Bactericidal activities of LB20304, a new fluoroquinolone

The time-kill curves of LB20304, a novel fluoroquinolone that has potent antibacterial activity against gram-positve and gram-negative bacteria, were calculated at the concentration of 1/4-, 1/2-, 1-, 2- and 4-times the MIC againstStaphylococcus aureus 77, Escherichia coli, 3739E,Pseudomonas aeruginosa 1912E. The bactericidal activity of LB20304 for these strains was very rapid and comparable to that of ciprofloxacin. LB20304 produced a decrease in the log₁₀ CFU per milliliter of ≥3 within 2 h at 4-times the MIC for all strains and consitently prevented regrowth of bacteria after 24 h of incubation. The MBCs (Minimal Bactericidal Concentration) of LB20304 against test organisms were equal to or at most four-times higher than the MICs.     

In vitro activities of LB20304, a new fluoroquinolone

Thein vitro activity of LB20304 was evaluated against clinical isolates and compared with those of Q-35, ciprofloxacin, sparfloxacin, lomefloxacin and ofloxacin. LB20304 demonstrated 16- to 64-fold more potent activity than ciprofloxacin against gram-positive bacteria. LB20304 inhibited 90% of the isolates of methicillin-susceptibleStaphylococcus aureus (MSSA) at a concentration of 0.016 μg/ml (MIC₉₀). MIC₉₀ values of LB20304 against methicillin-resistantStaphylococcus aureus (MRSA), methicillin-susceptibleStaphylococcus epidermidis (MSSE), methicillin-resistantS. epidermidis (MRSE) andStreptococcus pneumoniae were 2 μg/ml, 0.016 μg/ml, 0.5 μg/ml and 0.031 μg/ml, respectively. LB20304 was also very active against gram-negative bacteria. AgainstEscherichia coli, Klebsiella pneumoniae, Serratia marcescens, Pseudomonas aeruginosa andAcinetobacter calcoaceticus, MIC₉₀S of LB20304 were 0.031 μg/ml, 0.25 μg/ml, 2 μg/ml, 8 μg/ml and 0.5 μg/ml, respectively. Its activity was comparable to that of ciprofloxacin but much better than those of Q-35, sparfloxacin, ofloxacin and lomefloxacin. LB20304 also exhibited the most potent activity among quinolones tested against laboratory standard strains, ofloxacin-resistant strains, β-lactamase-producing strains and anaerobic strains. The inhibitory effect (IC₅₀) of LB20304 on DNA gyrase fromMicrococcus luteus, determined by the supercoiling assay, was 8-fold more potent than that of ciprofloxacin. LB20304 did not induce topoisomerase-associated DNA cleavage even at a concentration of 10 mg/ml, although ciprofloxacin induced DNA cleavage at a concentration of 1 mg/ml.     

Bacterial resistance to LB20304, a new fluoroquinolone antibiotic

In vitro studies were conducted to determine the frequency rate of spontaneous resistance to LB20304 and to dertemine whether cross-resistance to other antimicrobial agents develops. In eight strains of bacteria, the frequency of mutation to LB20304 at the concentrations of four and eight times the minimal inhibitory concentration (MIC) ranged from less than 4.0×10^?10 to 2.2×10^?8. These results were similar to those found for other new fluoroquinolones. The development of stepwise resistance was determined by repeated subculture in broth in the presence of increasing concentration of the compounds. Exposure of bacteria to increasing concentrations of LB20304 resulted in the selection of organisms with higher MICs. There were 4-to 128-fold increases in the MIC of LB20304 for bacterial strains ofStaphylococcus aureus, Streptococcus pneumoniae, Escherichia coli andPseudomonas aeruginosa. However, those strains, selected after repeated exposure were well within the susceptibility range for LB20304 except forPseudomonas aeruginosa. The resistant isolates selected with LB20304 showed cross-resistance when tested against ciprofloxacin andvice versa.     

