HPLC detection of miltefosine using an evaporative light scattering detector.

Miltefosine has recently been introduced as leishmanicidal drug for oral administration (Impavido). Previous communications report about the use of liquid chromatography coupled with mass spectrometry detection to detect miltefosine in pharmaceutical preparations and biological fluids. We report about a new method to detect miltefosine using an evaporative light scattering detector (ELSD). The absolute recovery of the analyte was greater than 98.0%. The limit of quantification for miltefosine in plasma at a signal-to-noise ratio of 7.3 was 0.34 microg/ml. The precision of the assay yielded coefficients of variation ranging from 1.8 to 4.5% and an accuracy of 97-107%. Our method advances the qualitative and quantitative detection of miltefosine by combining rapid and efficient solid phase extraction and analysis with an evaporative light-scattering detector.

Amphotericin B.

Invasive fungal infections are a major cause of morbidity and mortality in immunodeficient individuals (such as AIDS patients) and in transplant recipients or tumor patients undergoing immunosuppressive chemotherapy. Amphotericin B is one of the oldest, yet most efficient antimycotic agents. However, its usefulness is limited due to dose-dependent side-effects, notably nephrotoxicity. In order to improve its safety margin, new pharmaceutical formulations of amphotericin B have been designed especially to reduce its detrimental effects on the kidneys. Since the 1980s, a wide variety of new amphotericin B formulations have been brought forward for clinical testing, many of which were approved and reached market value in the 1990s. This review describes and discusses the molecular genetics, pharmacological, toxicological, and clinical aspects of amphotericin B itself and many of its innovative formulations.

The impact of nanobiotechnology on the development of new drug delivery systems.

Nanotechnology, or systems/devices manufactured at the molecular level, is a multidisciplinary scientific field undergoing explosive development. A part of this field is the development of nanoscaled drug delivery devices. Nanoparticles have been developed as an important strategy to deliver conventional drugs, recombinant proteins, vaccines and more recently nucleotides. Nanoparticles and other colloidal drug delivery systems modify the kinetics, body distribution and drug release of an associated drug. Other effects are tissue or cell specific targeting of drugs and the reduction of unwanted side effects by a controlled release. Therefore nanoparticles in the pharmaceutical biotechnology sector improve the therapeutic index and provide solutions for future delivery problems for new classes of so called biotech drugs including recombinant proteins and oligonucleotides. This review discusses nanoparticular drug carrier systems with the exception of liposomes used today, and what the potential and limitations of nanoparticles in the field of pharmaceutical biotechnology are.

Solid lipid nanoparticles for parenteral drug delivery.

This review describes the use of nanoparticles based on solid lipids for the parenteral application of drugs. Firstly, different types of nanoparticles based on solid lipids such as "solid lipid nanoparticles" (SLN), "nanostructured lipid carriers" (NLC) and "lipid drug conjugate" (LDC) nanoparticles are introduced and structural differences are pointed out. Different production methods including the suitability for large scale production are described. Stability issues and drug incorporation mechanisms into the particles are discussed. In the second part, the biological activity of parenterally applied SLN and biopharmaceutical aspects such as pharmacokinetic profiles as well as toxicity aspects are reviewed.

Natural products as antiparasitic drugs.

Natural products are not only the basis for traditional or ethnic medicine. Only recently, they have provided highly successful new drugs such as Artemisinin. Furthermore, screening natural products found in all sorts of environments such as the deep sea, rain forests and hot springs, and produced by all sorts of organisms ranging from bacteria, fungi and plants to protozoa, sponges and invertebrates, is a highly competitive field where all of the major pharmaceutical companies are encountered. Already, many new natural product groups have revealed antiparasitic properties of surprising efficacy and selectivity, as will be shown in this review for plant-derived alkaloids, terpenes and phenolics. Many novel lead structures, however, have severe chemico-physical drawbacks such as poor solubility. Here, innovative drug formulations and carrier systems might help, as discussed by the authors in another article of this series.

Formulation and biopharmaceutical issues in the development of drug delivery systems for antiparasitic drugs.

The development of really new antiparasitic drugs to market level is a very rare event. A large number of lead structures have already been screened and discarded, the market is large but poor, and the administrative barriers are increasingly high and costly. Novel antiparasitics must not only be better, they must also be substantially safer than the existing repertoire. There are two major aspects to drug development. One is the strategy of pathogen-specific biochemical intervention, the other the strategy of optimal formulation and application. This review focuses on the latter. In finding and adapting innovative and "intelligent", i.e. parasite- and disease-specific formulations and delivery systems, established but deficient drugs might be optimised, enhancing their efficiency and reducing negative side effects at relatively low cost. Further, many promising new ideas are severely hampered by the low water solubility of the antiparasitic drug. Here as well, some of the innovative drug formulation and delivery systems discussed below might offer highly efficient, while technologically simple, solutions.

