[Show abstract][Hide abstract] ABSTRACT: The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuT(Aa)) has been used as a model for mammalian Na(+)/Cl(-)-dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuT(Aa) liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homology modeling and docking studies. Two approaches were used subsequently to differentiate between three clusters of potential docking poses: 1) a diagnostic SERT(Y95F) mutation, which greatly reduced the affinity for [(3)H]imipramine but did not affect substrate binding; 2) competition binding experiments in the presence and absence of carbamazepine (i.e., a tricyclic imipramine analog with a short side chain that competes with [(3)H]imipramine binding to SERT). Binding of releasers (para-chloroamphetamine, methylene-dioxy-methamphetamine/ecstasy) and of carbamazepine were mutually exclusive, but Dixon plots generated in the presence of carbamazepine yielded intersecting lines for serotonin, MPP(+), paroxetine, and ibogaine. These observations are consistent with a model, in which 1) the tricyclic ring is docked into the outer vestibule and the dimethyl-aminopropyl side chain points to the substrate binding site; 2) binding of amphetamines creates a structural change in the inner and outer vestibule that precludes docking of the tricyclic ring; 3) simultaneous binding of ibogaine (which binds to the inward-facing conformation) and of carbamazepine is indicative of a second binding site in the inner vestibule, consistent with the pseudosymmetric fold of monoamine transporters. This may be the second low-affinity binding site for antidepressants.
Full-text · Article · Dec 2010 · Molecular pharmacology
[Show abstract][Hide abstract] ABSTRACT: Duramycin (Moli1901) is being developed for the treatment of reduced mucociliary clearance in cystic fibrosis. This study was conducted to estimate lung residence time and systemic exposure and to assess whether duramycin causes an inflammatory response. Six volunteers were administered a single dose (7.5 mg) of nebulized duramycin and underwent bronchoscopies to obtain a composite data set for pharmacokinetic analysis; duramycin was measured in the cellular fraction of bronchoalveolar lavage fluid (BALF) (mainly alveolar macrophages) and brush biopsies (bronchial epithelial cells). The estimated t1/2 of duramycin was ∼5 days in brush biopsies and 25 to 91 days in BALF cells. Levels of duramycin in BALF (C
max 800 ng/mg) exceeded those in brush biopsies by ∼20-fold. Duramycin was absent from plasma and did not cause any detectable inflammatory response in pulmonary tissue as judged from the BALF profile of 14 relevant cytokines. Our data suggest that duramycin qualifies for intrapulmonary administration in cystic fibrosis (CF) patients.
Full-text · Article · Jun 2008 · Archiv für Experimentelle Pathologie und Pharmakologie
[Show abstract][Hide abstract] ABSTRACT: To evaluate the effect of fosfomycin on proinflammatory cytokines, a bolus of 2 ng of bacterial lipopolysaccharide/kg of body
weight was injected intravenously into healthy volunteers. After 2 h, subjects received 8 g of fosfomycin or placebo in a
randomized crossover study design. The resulting concentrations of tumor necrosis factor alpha, interleukin-1β (IL-1β), and
IL-6 expressed as protein and mRNA levels were almost identical with and without fosfomycin.
Preview · Article · Jun 2007 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: Although a wide range of therapeutic strategies have been developed to improve the outcome of severe sepsis, a convincing reduction in mortality is lacking. Recently, increasing attention has been paid to immunomodulatory effects of antimicrobials. This study set out to explore the immunomodulatory effects of fosfomycin, a broad-spectrum antibiotic frequently used in septic patients, at the protein and molecular levels in vitro.
Whole blood from 11 healthy volunteers was incubated with 50 pg/mL endotoxin and 100 microg/mL fosfomycin or physiological sodium chloride for 4 h. Real-time RT-PCR was performed for various pro- and anti-inflammatory cytokines. Concentrations of tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 in the supernatant were measured using a commercially available ELISA.
