Ultra high performance liquid chromatography method for the determination of pentamidine and analog in rat biological fluids.

Pentamidine isethionate (PTMD) is an antiprotozoal agent used in different parasitic diseases as Human African Trypanosomiasis or Pneumocystis pneumonia. Given its side effects, numerous analogs are still under development worldwide. PTMD has been recently described having a potential activity in myotonic dystrophy (type 1). Here we present an UPLC method coupled to fluo or PDA detection for PTMD and one analog determination in rat plasma or urine. The chromatographic separation was achieved on a Acquity UPLC® HSS T3 analytical column using a mobile phase combining formic acid 0.1% (v/v) and acetonitrile (ACN) at a constant flow rate of 0.4 mL/min. Preliminary, an innovative µSPE (solid phase extraction) procedure using Oasis® WCX sorbent was processed and gave satisfying and reproducible results in terms of extraction yields. Additionally, the methods were successfully validated using the accuracy profiles approach (ß=95% and acceptance limits=15%) over the ranges 2.88-287.52 ng/mL and from 143.76 ng/mL to 1.72 µg/mL in rat plasma and urine for PTMD and for EBAB, from 4.23 to 423.39 ng/mL and from 211.69 ng/mL to 2.54 µg/mL for plasma and urine, respectively. The validated protocols were applied to a pharmacokinetic (PK) study on rats and permitted to point out some relevant PK parameters on PTMD and its studied analog.

Estudo comparativo entre o antimoniato-n-metilglucamina (glucantime) e o isotionato de pentamidina (pentacarinat) em lesões cutâneas da leishmaniose tegumentar / Comparative study of antimony-n-methylglucamine (glucantime) and pentamidine isethionate (Pentacarinat) in skin lesions of cutaneous leishmaniasis

O prognóstico da leishmaniose cutânea (LC) com o uso dos antimoniais pentavalentes (Sb+5) de um modo geral é considerado bom, embora alguns casos tornem-se refratários à terapêutica tradicional. Infelizmente, não existem marcadores de gravidade da doença ou marcadores de resposta terapêutica, limitando a utilização de formas de tratamento mais efetivas. Em alguns casos, porém, existe a necessidade de utilizar outras drogas, como a anfotericina B (desoxocolato) e as pentamidinas (isotionato e mesilato), consideradas como drogas de 2ª escolha no tratamento das leishmanioses, sendo de fundamental importância à busca de novos esquemas terapêuticos. O objetivo do estudo foi comparar a eficácia entre o antimoniato-N-metilglucamina (Glucantime®) e o isotionato de pentamidina (Pentacarinat®) no tratamento da forma cutânea da leishmaniose tegumentar (LT)...

The prognosis of cutaneous leishmaniasis (CL) with the use of pentavalents antimonials (Sb+5) is generally considered good, although in some cases have become refractory to conventional therapy. Unfortunately there are no markers of disease severity or markers of therapeutic response, limiting the use of more effective forms of treatment. In some cases, however, there is a need for other drugs such as amphotericin B (desoxycholate) and pentamidine (isethionate and mesylate), which are considered as the second choice in the treatment of leishmaniasis, since few studies with reduced doses of these drugs demonstrated encouraging results in tegumentary leishmaniasis (TL), which is paramount in the search for new therapeutic regimens using proven antileishmanial drugs. We have compared the effectiveness between the N-methylglucamine antimoniate (Glucantime®) and pentamidine isethionate (Pentacarinat®) in the treatment of CL in an endemic area of tegumentary leishmaniasis (TL)...

A novel HPLC assay for pentamidine: comparative effects of creatinine and inulin on GFR estimation and pentamidine renal excretion in the isolated perfused rat kidney.

