Development, validation and application of a novel HPLC-MS/MS method for the measurement of minocycline in human plasma and urine.

New treatments are urgently required to treat infections caused by multi-drug resistant Acinetobacter baumanni,. To address this need, a new formulation of Minocin®, (minocycline for injection) has been developed that allows for higher doses of minocycline to be administered. Phase 1 clinical trials were conducted in healthy volunteers to assess the safety and pharmacokinetics (PK) of this new formulation at higher doses. In order to generate PK data, novel, selective and simple HPLC-MS/MS based assays were developed and validated for the determination of minocycline (MC) in human plasma and urine. The respective working ranges were 0.05 to 30 mg/L and 0.1 to 30 mg/L. Removal of endogenous proteins with trichloroacetic acid was used as a simple means of extracting MC from the samples. An analogue, tetracycline was used as the internal standard (IS). Chromatographic separation, including that of MC from its 4-epimer (4-EMC), was achieved on a Waters XBridge BEH C18 column (50 x 4.6 mm ID, 5 µm) with gradient elution. The mobile phases comprised water containing 5 mM ammonium formate at a pH of 2.5, and methanol containing 5 mM ammonium formate. The internal standard (IS) was tetracycline, a structural analogue of minocycline. The methods were fully validated and met regulatory acceptance criteria for intra-run and inter-run accuracy and precision, carryover, dilution integrity and matrix effects. Mean extraction recoveries ranged between 64.3% and 84.6% for MC and 64.3% for the IS. There was no significant ion suppression or enhancement for MC or the IS. The validated assays were successfully applied to 1423 plasma and 689 urine samples from a Phase 1 clinical study. There was no evidence of instability, or significant interconversion between MC and 4-EMC, in stored clinical samples, spiked plasma and urine samples, or their extracts, under various test conditions.

Effect of feeding on the pharmacokinetics of oral minocycline in healthy adult horses.

Minocycline is commonly used to treat bacterial and rickettsial infections in adult horses but limited information exists regarding the impact of feeding on its oral bioavailability. This study's objective was to compare the pharmacokinetics of minocycline after administration of a single oral dose in horses with feed withheld and with feed provided at the time of drug administration. Six healthy adult horses were administered intravenous (2.2 mg/kg) and oral minocycline (4 mg/kg) with access to hay at the time of oral drug administration (fed) and with access to hay delayed for 2 hr after oral drug administration (fasted), with a 7-day washout between treatments. Plasma concentration versus time data was analyzed based on noncompartmental pharmacokinetics. Mean ± SD bioavailability (fasted: 38.6% ± 4.6; fed: 15.7% ± 2.3) and Cmax (fasted: 1.343 ± 0.418 µg/ml; fed: 0.281 ± 0.157 µg/ml) were greater in fasted horses compared to fed horses (p < .05 both). Median (range) Tmax (hr) in fasted horses was 2.0 (1.5-3.5) and in fed horses was 5.0 (1.0-8.0) and was not significantly different between groups. Overnight fasting and delaying feeding hay 2 hr after oral minocycline administration improve drug bioavailability and thus plasma concentrations.

Pharmacokinetics of tigecycline in turkeys following different routes of administration.

The aim of this research had been to determine the pharmacokinetics of tigecycline (TIG) in turkey after intravenous (i.v.), intramuscular (i.m.), subcutaneous (s.c.), and oral (p.o.) administration at a dose of 10 mg/kg. TIG concentrations in plasma were determined using high-performance liquid chromatography with tandem mass spectrometry. Mean concentrations of TIG in turkey plasma in the i.v. group were significantly higher than concentrations of this drug obtained after using the other administration routes. No significant differences were demonstrated in respect to the concentrations achieved after i.m. and s.c. administration. The bioavailability of TIG after i.m., s.c., and p.o. administration was 32.59 ± 5.99%, 34.91 ± 9.62%, and 0.97 ± 0.57%, respectively. Values of half-life in the elimination phase were 23.49 ± 6.51 hr, 25.42 ± 4.42 hr, and 26.62 ± 5.19 hr in i.v., i.m., and s.c. groups, respectively, values of mean residence time were 7.92 ± 1.41 hr, 19.62 ± 2.82 hr, and 17.55 ± 2.59 hr in i.v., i.m., and s.c. groups, respectively, whereas the volume of distribution was 14.85 ± 5.71 L/kg, 14.68 ± 2.56 L/kg, and 15.37 ± 3.00 L/kg in i.v., i.m., and s.c. groups, respectively. Because TIG is not absorbed from the gastrointestinal tract in turkeys to a clinically significant degree, this drug given p.o. could find application in commercial turkey farms only to treat gastrointestinal tract infections.