Incubation of human leucocytes with endotoxin increased messenger RNA (mRNA) levels of cytokines several thousand fold compared with baseline. The addition of fosfomycin significantly inhibited mRNA levels of pro-inflammatory cytokines such as IL-1-alpha, IL-6 and TNF-alpha after 2 h (P < 0.01), while no significant reduction was observed for the anti-inflammatory cytokines IL-4, IL-10 and IL-13 (P = 0.26). At the protein level, the concentrations of IL-6 and TNF-alpha increased approximately 3000- and 600-fold after 4 h of incubation with lipopolysaccharide as compared with baseline, respectively. Addition of fosfomycin significantly reduced cytokine levels by 56% and 73% for IL-6 and TNF-alpha, respectively.
Fosfomycin extensively decreased mRNA levels and release of pro-inflammatory cytokines in human blood. The broad antimicrobial coverage of fosfomycin and its immunosuppressive effects could be clinically useful in patients with sepsis.
No preview · Article · Mar 2007 · Journal of Antimicrobial Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: Microdialysis is an increasingly employed technique for the determination of tissue pharmacokinetics. A high-performance liquid chromatography method for the quantitative determination of caspofungin in human microdialysates with amperometric detection is described. Since microdialysis of caspofungin is performed with a 100,000 molecular mass cut-off membrane, microdialysates contain protein that was precipitated at pH 4 with acetonitrile. Addition of 1-propanol (33%, v/v) to the sample extract improved the analytical recovery to 81-89%. Caspofungin and the internal standard clarithromycin were separated isocratically on a cyanopropyl silica column using acetonitrile-0.05 M citrate (33:67, v/v), adjusted to an apparent pH of 6.9, at a flow rate of 1.0 ml/min, and amperometric detection at +950 mV oxidation potential. Within-day and between-day imprecision and inaccuracy were <11%. The lower limit of quantification was 0.07 microg/ml. The method was applied to in vitro microdialysis experiments. Ringer's solution containing 1% (w/v) human albumin was used for the perfusing and surrounding medium, respectively. Albumin did not entirely prevent adsorption of caspofungin to the surface of membrane and/or tubing. When the binding-sites were saturated with albumin plus caspofungin prior to the start of sampling, the percentage of drug appearing in the microdialysate ("recovery") remained stable over the concentration range tested.
No preview · Article · Nov 2006 · Journal of Chromatography B
[Show abstract][Hide abstract] ABSTRACT: Desmopressin is usually administered intranasally in the treatment of central diabetes insipidus or nocturnal enuresis. The sublingual administration of desmopressin is expected to be an alternative to the intranasal route with advantages to children and to patients with allergic rhinitis or chronic rhinosinusitis. Therefore, the present study was carried out to explore the time-versus-concentration profile of desmopressin in plasma after sublingual administration to healthy volunteers.
A total of 16 healthy male volunteers were enrolled in this open, exploratory, 1-period, randomized, dose-escalation study. Volunteers received a single sublingual dose of either 20, 40, 80, 160, 240 or 320 microg of desmopressin acetate. Desmopressin plasma concentrations were measured over a 12-hour period using a validated radioimmunoassay method. Safety and tolerability were assessed simultaneously.
Plasma concentrations of desmopressin were below the lower limit of quantification (LLOQ) of 5 pg/ml for doses lower than 80 microg. For the doses of 160 - 320 microg the time-versus-concentration profiles were higher than the LLOQ. The area under the curve from 0 - 12 h (AUC0-12h) was 54.66 +/- 25.92 pg x h/ml after the 160 microg dose, 104.38 +/- 79.10 pg x h/ml following the 240 microg dose and 133.18 +/- 181.84 pg x h/ml following the 320 microg dose. Given the data from previous experiments, the time-versus-concentration profile of desmopressin in plasma after a sublingual dose of 240 microg appeared to be in the range of previously published data on an intranasal dose of 20 microg. Sublingual administration of desmopressin proved to be safe and was well tolerated by all volunteers.