PURPOSE: 1. To develop and validate an analytical method for pentamidine (PTM) by reversed-phase HPLC. 2. To compare the effects of creatinine and inulin on PTM excretion in the isolated perfused rat kidney. METHODS: The HPLC method utilized a base deactivated, 5 micro, C18 column and a mobile phase containing acetonitrile (24%) and 0.025 M monobasic phosphate buffer, pH 3.2 (76%). Mobile phase flow rate and UV detection wavelength were 1 mL/min and 270 nm, respectively. Sulfadiazine (SDZ) was used as the internal standard. The method was used to measure pentamidine in perfusate and urine samples generated from studies with the isolated perfused rat kidney (IPK) model. Perfusion experiments were conducted in the presence of two different GFR markers: creatinine and inulin (PTM dose 800 micro g). Both creatinine and inulin were assayed using colorimetric methods. RESULTS: The HPLC assay is rapid, sensitive and reproducible. The method was validated over two standard concentration ranges: 0.1 to 1 micro g/mL, and 1 to 10 micro g/mL. In control (drug-naïve) IPK perfusions, creatinine clearance was approximately 15% greater than inulin clearance (0.80+/- 0.21 mL/min vs. 0.69+/-0.17 mL/min, p > 0.05). In the presence of PTM, however, creatinine clearance was reduced to 0.56+/-0.27 (p < 0.05 compared to control). Inulin clearance was not altered by PTM administration (0.76+/-0.26 mL/min). Cumulative urinary excretion of PTM (% dose) was 3.0+/-0.47% and 9.6+/-4.2% in the presence of creatinine and inulin, respectively. PTM clearance was significantly reduced (0.06+/-0.01 mL/min vs. 0.13+/-0.01 mL/min, p < 0.05) and % kidney accumulation significantly enhanced (66+/-4.7% vs. 37+/-9.7%, p < 0.05) by creatinine. CONCLUSIONS: Creatinine overestimated GFR in the IPK. The altered renal excretion of PTM by creatinine is consistent with inhibition of PTM tubular secretion. Because of increased kidney accumulation, detrimental effects of PTM on renal function were observed. Based on these findings, creatinine should be used cautiously as an indicator of GFR in IPK experimentation.

Development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates.

Components within synthetic chemical and natural product extract libraries often interfere with fluorescence-based assays. Fluorescence interference can result when the intrinsic spectral properties of colored compounds overlap with the fluorescent probes. Typically, fluorescence-based protease assays use peptide amidomethylcoumarin derivatives as substrates. However, because many organic compounds absorb in the ultraviolet region, they can interfere with coumarin-based fluorescence assays. Red-shifted fluorescent dyes such as peptidyl rhodamine derivatives are useful because there is generally less interference from organic compounds outside the ultraviolet wavelengths. In this report, rhodamine-based fluorogenic substrates, such as bis-(Leu)(2)-Rhod110 and bis-(Ala-Pro)-Rhod110, were developed for leucine aminopeptidase and dipeptidyl aminopeptidase. Novel, tandem rhodamine substrates such as Ala-Pro-Rhod110-Leu were designed with 2 protease cleavage sites and used to assay 2 proteases in a multiplex format. General endpoint high-throughput screening (HTS) assays were also developed for leucine aminopeptidase, dipeptidyl aminopeptidase, and trypsin that incorporated both amidomethylcoumarin and rhodamine-based fluorogenic substrates into a single screening format. These dual-substrate assays allowed for the successful screening of the LOPAC trade mark collection and natural product extracts despite high levels of fluorescence interference.

Immunoassays for pentamidine and related compounds: development of a facile inhibitory ELISA suitable for clinical use.

Aromatic dicationic drugs have a broad spectrum of activity against protozoal and fungal pathogens including Pneumocystis carinii, Leishmania mexicana amazonensis, Cryptosporidium parvum and Cryptococcus neoformans. Pentamidine serves as the exemplar for an extensive collection of newly synthesized related compounds, which have reduced toxicity and a wider range of target organisms. Assays of pentamidine and related compounds have depended on HPLC-tandem mass spectrometry (HPLC-TMS) for the quantitation and identification of drug and metabolites. Immunoassays for pentamidine would have many advantages over the HPLC methods including relative simplicity of assay format and required equipment, convenience in sample preparation and reduction in time and cost of assays. In this report we describe a simple ELISA based immunoassay for pentamidine and pentamidine-like drugs with requisite sensitivity and specificity for use as a clinical assay (EC50 value of about 50 nanomolar). Immunogen was synthesized by coupling the hapten aminopentamidine to ovalbumin (chemically modified to provide an optimal number of -SH groups) using sulfo-MBS. Maleic-anhydride activated ELISA plates were covalently sensitized using the aminopentamidine hapten and used in an inhibitory ELISA assay format whereby the ability of analyte to suppress antibody binding to sensitized plate was measured. The assay detects primarily the phenolic amidine of pentamidine when in a para position and hence can also detect structurally related derivatives of pentamidine of potential interest as new therapeutic agents.

