Determination of the trypanocidal drug melarsoprol and its conversion products in biological fluids with HPLC-ICPMS/ESMS.

Although melarsoprol, an organoarsenic compound, is widely used for the treatment of trypanosomiasis (human African sleeping sickness), very little is known about its fate in the human body, its active metabolites passing the blood-brain barrier and the mode of action. Previous pharmacological studies based on the determination of melarsoprol by HPLC-UV or by a bioassay method produced different results. We report a HPLC-ICPMS method suitable for determining melarsoprol and its metabolites in biological fluids. The arsenic selective capability of the method allowed the quantitative measurement of melarsoprol and two arsenic-containing conversion products produced when melarsoprol was incubated with human serum and blood. The major product was identified as melarsen [4-[(4,6-diamino-1,3,5-triazin-2-yl)amino]phenyl]arsonic acid by HPLC/electrospray MS, and by accurate mass measurements. Investigations about the stability of melarsoprol in serum showed that within 30 h about 10% of melarsoprol is converted to melarsen. In blood, however, most of the melarsoprol was bound to proteins and only 1% was converted to melarsen after 30 hours. The limit of detection for melarsoprol and its conversion products were in the range of 1 µg AsL(-1) (13 nmol As L(-1)) based on signal to noise ratio of 3 with a 10 µL injection volume allowing direct determination of the compounds in blood and serum (after protein precipitation) at therapeutically realistic concentrations.

The phenomenon of treatment failures in Human African Trypanosomiasis.

Treatment of Human African Trypanosomiasis (HAT or sleeping sickness) relies on a few drugs which are old, toxic and expensive. The most important drug for the treatment of second stage infection is melarsoprol. During the last 50 years treatment failures with melarsoprol were not a major problem in Trypanosoma brucei gambiense patients. Commonly a relapse rate of 5-8% was reported, but in recent years it has increased dramatically in some important foci of T. b. gambiense sleeping sickness. Treatment failures for T. b. rhodesiense are much less of a problem apart from some reports between 1960 and 1985 of refractoriness in T. b. rhodesiense patients in East Africa. Analysis of those isolates revealed that their in vitro sensitivity to melarsoprol was one-tenth that of sensitive isolates, and complete failure to cure the infection in the acute mouse model with melarsoprol levels comparable with those in human patients. There was very little indication of resistance in T. b. gambiense isolates from Côte d'Ivoire and NW Uganda. The in vitro melarsoprol sensitivities for populations from relapsing and from curable patients were in the same range. Melarsoprol concentrations in the plasma and cerebrospinal fluid of patients 24 h after treatment did not show any difference between patients who relapsed and those who could be cured. The reason for relapses in the recent T. b. gambiense epidemics are not known. Other parasite-related factors might be involved, e.g. affinity to extravascular sites other than the CNS which are less accessible to the drug. In conclusion, a combination of factors rather than a single one may be responsible for the phenomenon of melarsoprol treatment failures in T. b. gambiense patients.

Melarsoprol refractory T. b. gambiense from Omugo, north-western Uganda.

Culture adapted T. b. gambiense isolated from Northwest Uganda were exposed to 0.001-0.14 microg/ml melarsoprol or 1.56-100 microg/ml DL-alpha-difluoromethylornithine (DFMO). Minimum inhibitory concentrations (MICs) of each drug were scored for each isolate after a period of 10 days drug exposure. The results indicate that T. b. gambiense isolates from Northwest Uganda had elevated MIC values for melarsoprol ranging from 0.009 to 0.072 microg/ml as compared with T. b. gambiense isolates from Cote d'Ivoire with MIC values ranging from 0.001 to 0.018 microg/ml or with T. b. rhodesiense from Southeast Uganda with MIC values from 0.001 to 0.009 microg/ml. All MIC values obtained fell below expected peak melarsoprol concentrations in serum of treated patients. However, it may not be possible to maintain constant drug concentrations in serum of patients as was the case in our in vitro experiments. Importantly, the MIC of 0.072 microg/ml exhibited by one of the isolates from Northwest Uganda was above levels attainable in CSF indicating that this isolate would probably not be eliminated from CSF of treated patients. PCR amplification of the gene encoding the P2-like adenosine transporter followed by restriction digestion with Sfa NI enzyme revealed presence of fragments previously observed in a trypanosome clone with laboratory-induced arsenic resistance. From our findings it appears that reduced drug susceptibility may be one factor for the frequent relapses of sleeping sickness after melarsoprol treatment occurring in Northwest Uganda.