High performance liquid chromatographic assay of a new fluoroquinolone,LB20304, in the plasma of rats and dogs

High-performance liquid chromatographic method was developed for the determination of LB 20304 (compound 1) in the plasma of rats and dogs. The analyte was deproteinized with 1 volume of methanol and 1/2 volume of 10% zinc sulfate, and the supernatant was injected onto a reversed-phase HPLC column. The mobile phase was a mixture of 24 parts of acetonitrile and 76 parts of 0.1% trifluoroacetic acid. The flow rate was 1 ml/min, and the effluent was monitored by fluorescence detector at an excitation wavelength of 337 nm and an emission wavelength of 460 nm. The retention time of compound 1 was 6.3 min. The assay of compound 1 was linear over the concentration range of 0.2–100 μl/ml in the plasma of rats and dogs. The lower limit of quantification was 0.2 μg/ml using 100 μl of plasma with a 97–99% accuracy and a 12–14% precision. In the 0.5, 5, and 50 μg/ml quality control samples, the intra- and inter-day accuracy were 88–95% and 88–97%, whereas intra- and inter-day precision were 0.5–6.6% and 0.2–9.3%, respectively, in the plasma of rats and dogs. The recoveries were 68–71% independent of concentration and species in the plasma. No interferences from endogenous substances were observed. Taken together, the above HPLC assay method by deproteinization and fluorescence detection was suitable for the determination of compound 1 in the preclinical pharmacokinetics.     

Factors affectingin vitro activity of LB20304, a new fluoroquinolone

LB20304 is a novel fluoroquinolone that exhibits a potent broad spectrum antibacterial activity against both gram-positve and gram-negative bacteria. The MICs (Minimal Inhibitory Concentration) of LB20304 were determined against both gram-positve and gram-negative bacteria under various conditions including several media, pHs, and inoculum concentrations. Thein vitro activity of LB20304 was not significantly affected by the changes in testing conditions such as components of media and inoculum concentrations, but it was slightly reduced by acid condition. The MICs and MBCs (Minimal Bactericidal Concentration) of LB20304 againstStaphylococcus aureus, Escherichia coli andPseudomonas aeruginosa were hardly affected by the presence of 50% human serum, mouse serum, guinea pig serum or horse serum, and the MBCs were equal to or at most four-times higher than the MICs. The activities of LB20304 were decreased by the presence of high concentraion of Mg⁺⁺ or human urine (pH, 5.5) in the test media. The frequencies of mutants resistant to LB20304 were similar to or lower than those found in ciprofloxacin and sparfloxacin.     

Pharmacokinetics and dose-dependency of LB30870 in rats, dogs, and monkeys

This study was to examine the pharmacokinetics of LB30870, a thrombin inhibitor, after IV and oral administration to rats, dogs, and monkeys. In rats and dogs, LB30870 showed linear pharmacokinetics after IV and oral administration. The oral bioavailability (BA) in rats was about 30 % with high inter-subject variability in the time to reach peak plasma concentration (T_max). Oral absorption of a solution and prototype tablet formulations of LB30870 were tested in dogs. T_max was 30 min and the BA values were 40.8–43.1 % with solution formulation. BA values after oral administration of the two tablet formulations at the dose of 100 mg/dog were 27.0 and 30.8 %. T_max were 60 min in the tablet formulation, indicating that the disintegration and dissolution of tablets caused delay in T_max compared to solution formulation. After IV administration of LB30870 to monkeys, the plasma concentrations decreased bi-exponentially and BA was 15.0 % after oral (20 mg/kg) dosing. In summary, linear pharmacokinetics of LB30870 were observed in both rats and dogs. The differences in BA among species could be due to difference in absorbed fraction and/or the first pass extraction (pre-systemic elimination) of LB30870.     