Formulation of amphotericin B as nanosuspension for oral administration.

Amphotherin B was formulated in a nanosuspension as a new oral drug delivery system for the treatment of experimental visceral leishmaniasis. Amphotericin B (AmB) nanosuspensions were produced by high pressure homogenisation obtaining particles with a PCS diameter of 528 nm. Environmental stability was determined in artificial gastrointestinal fluids at different pH and electrolyte concentrations. In vivo efficacy was determined in a mouse model of visceral leishmaniasis. Following oral administration (5 mg kg(-1)), micronised amphotericin B did not show any curative effect. However, administrations of amphotericin B nanosuspension, reduced liver parasite load by 28.6% compared to untreated controls.

Antileishmanial activity of hydrolyzable tannins and their modulatory effects on nitric oxide and tumour necrosis factor-alpha release in macrophages in vitro.

A series of 27 hydrolyzable tannins and related compounds was tested for antiparasitic effects against both extracellular promastigote and intracellular amastigote Leishmania donovani organisms. In parallel, the compounds were evaluated for their immunomodulatory effects on macrophage functions, including release of nitric oxide (NO), tumour necrosis factor-alpha (TNF-alpha) and interferon (IFN)-like properties using several functional assays. Of the series of polyphenols tested, only gallic acid (54 microM NO) and its methyl ester (32 microM NO) induced murine macrophage-like RAW 264.7 cells to release NO in appreciable amounts (IFN-gamma/LPS 119 microM NO). The in vitro TNF-inducing potential of the polyphenols examined increased in the order of oligomeric ellagitannins (EC(50) > 25 microg/ml) < monomeric ellagitannins, gallotannins (EC(50) 8.5 to > 25 microg/ml) < C-glucosidic ellagitannins, dehydroellagitannins (EC(50) 0.6 - 2.8 microg/ml) at the host cell subtoxic concentration of 50 microg/ml. Furthermore, promastigotes of Leishmania donovani were assayed in the presence of these polyphenols and the results showed that none of the compounds was significantly toxic (EC(50) > 25 microg/ml) to the extracellular forms. In contrast, all polyphenols showed pronounced antileishmanial activities (EC(50) < 0.4 - 12.5 versus 7.9 microg/ml for Pentostam) against intracellular amastigotes of L. donovani residing within RAW cells. Noteworthy, most compounds exhibited low cytotoxicity against the murine host cells (EC(50) >25 microg/ml). Furthermore, some ellagitannins and the majority of dehydroellagitannins induced potent interferon-like activities as reflected by inhibition of the cytopathic effect of encephalomyocarditis virus on fibroblast L929 cells. This is the first report on hydrolyzable tannins as a new class of natural products with leishmanicidal activity including their potential for inducing the release of NO, TNF and IFN-like activity in macrophage-like RAW cells.

In vitro activity of aurones against Plasmodium falciparum strains K1 and NF54.

A series of naturally occurring aurones (e. g., Rubiaceae, Cyperaceae) was synthesized and tested for the ability to inhibit erythrocytic stages of Plasmodium falciparum strains in vitro. Some of the these compounds exhibited antiplasmodial activity in the micromol range, determined as fifty percent-inhibitory concentrations (IC(50)). Drug activity was not associated with cytotoxicity for the mammalian tumor cell lines KB and SKMel (IC(50) > 3.0 microM). The most active compound was 4,6,4'-triacetyl-3',5'-dimethoxy-2-aurone with IC(50) values of 0.007 microM and 0.18 microM for the P. falciparum strains K1 and NF54, respectively. Interestingly, the multiple drug-resistant P. falciparum strain K1 was more sensitive to tested aurones than the drug-susceptible strain NF54.

Evaluation of in vitro activity of aurones and related compounds against Cryptosporidium parvum.

The efficacy of a series of aurones, auronols and 4-methoxy-alpha-pyrones has been screened for the ability to inhibit the intracellular growth of the parasitic protist Cryptosporidium parvum using an in vitro enzyme linked immunosorbent assay (ELISA). All aurones of this series were active at 25 to 100 microM. 10 of 19 aurones inhibited the intracellular growth of C. parvum by > 90 % with moderate to no toxicity. The most active of these was 3',4',6-trihydroxy-2-[phenylmethylene]-3(2H)-benzofuranone.

