Chemical and biochemical characterization and in vivo safety evaluation of pharmaceuticals in drinking water.

The water constituents that are currently subject to legal control are only a small fraction of the vast number of chemical substances and microorganisms that may occur in both the environment and water resources. The main objective of the present study was to study the health impact resulting from exposure to a mixture of pharmaceuticals that have been detected in tap water at low doses. Analyses of atenolol, caffeine, erythromycin, carbamazepine, and their metabolites in blood, urine, feces, fat tissue, liver, and kidney after exposure to a mixture of these pharmaceuticals in treated drinking water were performed. The effects of this exposure were assessed in rats by measuring biochemical markers of organ injury or dysfunction. Simultaneously, the selected pharmaceuticals were also quantified in both physiological fluids and organ homogenates by liquid chromatography-tandem mass spectrometry (performed in multiple reaction monitoring mode and full scan mode). Following exposure of rats to a concentration of a pharmaceutical which was 10 times higher than the concentration known to be present in tap water, trace levels of some pharmaceuticals and their metabolites were detected in biological samples. This exposure did, however, not lead to significant organ injury or dysfunction. Thus, the authors report an experimental model that can be used to characterize the safety profile of pharmaceuticals in treated drinking water using a multiorgan toxicity approach. Environ Toxicol Chem 2016;35:2674-2682. © 2016 SETAC.

Effect of mibefradil on CYP3A4 in vivo.

Mibefradil, a calcium channel blocker, was removed from the market because of adverse drug interactions with coadministered CYP3A4 substrates. This study examined the effect of mibefradil on the activity of hepatic and intestinal CYP3A4 in vivo, employing the erythromycin breath test (EBT) and oral midazolam pharmacokinetics. This was a two-period, single-blind, placebo-controlled crossover study in which 8 male volunteers were randomized to the order of receiving placebo and a single 100-mg oral dose of mibefradil. Oral midazolam was coadministered with intravenous [14C N-methyl] erythromycin 1 hour after mibefradil/placebo administration. The EBT was performed 20 minutes following erythromycin administration. Blood and urine were collected during the 36 hours following probe drug administration for analysis of midazolam pharmacokinetics. Coadministration of mibefradil increased the Cmax of midazolam 3-fold, the AUC 8- to 9-fold, and the t1/2 4-fold. Mibefradil coadministration decreased the amount of exhaled 14CO2 in 6 of 8 subjects, with a mean decrease of 25%. It was concluded that a single oral dose of mibefradil significantly inhibits CYP3A4 in intestine and liver. These data support that adverse drug interactions involving mibefradil reflect inhibition of CYP3A4 in intestine and liver. Also, they suggest that the EBT, while a valid probe of in vivo hepatic CYP3A4 activity, is a single time point measurement and may be less sensitive than oral midazolam pharmacokinetics in detecting CYP3A4 inhibition.

Inhibitory effect of erythromycin on P-glycoprotein-mediated biliary excretion of doxorubicin in rats.

The macrolide antibiotic erythromycin has recently been shown to overcome the resistance to anticancer drugs that results from overexpression of P-glycoprotein. The present study, using erythromycin lactobionic acid as a model drug, investigated the inhibitory effects of erythromycin on the efflux of doxorubicin from P388/ADR cells expressing P-glycoprotein and on the biliary excretion mechanism of doxorubicin in rats, which is primarily mediated by P-glycoprotein. Erythromycin lactobionic acid was found to inhibit the efflux of doxorubicin (5 microM) from P388/ADR cells in a concentration-dependent manner. In rats receiving constant-rate infusion of doxorubicin (30 micrograms/min), both the biliary and renal clearance of this drug dramatically decreased and its plasma concentrations increased after an intravenous injection of erythromycin lactobionic acid (100 mg/kg as erythromycin). These results suggest that erythromycin competitively inhibits P-glycoprotein-mediated biliary and renal excretion of doxorubicin. The effect of erythromycin on the biliary secretion of doxorubicin was also analyzed quantitatively by the competitive inhibition model. The computer-estimated values of Vmax/Km, Km and Ki were 8.79 ml/minute, 0.82 microgram/ml and 0.41 microgram/ml, respectively. The findings of these experiments suggest that the inhibitory effect of erythromycin on the P-glycoprotein-mediated biliary excretion of doxorubicin is competitive and that combination chemotherapy of doxorubicin with erythromycin may induce toxicity as a result of increased plasma concentrations of doxorubicin.