Investigations of the metabolites of the trypanocidal drug melarsoprol.

BACKGROUND: Melarsoprol remains the first-choice drug for trypanosomiasis (human African sleeping sickness). To contribute to the sparse pharmacologic data and to better understand the cause of the frequent serious adverse reactions, we investigated the metabolism of this 50-year-old organoarsenic compound. RESULTS: The half-life of melarsoprol determined by HPLC was <1 hour compared with 35 hours determined by bioassay and atomic absorption spectroscopy, indicating the existence of active metabolites. One metabolite, melarsen oxide, was identified by ultraviolet HPLC after incubation of melarsoprol with microsomes. The maximum plasma concentration of melarsenoxide was reached 15 minutes after administration; the clearance was 21.5 mL/min/kg and the half-life of free melarsen oxide was 3.9 hours. Either melarsen oxide or a yet-undiscovered active metabolite is irreversibly bound to proteins, as shown by ultrafiltration, precipitation experiments, and atomic absorption spectroscopy. Because of the poor pharmaceutical properties of melarsoprol, the therapeutic potential of melarsen oxide was investigated. In a rodent model of acute infection, 20 of 20 mice were cured (0.1 to 1 mg/kg intravenously or 2.2 mg/kg intraperitoneally). In a rodent model of central nervous system infection, five of six mice survived for more than 180 days (5 mg/kg intravenously), indicating a sufficient melarsen oxide penetration across the blood-brain barrier. CONCLUSION: The prospects for the future of trypanosomiasis treatment are deplorable. Investigations on the improvement of the use of the old drugs are therefore required. The results of this study may build a basis for further research on the cause of severe adverse reactions.

An automated biological assay to determine levels of the trypanocidal drug melarsoprol in biological fluids.

For the investigation of the pharmacokinetic properties of a drug, methods for sensitive and precise quantification are a prerequisite. Only few functional methods exist for the determination of the trypanocidal drug melarsoprol in biological fluids: A bioassay which requires microscopical evaluation and two HPLC methods, which require sample extraction and are difficult to automatize due to the drug's properties. We report the development of an automated biological assay, based on the fluorescent dye Alamar blue. To validate the assay for melarsoprol, 108 serum and 37 cerebrospinal fluid (CSF) samples were spiked with melarsoprol at concentrations of 17-92 ng/ml for CSF and 17 ng/ml-2.2 microg/ml for serum. The precision (repeatability) expressed as the interday average coefficient of variation was 9.9% for serum and 18.8% for CSF samples over the respective concentration range. The accuracy (measurement for the systematic error) of the test was 99.4% for serum and 96.4% for CSF. The assay's limit of quantitation with the use of the trypanosome stock STI 704 BABA was 4 ng/ml for both serum and CSF samples.

Clinical study of an organic arsenical, melarsoprol, in patients with advanced leukemia.

