Fast determination of antibiotics in whole blood.

There is a need for analytical methods capable of monitoring blood antibiotic levels in real time. Here we present a method for quantifying antibiotic levels in whole blood that does not require any sample pretreatment. The tests employ the enzyme penicillinase to assay for penicillin G, penicillin V and ampicillin using a flow-injected biosensor, the Enzyme Thermistor. Optimal flow rates, sample volumes and pH were determined to be 0.5 mL/min, 100 µL and 7.0, respectively. Analysis of the antibiotics diluted in buffer gave a linear range of 0.17-5.0 mM. Calibration curves prepared using blood spiked with the antibiotics gave a linear range of 0.17-2.0 mM. Linear regression values for all of the calibration curves were 0.998 or higher. Assay cycle time was 5 min. The relative standard deviation value for 100 determinations of a mock blood sample spiked with penicillin G was 6.71%. Despite the elimination of sample pretreatment, no detectable clogging or signal drift was observed. The assay provides a fast, simple, reliable analytical method for determining antibiotic concentrations in blood without the need for any sample pretreatment. This is an important first step towards developing a device capable of real-time monitoring of antibiotic levels in whole blood. The technology has the potential to significantly improve the outcomes of patients undergoing critical care.

Comparative bioavailability study of two phenoxymethylpenicillin potassium tablet formulations in healthy volunteers.

OBJECTIVE: The aim of this study was to evaluate the performance of 2 phenoxymethylpenicillin 500,000 UI tablet formulations in healthy human volunteers. MATERIAL AND METHODS: The study was conducted using an open, randomized crossover design with a 7-day washout interval. A single dose of each formulation was administered to 26 healthy volunteers as assessed by clinical and laboratory test evaluations. The plasma samples were obtained over an 8-h interval and phenoxymethylpenicillin concentrations were quantified by a suitable and validated HPLC-UV method with detection at 220 nm. Systolic and diastolic blood pressure and pulse rate measurement were taken pre dose and at intervals up to 8 h. RESULTS: Tolerance of both products was adequate. The mean of Meracilina/Pen-Ve-Oral 500,000 UI% geometric mean was 99.89% for AUC0-t, 100.86% for AUC0-infinity and 101.11% for Cmax. The 90% confidence intervals were 94.62 - 105.46%, 95.22 - 106.83% and 98.61 - 103.87%, respectively. The mean recovery of phenoxymethylpenicillin was 94.8%, while the retention time observed for phenoxymethylpenicillin and phenytoin (internal standard) was 4 and 10 min, respectively. The limit of quantification was 0.10 mg/l. CONCLUSION: Since the 90% CI for AUC0-t, AUC0-infinity and Cmax ratios were all within the 80 - 125% interval proposed by the US FDA and accepted by ANVISA, it was concluded that the Meracilina formulation (manufactured by AchA(c) S.A.) is bioequivalent to Pen-Ve-Oral (manufactured by Eurofarma) for both the rate and the extent of bioavailability.

The influence of caprate on rectal absorption of phenoxymethylpenicillin: experience from an in-vivo perfusion in humans.

The aim of this in-vivo perfusion study in humans was to investigate the influence of a penetration enhancer, sodium caprate, on the rectal absorption of phenoxymethylpenicillin and antipyrine. Six subjects, 3 male and 3 female, were included in two separate studies using perfusion solution of different pH (T1 and T2, respectively). Each in-vivo rectal perfusion investigation lasted for 200 min and consisted of two periods of 100 min, the first serving as a control, and sodium caprate being added in the second period in both T1 and T2. The concentrations of phenoxymethylpenicillin, antipyrine and sodium caprate in the outlet perfusate were assayed by HPLC, as was the plasma concentrations of phenoxymethylpenicillin. At pH 6.0 (0-100 min) the fraction absorbed (f(abs)) and effective permeability (P(eff)) of phenoxymethylpenicillin were 0.3% and 0.06 x 4 cm s(-1), respectively, and remained unaffected by the addition of sodium caprate. When the same subjects were perfused at pH 7.4, the f(abs) and P(eff) of phenoxymethylpenicillin were 2.4% and 0.11 x 10(-4) cm s(-1) (0-100 min), respectively, also remaining unchanged by addition of sodium caprate (100-200 min). It was possible to determine the plasma AUC of phenoxymethylpenicillin after addition of sodium caprate in three subjects at both pHs; this was in the range of 14.0-62.8 and 56.4-231 (min micromol L(-1)) at pH 6.0 and 7.4, respectively. Interestingly, there was a correlation between P(eff) for sodium caprate and the individual plasma AUC and C(max) of phenoxymethyl-penicillin, which indicates that the permeability of the enhancer in the tissue upon which it should act is crucial for achieving an effect. The f(abs) and the P(eff) of antipyrine were not affected at either pH when sodium caprate was added to the perfusion solution. In conclusion, the plasma pharmacokinetics of phenoxymethylpenicillin suggested a slightly increased rectal absorption at pH 7.4 in subjects where sodium caprate was transported into the rectal tissue. However, the increased P(eff) for phenoxymethylpenicillin wastoo small to detectfrom the outlet perfusate, which suggests that sodium caprate alone has a limited effect on the permeability in-vivo across the rectal epithelium when it is presented in a solution.