A very high inter-individual variability in desmopressin plasma concentrations was detected after sublingual administration. A sublingual dose of 240 microg of desmopressin appeared to result in a pharmacokinetic profile comparable to 20 microg administered intranasally. These data, however, need to be verified in a separate well-designed prospective clinical study.
No preview · Article · May 2006 · International journal of clinical pharmacology and therapeutics
[Show abstract][Hide abstract] ABSTRACT: The present study was carried out to test bioequivalence between two different oral desmopressin formulations. Sixty healthy volunteers were enrolled in the study and were randomly assigned to receive the test (T) and reference (R) drug in a two-period two-sequence, crossover, analyst-blinded study design. Subjects received an oral dose of 400 mug of desmopressin acetate separated by a wash-out period of at least 7 days. The area under the concentration-time curve (AUC) over 12 h in plasma and the maximum concentration (C(max)) were compared by analysis of variance (ANOVA) after log transformation. The mean ratios of the T to R drug were within the bioequivalence boundaries with mean values of 1.00 (90% CI: 0.87-1.14) and 1.03 (90% CI: 0.92-1.15) for AUC(0-t) and AUC(0-inf), respectively. For the C(max), the mean ratio of the T to R drug was 0.97 (90% CI: 0.87-1.08). The rate and the extent of oral desmopressin absorption were identical for both formulations. Hence, the desmopressin test tablet met all bioequivalence criteria of the marketed reference desmopressin tablet.
[Show abstract][Hide abstract] ABSTRACT: Single nucleotide polymorphisms in the human multidrug-resistance gene ABCB1 have been reported to be associated with altered expression and function of P-glycoprotein, an efflux transporter, expressed at the blood-brain barrier. To test whether certain ABCB1 haplotypes contribute to interindividual differences in central nervous system drug distribution, brain distribution of a model P-glycoprotein substrate, the calcium channel inhibitor verapamil, was measured by positron emission tomography (PET) in 2 groups of healthy volunteers.
Ten homozygous carriers (cases) of the TTT haplotype (3435T, 1236T, and 2677T) and 10 controls homozygous for the wild-type CGC haplotype (3435C, 2677G, and 1236C) were administered a mean intravenous bolus of 412 +/- 114 MBq carbon 11-labeled verapamil containing less than 15 nmol of unlabeled verapamil. PET imaging of brain tissue and venous blood sampling were performed for 1 hour after dosing.
As a measure of brain penetration, the ratio of PET area under the time-radioactivity curve (AUC) to plasma AUC was calculated from time-radioactivity curves, with a mean ratio of 1.1 +/- 0.3 (SD) (95% confidence interval, 0.9-1.3) for cases and 1.1 +/- 0.2 (95% confidence interval, 0.9-1.2) for controls, respectively (P = .96). Mean brain AUC values were 31.2 +/- 3.9 and 35.7 +/- 5.7 for the TTT and CGC haplotype, respectively (P = .11). Plasma AUCs were not significantly different.
No difference in the brain distribution of [(11)C]verapamil could be detected in healthy volunteers differing in ABCB1 haplotypes.
[Show abstract][Hide abstract] ABSTRACT: The objective of the present study was to evaluate whether cefpirome, a member of the latest class of broad-spectrum cephalosporins,
sufficiently penetrates subcutaneous adipose tissue in septic patients. After the administration of the drug at 2 g, tissue
cefpirome concentrations in septic patients (n = 11) and healthy controls (n = 7) were determined over a period of 4 h by means of microdialysis. To assess the antibacterial effect of cefpirome at the
target site, the measured pharmacokinetic profiles were simulated in vitro with select strains of Staphylococcus aureus and Pseudomonas aeruginosa. The tissue penetration of cefpirome was significantly impaired in septic patients compared with that in healthy subjects.