Determination of tigecycline in human plasma by LC-MS/MS and its application to population pharmacokinetics study in Chinese patients with hospital-acquired pneumonia.

A selective, sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of tigecycline (TGC) in human plasma, using tigecycline-d9 as an internal standard (IS). Analytical samples were prepared using a protein precipitation method coupled with a concentration process. The analyte and IS were separated on a reversed-phase Waters Acquity UPLC® BEH-C18 column (2.1 × 50 mm i.d., 1.7 µm) with a flow rate of 0.25 mL/min. The mobile phase consisted of water, containing 0.2% formic acid (v/v) with 10 mm ammonium formate (A) and acetonitrile (B). The mass spectrometer was operated in selected reaction monitoring mode through electrospray ionization ion mode using the transitions of m/z 586.2 → 513.1 and m/z 595.1 → 514.0 for TGC and IS, respectively. The linearity of the method was in the range of 10-5000 ng/mL. Intra- and inter-batch precision (CV) for TGC was <9.27%, and the accuracy ranged from 90.06 to 107.13%. This method was successfully applied to the analysis of samples from hospital-acquired pneumonia patients treated with TGC, and a validated population pharmacokinetic model was established. This developed method could be useful to predict pharmacokinetics parameters and valuable for further pharmacokinetics/pharmacodynamics studies.

The presence of minocycline in the tear film of normal horses following oral administration and its anticollagenase activity.

PURPOSE: Tetracyclines have activity against matrix metalloproteinases (MMP). Oral medications with effects on the ocular surface are of interest in patients where repeated topical dosing is limited. The aim of this study was to characterize the concentration of minocycline in the tears of normal horses after oral administration and to determine if this level directly inhibits MMP activity. METHODS: Five healthy adult ponies were administered oral minocycline (Wedgewood Pharmacy; Swedesboro, NJ) at 4 mg/kg every 12 h for 5 days. Tears were collected at T = 2, 26, 50, 56, 74, 80, and 98 h. Tear minocycline concentrations were analyzed using high performance liquid chromatography. The inhibition of recombinant human MMP-2 and MMP-9 by minocycline was investigated using fluorescence resonance energy transfer. RESULTS: Minocycline was present in the tears of each pony at every measurement but with interpony variability. A mean concentration of 11.8 µg/mL was present 2 h after administration of the first dose. Minocycline did not directly inhibit MMP-2 or MMP-9 function at a concentration achieved in the pony tear film. CONCLUSIONS: Minocycline was present in the tears of all ponies at each sampling point following oral administration. One pony of the five had consistently lower levels of minocycline secretion (P ≤ 0.05). The concentration secreted in the tears did not directly inhibit MMP-2 or MMP-9 when tested in vitro. The inconsistencies in the tear concentration and the inhibition activity suggest topical application may be necessary to attain direct inhibition of MMP with minocycline.

Minocycline in Acute Cerebral Hemorrhage: An Early Phase Randomized Trial.

BACKGROUND AND PURPOSE: Minocycline is under investigation as a neurovascular protective agent for stroke. This study evaluated the pharmacokinetic, anti-inflammatory, and safety profile of minocycline after intracerebral hemorrhage. METHODS: This study was a single-site, randomized controlled trial of minocycline conducted from 2013 to 2016. Adults ≥18 years with primary intracerebral hemorrhage who could have study drug administered within 24 hours of onset were included. Patients received 400 mg of intravenous minocycline, followed by 400 mg minocycline oral daily for 4 days. Serum concentrations of minocycline after the last oral dose and biomarkers were sampled to determine the peak concentration, half-life, and anti-inflammatory profile. RESULTS: A total of 16 consecutive eligible patients were enrolled, with 8 randomized to minocycline. Although the literature supports a time to peak concentration (Tmax) of 1 hour for oral minocycline, the Tmax was estimated to be at least 6 hours in this cohort. The elimination half-life (available on 7 patients) was 17.5 hours (SD±3.5). No differences were observed in inflammatory biomarkers, hematoma volume, or perihematomal edema. Concentrations remained at neuroprotective levels (>3 mg/L) throughout the dosing interval in 5 of 7 patients. CONCLUSIONS: In intracerebral hemorrhage, a 400 mg dose of minocycline was safe and achieved neuroprotective serum concentrations. However, oral administration led to delayed absorption in these critically ill patients and should not be used when rapid, high concentrations are desired. Given the safety and pharmacokinetic profile of minocycline in intracerebral hemorrhage and promising data in the treatment of ischemic stroke, intravenous minocycline is an excellent candidate for a prehospital treatment trial. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01805895.