Determination of erythromycin in serum and urine by high-performance liquid chromatography with ultraviolet detection.

A high-performance liquid chromatographic analysis of erythromycin in human serum and urine with UV detection at 200 nm is presented. The method involves a solid-phase extraction procedure followed by a simple phase separation step and chromatography on a reversed-phase column. The method has sensitivity limits of 0.25 and 1.0 micrograms/mL in serum and urine, respectively, and is sufficiently sensitive to monitor concentrations of erythromycin in human serum and urine after the administration of a single 500-mg erythromycin stearate tablet.

Delivery of erythromycin to subcutaneous tissues in rats by means of a trans-phase delivery system.

Topical administration of antibiotics is associated with reduced risk of systemic side-effects and alteration of gut microflora, and results in higher concentrations of antibiotics at the site of application (and so a lower dose of the drug is required). In conditions such as acne vulgaris, infiltration of the antibiotics into the infected subcutaneous layers is highly desirable. A trans-phase delivery system (TPDS), a mixture of benzyl alcohol, acetone and isopropanol, has been shown to enhance the effective transport of the antibiotic erythromycin across the epidermal barrier and enhance accumulation in the dermis. Two formulations containing N-methyl[14C]erythromycin were compared, a TPDS solution and a propylene glycol solution. They were applied to the dorsal areas of 4-6 week old Fischer rats and tissues were removed for analysis of radioactivity after 2, 4, 8, 12 or 24 h and skin was biopsied and sectioned for autoradiography. The erythromycin dissolved in the TPDS solvent mixture penetrated the stratum corneum and a relatively high concentration was maintained in adjacent tissues for up to 24 h. Penetration was very effective and the erythromycin was detected in significant amounts in the underlying muscle, various organs and later in the urine. In contrast the propylene glycol carrier, probably because of its primarily hydrophilic character, caused the erythromycin to traverse tissue barriers rapidly and appear in the urine. Microautoradiographs qualitatively revealed progressive disappearance of radioactivity from the surface; this correlated with results obtained by direct isotope counting. The route of penetration, in addition to following the interkeratinocyte spaces, seemed to include the perimeter of the pilosebaceous glands and their appendages before diffusion into the capillaries. The propylene glycol solution seemed to traverse the epidermis and the papillary and reticular dermis more rapidly, which might explain its rapid appearance in the urine. These data suggest that the different solutions penetrate the skin by different mechanisms.

Determination of erythromycin A in rat plasma and urine by high-performance liquid chromatography with chemiluminescence detection using Tris(2,2'-bipyridine)ruthenium(II).

A simple and sensitive method was developed for the determination of erythromycin A (EA), decladinosyl erythromycin A (dClEA) and erythromycin B (EB) in rat plasma and urine by high-performance liquid chromatography with electrogenerated chemiluminescence detection using Tris(2,2'-bipyridine)ruthenium(II). The recovery rates of EA, dClEA and EB were 97, 94 and 85% from rat plasma and 89, 83 and 93% from rat urine, respectively. The calibration curves were linear over the concentration ranges 0.05-5 microg/mL for plasma and 0.5-50 microg/mL for urine. The precision and accuracy for all analytes in rat plasma were < or =9.0 and -6.3-7.2%, and those in urine were < or =9.4% and -6.1-7.6%, respectively. This method proved to be a powerful tool for determination of EA, dClEA and EB concentrations in samples from rats.

High-performance liquid chromatography of erythromycin propionyl ester and erythromycin base in biological fluids.