Inorganic arsenic trioxide (As(2)O(3)) induces a high proportion of complete remissions in relapsed patients with acute promyelocytic leukemia (APL). Previously, we have shown that both As(2)O(3 )and melarsoprol, an organic arsenical used for the treatment of trypanosomiasis, exhibit broad antileukemic activity against both chronic and acute myeloid and lymphoid leukemia cell lines. Given the breadth of this activity, we initiated a clinical study to evaluate the pharmacokinetics, safety, and potential efficacy of melarsoprol in patients with refractory or resistant leukemia. Using the antitrypanosomal dose and schedule, patients received escalating intravenous doses daily for 3 days, repeated weekly for 3 weeks. Doses were 1 mg/kg on day 1, 2 mg/kg on day 2, and 3.6 mg/kg on day 3 and on all days thereafter, up to a maximum daily dose of 200 mg. Eight patients [6 AML (2 morphologic APL), 1 CML, 1 CLL] were treated. Mean peak plasma concentrations of the parent drug were obtained immediately after injection, ranged from 1.2 microg/ml on day 1 to 2.4 microg/ml on day 3, were dose proportional, and decayed with a t(1/2) congruent with 15 min. A minor clinical response (regression of splenomegaly and lymphadenopathy) was observed in a patient with chronic lymphocytic leukemia. Central nervous system (CNS) toxicity proved limiting on this dose and schedule. Three patients experienced generalized grand mal seizures during the second week of therapy. We concluded that this dose and schedule of melarsoprol is associated with excessive CNS toxicity and that verification of the striking preclinical activity in patients with leukemia will require developing an alternative dose and schedule.

Discrepancy in plasma melarsoprol concentrations between HPLC and bioassay methods in patients with T. gambiense sleeping sickness indicates that melarsoprol is metabolized.

OBJECTIVE: With the use of a specific high-performance liquid chromatography (HPLC) method and a bioassay which determines trypanocidal activity, concentrations of melarsoprol were assessed in plasma, urine and cerebrospinal fluid (CSF) from 8 patients with late-stage Trypanosoma gambiense sleeping sickness. The aim was to unravel to what extent the bioassay codetermines biologically active metabolites of melarsoprol. METHODS: Subjects were given one dose of melarsoprol i.v. per day for 4 days (1.2, 2.4, 3.0-3.6, 3.0-3.6 mg per kg b.w., respectively). Plasma samples were obtained before the first melarsoprol injection, immediately after and at 1 h, 24 h and 5 days after the 4th injection. Urine was collected before melarsoprol therapy and at 24 h after the 4th injection. CSF samples were taken once before treatment and at 24 h after the 4th injection. RESULTS: HPLC analyses showed that plasma concentrations immediately after the 4th injection varied from 2200 to 15,900 nmol/l; dropping to 0-1800 nmol/l at 1 h; and to undetectable levels at 24 h. In urine small amounts of melarsoprol were recovered. Melarsoprol could not be detected in CSF by HPLC. Immediately after injection, bioassay analyses showed plasma concentrations of the same magnitude as HPLC assays but at 1 h they were 4-65-fold higher than the levels assessed by HPLC. Even 24 h and 5 days after the 4th injection there was significant but decreasing activity. Urine levels were 40-260-fold higher than the measured HPLC concentrations, whereas there was low but detectable activity in CSF. CONCLUSION: Results indicate that melarsoprol is rapidly eliminated from plasma. The significant trypanocidal activity determined by bioassay and simultaneous low or undetectable levels of melarsoprol assayed by HPLC indicate that the compound is transformed into metabolites with parasiticidal activity.

Determination of melarsoprol in biological fluids by high-performance liquid chromatography and characterisation of two stereoisomers by nuclear magnetic resonance spectroscopy.

The analysis of melarsoprol in whole blood, plasma, urine and cerebrospinal fluid is described. Extraction was made with a mixture of chloroform and acetonitrile followed by back-extraction into phosphoric acid. A reversed-phase liquid chromatography system with ultraviolet detection was used. The relative standard deviation was 1% at concentrations around 10 mumol/l and 3-6% at the lower limit of determination (9 nmol/l in plasma, 93 nmol/l in whole blood, 45 nmol/l in urine and 10 nmol/l in cerebrospinal fluid). Melarsoprol is not a stable compound and samples to be stored for longer periods of time should be kept at -70 degrees C. Plasma samples can be stored at -20 degrees C for up to 2 months. Chromatography showed that melarsoprol contains two components. Using nuclear magnetic resonance spectroscopy the two components were shown to be diastereomers which slowly equilibrate by inversion of the configuration at the As atom.