Proanthocyanidins and related compounds: antileishmanial activity and modulatory effects on nitric oxide and tumor necrosis factor-alpha-release in the murine macrophage-like cell line RAW 264.7.

A series of 17 proanthocyanidins and structurally related compounds was tested for activity against Leishmania donovani amastigotes and promastigotes in vitro. Most of the polyphenols significantly inhibited the intracellular survival of L. donovani amastigotes (EC50 0.8-10.6 nM) when compared with the antileishmanial drug Pentostam (EC50 10.6 nM), but all were inactive against the extracellular form (EC50 7.8 to >86 nM). Noteworthy is that all compounds exhibited only moderate or no cytotoxicity against the murine host cells (EC50 7.8 to >56 nM; >25 microg/ml). These polyphenols were further evaluated for immunomodulatory effects on macrophage functions, including release of nitric oxide (NO), tumor necrosis factor-alpha (TNF) and interferon (IFN)-like properties using several functional assays. The results showed that all compounds induced murine RAW 264.7 cells only moderately to release NO (7-26 microM) relative to the reference stimulus IFN-gamma/LPS (119 microM). The TNF-inducing potential of the polyphenols producing 50% lysis in murine L929 cells ranged from absent to 138 U/ml at the host cell subtoxic concentration of 50 microg/ml. The highest TNF-inducing activity was associated with those flavan-3-ols with galloyl groups (98-127 U/ml). For proanthocyanidins, it appeared that an increase in the flavanyl chain length did not enhance the induction of TNF-release (32-86 U/ml and below detection limits for oligomers and polymers, respectively). With interferon-like activities, phylloflavan and a prodelphinidin polymer showed appreciable cytoprotective effects, as reflected by the inhibition of the cytopathic effect of encephalomyocarditis virus on L929 fibroblast cells (38 and 36 U/ml, respectively). All remaining compounds displayed only negligible or moderate protective effects at subtoxic concentrations up to 25 microg/ml (<5 to 12 U/ml). These results indicate that proanthocyanidins and related compounds have favorable antileishmanial activity in vitro and might be considered as beneficial immunological response modifiers provided there are no bioavailability problems.

Tannins and related compounds: killing of amastigotes of Leishmania donovani and release of nitric oxide and tumour necrosis factor alpha in macrophages in vitro.

The antileishmanial and immunomodulatory potencies of a series of 28 polyphenols were evaluated in terms of extra- and intracellular leishmanicidal activity and macrophage activation for release of nitric oxide (NO), tumour necrosis factor (TNF) and interferon (IFN)-like properties. For this, several functional bioassays were employed including an in vitro model for leishmaniasis in which murine bone marrow-derived macrophages (BMMphi) were infected with the obligate intracellular parasite Leishmania donovani, an extracellular Leishmania proliferation assay, a fibroblast-lysis assay (TNF-activity), and a biochemical assay for NO. Except for gallic acid, its methyl ester, shikimic acid and catechin (EC50 25.8-67.9 nM) all polyphenols tested significantly inhibited the intracellular survival of L. donovani amastigotes (EC50 0.4-13.9 nM) when compared with the clinically used agent, sodium stibogluconate (EC50 10.6 nM). In contrast, none of the samples proved to be directly toxic for the extracellular promastigote form of the parasite. Noteworthy, the phenolic samples showed only moderate or no cytotoxicity against the murine host cells (EC50 10 to >144 nM). Although NO is an important effector molecule in macrophage microbicidal activity, the inducing potential of the test compounds for its release was found to be very moderate ranging from 7-54 microM (IFN-gamma/LPS 119 microM). On the other hand, inhibition of NO production had no apparent effect on intracellular leishmanicidal activity of polyphenols. Their in vitro TNF-inducing potential producing 50% lysis in murine L929 cells increased in the order of simple phenols and flavanols (34-48 U/ml) < A-type proanthocyanidins (53-80 U/ml) < B-type proanthocyanidins (64-200 U/ml) < hydrolyzable tannins (287-350 U/ml) at the host cell subtoxic concentration of 50 microg/ml. Furthermore, gallic acid and some hydrolyzable tannins showed appreciable IFN-like activities (14-23 U/ml) as reflected by inhibition of the cytopathic effect of encephalomyocarditis virus on fibroblast L 929 cells. The results provide a rational basis for the recorded anti-infectious efficacy of traditionally used herbal medicines containing tannins in vivo, in the light of both only moderate direct antimicrobial activities of distinct polyphenols in vitro and the limited knowledge on their uptake in humans.