Determination of tigecycline in turkey plasma by LC-MS/MS: validation and application in a pharmacokinetic study.

Tigecycline (TIG), a novel glycylcycline antibiotic, plays an important role in the management of complicated skin and intra-abdominal infections. The available data lack any description of a method for determination of TIG in avian plasma. In our study, a selective, accurate and reversed-phase high performance liquid chromatography-tandem mass spectrometry method was developed for the determination of TIG in turkey plasma. Sample preparation was based on protein precipitation and liquid-liquid extraction using 1,2-dichloroethane. Chromatographic separation of TIG and minocycline (internal standard, IS) was achieved on an Atlantis T3 column (150 mm × 3.0 mm, 3.0 µm) using gradient elution. The selected reaction monitoring transitions were performed at 293.60 m/z → 257.10 m/z for TIG and 458.00 m/z → 441.20 m/z for IS. The developed method was validated in terms of specificity, selectivity, linearity, lowest limit of quantification, limit of detection, precision, accuracy, matrix effect, carry-over effect, extraction recovery and stability. All parameters of the method submitted to validation met the acceptance criteria. The assay was linear over the concentration range of 0.01-100 µg/ml. This validated method was successfully applied to a TIG pharmacokinetic study in turkey after intravenous and oral administration at a dose of 10 mg/kg at various time-points.

Comparison of Omadacycline and Tigecycline Pharmacokinetics in the Plasma, Epithelial Lining Fluid, and Alveolar Cells of Healthy Adult Subjects.

The steady-state concentrations of omadacycline and tigecycline in the plasma, epithelial lining fluid (ELF), and alveolar cells (AC) of 58 healthy adult subjects were obtained. Subjects were administered either omadacycline at 100 mg intravenously (i.v.) every 12 h for two doses followed by 100 mg i.v. every 24 h for three doses or tigecycline at an initial dose of 100 mg i.v. followed by 50 mg i.v. every 12 h for six doses. A bronchoscopy and bronchoalveolar lavage were performed once in each subject following the start of the fifth dose of omadacycline at 0.5, 1, 2, 4, 8, 12, or 24 h and after the start of the seventh dose of tigecycline at 2, 4, 6, or 12 h. The value of the area under the concentration-time curve (AUC) from time zero to 24 h postdosing (AUC0-24) (based on mean concentrations) in ELF and the ratio of the ELF to total plasma omadacycline concentration based on AUC0-24 values were 17.23 mg · h/liter and 1.47, respectively. The AUC0-24 value in AC was 302.46 mg · h/liter, and the ratio of the AC to total plasma omadacycline concentration was 25.8. In comparison, the values of the AUC from time zero to 12 h postdosing (AUC0-12) based on the mean concentrations of tigecycline in ELF and AC were 3.16 and 38.50 mg · h/liter, respectively. The ratio of the ELF and AC to total plasma concentrations of tigecycline based on AUC0-12 values were 1.71 and 20.8, respectively. The pharmacokinetic advantages of higher and sustained concentrations of omadacycline compared to those of tigecycline in plasma, ELF, and AC suggest that omadacycline is a promising antibacterial agent for the treatment of lower respiratory tract bacterial infections caused by susceptible pathogens.

Simultaneous UHPLC-UV analysis of hydroxychloroquine, minocycline and doxycycline from serum samples for the therapeutic drug monitoring of Q fever and Whipple's disease.

A fast UHPLC-UV method was developed for the simultaneous analysis of Hydroxychloroquine, Minocycline and Doxycycline drugs from 100µL of human serum samples. Serum samples were extracted by liquid-liquid extraction and injected into a phenyl hexyl reverse phase column. Compounds were separated using a mobile phase linear gradient and monitored by UV detection at 343nm. Chloroquine and Oxytetracycline were used as internal standards. Lower and upper limits of quantifications, as well as the other levels of calibration, were validated with acceptable accuracy (<15% deviation) and precision (<15% coefficient of variation) according to the European Medicines Agency guidelines. This new method enables cost and time reduction and was considered suitable for the clinical laboratory. It is the first published assay for the therapeutic drug monitoring of patients diagnosed with Q fever or Whipple's disease.

