Preliminary Assessment of Intramuscular Depot of Lipid-Based Decoquinate Formulation for Long-Term Chemoprophylaxis of Malaria

Abstract

Sustained-release formulations of decoquinate were evaluated for the long-term prophylaxis of malaria. In the initial experiment, mice were protected from liver-stage Plasmodium infection by intramuscular administration of a lipids-based formulation at a dose of decoquinate 200 mg/kg. The mice that were inoculated with Plasmodium berghei sporozoites 34 days after the administration of a one-time drug dose were continuously monitored for 60 days and shown to be free of Plasmodium parasites. The optimized formulation for the sustained release of decoquinate was prepared by hot melt extrusion, constructed by lipids including cholesterol and mono or diglycerides, and had a drug load of 20 to 40% and particle size of 30 to 50 μm. Decoquinate of the lipids-based formulation was slowly released in vitro at a constant rate for the duration of two months, and was examined and continuously exposed at a therapeutic level in the blood for as long as 4 to 6 months. Further evaluation showed that the lipids-based formulation at doses of decoquinate 100 to 150 mg/kg could protect mice from Plasmodium infection for a period of 120 days. It is the first time that cholesterol has been used for a controlled drug delivery system of decoquinate. The results may provide useful information, not only for preparing a formulation of long-acting decoquinate but also in general for developing a controlled drug release system. The one-time administration of pharmaceutical agents in such a slow-release system may serve patients with no concerns about compliance.

Keywords: Plasmodium infection; cholesterol; decoquinate; sustained release.

Figure 1

In vitro dissolution and in vivo efficacy assessment of sustained-release formulation of decoquinate (SRFD). (A) In vitro release of DQ from the solid form of SRFD. Each data point represents the percentage of cumulative drug released from the solid formulation of DQ, the drug released and accumulated from all previous times. (B) Microscopic examination of parasitemia in mice. The examination was carried out 4 days after the mice were infected by the Plasmodium berghei sporozoites. Lipids-based formulation of DQ was placed 34 days before Plasmodium infection. F1, F2, F3, and F8 were each suspended in saline and given to mice intramuscularly (IM). F4 was injected in solid form (stick) subcutaneously; F3 was also given in solid stick. F9 was prepared and injected as an emulsion. (C) Survival rates. The mice from the experiments shown in (B) were raised for 60 days and survival rates were counted 60 days post-inoculation.

Figure 2

Evaluation of SRFD (F3) in malaria prophylaxis. (A) A schematic depiction of animal experimental design for the data shown in (B–F). (B) F3 at a dose of DQ 200 mg/kg, vehicle control of F3 (no DQ), F10 dissolved in NMP, and primaquine phosphate (PQ) at a dose of 30 mg/kg dissolved in saline, were given to mice by IM. Six weeks later, mice were inoculated with SPZ of Plasmodium berghei ANKA 868 expressing firefly luciferase via the tail vein. Images were acquired by using In Vivo Image Systems (IVIS) 46 h after the inoculation. (C) Counts presented as the flux of photons, corresponding to images in (B) from IVIS detection. (D) Parasitemia detection 6 days after Plasmodium berghei SPZ inoculation. (E) Parasitemia was detected periodically over 60 days after SPZ inoculation. The lines were interrupted at the time when animals were seriously ill and euthanized. Note that one animal in the F3 group was infected but recovered, parasitemia level peaked at day fifteen, then down to zero on day 35, and no recrudescence occurred. (F) The percentage of animals that survived 60 days after SPZ injection.

Figure 3

Physical features of SRFD. (A) X-ray diffraction graph of pure DQ powder; (B) X-ray diffraction graph of F2 made by HME; (C) X-ray diffraction graph of physical mixture of the same components of F2 prior to HME process. The peak heights of DQ from HME extrudates accounted for only a small percentage of the peaks of pure DQ.

Figure 4

Duration of malaria prophylaxis provided by SRFD in mice with Plasmodium infection. HME extrudates (F2) were suspended in saline and given to separate groups of mice (C57) at a dose of DQ 200 mg/kg by IM. After SRFD replacement, mice were, respectively, inoculated with Plasmodium berghei SPZ at different time lengths. New mice for saline and vehicle controls were replaced and matched with experimental mice in each group. (A) Images shown are from experimental groups with the SPZ inoculation on days 90, 120, 150, and 180. Images from the early time points before 90 days are not shown. Mice were monitored by IVIS 24, 48, and 72 h after SPZ injection. At 24 and 48 h, the signals if any were localized in the abdominal area (liver) and then spread to the whole body at 72 h or after 48 h. (B) Parallel experiments were performed in the same kind of animals (C57) as the mice in (A). The same SRFD (F2) was administered at a dose of DQ 200 mg/kg by IM to the mice. Blood samples were drawn at different days by cardiac puncture and the blood concentrations of DQ were measured by LC-MS. (C) Mean particle size of HME extrudates (F2).

Figure 5

In vivo efficacy validation of effective dose of DQ in SRFD. SRFD (F2) with different doses of DQ were placed one month before P. berghei 868 SPZ inoculation. (A) Parasitemia was detected by examining blood smears from mice 6 days after the SPZ inoculation. (B) Percent survival rates of animals 75 days after the SPZ inoculation.