Determination of pentamidine in serum and urine by micellar electrokinetic chromatography.

A number of parameters influencing the electrokinetic processing of pentamidine by micellar electrokinetic chromatography (MEKC) were studied in order to develop an analytical method for this compound. The parameters considered were: pH, ionic strength, and SDS concentration of electrolyte, temperature and working voltage. On the basis of the results obtained, the best analytical conditions for the detection of pentamidine in serum and urine by MEKC were determined. Analysis by MEKC permitted determination of the drug in 10 min. Good linearity, reproducibility and accuracy were obtained in the range 0-30 micrograms/ml for both samples, with a correlation coefficient r > or = 0.9998 and a recovery of 87-92% in serum and 90-108.9% in urine. We examined the metabolism of pentamidine using rat liver homogenates in order to exclude any possible interference of metabolites in the analysis of pentamidine.

High-performance liquid chromatographic assay detects pentamidine metabolism by Fisher rat liver microsomes.

Fisher rat liver microsomes metabolized the antimicrobial drug pentamidine to four new compounds detected by gradient elution reversed-phase high-performance liquid chromatography with variable wavelength detection. Coelution experiments with pentamidine metabolite standards determined the new peaks to be previously identified hydroxylated metabolites of pentamidine, with 1,5-bis(4'-amidinophenoxy)-3-pentanol and 1,5-di-(4'-amidinophenoxy)-2-pentanol formed in the greatest amount. The data contradict a previous report that Fisher rat liver homogenates do not metabolize pentamidine. Pentamidine and its known primary metabolites have almost identical absorption spectra; thus, pentamidine metabolism must be evaluated using gradient elution HPLC to resolve pentamidine from its metabolites. The current assay has now been used to demonstrate that Fisher and Sprague-Dawley rat, mouse, rabbit and human liver microsomes all metabolize pentamidine in vitro.

Pentamidine aerosols and environmental contamination: health-care workers at risk.

In our hospital safety guidelines are available for the handling of pentamidine in the day-care department, but no safety ventilation cabin is used because only one patient a day has been treated, The number of patients to be treated, however, is growing, resulting in the need to treat more than one patient a day. To determine the environmental contamination and exposure of health-care workers during and after aerosolised pentamidine treatment of more than one patient with the acquired immunodeficiency syndrome a day a high-performance liquid chromatographic method is used for the detection and quantification of pentamidine. High volume air samples were taken before, immediately after and the day after a treatment session of up to three patients. Also, sediment samples and personal air samples close to the mouth of the health-care workers were taken. Immediately after a treatment session the air in the room contains 1.0-99.7 micrograms pentamidine per m3 of air. Before and the morning after treatment no pentamidine could be detected in the air. Sediment samples vary in detectable amounts of pentamidine from < 5 to 1165 micrograms. pentamidine/cm2. The personal air samples also show a large variation in quantities of pentamidine: < 5-170 ng a filter. When large amounts of pentamidine in the high volume air samples are found high amounts of pentamidine on the sediment samples and the personal air samples are found as well. This means that the patients treated should be instructed well on how to use the nebulizer correctly and be monitored during treatment. Additional safety measures (for example the use of a safety ventilation cabin) should be taken when more than one patient is treated a day.

Lobar pentamidine levels and Pneumocystis carinii pneumonia following aerosolized pentamidine.

Recent studies have suggested that failure of pentamidine prophylaxis against Pneumocystis carinii pneumonia (PCP) may be due to reduced deposition of pentamidine in the upper lobes. In this study, we performed bronchoalveolar lavage from the apical segment of the upper lobe and the middle lobe in 51 HIV-positive patients, all of whom were receiving prophylaxis with aerosolized pentamidine, who had presented with acute respiratory symptoms. Lavage fluid from each lobe was assayed for pentamidine using high-performance liquid chromatography (HPLC). The number of clusters of P carinii were counted after staining with a Wright-Giemsa stain. The patients were subclassified as PCP-positive (32 patients) and PCP-negative (19 patients) on the basis of the presence/absence of P carinii clusters in their BAL fluid. The concentration of pentamidine in the upper lobe compared with the middle lobe was no different (using paired Student's t tests) for either PCP-positive patients or PCP-negative patients. In comparing the positive with the negative subjects, using unpaired Student's t test, there was no difference in the concentration of pentamidine in the upper lobe or the middle lobe. For PCP-positive patients, the numbers of P carinii clusters were on average higher in the upper lobes (mean +/- SD: upper = 14.9 +/- 16.6, middle 7.5 +/- 10.8, p = 0.013, paired Student's t test), but there was no correlation between lobar P carinii cluster counts and pentamidine levels. We conclude that the absence of a relationship between cluster count and pentamidine level, the similarity in regional pentamidine levels between upper and middle lobes, as well as the similarity in pentamidine levels between the PCP-positive and PCP-negative groups indicate that the regional dose of pentamidine is not the determining factor as to whether aerosolized pentamidine prophylaxis will succeed or fail.

