Karin Seifert

Antiprotozoal activities of phospholipid analogues

The antiprotozoal activity of phospholipid analogues, originally developed as anti-cancer drugs, has been determined in the past decade. The most susceptible parasites are Leishmania spp. and Trypanosoma cruzi with activity also shown against Trypanosoma brucei spp., Entamoeba histolytica and Acanthamoeba spp. Miltefosine, an alkylphosphocholine, was registered for the oral treatment of visceral leishmaniasis (VL) in India in March 2002. This review will focus on the biological activities of phospholipid analogues. Biochemical and molecular targets and mechanism(s) of action have been studied extensively in tumor cells but have not been determined in protozoa.

Characterisation of Leishmania donovani promastigotes resistant to hexadecylphosphocholine (miltefosine)

Leishmania donovani promastigote lines resistant to hexadecylphosphocholine (HePC, miltefosine) at 2.5, 5.0, 10.0, 20.0 and 40.0 microM were developed in vitro by continuous step-wise drug pressure. The 40 microM line was 15 times more resistant to HePC than the wild-type clone and showed cross-resistance to the ether lipid ET-18-OCH3 (edelfosine) but not to the standard anti-leishmanial drugs. Resistance was stable up to 12 weeks in drug-free culture medium. No amplification of specific genes, including the multidrug resistance P-glycoprotein gene, could be detected in the resistant parasites.

In Vitro and In Vivo Interactions between Miltefosine and Other Antileishmanial Drugs

ABSTRACT The interaction of miltefosine with amphotericin B, sodium stibogluconate, paromomycin, and sitamaquine was assessed in vitro and additionally for the first three combinations in vivo. In vitro interactions were indifferent for miltefosine combined with amphotericin B (mean sums of fractional inhibitory concentrations [mean ∑FICs] ranging from 1.22 to 1.51 at the 50% effective concentration [EC50] level and 1.08 to 1.38 at the EC90 level), sitamaquine (mean ∑FICs from 1.33 to 1.38 and 1.0 to 1.02, respectively), and paromomycin (mean ∑FICs from 0.79 to 0.93 at the EC50 and 0.77 to 1.35 at the EC90 level). Some synergy was observed for miltefosine combined with sodium stibogluconate (mean ∑FICs from 0.61 to 0.75 at EC50 and 0.49 to 0.97 at EC90). Different interactions were found in vivo, where the highest potentiation of miltefosine activity was achieved with amphotericin B (activity enhancement index [AEI] of up to 11.3). No significant interaction was observed when miltefosine was combined with sodium stibogluconate (AEI of up to 2.38). The potentiation of miltefosine in vivo was also achieved with the combination of miltefosine and paromomycin (AEI of up to 7.22).

Cytotoxic Activities of Alkylphosphocholines against Clinical Isolates of Acanthamoeba spp.

ABSTRACT Free-living amoebae of the genus Acanthamoeba are causing serious chronic conditions such as destructive keratitis in contact lens wearers or granulomatous amoebic encephalitis in individuals with compromised immune systems. Both are characterized by the lack of availability of sufficiently effective and uncomplicated, manageable treatments. Hexadecylphosphocholine (miltefosine) is licensed for use as a topical antineoplastic agent, but it is also active in vitro against several protozoan parasites, and it was applied very successfully for the treatment of human visceral leishmaniasis. The aim of our study was to evaluate the efficacy of hexadecylphosphocholine and other alkylphosphocholines (APCs) against Acanthamoeba spp. The in vitro activities of eight different APCs against three Acanthamoeba strains of various pathogenicities were determined. All substances showed at least amoebostatic effects, and some of them disrupted the amoebae, as shown by the release of cytoplasmic enzyme activity. Hexadecylphosphocholine exhibited the highest degree of cytotoxicity against trophozoites, resulting in complete cell death at a concentration as low as 40 μM, and also displayed significant cysticidal activity. Hexadecylphosphocholine may be a promising new candidate for the topical treatment of Acanthamoeba keratitis and, conceivably, even for the oral treatment of granulomatous amoebic encephalitis.

In vitro activity of anti-leishmanial drugs against Leishmania donovani is host cell dependent.