Pharmacokinetics, tissue distribution, and excretion of sitafloxacin, a new fluoroquinolone antibiotic, in rats, dogs, and monkeys

The pharmacokinetics, tissue distribution and excretion of sitafloxacin (CAS 127254-12-0, DU-6859a) were investigated in rats, dogs, and monkeys following single intravenous or single oral administration of 14C-labelled sitafloxacin at a dose of 4.69 mg/kg. Following single administration of the oral dose, serum concentrations of radioactivity peaked at 0.5 h in rats, 2.3 h in dogs, and 2.5 h in monkeys. The apparent absorption ratios of 14C-sitafloxacin based on the AUC0-infinity were 31%, 51%, and 93% in rats, dogs, and monkeys, respectively. In rats, the drug-related radioactivity had been distributed to most organs and tissues 30 min after oral dosing, and had been essentially eliminated after 24 h. The highest levels of radioactivity were observed in the kidneys and liver, whereas the concentrations in the cerebrum and spinal cord were much lower than the serum value. The urinary recoveries of radioactivity after intravenous dosing were 45.5 % in rats, 32.3 % in dogs, and 77.8 % in monkeys. In bile duct-cannulated rats, 57.8 % of the orally administered radioactivity was excreted in the bile within 48 h, and at least 45 % of the sitafloxacin-related material secreted in the bile was re-absorbed from the gastrointestinal tract. These results indicate that sitafloxacin is rapidly absorbed and widely distributed into various tissues. Sitafloxacin-related material is eliminated primarily through both renal and biliary excretion in rats, and possibly in dogs, whereas renal excretion is the major route of elimination in monkeys.     

Discovery of gemifloxacin (Factive, LB20304a): a quinolone of a new generation

Novel quinolone antibacterials, which bear an alkyloxime substituent in the 4-position and an aminomethyl substituent in the 3-position of the pyrrolidine ring, have been designed and synthesized. These fluoroquinolones were found to possess extremely potent antimicrobial activity against gram-positive organisms including resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA). Among these compounds our development candidate, Gemifloxacin (Factive, LB20304a), showed the best in vivo efficacy and pharmacokinetic profile in animals, as well as good safety pharmacological properties. Gemifloxacin was found to be especially effective against respiratory tract infections that account for over 70% of all infections. With once-a-day dosage, potency against respiratory tract infections such as chronic bronchitis and pneumonia was ensured without any significant side effect. In December 1999, Gemifloxacin filed a NDA for marketing approval to the US Food and Drug Administration.     

Pharmacokinetics of E-6087, a new anti-inflammatory agent, in rats and dogs.

The pharmacokinetics of E-6087, a newly developed cyclooxygenase-2 inhibitor, was studied in rats and dogs after single oral and intravenous doses. In both animal species, E-6087 was characterized by a long elimination half-life (20-35 h), a low plasma clearance (0.10-0.22 l h(-1) kg(-1)) and a relatively large volume of distribution (2-6 l kg(-1)). Oral bioavailability was lower in dogs than in rats whereas a faster elimination was found in rats. Multiple peaks were present regardless of administration route and animal species, suggesting the existence of enterohepatic circulation. Gender effect on the pharmacokinetics of E-6087 was only found in rats, with greater exposure and longer elimination in females than in males. Food intake reduced the bioavailability (approximately 22%) with no apparent changes in the absorption rate. After oral dosing of 1, 5 and 25 mg kg(-1) to rats, linearity was lost at the highest dose due to the low aqueous solubility of E-6087. Drug absorption was improved by micronization. E-6087 and E-6132, (a pharmacologically active metabolite), showed different pharmacokinetics. The higher percentage of E-6087 at early times suggests that E-6087 is the main compound responsible for in vivo activity, although E-6132 would contribute to the activity at later times.     

Pharmacokinetics of YM466, a new factor Xa inhibitor, in rats and dogs.