Population Pharmacokinetics of Tigecycline in Critically Ill Patients with Severe Infections.

We sought to describe the population pharmacokinetics of tigecycline in critically ill patients and to determine optimized dosing regimens of tigecycline for different bacterial infections. This prospective study included 10 critically ill patients given a standard dose of tigecycline. Blood samples were collected during one dosing interval and were analyzed using validated chromatography. Population pharmacokinetics and Monte Carlo dosing simulations were undertaken using Pmetrics. Three target exposures, expressed as ratios of the 24-h area under the curve to MICs (AUC0-24/MIC), were evaluated (≥17.9 for skin infections, ≥6.96 for intra-abdominal infections, ≥4.5 for hospital-acquired pneumonia). The median age, total body weight, and body mass index (BMI) were 67 years, 69.1 kg, and 24.7 kg/m2, respectively. A two-compartment linear model best described the time course of tigecycline concentrations. The parameter estimates (expressed as means ± standard deviations [SD]) from the final model were as follows: clearance (CL), 7.50 ± 1.11 liters/h; volume in the central compartment, 72.50 ± 21.18 liters; rate constant for tigecycline distribution from the central to the peripheral compartment, 0.31 ± 0.16 h-1; and rate constant for tigecycline distribution from the peripheral to the central compartment, 0.29 ± 0.30 h-1 A larger BMI was associated with increased CL of tigecycline. Licensed doses were found to be sufficient for Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and methicillin-resistant Staphylococcus aureus for an AUC0-24/MIC target of 4.5 or 6.96. For a therapeutic target of 17.9, an increased tigecycline dose is required, especially for patients with higher BMI. The dosing requirements of tigecycline differ with the indication, with pathogen susceptibility, and potentially with patient BMI.

Pharmacokinetics and Pharmacodynamics of Minocycline against Acinetobacter baumannii in a Neutropenic Murine Pneumonia Model.

Multidrug-resistant (MDR) Acinetobacter baumannii is increasingly more prevalent in nosocomial infections. Although in vitro susceptibility of A. baumannii to minocycline is promising, the in vivo efficacy of minocycline has not been well established. In this study, the in vivo activity of minocycline was evaluated in a neutropenic murine pneumonia model. Specifically, we investigated the relationship between minocycline exposure and bactericidal activity using five A. baumannii isolates with a broad range of susceptibility (MIC ranged from 0.25 mg/liter to 16 mg/liter). The pharmacokinetics of minocycline (single dose of 25 mg/kg of body weight, 50 mg/kg, 100 mg/kg, and a humanized regimen, given intraperitoneally) in serum and epithelial lining fluid (ELF) were characterized. Dose linearity was observed for doses up to 50 mg/kg and pulmonary penetration ratios (area under the concentration-time curve in ELF from 0 to 24 h [AUCELF,0-24]/area under the concentration time curve in serum from 0 to 24 h [AUCserum,0-24]) ranged from 2.5 to 2.8. Pharmacokinetic-pharmacodynamics (PK-PD) index values in ELF for various dose regimens against different A. baumannii isolates were calculated. The maximum efficacy at 24 h was approximately 1.5-log-unit reduction of pulmonary bacterial burdens from baseline. The AUC/MIC ratio was the PK-PD index most closely correlating to the bacterial burden (r2 = 0.81). The required AUCELF,0-24/MIC for maintaining stasis and achieving 1-log-unit reduction were 140 and 410, respectively. These findings could guide the treatment of infections caused by A. baumannii using minocycline in the future. Additional studies to examine resistance development during therapy are warranted.

The complexity of minocycline serum protein binding.

