmidomycin was given with clindamycin (900 mg fosmidomycin in combination with clindamycin at 600 mg administered orally every 12 hours for 7 days) on the other hand, radical cure was achieved in all patients even with the trough concentrations of as low as 0.2 μg/ml. This could be explained by pharmacodynamic synergistic interaction between fosmidomycin and clindamycin on parasite enzyme DOXP reductoisomerase. Clindamycin targets the prokaryote-like ribosomes of the apicoplast and by this means inhibits self-replication of the organelle [
27,
28]. As a consequence of this mechanism, the drug displays a typical delayed kill kinetic effect, the growth of parasites being unaffected until the second replication after drug exposure. Under such conditions, the
in vitro growth of
P. falciparum is inhibited with an IC
50 and IC
90 of approximately 25 and 50 nM, respectively. As a consequence of its high activity but slow onset of activity, clindamycin is recommended for the treatment of asymptomatic malaria or in combination with other antimalarials. It has been suggested that time-dependent antimicrobial agents should remain above the MIC of pathogens for at least three malaria cycle of seven days. Unlike fosmidomycin, recent studies have suggested that clindamycin displays concentration-dependent bacteriocidal activity [
17,
29] while exhibiting an
in vitro post antibiotic effect (PAE) for certain period.
In vivo, the PAE is generally longer and due to effects such as post antibiotic leukocyte enhancement and post-antibiotic sub-MIC effect. Frequent dosing interval of clindamycin regimen is therefore not necessary. The recommended oral dosing of clindamycin for bacterial infection was 600 mg every 6 hr or 900 mg every 8 hr. However, it was later shown that the pharmacokinetics and concentration-time profiles were comparable with the dosing regimen 1,200 mg every 12 hr which is more practical for clinical application. This may explain the sustained antimalarial efficacy when clindamycin was used with fosmidomycin despite low minimum plasma concentrations of fosmidomycin at steady-state. Whether the antimalarial activity of clindamycin bioactive metabolites should also be considered when performing a pharmacokinetic/pharmacodynamic evaluation has not yet been elucidated. In human, the formation of
N-desmethyl clindamycin and its excretion in urine and faece have been confirmed [
25] but the metabolite could not be detected in the plasma of patients [
30]. The currently suggested dose regimens are based on parent compound concentrations only and the presence of metabolite activity (if any) would only amplify the positive therapeutic outcome. Further study is required for dose optimization of the appropriate ratio of fosmidomycin and clindamycin combination including dosing interval with shorter course of at least 3 days that is safe and effective (produces 100% cure) for treatment of multidrug resistant falciparum.