The pharmacokinetics of YM466, a selective inhibitor for factor Xa, was investigated after single intravenous and oral dosing to rats and dogs. After i.v. dosing, plasma YM466 concentration declined in a bi-phasic manner with a terminal elimination half-life of 1.4 h in rats and 0.8 h in dogs. Total plasma clearance values were 884 and 1212 ml/h/kg in rats and dogs, respectively. After oral dosing, plasma YM466 concentrations reached maximum within 2 h and increased in a dose-proportional manner in rats while increase was nonlinear in dogs. The absolute bioavailability of YM466 was 2.7-4.5% in rats, almost constant regardless of the dose levels investigated, while it was 6.9-24.6% in dogs, indicating nonlinear pharmacokinetics. The plasma protein binding of YM466 was 54.7-56.5% in rats and 45.2-49.0% in dogs and almost constant regardless of the concentration. No metabolism of YM466 was observed in an in vitro liver microsome study. These findings suggest that the low bioavailability of YM466 is attributable to the poor absorption not to the extensive metabolism.     

Pharmacokinetics of nilvadipine, a new dihydropyridine calcium antagonist, in mice, rats, rabbits and dogs

1. The pharmacokinetics of nilvadipine in male and female rats, and in male mice, rabbits and dogs were studied after i.v. and oral dosing. 2. After i.v. dosing (0.1 mg/kg), the plasma concentrations of nilvadipine declined two- or three-exponential with terminal half-lives of 0.73 h in mice, 1.2 h in male and female rats, 3.7 h in rabbits and 5.0 h in dogs. Sex difference in pharmacokinetics after i.v. dosing in rats was not found. The systemic plasma clearance was in the order of mice greater than rats greater than rabbits greater than dogs, and nearly equalled the hepatic blood flow in each species. The volume of distribution at steady-state was high (greater than 4 L/kg) in all species. 3. After oral dosing, plasma concentrations of nilvadipine peaked within 1 h in all species except for middle and higher doses (4 and 16 mg/kg) in dogs. The area under the plasma concentration-time curves in male rats (3.2-100 mg/kg) and dogs (1-16 mg/kg) increased in proportion to the dose. Bioavailability was low in male rats (3-4%) and rabbits (2%), but in other species was 29-44%. The oral clearance in male rats was about 8 times higher than in female rats. 4. The free fraction of nilvadipine in plasma was 1.94% in mice, 1.89% in rabbits and 0.85% in dogs, with no dependence on plasma concentration over a range of 10-100 ng/ml.     

Pharmacokinetics of nicorandil, a new coronary vasodilator, in dogs

The kinetic disposition of nicorandil, N-[2-( nitroxy )ethyl]-3- pyridinecarboxamide (1), and its main metabolic product, N-[2-(hydroxy)-ethyl]-3- pyridinecarboxamide (II), was studied after administering intravenous and oral doses (2.5 mg/kg) of nicorandil to the same beagle dogs. The plasma concentrations were measured using a high-performance liquid chromatographic method. The pharmacokinetic data derived from intravenous administration of nicorandil were: t1/2, 0.73 plus/minus 0.11 h; Vdarea , 0.67 plus/minus 0.04 L/kg; and total plasma clearance, 13.50 plus/minus 1.05 mL/min/kg. After oral administration, nicorandil was rapidly absorbed (tmax, 0.58 plus/minus 0.11 h). The oral bioavailability was calculated as 0.72 plus/minus 0.07. The metabolic formation of the corresponding alcohol after intravenous and oral administration of the parent compound appeared to occur quite efficiently, and its elimination half-life (3.09 plus/minus 0.25 and 3.69 plus/minus 0.88 h after intravenous and oral administration of nicorandil, respectively) was longer than that of the parent compound. Since the dose employed in this study was much higher than the expected therapeutic doses, whether such a good bioavailability after a lower dose of the drug would be obtained in humans remains unanswered.     