OBJECTIVES To evaluate the in vitro activity of anti-leishmanial drugs against intracellular Leishmania donovani amastigotes in different types of macrophages. METHODS Mouse peritoneal macrophages (PEMs), mouse bone marrow-derived macrophages (BMMPhi), human peripheral blood monocyte-derived macrophages (PBM Phi) and differentiated THP-1 cells were infected with L. donovani. Cultures were incubated with sodium stibogluconate, amphotericin B deoxycholate (Fungizone), miltefosine or paromomycin sulphate over six concentrations in 3-fold serial dilutions for 5 days. Analysis was based on percentage inhibition of infected macrophages and EC(50)/EC(90) values estimated using sigmoidal curve-fitting. RESULTS The rank order of drug activity was the same in the different macrophage populations: amphotericin B > miltefosine > sodium stibogluconate > paromomycin. However, significant (P < 0.05) differences were observed between populations. Amphotericin B was more active in PEMs and BMM Phi (EC(50) 0.02-0.06 microM) compared with PBM Phi and differentiated THP-1 cells (EC(50) 0.08-0.40 microM) and miltefosine was more active in PBM Phi (EC(50) 0.16-0.74 microM) compared with PEMs and BMM Phi (EC(50) 2.60-7.67 microM). Sodium stibogluconate displayed highest activity in PBM Phi (EC(50) 1.38-1.89 microg Sb(v)/mL), followed by PEMs (EC(50) 21.75-27.79 microg Sb(v)/mL) and BMM Phi and differentiated THP-1 cells (EC(50) 28.96-112.77 microg Sb(v)/mL). Paromomycin showed highest activity in PBM Phi (EC(50) 80.03-104.38 microM) and PEMs (EC(50) 75.42-201.63 microM). CONCLUSIONS In vitro activity of anti-leishmanial drugs is host cell dependent. This has implications for: (i) the evaluation of in vitro drug activity; (ii) the evaluation of drug susceptibility of clinical isolates; and (iii) the standardization of anti-leishmanial drug assays.

Effects of Miltefosine and Other Alkylphosphocholines on Human Intestinal Parasite Entamoeba histolytica

ABSTRACT The protozoan parasite Entamoeba histolytica is the cause of amoebic dysentery and liver abscess. It is therefore responsible for significant morbidity and mortality in a number of countries. Infections with E. histolytica are treated with nitroimidazoles, primarily with metronidazole. At this time, there is a lack of useful alternative classes of substances for the treatment of invasive amoebiasis. Alkylphosphocholines (alkyl-PCs) such as hexadecyl-PC (miltefosine) were originally developed as antitumor agents, but recently they have been successfully used for the treatment of visceral leishmaniasis in humans. We examined hexadecyl-PC and several other alkyl-PCs with longer alkyl chains, with and without double bond(s), for their activity against two strains of E. histolytica. The compounds with the highest activity were oleyl-PC, octadecyl-PC, and nonadecenyl-PC, with 50% effective concentrations for 48 h of treatment between 15 and 21 μM for strain SFL-3 and between 73 and 98 μM for strain HM-1:IMSS. We also tested liposomal formulations of these alkyl-PCs and miltefosine. The alkyl-PC liposomes showed slightly lower activity, but are expected to be well tolerated. Liposomal formulations of oleyl-PC or closely related alkyl-PCs could be promising candidates for testing as broad-spectrum antiprotozoal and antitumor agents in humans.

Structures, Targets and Recent Approaches in Anti-Leishmanial Drug Discovery and Development

Recent years have seen a significant improvement in available treatment options for leishmaniasis. Two new drugs, miltefosine and paromomycin, have been registered for the treatment of visceral leishmaniasis (VL) in India since 2002. Combination therapy is now explored in clinical trials as a new treatment approach for VL to reduce the length of treatment and potentially prevent selection of resistant parasites. However there is still a need for new drugs due to safety, resistance, stability and cost issues with existing therapies. The search for topical treatments for cutaneous leishmaniasis (CL) is ongoing. This review gives a brief overview of recent developments and approaches in anti-leishmanial drug discovery and development.

N-(2-hydroxypropyl)methacrylamide–amphotericin B (HPMA–AmB) copolymer conjugates as antileishmanial agents