Objectives: Serum protein binding is critical for understanding the pharmacology of antimicrobial agents. Tigecycline and eravacycline were previously reported to have atypical non-linear protein binding; the percentage of free fraction decreased with increasing total concentration. In this study, we extended the investigation to other tetracyclines and examined the factors that might impact protein binding. Methods: Different minocycline concentrations (0.5-50 mg/L) and perfusion media (saline, 0.1 M HEPES buffer and 0.1 and 1 M PBS) were examined by in vitro microdialysis. After equilibration, two dialysate samples were taken from each experiment and the respective antimicrobial agent concentrations were analysed by validated LC-MS/MS methods. For comparison, the serum protein bindings of doxycycline and levofloxacin were also determined. Results: The free fraction of minocycline decreased with increasing total concentration, and the results depended on the perfusion media used. The trends of minocycline protein binding in mouse and human sera were similar. In addition, serum protein binding of doxycycline showed the same concentration-dependent trend as minocycline, while the results of levofloxacin were concentration independent. Conclusions: The serum protein bindings of minocycline and doxycycline are negatively correlated with their total concentrations. It is possible that all tetracyclines share the same pharmacological property. Moreover, the specific perfusion media used could also impact the results of microdialysis. Additional studies are warranted to understand the mechanism(s) and clinical implications of serum protein binding of tetracyclines.

Meropenem for treating KPC-producing Klebsiella pneumoniae bloodstream infections: Should we get to the PK/PD root of the paradox?

The objective of this study was to assess the achievement of pharmacokinetic/pharmacodynamic (PK/PD) targets of meropenem (MEM) in critically-ill patients with bloodstream infections (BSI) due to Klebsiella pneumoniae-carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) with MEM minimum inhibitory concentrations (MICs) ≥16 mg/L. Nineteen critically-ill patients with KPC-Kp BSI were given combination therapy including MEM, tigecycline, plus colistin or gentamicin (according to susceptibility testing). MEM was administered as an extended 3-hour infusion of 2 g every 8 hours, or adjusted according to renal function. MEM plasma concentrations were determined by high-performance liquid chromatography. PK/PD targets for MEM were defined as T > 40% 1×MIC and T > 40% 4×MIC. Possible synergisms between MEM and coadministered agents were assessed by time-kill assays based on plasma levels for MEM and on fixed plasma concentrations for the other agents. In none of 19 patients MEM reached any PK/PD target. The actual MEM MICs were 256, 512, and 1024 mg/L in 1, 3, and 15 isolates, respectively. However, theoretically, the PK/PD target of T > 40% 1×MIC could have been achieved in 95%, 68%, 32% and 0% of the isolates for MIC equal to 8, 16, 32, and 64 mg/L, respectively. No synergisms were observed between MEM and coadministered agents. In conclusion, high-dose MEM failed to reach PK/PD targets in 19 patients with BSI due to KPC-Kp with very high MEM MICs. On a theoretical basis, our results suggest a possible usefulness of MEM against resistant blood isolates with MICs up to 32 mg/L.

Development and validation of an LC-MS/MS method for the determination of tigecycline in human plasma and cerebrospinal fluid and its application to a pharmacokinetic study.

Tigecycline (TGC) is an important antibiotic in treating various drug-resistant bacteria. The dosage regimen for cerebral intraventricular TGC is still unknown. The aim of the study was to develop and validate liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the determination of TGC in human plasma and cerebrospinal fluid (CSF) to obtain an applicable regimen. The ion transitions under ESI positive model were performed at m/z 586.3 > 513.2 and m/z 595.3 > 514.3 for TGC and d9-TGC internal standard (IS). For plasma and CSF samples, the calibration curve of TGC was linear within the ranges 25-2000 and 250-100,000 ng/mL; the IS normalized matrix effect was within the ranges 96.46-101.06% and 101.13-103.58%, respectively, for all. TGC was stable under all tested conditions. The patient received 1 mg intraventricular and 49 mg intravenous administration of TGC. The AUC0-12 in plasma and CSF calculated according to our noncompartment model were 4713 and 23,0238 h ng/mL, respectively. Given our findings cerebral intraventricular TGC may be a choice for clinicians to treat drug-resistant Gram-negative bacterial-induced meningitis and the safety and efficacy of this administration route warrants further study.

Effect of feeding on the pharmacokinetics of oral minocycline in healthy research dogs.