The simultaneous determination of erythromycin propionate and erythromycin base in serum and urine by high-performance liquid chromatography using oleandomycin as internal standard is described. The separation was achieved on a reversed-phase C18 column employing acetonitrile-0.05 M phosphate buffer (65:35), adjusted to pH 7.0, as the mobile phase with coulometric detection. Hydrolysis of the ester during blood sample collection was minimised by immediate high-speed centrifugation of collected blood samples, followed by separation and immediate freezing of the serum fraction. A solid-phase extraction procedure, combined with a simple phase-separation step was used prior to chromatographic analysis. The method has the necessary precision, sensitivity and accuracy to allow the simultaneous determination of both components in serum and urine following a single 500-mg oral dose of erythromycin estolate.

Simultaneous determination of clarithromycin and 14(R)-hydroxyclarithromycin in plasma and urine using high-performance liquid chromatography with electrochemical detection.

A sensitive method for the simultaneous high-performance liquid chromatographic determination of clarithromycin and its active metabolite in plasma and urine is described. Alkalinized samples were coextracted with an internal standard and analyzed on a C8 column using electrochemical detection. Recoveries were greater than or equal to 85% and consistent. Standard curves for plasma were linear in the range 0-2 micrograms/ml for both compounds (r greater than 0.99), with limits of quantification of approximately 10.03 micrograms/ml (0.5-ml sample). Within-day and day-to-day precision were good, with coefficients of variation mostly within +/- 5%; accuracy for both compounds were routinely within 90-110% of theoretical values. Standard curves for urine were linear in the range 0-100 micrograms/ml with limits of quantification of 0.5 micrograms/ml (0.2-ml sample). Urine assays also had similar within-day and day-to-day precisions and accuracy.

Sensitive high-performance liquid chromatographic determination of EM523, a new erythromycin derivative, in human plasma and urine with ultraviolet detection using column switching.

A sensitive high-performance liquid chromatographic (HPLC) method using column switching is described for the determination of EM523 (I), a new erythromycin derivative, in human plasma and urine. The analyte was extracted from alkalinized plasma or urine with a mixture of n-hexane and acetone. After the evaporation of the organic layer, the reconstituted residue was injected into the HPLC system and separated on the first column. After column switching, the heart-cut fraction contamination the analyte was further separated on the second column and monitored by ultraviolet absorbance at 210 nm. The detection limits were 1 ng/ml in plasma and 10 ng/ml in urine. The method was applied to the clinical trials of I.

Analysis of erythromycin in biological fluids by high-performance liquid chromatography with electrochemical detection.

A simple and sensitive high-performance liquid chromatographic assay was developed for the quantitative determination of major erythromycin components and their potential metabolites or degradation products in plasma and urine. An ether extract of alkalized plasma sample was chromatographed on a reversed-phase column and the components in the column effluent were monitored by an electrochemical detector. The recovery of the drug from extraction was virtually 100%. The detection limits for erythromycin A in plasma were 5-10 ng/ml and 30 ng/ml using 1 and 0.2 ml of sample, respectively. For urine samples, a simple one-step deproteinization with two volumes of acetonitrile was satisfactory for analysis. The method has been evaluated in plasma and urine from dogs receiving oral or intravenous erythromycin A. The standard curves for potential metabolites or degradation products were not constructed due to the lack of sufficient samples.

N-Didemethyl-n-propionyl-6,9;9, 12-erythromycin A-spiroketal, a new metabolite of erythromycin ethyl succinate in man.

A novel, neutral metabolite of erythromycin was isolated from the urine of a patient treated with erythromycin ethyl succinate. After separation and purification on Sephadex LH-20 column chromatography, the structure of the metabolite was deduced from spectral data to be the propionamide of the didemethylated 6,9;9, 12-spiroketal of erythromycin A.

Pharmacokinetic advantages of erythromycin estolate over ethylsuccinate as determined by high-pressure liquid chromatography.