Saliva-resistant coating of tablets prevents oral release of penicillin: plasma but not saliva equivalence.

OBJECTIVE: To investigate saliva and plasma concentrations of penicillin after the intake of a conventional phenoxymethylpenicillin (PcV) tablet and a tablet with saliva-resistant coating (PcVsr), both containing 1 g penicillin. METHODS: The study had an open randomized crossover design and involved 24 healthy subjects. Saliva and blood were sampled intermittently for 6 h after tablet intake. RESULTS: Within the first 10 min after tablet intake penicillin was detected in saliva in ten subjects taking PcV and in none taking PcVsr (P < 0.001). These initial saliva concentrations were short-lasting, but in some subjects 50 to 100 times higher than those following the peak concentration in plasma, i.e. at 40 min or more after swallowing. From 40 min and onwards the saliva concentrations of penicillin were very similar for the two formulations. The elimination of high initial saliva concentrations may diminish ecological disturbances of the mouth flora as well as removing the unpleasant taste of penicillin. The plasma concentrations of penicillin were similar for the two formulations throughout the 6-h sampling period and the mean ratio of the area under the plasma concentration-time curve was 99% for PcVsr in relation to PcV, the 90% confidence interval being 86-115%. The corresponding values for the maximum plasma concentration were 108% and 93 127%. The time to maximum concentration was 45 min for PcVsr and 41 min for PcV. Thus, with regard to standard criteria which are based on systemic (plasma) concentrations, the formulations were bioequivalent despite the substantial difference in initial local (saliva) concentrations. CONCLUSION: Saliva-resistant coating of tablets can prevent oral release of penicillin without affecting the plasma concentrations. From a clinical point of view both local and systemic equivalence should be established before bioequivalence is assumed.

Determination of penicillin-V in human plasma by high-performance liquid chromatography and solid-phase extraction.

A high-performance liquid chromatographic method has been developed for the determination of penicillin-V concentrations between 0.1 and 19 micrograms/ml in human plasma. Penicillin-V was isolated from plasma by solid-phase extraction on a C18/OH cartridge. The extracts were injected onto a reversed-phase HPLC system. A 125 x 4 mm C18 column was used to separate penicillin-V from its main metabolites, 5R- and 5S-penicilloic acid and endogenous compounds. The eluent consisted of 66% 0.02 M phosphoric acid buffer, to which tetrabutylammonium dihydrogenphosphate and 34% acetonitrile were added. The column effluent was monitored by ultraviolet spectrophotometry at 269 nm. Using this method, penicillin-V concentrations in plasma could be determined with an accuracy between -5.4 and 5.2% and a precision between 0.8 and 1.6%. The method has proved to be reliable and was used in bioavailability studies for the development of a new oral penicillin-V formulation.

Penetration of penicillin V to tonsillar surface fluid in healthy individuals and in patients with acute tonsillitis.

In the treatment of group A streptococcal tonsillitis, as the bacteria are located on the epithelial surface, an important determinant of outcome is the concentration of penicillin in extracellular tonsillar surface fluid. Accordingly, we investigated the concentration of penicillin in serum, and penetration to tonsillar surface fluid and saliva in nine patients with acute group A streptococcal tonsillitis and in nine healthy controls. Among the healthy subjects, despite high serum penicillin concentrations (mean, 2.04 micrograms/ml), there was no penetration to tonsillar surface fluid or to saliva, whereas erythromycin penetrated to tonsillar surface fluid in 3/6 cases. Of the nine patients with acute tonsillitis, on the first day of treatment eight manifested high concentrations of penicillin in tonsillar surface fluid (mean, 0.34 micrograms/ml--i.e. well above the minimal inhibitory concentration (MIC) for group A streptococci), but penetration to saliva was found in only two patients. On the tenth day of treatment, penicillin was not present in the saliva of any of the patients and was present in the tonsillar surface fluid of only one. The results suggest that measurable concentrations of penicillin in tonsillar surface fluid can only be obtained in the presence of inflammation with fluid exudation through the tonsillar epithelium.