Determination of 1,3-di(4-imidazolino-2-methoxyphenoxy)propane in rat, dog and human plasma and urine by high-performance liquid chromatography with fluorescence detection.

A sensitive and selective high-performance liquid chromatographic (HPLC) method was developed for the determination of 1,3-di(4-imidazolino-2-methoxyphenoxy)propane (DMP) in rat, dog and human plasma (50-5000 ng/ml) and urine (0.1-10 micrograms/ml). DMP and DMPent (dimethoxyimidizolinopentamidine, the internal standard), are extracted from alkanized plasma with n-butyl chloride-n-butanol (9:1, v/v). The organic phase is dried under nitrogen, reconstituted in mobile phase, and washed with hexane. Separation is achieved by ion-pair chromatography on a Zorbax Rx C8 column with fluorescence detection. The analysis of pooled plasma (80, 400, and 4000 ng/ml) and urine controls (0.3, 1.6, and 8 micrograms/ml) demonstrated excellent precision and accuracy over a three-day period. The recovery of DMP is > 90% from rat, dog, and human plasma and > 85% from rat and human urine, and 60-70% from dog urine. The limit of quantitation (LOQ) of the assay is 50 ng/ml in rat, dog and human plasma. Using the high-sensitivity assay, the limit of quantitation was decreased to 5, 2 and 0.6 ng/ml in rat, dog and human plasma, respectively. The LOQ of the assay is 0.1 microgram/ml in rat, dog and human urine. The assay was used to determine plasma and urine concentrations of DMP in pharmacokinetic studies in rat and dog.

Determination of pentamidine isethionate in air.

A concern currently exists regarding the potential for exposure of health care workers to pentamidine isethionate, a drug used for prevention and treatment of Pneumocystis carinii pneumonia in immunocompromised patients, including those with human immunodeficiency virus infection. In order to evaluate worker exposures, a sampling and analytical method for pentamidine isethionate in air has been developed. This method involves sampling with a 37-mm PVC membrane filter at 1 to 2 L/min, recovery with 3 mL of 50:50 ethanol:water with 0.085% phosphoric acid and 0.04% tetramethylammonium chloride in an ultrasonic bath for 10 min, and analysis by high performance liquid chromatography with fluorescence detection. The limit of detection is about 18 ng per sample, and the lower limit of quantitation is 50 ng per sample. Recoveries of pentamidine isethionate from PVC filters were 0.76 to 0.91 at 50 to 8820-ng levels of fortification. Samples were stable on PVC filters during 27 days of storage at room temperature. Because patients who are treated with pentamidine isethionate are at increased risk of contracting tuberculosis (TB), safety precautions for handling samples contaminated with TB were included in the sampling and analytical method.

Health effects and exposure assessment of aerosolized pentamidine handlers.

Nurses administering aerosolized pentamidine (AP) were studied to determine any effect AP may be having on their health. Exposure was determined by each nurse's self-report of treatment given as recorded in a daily log and personal and area pentamidine sampling. Outcome measures were self-reported symptoms recorded in a daily log and peak expiratory flow rates (PEFR) and cross-shift and cross-week pulmonary function tests (PFTs). Results revealed no dose-response effect of pentamidine exposure on cross-shift and cross-week PFTs. However, declines in cross-shift PEFRs, diffusion capacities, and increased symptom complaints were observed for a subset of the study population. This suggested that outcomes were modulated by host factors (history of hay fever and allergy) as well as exposure doses. Treatment both efficacy in containing fugitive AP aerosol was also corroborated as a means of minimizing worker exposure.

Lung deposition of nebulised pentamidine in children.