Pharmacokinetics of melarsoprol in uninfected vervet monkeys.

The level of the trypanocidal drug melarsoprol was determined in serum and cerebrospinal fluid (CSF) of six healthy vervet monkeys after intravenous application of the drug following a standard treatment schedule and a recently suggested alternative protocol. The maximum serum levels measured were about 3 micrograms/ml. A three-compartment model was used to analyze the serum data. The mean residence time calculated for melarsoprol in serum was 18 h, the volume of distribution was 3.6 l/kg and the clearance was 3.5 ml/min*kg. In the CSF the drug levels were generally very low, not exceeding 55 ng/ml, and the adaptation of the drug levels was found to be very low. The comparison of the drug concentrations required to eliminate trypanosomes in vitro and the drug concentrations reached in the CSF during treatment revealed that the latter might be insufficient in some cases to eliminate all trypanosomes from this site. The peak serum levels during alternative application of the drug were lower compared to those during empirical treatment. No evidence for drug cumulation in the body was found. The results of this study are compared with recent pharmacokinetic data from human patients, and discussed in the context of the problem of relapses and reactive encephalopathy occurring after treatment of sleeping sickness.

Pharmacokinetic properties of the trypanocidal drug melarsoprol.

With a biological assay and atomic absorption spectrometry we determined the level of melarsoprol in the serum and cerebrospinal fluid of 19 patients treated with melarsoprol in Daloa, Ivory Coast. Most serum levels were between 2 and 4 micrograms/ml 24 h after administration, and were still > or = 0.1 microgram/ml after 120 h. Levels in the cerebrospinal fluid were between 0 and 0.1 microgram/ml. Elimination was biphasic, with a pronounced beta 1 phase. Mean terminal elimination half-life of melarsoprol was about 35 h, volume of distribution was about 100 l and total clearance was about 50 ml/min. The results of these first pharmacokinetic studies on melarsoprol were used to simulate possible alternative therapy schemes which might avoid some of the problems that arise with melarsoprol use.

An in vitro bioassay for quantification of melarsoprol in serum and cerebrospinal fluid.

A biological assay was developed for measuring melarsoprol in serum and cerebrospinal fluid of patients with human African trypanosomiasis. Trypanosomes were cultivated in microtiter plates for 72 hours with melarsoprol (Mel B) in concentrations of 1.25 micrograms/ml to 2.2 ng/ml. The minimum inhibitory concentration of Mel B for a reference Trypanosoma brucei rhodesiense clone was determined by microscopical examination. Samples of serum or cerebrospinal fluid were incubated under the same conditions and the highest dilution determined which caused death of all trypanosomes. The melarsoprol concentration of the sample was then calculated using the sample dilution and the determined minimal inhibitory concentration of the trypanosome population used for the assay. The test was validated using a number of reference samples and it was used for melarsoprol determination in serum- and cerebrospinal fluid samples taken from two treated patients. A sample size of 100 microliters was sufficient to perform the assay. The lower detection limit was 9 ng/ml (22.6 nmol/ml). The assay has potential for measuring other trypanocidal drugs in body fluids.

ELISA assay for melarsoprol.

A sensitive ELISA method has been developed for measuring the trypanocidal drug melarsoprol. The test allows for the detection of the drug in human sera and in cerebrospinal fluid at the ng/ml level. Preliminary analyses on patient sera and CSF confirm the feasibility of the method. Further application of the test will enable to conduct the necessary pharmacokinetic and metabolic studies; the drug monitoring should hopefully result in improved treatment schedules minimizing the undesired side effects, e.g. lethal encephalopathies.