Pharmacokinetics of flunoxaprofen in rats, dogs, and monkeys

The kinetics of flunoxaprofen, an anti-inflammatory nonsteroidal drug, was studied in rats (Charles River), dogs (beagles), and monkeys (Macaca fascicularis). Plasma levels, after oral and iv administration of 20-40 mg/kg, and urinary excretion were followed for 24-72 h; the determinations were performed by gas chromatography. Levels in various organs and in rat bile were also determined. The pharmacokinetic parameters show noteworthy similarities in the three species studied: high bioavailability, extensive biotransformations with small urinary excretion of unmodified drug, total clearance between 40 and 50 mL/h/kg, and peak plasma levels of approximately 200 micrograms/mL. Rats show a high value in volume of distribution (2 L/kg), whereas dogs and monkeys show a volume of distribution between 0.13 and 0.18 L/kg. In the rat, the half-life of the drug is approximately 70 h, whereas in the dog and monkey, a half-life of approximately 2 h was found.     

Metabolism and excretion study of DW116, a new fluoroquinolone, in rats

Metabolite identification and urinary and biliary excretion of the new fluoroquinolone antibacterial agent DW116 [1-(5-fluoro-2-pyridyl)-6-fluoro-7-(4-methyl-1-piperazinyl)-1,4-dihydro-4-oxoquinoline-3-carboxylic acid, hydrochloride] after oral administration have been studied in Sprague-Dawley rats. The excretion kinetics were monoexponential. Most of the drug was eliminated via the hepatic and renal routes. Mean renal clearance of DW116 was 73.4 ml/hr/kg and mean biliary clearance was 83.8 ml/hr/kg. The major metabolite excreted in the bile was identified as the glucuronide ester of the parent drug using base-hydrolysis of the conjugate metabolite followed by co-HPLC with standard compound,¹⁹F-NMR and LC-MS methods. The glucuronide conjugate was also found in urine. The mean urinary recoveries of free and total (free plus glucuronide ester) DW116 were 28.6±2.7% and 36.4±1.8% of the administered dose and the corresponding biliary recoveries were 14.4±5.5% and 37.0±7.6%, respectively.     

Pharmacokinetics of besulpamide in rats and dogs

The pharmacokinetics of besulpamide were studied in rats and its urinary excretion in rats and dogs. Kinetic characteristics were the same for both male and female rats according to a two-compartment open model. Biological half-life was 1-4 hr, absorption half-life was approximately 10 min and absolute bioavailability was nearly complete (F = 86.4%). In rats, urinary excretion of unchanged besulpamide was 30% after i.v. administration and 7% after oral administration, whereas in dogs after oral administration it was 54%. Values of the area under the plasma concentration-time curve in rats and percent of urinary excretion in dogs show that the kinetics of besulpamide are not dose-related when the drug is administered at doses of 10-50 mg/kg.     

Pharmacokinetics of nizatidine in dogs and rats

1. Plasma levels of nizatidine and two metabolites (N-desmethyl nizatidine and nizatidine S-oxide) were studied in dogs and rats.

2. The time-courses of plasma concentration of nizatidine after i.v. and oral administration to dogs well fitted a two-compartment model. Absorption after oral administration was rapid, and the peak plasma level (C_max) and area under the plasma concentration-time curve (AUC) were proportional to doses. Absolute bioavailability was close to 100%, indicating that nizatidine has a negligible first-pass effect.

3. Nizatidine was eliminated by apparent first-order kinetics after i.v. administration to rats, and the elimination in rats was faster than in dogs. Bioavailability in rats was 72.4%, indicating a slight first-pass effect in rats.

4. When plasma clearance (Cl_p), or volume of distribution (V_d.β), of nizatidine were plotted against animal body weight on a log-log scale, good correlations were obtained for rats, dogs and humans.

5. Peak levels (C_max) of two metabolites were reached at 0.5–1.0?h after oral and i.v. administration of nizatidine to dogs. The elimination curves of the two metabolites were similar to that of nizatidine. Significant relationships were found between doses of nizatidine and C_max or AUC of both metabolites.     