BACKGROUND: The effect of food on minocycline oral absorption in dogs is unknown. OBJECTIVE: The objective was to determine the pharmacokinetics of minocycline after administration of a single oral dose in fed and fasted dogs. METHODS: Ten research hounds were administered oral minocycline (approximately 5 mg/kg) with and without food, in a crossover study, with a one-week wash-out between treatments. Blood samples were collected immediately prior to minocycline administration and over 24 h. Minocycline plasma drug concentrations were measured using high-performance liquid chromatography using ultraviolet detection and were analysed with compartmental modelling to determine primary pharmacokinetic parameters. Each dog was analysed independently, followed by calculation of means and variation of the dogs. The Wilcoxon signed-rank test [analysing secondary pharmacokinetic parameters - peak concentration (CMAX ), area under the concentration versus time curve (AUC)] was used to compare the two groups. A population pharmacokinetic modelling approach was performed using nonlinear mixed effects modelling of primary parameters for the population as fixed effects and the difference between subjects as a random effect. Covariate analysis was used to identify the source of variability in the population. RESULTS: No significant difference was found between treatments for AUC (P = 0.0645), although AUC was higher in fasted dogs. A significant difference was found for CMAX (P = 0.0059), with fasted dogs attaining a higher CMAX . The covariate of fed versus fasted accounted for a significant variation in the pharmacokinetics. CONCLUSIONS AND CLINICAL IMPORTANCE: Because feeding was a significant source of variation for the population's primary pharmacokinetic parameters and fasted dogs had higher minocycline concentrations, we recommend administering minocycline without food.

Identification of Acinetobacter baumannii serum-associated antibiotic efflux pump inhibitors.

Adaptive antibiotic resistance is a newly described phenomenon by which Acinetobacter baumannii induces efflux pump activity in response to host-associated environmental cues that may, in part, account for antibiotic treatment failures against clinically defined susceptible strains. To that end, during adaptation to growth in human serum, the organism induces approximately 22 putative efflux-associated genes and displays efflux-mediated minocycline tolerance at antibiotic concentrations corresponding to patient serum levels. Here, we show that in addition to minocycline, growth in human serum elicits A. baumannii efflux-mediated tolerance to the antibiotics ciprofloxacin, meropenem, tetracycline, and tigecycline. Moreover, using a whole-cell high-throughput screen and secondary assays, we identified novel serum-associated antibiotic efflux inhibitors that potentiated the activities of antibiotics toward serum-grown A. baumannii. Two compounds, Acinetobacter baumannii efflux pump inhibitor 1 (ABEPI1) [(E)-4-((4-chlorobenzylidene)amino)benezenesulfonamide] and ABEPI2 [N-tert-butyl-2-(1-tert-butyltetrazol-5-yl)sulfanylacetamide], were shown to lead to minocycline accumulation within A. baumannii during serum growth and inhibit the efflux potential of the organism. While both compounds also inhibited the antibiotic efflux properties of the bacterial pathogen Pseudomonas aeruginosa, they did not display significant cytotoxicity toward human cells or mammalian Ca(2+) channel inhibitory effects, suggesting that ABEPI1 and ABEPI2 represent promising structural scaffolds for the development of new classes of bacterial antibiotic efflux pump inhibitors that can be used to potentiate the activities of current and future antibiotics for the therapeutic intervention of Gram-negative bacterial infections.

Minocycline pharmacokinetics and pharmacodynamics in dogs: dosage recommendations for treatment of meticillin-resistant Staphylococcus pseudintermedius infections.

BACKGROUND: Although minocycline is not licensed for use in dogs, this tetracycline has therapeutic potential against meticillin-resistant Staphylococcus pseudintermedius. HYPOTHESIS/OBJECTIVES: The aim of this study was to establish rational dosage recommendations for minocycline use in dogs. Specific objectives were to generate and analyse minocycline pharmacokinetic (PK) data on plasma and interstitial fluid (ISF) concentrations, plasma protein binding and pharmacodynamic (PD) data on antimicrobial activity against S. pseudintermedius. ANIMALS: Six healthy dogs from a research colony were used in this study. METHODS: Dogs were administered 5 mg/kg intravenously and 10 mg/kg orally (p.o.) of minocycline hydrochloride in separate crossover experiments. In vivo drug concentrations in plasma and in ISF collected by ultrafiltration were measured by high-performance liquid chromatography. Pharmacokinetic analysis was performed on plasma and ISF concentrations. PK/PD analysis was completed using in vitro data on plasma protein binding and minocycline susceptibility in 168 S. pseudintermedius isolates. RESULTS: Minocycline distributed to the ISF to a higher degree than predicted by the protein-unbound fraction in plasma. A large volume of distribution after oral administration, with plasma and ISF elimination half-lives of 4.1 and 7.4 h, respectively, demonstrated that the ISF serves as a drug reservoir for sustained tissue concentrations. Monte Carlo simulation, used to assess target attainment at different drug dosages, indicated that p.o. administration of 5 mg/kg twice daily is sufficient to inhibit S. pseudintermedius strains with minimal inhibitory concentrations ≤0.25 µg/mL. CONCLUSIONS AND CLINICAL IMPORTANCE: Besides dosage recommendations for therapy of meticillin-resistant Staphylococcus pseudintermedius infections in dogs, the study also provides PK/PD data necessary to consider species-specific clinical breakpoints for minocycline susceptibility testing.