For subcutaneous adipose tissue, the area under the concentration-versus-time curve values from 0 to 240 min were 13.11 ±
5.20 g · min/liter in healthy subjects and 6.90 ± 2.56 g · min/liter in septic patients (P < 0.05). Effective bacterial growth inhibition was observed in all in vitro simulations. This was attributed to the significantly
prolonged half-life in tissue (P < 0.05), which kept the tissue cefpirome levels above the MICs for relevant pathogens for extended periods in the septic
group. By consideration of a dosing interval of 8 h, the values for the time above MIC (T > MIC) in tissue were greater than 60% for pathogens for which the MIC was ≤4 mg/liter in all septic patients. The present
data indicate that cefpirome is an appropriate agent for the treatment of soft tissue infections in septic patients. However,
due to the high interindividual variability of the pharmacokinetics of cefpirome in tissue, dosing intervals of not more than
8 h should be preferred to ensure that susceptible bacterial strains are killed in each patient.
Full-text · Article · Mar 2005 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: Antimicrobial agents have to cross the boundary of at least one body compartment to reach their site of action. Many factors determine how effective equilibration of the drug between different body compartments occurs. Drug exchange ranges from a persisting gradient to complete tissue-to-plasma equilibrium. Accordingly, the antimicrobial concentration in the plasma compartment can be higher than the minimal inhibitory concentration (MIC) of the relevant pathogen and, at the same time, significantly lower than the required MIC value in a peripheral compartment. Subtherapeutic levels of antimicrobial agents might have a serious impact on the therapeutic outcome such as pathogen persistency with subsequent therapeutic failure and the development of drug-resistances. Certain body compartments are considered to be poorly accessable to antimicrobial agents such as the central nervous system (CNS), bone and cartilage tissue as well as ischemic and encapsulated tissues. If an infection is located in one of these compartments, the dosage of the administered antimicrobial agent has to be high enough to reach effective levels in the affected target compartment. Therefore, the selection of the optimal antimicrobial agent, the adequate dosage and its application intervals are absolutely essential to antimicrobial efficacy. This has to happen in consensus with the patient's individual state of disease, the clinical appearance of the infection, and pharmacokinetic characteristics of the targeted compartment. If the necessary dosage of the antimicrobial agent exceeds the maximal permitted values due to poor tissue penetration properties, either alternative drugs or alternative routes of drug administration have to be applied.
No preview · Article · Oct 2004 · Chemotherapie Journal
[Show abstract][Hide abstract] ABSTRACT: To test whether norepinephrine (NOR) affects tissue microcirculation and impairs plasma-to-tissue equilibration of antimicrobial agents.
Eight healthy male volunteers were enrolled to an analyst-blinded, randomized, two-period two-sequence crossover study. A single intravenous dose of 2 g of cefpirome was administered simultaneously with starting a continuous infusion of NOR (0.16 microg/kg per min) or placebo (PL) over 180 min. The microdialysis technique was used for the assessment of unbound cefpirome concentrations in skeletal muscle tissue and subcutaneous adipose tissue. Free plasma concentrations were related to corresponding tissue concentrations. Haemodynamics were determined by the measurement of mean arterial blood pressure (MAP), heart rate and forearm blood flow (FBF).
Area under the concentration-time-curve (AUC) values of cefpirome for interstitium and plasma were not significantly different between the PL and NOR groups (P > 0.47). Tissue penetration of cefpirome as described by the ratios of the AUCs from 0 to 180 min for tissue to the AUC values for plasma were 0.81 +/- 0.34 for the PL group and 0.80 +/- 0.26 for the NOR group (P > 0.05). Baseline values of MAP, heart rate and FBF were not significantly different between study days. MAP increased significantly following NOR administration from 73.3 +/- 3.5 mmHg at baseline to 94.0 +/- 5.2 mmHg during infusion (P = 0.017). NOR exerted no significant effects on FBF.
We have shown that intravenous administration of NOR does not exert a significant effect on peripheral blood flow and tissue penetration of cefpirome in healthy men. This might be attributed to systemic regulatory mechanisms, which probably fully compensate for major changes in blood flow in peripheral tissues.
Preview · Article · Mar 2004 · Journal of Antimicrobial Chemotherapy