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.

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.

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.

In vitro leishmanicidal activity of monomeric and dimeric naphthoquinones.

A series of monomeric and dimeric naphthoquinones with potential for treatment of Leishmania infections was identified in vitro using both a direct cytotoxicity assay against extracellular promastigotes of Leishmania donovani, Leishmania infanturn, Leishmania enriettii, and Leishmania major and a test against intracellular amastigote L. donovani residing within murine macrophages. Several naphthoquinones proved to be active at concentrations in the microgram range (EC(50) 0.9-17.0 microg/ml). When tested against a panel of human cancer cell lines (KB, SKMel, A549, MDA) and murine bone marrow culture-derived macrophages (BMMPhi) as mammalian host cell controls, compounds with anti-Leishmania-activity showed moderate (EC(50)>25 microg/ml) to pronounced (EC(50)<10 microg/ml) toxic effects.

In vitro leishmanicidal activity of monomeric and dimeric naphthoquinones.

A series of monomeric and dimeric naphthoquinones with potential for treatment of Leishmania infections was identified in vitro using both a direct cytotoxicity assay against extracellular promastigotes of Leishmania donovani, L. infantum, L. enriettii, and L. major and a test against intracellular amastigote L. donovani residing within murine macrophages. Several naphthoquinones proved to be active at concentrations in the microgram range (EC(50) 0.9-17.0 microg/ml). When tested against a panel of human cancer cell lines (KB, SKMel, A549, MDA) and murine bone marrow culture-derived macrophages (BMMPhi) as mammalian host cell controls, compounds with anti-Leishmania-activity showed moderate (EC(50)>25 microg/ml) to pronounced (EC(50)<10 microg/ml) toxic effects.

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.

Nanosuspensions for the formulation of aphidicolin to improve drug targeting effects against leishmania infected macrophages.

A series of labdans and their derivatives have been identified as novel potential antileishmanial drugs using an in vitro test system against extracellular promastigotes and intracellular amastigotes of Leishmania donovani in murine macrophages (Kayser, O., Kiderlen, A.F., 1998. In vitro activity of leishmanicidal labdanes and related compounds. Proceedings of the Ninth International Congress of Parasitology, Monduzi Editore, Bologna, 925-929). Of these compounds, aphidicolin, a tetradecanhydro-3,9-dihydroxy-4,11b-dimethyl-8, 11a-methano-11aH-cyclo-hepta[a]naphthalin-4,9-dimethanol+ ++ (Fig. 1), was shown to be highly active at concentrations in the microgram range (EC(50) = 0.16 microg/ml). To improve drug targeting effects aphidicolin was formulated as nanosuspension and retested for its enhanced activity (EC(50) = 0.003 microg/ml).

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.

Nanosuspensions as a new approach for the formulation for the poorly soluble drug tarazepide.

Poorly soluble drugs are often a challenging problem in drug formulation, especially when the drug is not soluble in either aqueous media or organic solvents. Attempts to overcome the solubility problem are, e.g. solubilisation with mixed micelles or forming a complex using cyclodextrines, but these approaches are of limited success. Another problem with new high potential drug is that these drugs often show bioavailability problems. One tried to improve the in vivo performance of poorly soluble drugs by reducing the particles size of the drug thus leading to an increased surface area and an increased dissolution velocity (Müller et al., 1994, 1999). Some of these problems occurred with tarazepide and therefore it was tried to create a formulation with this drug as nanosuspension which is suitable for intravenous administration.

Nanosuspensions of poorly soluble drugs--reproducibility of small scale production.

The major problem of many newly developed pharmaceutical drugs is their poor solubility in water and simultaneously in organic media. To solve these problems formulation as nanosuspensions is an attractive alternative. During the drug development process screening for an optimal formulation by homogenisation is essential. Time and cost effective production in an initial phase of R&D can be conducted on lab scale by using the Micron Lab 40 in its discontinuous version. In this report reproducibility of small scale production parameters (particle size, size distribution, content of microparticles) was exemplary studied for the drug RMKP22.

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.

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.

Heavy metal contamination of nanosuspensions produced by high-pressure homogenisation.

High pressure homogenisation is a method for the production of nanosuspensions. In this process crystalline drug particles are pressed with high pressure through a narrow homogenisation gap. Due to the conditions in the gap it seems possible that metal erosion can occur. In this study the heavy metal (Fe) contamination of nanosuspensions produced by high pressure homogenisation was determined. Therefore nanosuspensions were analysed by atom absorption spectroscopy concerning their load of iron which is chosen as reference metal. The results show that the erosion of metal is below 1 ppm and will not cause any toxicological problems.

Antibacterial activity of simple coumarins: structural requirements for biological activity.

The antibacterial activity of a series of simple coumarins was evaluated against 8 microorganisms, including three Gram-positive (Staphylococcus aureus, beta-hemolytic Streptococcus and Streptococcus pneumoniae) and five Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis and Haemophilus influenzae), using the microdilution broth method. The coumarins tested showed broad diversity regarding growth inhibitory activity with minimum inhibitory concentrations ranging from 0.9 to > 12.4 microM. This study, presenting the first systematic analysis of structure-activity relationships among this group of coumarins, revealed some interesting structural requirements. While coumarins with a methoxy function at C-7 and, if present, an OH group at either the C-6 or C-8 position are invariably effective against the spectrum of tested standard bacteria (Gram-negative microorganisms including the Gram-positive bacterium Staphylococcus aureus), the presence of an aromatic dimethoxy arrangement is apparently favourable against those microorganisms which require special growth factors (beta-hemolytic Streptococcus, Streptococcus pneumoniae and Haemophilus influenzae). A combination of these structural features, two methoxy functions and at least one additional phenolic group as reflected by the highly oxygenated coumarins, identify promising candidates with antibacterial broad-spectrum activity.

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.

Structure-cytotoxicity relationships of a series of natural and semi-synthetic simple coumarins as assessed in two human tumour cell lines.

The cytotoxicity of 22 natural and semi-synthetic simple coumarins was evaluated in GLC4, a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values > 100 microM, following a continuous (96 h) incubation, most coumarins exhibited only low cytotoxicity. Several compounds, however, displayed significant potencies. As far as the structure--cytotoxicity relationship is concerned, it is conspicuous that all the potentially active natural compounds possess at least two phenolic groups in either the 6,7- or 6,8-positions. In addition, the 5-formyl-6-hydroxy substituted semi-synthetic analogue was found to be potent, reflecting the importance of at least two polar functions for high cytotoxicity.

Leishmanicidal activity of aurones.

This is the first report on aurones as a new class of natural products with leishmanicidal activity. A series of aurones with drug-potential for Leishmania infections was identified in vitro using both a direct cytotoxicity test against extracellular promastigotes of Leishmania donovani, L. infantum, L. enriettii, and L. major, and a test against intracellular amastigote L. donovani residing within murine macrophages. The compounds proved to be active at concentrations in the microgram range between 0.4 and 5.0 microg/ml. When tested against murine bone marrow-derived macrophages as a mammalian host cell control, all compounds showed only moderate cytotoxicity (EC50 2.32-25.0 microg/ml).

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.