Evaluating the minimum active concentrations of penicillin G and V and of retard forms in clinical conditions.

Following several signals indicating the inefficiency of the clinical treatment with various penicillin preparations in some cases, we decided to study the seric penicillin concentrations in the patients hospitalized in the "V. Babes" Hospital of Infectious Diseases, after administration of the various Romanian made forms of penicillin currently used in the therapy of streptococcal infections and in the prophylaxis of the sequelae of these infections. The data obtained on groups exceeding 30 persons by using two methods of determining the penicillin concentrations the dilutions and the diffusimetric methods revealed protective penicillin seric levels satisfactory for penicillin G and Efitard, according to the present treatment schemes. After 5 days from Moldamin administration only 45.4% of children and 43.3% of adults were found to have satisfactory penicillin concentrations. The administration of penicillin V reaches active penicillin concentrations in terms of the dose administered. The paper points out only one of the causes which together with others (such as beta-lactamase production and tolerance), contribute to the unsuccessful treatment with various forms of penicillin.

Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization.

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C18 solid-phase extraction column. After elution with 500 microliters of elution solution, the penicillins are derivatized with 500 microliters of 1,2,4-triazole-mercuric chloride solution at 65 degrees C for 30 min. The penicillin-mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C18 column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.

Pharmacokinetics of rheumatic fever prophylaxis regimens.

The pharmacokinetics of benzathine penicillin G (1,200,000 IU given intramuscularly), penicillin V (250 mg given orally twice daily), erythromycin stearate (250 mg given orally twice daily) and roxithromycin (150 mg given orally once daily) were investigated. The drugs were given prophylactically to prevent the recurrence of rheumatic fever to 20 patients attending a rheumatic fever clinic in a study of crossover design. Serum antibiotic concentrations were determined by microbiological assay at intervals of up to 28 days after intramuscular injection and immediately before and 1-2 h after the fifth oral dose. The concentrations of penicillin G in all serum samples obtained on day 28 after parenteral benzathine penicillin G administration were greater than 0.01 mg/dl. Most patients had no detectable penicillin V or erythromycin in blood samples drawn immediately before the fifth dose. The concentration of roxithromycin at 24 h after the dose was greater than 1.2 mg/dl in all patients. Based on the pharmacokinetic profiles, it is suggested that 1,200,000 IU benzathine penicillin G given every 4 weeks is an appropriate regimen for preventing the recurrence of rheumatic fever in Thai adults. Roxithromycin had much better pharmacokinetics than penicillin V and erythromycin stearate, and is probably the best alternative regimen to intramuscular penicillin G.

Concentration of phenoxymethylpenicillin in tonsillar tissue.

Seventeen patients underwent tonsillectomy 55-120 min after receiving oral phenoxymethylpenicillin 12.5 mg/kg bodyweight. The penicillin concentration in serum and tonsillar tissue in each patient was determined by a microbial assay. In 9 patients with tonsillar hyperplasia, but with no prior history of tonsillitis, the mean penicillin concentration in serum and in tonsillar tissue was 5.0 mg.l-1 and 1.32 mg.g-1, respectively, and in 8 patients with manifest tonsillitis the corresponding values were 3.01 mg.l-1 and 0.67 mg.g-1. It appears that the penicillin concentration in tonsillar tissue is about 1/5 to 1/4 of that in serum, regardless of whether the tonsils are inflamed or hypertrophied.

Bioavailability of a new liquid formulation of penicillin V (Isocillin syrup) and the influence of ethanol on bioavailability of penicillin V.

In an open, randomized four-way crossover study, four different formulations of penicillin V each containing 1.2 mega units were tested: A) new liquid formulation of Isocillin syrup, B) new liquid formulation of Isocillin syrup plus ethanol, C) commercially available penicillin V syrup containing ethanol as solubilizer and D) Isocillin film tablets. Six male and eight female healthy volunteers participated in the study. Serum concentration and urinary excretion of penicillin V were measured by HPLC or bioassay. The new liquid formulation of Isocillin syrup showed highest Cmax and AUC values. Correspondingly, urinary excretion of the new liquid formulation of Isocillin syrup was highest (38% of the given dose). Mean pharmacokinetic data of the new liquid formulation of Isocillin syrup were: tmax (h) = 0.81, Cmax (mg/l) = 9.92, AUC0-8h (mgh/l) = 13.67, lambda z (h) = 0.68, Cltot/f (ml/min) = 964. Ethanol (4.6%) had no absorption-enhancing effects. From a clinical point of view, all four formulations investigated in this trial showed serum concentrations well above bactericidal concentrations (greater than 1 mg/l) for more than 3 h. Although the interindividual variability of serum levels was high, the levels observed would guarantee therapeutic efficacy against penicillin sensitive bacterial strains.