Atovaquone nanosuspensions show excellent therapeutic effect in a new murine model of reactivated toxoplasmosis.

Immunocompromised patients are at risk of developing toxoplasma encephalitis (TE). Standard therapy regimens (including sulfadiazine plus pyrimethamine) are hampered by severe side effects. While atovaquone has potent in vitro activity against Toxoplasma gondii, it is poorly absorbed after oral administration and shows poor therapeutic efficacy against TE. To overcome the low absorption of atovaquone, we prepared atovaquone nanosuspensions (ANSs) for intravenous (i.v.) administration. At concentrations higher than 1.0 microg/ml, ANS did not exert cytotoxicity and was as effective as free atovaquone (i.e., atovaquone suspended in medium) against T. gondii in freshly isolated peritoneal macrophages. In a new murine model of TE that closely mimics reactivated toxoplasmosis in immunocompromised hosts, using mice with a targeted mutation in the gene encoding the interferon consensus sequence binding protein, i.v.-administered ANS doses of 10.0 mg/kg of body weight protected the animals against development of TE and death. Atovaquone was detectable in the sera, brains, livers, and lungs of mice by high-performance liquid chromatography. Development of TE and mortality in mice treated with 1.0- or 0.1-mg/kg i.v. doses of ANS did not differ from that in mice treated orally with 100 mg of atovaquone/kg. In conclusion, i.v. ANSs may prove to be an effective treatment alternative for patients with TE.

Production and characterisation of mucoadhesive nanosuspensions for the formulation of bupravaquone.

Bupravaquone is a new naphthoquinone antibiotic against Cryptosporidium parvum and other parasites. It has attracted interest for the treatment of C. parvum infections, because of the lack of a drug in the treatment of mostly AIDS patients. The bioavailability of bupravaquone is limited when given orally. To overcome the problem of the high elimination rate caused by diarrhoea, typical for C. parvum infections, bupravaquone was formulated as a mucoadhesive nanosuspension, i.e. combining the properties of mucoadhesive drug delivery systems, in this case hydro gels, with nanosuspensions. In this study different polymers/hydro gels were employed to create a prolonged retention time for the drug in the infected gastrointestinal tract (GIT). The second step to improve the bioavailability of bupravaquone was the formulation as nanosuspension. Therefore various concentrations of bupravaquone with different surfactants were tested. The production of these nanosuspensions was carried out by high pressure homogenisation. In addition to the classical stepwise production, about a new one step production method is described.

A new approach for targeting to Cryptosporidium parvum using mucoadhesive nanosuspensions: research and applications.

A new strategy to deliver antibiotics to the Cryptosporidium-infected gastrointestinal tract is presented. In an effort to augment the anticryptosporidial effect of clinically used drugs, mucoadhesive nanosuspensions were prepared. They have the ability to reside in the gastrointestinal tract for an extended period. The hydrogel contained bupravaquone nanosuspensions and an adhesive polymer (chitosan) powder dispersed in water. By the development of mucoadhesive nanosuspensions, a potential drug delivery system for poorly soluble drugs has been investigated to overcome bioavailability problems caused by the pathophysiological diarrhoeic situation in patients suffering from cryptosporidiosis. Adapting drug delivery systems to the situation of Cryptosporidium parvum infections in man allows increased retention times with a prolonged action at reduced elimination in the gastrointestinal tract. In this communication, in vivo data are presented to document the efficiency of bupravaquone formulated as mucoadhesive polymers to improve its activity against C. parvum.

The role of plasma proteins in brain targeting: species dependent protein adsorption patterns on brain-specific lipid drug conjugate (LDC) nanoparticles.

The in vivo organ distribution of particulate drug carriers is decisively influenced by the interaction with plasma proteins after i.v. administration. Serum protein adsorption on lipid drug conjugate nanoparticles, a new carrier system for i.v. application, was investigated by 2-dimensional electrophoresis (2-DE). The particles were surface-modified to target them to the brain. To assess the protein adsorption pattern after i.v. injection in mice prior to in vivo studies, the particles were incubated in mouse serum. Incubation in human serum was carried out in parallel to investigate similarities or differences in the protein patterns obtained from men and mice. Distinct differences were found. Particles incubated in human serum showed preferential adsorption of apolipoproteins A-I, A-IV and E. Previously, preferential adsorption of ApoE was reported as one important factor for targeting of Tween(R)80 modified polybutylcyanoacrylate nanoparticles to the brain. Preferential adsorption of ApoA-I and A-IV took place after incubation in mouse serum, adsorption of ApoE could not be clearly confirmed. In vivo localization of the LDC nanoparticles at the blood-brain barrier and diffusion of the marker Nile Red into the brain could be shown by confocal laser-scanning microscopy. Differences of the obtained adsorption patterns are discussed with regard to their relevance for correlations of in vitro and in vivo data obtained from different species.