BACKGROUND: Nebulised pentamidine is effective for preventing Pneumocystis carinii pneumonia in adults with acquired immunodeficiency syndrome. The nebuliser dose required to produce equivalent lung concentrations of pentamidine in children is unknown. This study was performed to measure pulmonary pentamidine deposition in children and to relate this to age, ventilation pattern, and body size. METHODS: Nebulised pentamidine (50 mg in 6 ml saline) was administered to 12 children (including one with lymphocytic interstitial pneumonitis) and to six adults with human immunodeficiency virus infection using a Respirgard II nebuliser. Technetium-99m labeled colloidal human serum albumin was used as an indirect marker for pentamidine and deposition in the lungs was detected by a gamma camera. RESULTS: Absolute deposition of pentamidine was not related to age, height, weight, spirometry, or ventilation characteristics. Deposition, as a mean (SD) percentage of nebuliser output, was similar in children aged 8-11 years (5.5(2.4)%), teenagers aged 12-15 years (7.2(2.2)%) and adults (7.1(2.6)%). Aerosol concentration within the lungs (% nebuliser output deposited/predicted total lung capacity) was therefore higher in children (1.9(1.5)%/1) and teenagers (1.9(0.7)%/1) than in adults (1.0(0.7%)/1), and was negatively correlated with height (r = -0.69) and weight (r = -0.50). Deposition of aerosol in the region of the large central airways was particularly marked in children. Small reductions in forced expiratory volume in one second and forced vital capacity after treatment did not differ significantly between adults and children and visual analogue scores of subjective adverse effects did not vary with age. CONCLUSIONS: These results suggest that children probably require lower nebuliser pentamidine doses to produce lung pentamidine concentrations equivalent to those found to be effective for preventing P carinii pneumonia in adults using the Respirgard II nebuliser.

Efficacy of engineering controls in reducing occupational exposure to aerosolized pentamidine.

Aerosolized pentamidine administration may pose potential risks to health care workers exposed to fugitive drug and to infectious respiratory pathogens (eg, tuberculosis) generated by pentamidine-induced cough. Classic infection control methods may be applied to this problem, although the effectiveness of these measures in mitigating environmental pentamidine exposure is unknown. Lack of data fully characterizing pentamidine's mechanism of action or potential mutagenicity, carcinogenicity, or teratogenicity raises concern and suggests worker exposed and environmental contamination be minimized. We report herein on the efficacy of an aerosol containment hood in containing fugitive pentamidine aerosol during administration.

Determination of pentamidine in whole blood, plasma, and urine by high-performance liquid chromatography.

The analysis of pentamidine in whole blood, plasma, and urine by liquid chromatography is described. Extraction was made with a mixture of acetonitrile and chloroform followed by back-extraction into phosphate buffer. A reversed-phase chromatographic system with fluorescence detection was used. The precision of the method was 5-7% at the lower limit of determination (16 nmol/L in plasma and hemolyzed whole blood, 27.7 nmol/L in urine).

An observation of polymorphism in pentamidine isethionate.

Two forms of pentamidine isethionate have been identified from two commercial samples of the compound. The lower melting form was produced by freeze drying and has a melting point of 133.4 degrees C whilst the higher melting form has a melting point of 192 degrees C. Powder x-ray diffraction studies showed that each modification was crystalline and the infra-red spectra showed differences that could be attributed to polymorphism. It was noted that when the freeze dried material was exposed to the atmosphere it converted to the higher melting form and was hygroscopic. Examination of this compound in a formal polymorphism screen was advocated.

Liquid chromatographic and spectroscopic characterization of pentamidine isethionate and impurities in bulk drug and injectables.

A liquid chromatographic (LC) method is described for evaluating purity of pentamidine isethionate (PI), a life-saving drug used in the treatment of Pneumocystis carinii pneumonia, which is a leading cause of death in persons with acquired immunodeficiency syndrome (AIDS). Six potential impurity compounds were synthesized to test the selectivity of the chromatographic system and to permit quantitation of impurities in various lots of PI products. The drug and impurities were separated with gradient elution on a cyano-bonded LC column. The analytic system provided information on the identities and levels of impurities in early experimental lots of PI. These results assisted the manufacturer in altering reaction conditions and purification procedures to ensure that succeeding lots were within the specification limits, i.e., no more than 0.4% of any single impurity or 0.7% of total impurities found in the final product.