[Penicillin VK absorption during fasting and after eating].

The absorption of penicillin VK (Rafapen) was evaluated in 12 children, 3-16 years.old, with infections due to penicillin-sensitive organisms. In each, 50 mg/kg of Rafapen was given after an overnight fast, and the next day the same amount was administered after a standard hospital breakfast. Blood samples were drawn before and 30, 60 and 90 min after the drug was given. Serum penicillin levels were determined by comparing the inhibition of growth on plates of Staphylococcus aureus (Oxford strain), with that of standard penicillin G concentrations. The serum levels after the overnight fast were higher, 4.75 U/ml, as compared to 3.38 U/ml 30 min after the drug was given following breakfast. This increase was abolished at 60 and 90 min, when the serum levels were 1.96 and 1.05 U/ml, respectively. We advise the use of oral penicillin VK in infections due to penicillin-sensitive organisms, without regard to feeding schedule.

[Comparative study of the penetration of penicillin V, amoxicillin, cefaclor and josamycin in the tonsils]. / Etude comparée de la pénétration de la pénicilline V, de l'amoxicilline, du céfaclor et de la josamycine dans les amygdales.

Fourty patients undergoing tonsillectomy for recurrent tonsillitis were administered penicillin V, amoxicillin, cefaclor or josamycin. Antibiotic concentrations in serum and tonsillar tissues were determined by microbiological assay. Cefaclor demonstrated a superior diffusion than penicillin V and amoxicillin, but nevertheless inferior to that of josamycin.

Efficacy of orally administered penicillin V for prophylaxis of experimentally induced streptococcal endocarditis.

Four oral penicillin V regimens were compared for the ability to prevent Streptococcus sanguis infection of experimentally induced valvular heart lesions in rabbits. Challenge doses of 10(4), 10(6), and 10(8) CFU of a penicillin-susceptible strain of S. sanguis were used in this study. Measured by recovery of test organisms from endocardial lesions, the lowest-concentration inoculum was infective for 53% of the recipients; the higher-concentration inocula were infective for all recipients. A single-oral-dose penicillin V regimen (36 mg/kg of body weight) prevented endocarditis when rabbits were challenged with 10(4) CFU, but protection diminished with increasing inoculum concentrations. In contrast, addition of a second penicillin V dose (18 mg/kg of body weight) administered with a 7-h interval between doses achieved fully effective prophylaxis against even the highest inoculum tested (10(8) CFU). A repeated set of experiments in which half the dose of penicillin V was administered showed significantly reduced protection against S. sanguis endocarditis.

Ethanol and the absorption of oral penicillin in man.

The effect of alcohol on the pharmacokinetics of phenoxymethylpenicillin was studied in six healthy volunteers. Ethanol had no influence on peak penicillin serum concentrations, times of the penicillin peaks, elimination half-lives (t1/2), AUC0-8h values and 24-h urinary excretion.

Effect of phenoxymethylpenicillin and erythromycin prophylaxis on anaerobic bacteraemia after oral surgery.

The effect of antibiotic prophylaxis on bacteraemia was assessed in 60 oral surgery operations. Fifty-one patients were divided in three groups receiving a 2 g dose of phenoxymethylpenicillin, a 0.5 g dose of erythromycin or no prophylaxis. During operation, the total numbers of bacteria were lower in the antibiotic groups than in the non-treatment groups (P less than 0.05). The number of patients with anaerobic bacteraemias, however, were similar in all three groups. The incidences of bacteraemia 10 min after operation were significantly lower in the antibiotic groups (P less than 0.05). The minimum inhibitory concentrations (MICs) of phenoxymethylpenicillin were below 2 mg/l in 93% of the strains and the minimum bactericidal concentrations (MBCs) were below this value in 80% of the strains. The MICs of erythromycin were below 2 mg/l in 80% of the isolates, and the MBCs were between 4-32 mg/l in 62% of the isolates.