Quantitative analysis and pharmacokinetics study of tigecycline in human serum using a validated sensitive liquid chromatography with tandem mass spectrometry method.

Tigecycline, a novel intravenously administered glycylcycline antibiotic, currently plays a key role in the management of complicated multiorganism infections. However, current liquid chromatography with tandem mass spectrometry methods briefly describe parameters and the only reported internal standard was sometimes difficult to obtain. In our study, an updated liquid chromatography with tandem mass spectrometry method for the quantitative analysis of tigecycline in human serum was developed. Sample preparation involved precipitation with 20% trichloroacetic acid. Chromatographic separation of tigecycline and tetracycline (internal standard) was achieved on a Hypersil GOLD C18 column using gradient elution. The selected reaction monitoring transitions were performed at m/z 586.1→513.2 for tigecycline and m/z 445.1→410.2 for tetracycline. The assay was linear over the concentration range of 5-2000 ng/mL. The intra- and interday precisions at three concentration levels (10, 100, and 1600 ng/mL) were <15% and their accuracies were within the range of 95.1-106.1%. The mean recovery ranged from 94.3 to 105.6% and the matrix effect from 92.1 to 97.6%. Tigecycline was stable under all tested conditions. This validated method was successfully applied to a pharmacokinetic study in critically ill patients. The data demonstrated that our method allows quantification of tigecycline in serum in a quick and reliable manner for widespread application.

Effect of sucralfate on oral minocycline absorption in healthy dogs.

Sucralfate and minocycline may be administered concurrently to dogs. The relative bioavailability of tetracyclines may be reduced if administered with sucralfate, but studies confirming these interactions in dogs are not available. This study evaluated the pharmacokinetics of oral minocycline in dogs (M), determined the effects of concurrent administration of sucralfate and minocycline (MS) on minocycline pharmacokinetics, determined the effects of delaying sucralfate administration by 2 h (MS+2) on minocycline pharmacokinetics, and established dosing recommendations based on pharmacodynamic indices. Oral minocycline (300 mg) and sucralfate suspension (1 g) were administered to five greyhounds in a randomized crossover design. Minocycline plasma concentrations were evaluated using liquid chromatography with mass spectrometry. The maximum plasma concentration (CMAX ) and area under the curve (AUC) of minocycline were 1.15 µg/mL and 8.0 h* µg/mL, respectively. The CMAX and AUC were significantly lower (P < 0.05) in the MS group (CMAX  = 0.33 µg/mL, AUC 3.0 h*µg/mL) compared with M or MS+2 (CMAX = 0.97 µg/mL, AUC 10.3 h*µg/mL). Delaying sucralfate by 2 h did not decrease oral minocycline absorption, but concurrent administration significantly decreased minocycline absorption. A dose of 7.5 mg/kg p.o. q12 h achieves the pharmacodynamic index for a bacterial minimum inhibitory concentration (MIC) of 0.25 µg/mL (AUC:MIC≥33.9).

Reassessment of tigecycline bone concentrations in volunteers undergoing elective orthopedic procedures.

The goal of the this study was to re-evaluate tigecycline bone concentrations in subjects undergoing elective orthopedic surgery, using multiple doses and a more robust bone assay than was used in a previous study. Each subject received three intravenous doses of tigecycline (one 100-mg infusion followed by two 50-mg infusions, each administered over 30 minutes). A single bone sample was collected from each subject at one of the following times: 1, 2, 4, 6, 8, or 12 hours after the third dose. Four blood samples were collected from each subject: before the first dose, before and after the third dose, and within 15 minutes of the collection time of the bone sample. Noncompartmental pharmacokinetic analysis serum and bone area under the curve for the given dose interval (AUCτ ) values were 2,402 ng h/mL and 11,465 ng h/g, and maximum concentration (Cmax ) values were 974 ng/mL and 2,262 ng/g, respectively. The bone to serum ratio calculated using the AUCτ values was 4.77, confirming tigecycline penetration into bone.