Leishmaniasis is a major health problem in many parts of the world, caused by various species of Leishmania. Amastigotes are the clinically relevant form of the parasite in the human host and reside in the parasitophorous vacuole within macrophages. Polymer–drug conjugates have been used for lysosomotropic drug delivery and have already shown potential in anticancer and antileishmanial chemotherapy. We synthesised N-(2-hydroxypropyl)methacrylamide–amphotericin B (HPMA–AmB) copolymer conjugates in which the AmB was attached to the polymer through a degradable GlyPheLeuGly linker. Antileishmanial activity was assessed in vitro against intracellular amastigotes in host macrophages [murine peritoneal exudate macrophages (PEMs), murine bone marrow-derived macrophages (BMMs) and differentiated THP-1 cells]. The most potent copolymers had 50% effective concentration (EC50) values of 0.03 μg/mL AmB equivalent against Leishmania donovani amastigotes in PEMs and BMMs and an EC50 of 0.57 μg/mL AmB equivalent against L. donovani in THP-1 cells. This activity was comparable with free AmB (EC50 = 0.03–0.07 μg/mL against L. donovani in PEMs and BMMs and 0.24–0.42 μg/mL against amastigotes in THP-1 cells) and Fungizone® (EC50 = 0.04–0.07 μg/mL against amastigotes in PEMs). Conjugates also showed potent in vivo activity with ca. 50% inhibition of parasite burden at 1 mg/kg body weight.

Modular Multiantigen T Cell Epitope–Enriched DNA Vaccine Against Human Leishmaniasis

A DNA vaccine for the induction of cell-mediated immunity against different types of leishmaniasis was developed and successfully tested in human cell culture systems for immunogenicity and an animal model for prophylaxis. Y Not?—A T Cell–Based Leishmania Vaccine Most successful vaccines against human diseases induce neutralizing antibodies—Y-shaped molecules that block toxins or prevent infection. However, some infections, such as leishmaniasis, are not effectively prevented with neutralizing antibodies. Yet, vaccines for these infections are urgently needed. Indeed, current treatment for Leishmania infection is expensive and has severe side effects, and prevention is limited to bed nets and insecticides. Now, Das et al. develop a DNA vaccine against leishmaniasis that induces T cell–based immunity. The authors began by screening for Leishmania antigens that were conserved across different species, endemic regions, and over time. They then confirmed that these antigens were immunogenic with blood from individuals who had been cured of Leishmania infection from different endemic regions. A DNA vaccine consisting of these antigens was protective in a rodent model of infection. This vaccine shows promise if confirmed in human testing, and the strategy of antigen selection is a proof of principle for development of other T cell–based vaccines. The leishmaniases are protozoal diseases that severely affect large populations in tropical and subtropical regions. There are only limited treatment options and preventative measures. Vaccines will be important for prevention, control and elimination of leishmaniasis, and could reduce the transmission and burden of disease in endemic populations. We report the development of a DNA vaccine against leishmaniasis that induced T cell–based immunity and is a candidate for clinical trials. The vaccine antigens were selected as conserved in various Leishmania species, different endemic regions, and over time. They were tested with T cells from individuals cured of leishmaniasis, and shown to be immunogenic and to induce CD4+ and CD8+ T cell responses in genetically diverse human populations of different endemic regions. The vaccine proved protective in a rodent model of infection. Thus, the immunogenicity of candidate vaccine antigens in human populations of endemic regions, as well as proof of principle for induction of specific immune responses and protection against Leishmania infection in mice, provides a viable strategy for T cell vaccine development.

In vitro interactions between sitamaquine and amphotericin B, sodium stibogluconate, miltefosine, paromomycin and pentamidine against Leishmania donovani

OBJECTIVES To evaluate in vitro interactions between sitamaquine and the current antileishmanial drugs amphotericin B, sodium stibogluconate, miltefosine, paromomycin and pentamidine against intracellular Leishmania donovani amastigotes in peritoneal mouse macrophages. A second objective was to evaluate the susceptibility of antimony-resistant L. donovani isolates to sitamaquine. METHODS Mouse peritoneal macrophages were infected with L. donovani amastigotes. Drug susceptibility was assessed in a standard 5 day assay and drug interactions with a modified fixed ratio isobologram method. Fractional inhibitory concentrations (FICs), sum FICs (∑FICs) and an overall mean ∑FIC were calculated for each combination. The nature of interaction was classified on the basis of the mean ∑FIC as follows: synergy as mean ∑FIC≤0.5, indifference as mean ∑FIC between >0.5 and ≤4 and antagonism as mean ∑FIC>4. RESULTS Interactions between sitamaquine and amphotericin B, sodium stibogluconate, paromomycin and miltefosine were classified as indifferent at the 50% and 90% effective concentration (EC50 and EC90, respectively) levels. The sitamaquine/pentamidine combination was synergistic, with overall mean ∑FICs from 0.5 to 0.6 at the EC50 level and from 0.3 to 0.7 at the EC90 level. Sitamaquine displayed in vitro activity against L. donovani isolates resistant to sodium stibogluconate. CONCLUSIONS This study expands the preclinical data on drug combinations and provides the basis for further studies as antileishmanial chemotherapy is moving towards multidrug treatment regimens.