Immunomodulatory principles of Pelargonium sidoides.

Extracts and isolated constituents (coumarins and phenols) of Pelargonium sidoides DC, a plant species used in folk medicine by the Southern African native population, were evaluated for their effects on nonspecific immune functions. Although this herbal medicine is also successfully employed in modern phytotherapy in Europe to cure infectious diseases of the respiratory tract, the scientific basis of its remedial effects is still unclear. Thus, functional bioassays including an in vitro model for intracellular infection with Leishmania parasites, an extracellular Leishmania growth assay, a fibroblast-virus protection assay (IFN activity), a fibroblast-lysis assay (TNF activity) and a biochemical assay for inorganic nitric oxides (iNO) were employed. None of the test samples revealed significant activity against extracellular, promastigote Leishmania donovani, the causative agent of human visceral leishmaniasis. In contrast, apart from the coumarin samples, all the Pelargonium extracts (EC(50) <0.1-3.3 microg/mL), gallic acid (EC(50) 4.4 microg/mL) and its methyl ester (EC(50) 12.5 microg/mL) significantly reduced the intracellular survival of L. donovani amastigotes within murine macrophages. These data indicate that the samples acted indirectly on Leishmania parasites, possibly by activating leishmanicidal macrophage functions. Macrophage activation was confirmed by detection of tumour necrosis factor (TNF-alpha) and inorganic nitric oxides (iNO) in supernatants of sample-treated macrophage cultures. Synthesis of iNO is a well-known effector mechanism of macrophages against microorganisms such as Leishmania. Interestingly, blocking iNO-synthase with L-NMMA had no substantial effect on sample-induced intracellular Leishmania kill. From bioassay-guided fractionation, gallic acid and its methyl ester present in large amounts in P. sidoides and in its active extracts, were identified as the prominent immunomodulatory principle for this herbal medicine. The results, when taken together with recent reported antibacterial activity, provide a rational basis for both the traditional and the present utilization of P. sidoides in the claimed conditions.

In vitro leishmanicidal activity of naturally occurring chalcones.

A variety of chalcones have been shown to exhibit activity against Leishmania parasites. In contrast to synthetic or semisynthetic chalcones, only a few plant-derived compounds have been investigated. To provide a scientific rational for the antiprotozoal potency of plants used in ethnomedicine and containing chalcones, and in the search for new antiprotozoal drugs, we have carried out a primary screening for in vitro leishmanicidal activity of 20 chalcones isolated from plants. The compounds were tested against extracellular promastigotes of Leishmania donovani, L. infantum, L. enrietii and L. major, and against intracellular amastigote L. donovani residing within murine macrophages. Against the extracellular Leishmania (L. donovani), most compounds were active with EC(50) values between 0.07 and 2.01 microg/mL. Some of these chalcones, 2',4'-dihydroxy-4-methoxychalcone, 2'-hydroxy-3,4-dimethoxychalcone and 2-hydroxy-4,4'-dimethoxychalcone also significantly inhibited the intracellular survival of L. donovani parasites with EC(50) values between 0.39 and 0.41 microg/mL. When tested against murine bone marrow-derived macrophages as a mammalian host cell control, all compounds with antileishmanial activities also proved to be cytotoxic to varying extents (EC(50) 0.19-2.06 microg/mL). Correlations between molecular structures and antileishmanial activity are discussed in detail. Specific compounds are illustrated with emphasis on their mode of action and potential for the development of selective antiprotozoal agents.

Nanosuspensions as particulate drug formulations in therapy. Rationale for development and what we can expect for the future.

An increasing number of newly developed drugs are poorly soluble; in many cases drugs are poorly soluble in both aqueous and organic media excluding the traditional approaches of overcoming such solubility factors and resulting in bioavailability problems. An alternative and promising approach is the production of drug nanoparticles (i.e. nanosuspensions) to overcome these problems. The major advantages of this technology are its general applicability to most drugs and its simplicity. In this article, the production of nanoparticles on a laboratory scale is presented, special features such as increased saturation solubility and dissolution velocity are discussed, and special applications are highlighted, for example, mucoadhesive nanosuspensions for oral delivery and surface-modified drug nanoparticles for site-specific delivery to the brain. The possibilities of large scale production -- the prerequisite for the introduction of a delivery system to the market -- are also discussed.