The pharmacokinetics of erythromycin estolate (500 mg) and erythromycin ethylsuccinate (600 mg) were compared in 12 healthy volunteers after single doses and after repeated oral doses (every 8 h). High-pressure liquid chromatography with electrochemical detection was used to determine concentrations in plasma and urine of estolate, ethylsuccinate, and erythromycin base. The maximum concentration of drug in the serum, the half-life, and the area under the curve for erythromycin estolate were significantly greater than those of erythromycin ethylsuccinate after both regimens. After single and multiple doses, the respective areas under the curve of erythromycin base generated by estolate formulation were 3 and 1.6 times greater (P less than 0.05) than those of ethylsuccinate. The lower percentage of hydrolysis of erythromycin estolate (41 versus 69%) combined with its longer half-life (5.47 versus 2.72 h) and its larger area under the curve (30.61 versus 4.68 micrograms/h/ml, after multiple doses) could explain these differences. This study underscores the need for a specific high-pressure liquid chromatography assay and the importance of wide variability, rate-limited processes, changes with multiple doses, and the appearance of a second peak when one studies the pharmacokinetics of erythromycin esters. The pharmacokinetic data presented in this study reinforce the clinical advantages of erythromycin estolate over erythromycin ethylsuccinate.

Metabolism and disposition of clarithromycin in man.

The metabolic fate and pharmacokinetics of clarithromycin following a single 250- or 1200-mg oral dose of 14C-clarithromycin were studied in six healthy adult males. Peak plasma levels of clarithromycin averaged 0.6 microgram/ml after the low dose and 2.7 micrograms/ml after the high dose. The AUC of clarithromycin increased 13-fold, with the 4.8-fold increase in dose, while the plasma half-life increased from 4.4 hr to 11.3 hr. The major metabolite in plasma and urine was the microbiologically active 14-hydroxylated-R epimer of clarithromycin. After 5 days, a mean of 38% of the low dose (18% as clarithromycin) and 46% of the high dose (29% as clarithromycin) was recovered in the urine, with approximately one-third eliminated during the first 24 hr. The nature of the urinary and fecal metabolites revealed the involvement of three metabolic pathways, viz. 1) hydroxylation at the 14-position to form the R and S epimers, 2) N-demethylation, and 3) hydrolysis of the cladinose sugar. Secondary metabolism via these pathways was also evident. The overall recovery of metabolites, but not total radioactivity, decreased 42% after the high dose. The nonlinear pharmacokinetic behavior of clarithromycin and the decrease in metabolite production suggest that clarithromycin metabolism can be saturated at high doses.

Removal of interference by erythromycin, phenazopyridine, and methenamine mandelate in the Porter-Silber reaction.

Erythromycin, phenazopyridine, methenamine mandelate, acetazolamide, and ascorbic acid reportedly interfere with the Porter-Silber reaction in the determination of urinary 17-hydroxycorticosteroids. Sodium bisulfite, added to the urine after hydrolysis with glucuronidase, removed almost all nonsteroidal interferences in urine from healthy adults medicated with the first three drugs, but such studies proved that acetazolamide and ascorbic acid do not interfere with the Porter--Silber reaction, whether or not sodium bisulfite is added.

Separation of erythromycin and related substances by high-performance liquid chromatography on poly(styrene-divinylbenzene) packing materials.

A comparative evaluation of three brands of poly(styrene-divinylbenzene) copolymers, Hamilton PRP-1 (10 micron), Rogel (8 micron) and TSK-Gel (10 micron), as column packing materials for high-performance liquid chromatographic separation of erythromycins is presented. Erythromycins A, B and C, anhydroerythromycin A, erythromycin A enol ether, N-demethylerythromycin A, anhydro N-demethylerythromycin A and N-demethylerythromycin A enol ether were chromatographed. The effects of column temperature, concentration of organic modifier in the mobile phase, concentration of phosphate buffer, the addition of quaternary ammonium salts and pH are described. The best separations were obtained on TSK-Gel with the mobile phase acetonitrile-methanol-0.2 M tetramethylammonium hydroxide pH 8.0-0.2 M phosphate buffer pH 8.0-water (30:15:25:5:25). PRP-1 and Rogel gave equally good separations but with higher retention volumes.