Pharmacokinetics of fosfosal in rats and dogs

Fosfosal (Disdolen) is a non acetyl derivative of salicylic acid with antiinflammatory and analgesic properties and a greater tolerance than acetylsalicylic acid and lysine acetylsalicylate. In the present work the pharmacokinetics of fosfosal have been studied after oral and intravenous administrations of 100 and 80 mg/kg in rats and dogs, respectively. Plasma concentrations of fosfosal and salicylic acid were determined by an HPLC method. After intravenous administration fosfosal plasma levels decreased rapidly showing a half-life of 2.7 min in rats and 6.7 min in dogs. Fosfosal in plasma is quickly hydrolyzed into salicylic acid producing high concentrations in few minutes after administration. The half-life of salicylic acid was 13.8 and 7.1 h for rats and dogs, respectively. After oral administration only salicylic acid was detected in plasma, indicating that fosfosal, when orally administered, behaves as a prodrug. From the comparison of the AUC for salicylic acid obtained after oral and intravenous administrations it can be deduced that fosfosal it totally absorbed in the two species studied.     

Pharmacokinetics and absolute bioavailability of sitafloxacin,a new fluoroquinolone antibiotic,in healthy male and female Caucasian subjects

1. The aim was to compare the pharmacokinetics of sitafloxacin from a capsule formulation (dose of 500mg sitafloxacin) and an intravenous (i.v.) formulation infused over 1?h (dose of 400?mg sitafloxacin) in healthy male and female subjects and to estimate the absolute bioavailability of sitafloxacin from the capsule formulation. 2. Following oral administration, sitafloxacin was rapidly absorbed, with a mean maximum concentration in plasma (C max) of 4.65 μg ml -1 occurring at a median t max = 1.25?h giving a mean AUC(0- ∝) = 28.1 μg?h ml -1. For the i.v. administration, a mean C -max = 5.53 μ ml -1 occurred at the end of the 1-h infusion with a mean AUC(0- ∝) = 25.4μg?h ml -1. The mean terminal elimination half-life was 7.0?h (oral) and 6.6?h (i.v.). For the oral and i.v. formulations, the mean total plasma clearance was 296 and 263 ml?min -1, respectively and the mean volume of distribution was 180 and 150 litres, respectively. 3. Within 48h post-dose, 61% (range 26-86%) of the administered dose was excreted unchanged in urine following capsule administration, compared with 75% (range 42-101%) following the i.v. formulation. For both formulations, the renal clearance of sitafloxacin (means of 181 and 198 ml min-1 for the capsule and i.v. doses, respectively) implies active tubular secretion of the drug. 4. The absolute bioavailability of sitafloxacin from the capsule formulation was high at 89%, with a 95% CI of 84-94%. The intersubject variability (CV%) in the sitafloxacin AUC(0- ∝) for the capsule was low at 18.6%. 5. Gender differences in the pharmacokinetics of sitafloxacin were small and would not warrant dose adjustment. 6. The findings show that the capsule formulation offers good oral bioavailability and merits further clinical evaluation of sitafloxacin as an orally effective fluoroquinolone antibacterial.     

Pharmacokinetics of FK027 in rats and dogs

The pharmacokinetics of FK027, a new oral cephalosporin, were investigated in rats and dogs and compared with those of cefaclor, cephalexin and amoxicillin. Upon oral administration to either rats or dogs, FK027 produced higher and more sustained serum levels than the reference drugs, hence a longer half-life. After both oral and intravenous administration, the half-life of FK027 in dogs was approximately three fold that in rats. Although the concentrations of FK027 in rat kidney, liver and spleen were lower than those of cephalexin and amoxicillin, they were sustained similarly to the serum levels. The 24-hour urinary and biliary recovery rates of FK027 in rats after oral dosing with 100 mg/kg were 34.1 and 21.9%, respectively. The urinary excretion of FK027 was significantly lower than that of the reference drugs, however, the biliary excretion was higher. In dogs, 23.4 and 0.2% of the given dose of 40 mg/kg of FK027 was excreted in the 24-hour urine and bile, respectively. Bioavailability of FK027 after oral dosing was 38% in rats and 47% in dogs, as calculated from intravenous data. Binding of FK027 to serum protein in all species was the highest of the test drugs: 63% for human, 93% for dog, 61% for rat serum.