A Study On The Penetration Of Levofloxacin To Bronchi And Lung Tissue In The Clinical Patients

The evidence from the investigation on Pharmacokinetics/Pharmacokinetics profile of Levofloxacin(LVFX),an analogue of fluoroquinolones shows that this agent exerts its bactericidal effect in concentration fashion,i.e.the its effects against pathogens increases with its concentration.The two variables,the ratios of C_max(plasma peak Concentration) to MIC (Minimal bacteria-Inhibitory Concentration),C_max/MIC,and AUC_0-24h(Area Under time-plasma concentration Curve from zero to 24 hours) to MIC,AUC_0-24h/MIC are the most key parameters associated with the its therapeutic efficacy,bacterial eradication and prevention from bacterial resistance.The public investigations suggested that the values of C_max/MICs for fluoroquinolones should achieve up to a range of 5～10 to prevent Streptococcus pneumonia from the resistance. The,the common dose regime is 500 mg,daily once in the US and Europe.However,the dose regime used is 200 mg,twice daily in China and 100 mg,thrice daily in Japan,respectively. Based on the MIC₉₀ value of 1μg/mL for LVFX against Streptococcus pneumoniae,the C_max/MICs values associated with the two later dose regimes in the above are less than 5 which cannot achieve the level for resistance prevention.Although in the recent,the dose regime of LFVX was switched to that of 500 mg,once daily domestically,there are no reports on the bacteria-inhibitory or bactericidal effects of the drug concentration with this therapeutic regime in biologic fluid of the patients with lower respiratory tract in China or other countries.This study is aimed to investigate the profile of LVFX penetration into bronchi and lung tissue fluid and to provide an evidence for a rational therapeutic regime for LVFX in lower respiratory infections.This study consists of two parts as follows:1.To establish a HPLC method appropriate to detect in quantity the concentration of LVFX in plasma,bronchoalyeolar lavage fluid(BALF),lung and bronchia mucous membrane;2.To investigate the profile of LVFX into bronchi and lung tissue fluid Part 1 The HPLC of determination of concentration of Levofloxain in biologic samplesA HPLC(High Performance Liquid Chromatography) method appropriate to detect the concentration of LVFX in plasma,BALK sputum,lung tissue and bronchia mucous membrane was established.The HPLC system consists of a TSK-gel ODS-80TM C₁₈(4.6mm^* 150mm,5μm) column,and mobile fluid containing 50mM KH₂PO₄(pH2.0)-THF-1mMCH₃COONH₄(92/7/1, V/V/V).A fluorescence detector with 296nm/504nm of Ex/Em was used and the flow rate was 1.0 ml/min.A solid extraction was used for extraction of drug components in the biologic samples.Results:(1) the components from blank plasma,BALK sputum,lung tissue and bronchi menbrance did not interfere with the peaks of LVFX and internal reference;(2) the extraction recovery of LVFX were(99.21±1.28)%for plasma,(100.7±1.37)%for BALF, (99.76±0.95)%for sputum,(101.4±2.28)%for lung tissue and(100.4±1.25)%for bronchia membrane,respectively;(3) A linear relationship of LVFX concentration in the samples of plasma,BALF,sputum and biologic tissue extractive fluids to peak area in the concentration of 0.010～5.000μg/ml was shown(r＞0.999),respectively.The lowest quantity limit was 0.010μg/mL for all biologic samples;(4) The relative standard deviations for intra-day were 2.9%or less for plasma,4.5%or less for BALF,4.3%or less for sputum and 1.9%or less for other biologic tissue extractive fluids,respectively;(5) The relative standard deviations for inter-days were 2.5%or less for plasma,6.6%or less for BALK 6.7%or less for sputum and 5.0%or less for other biologic tissue extractive fluids,respectively;(6) the relative recoveries for the above biologic samples were(97.3～101.9)%,(97.3～101.3)%,(99.9～103.2)%and(96.3～102.9)%, respectively;(7) The LVFX samples of plasma,BALF,sputum and other biologic tissue extractive fluids were stable within 3 hours in room temperature,4 hours in room temperature post pre-treatment and 2 repeats of frozen-throwing under -40℃.Conclusion:The HPLC used in this study for determination of LVFX concentration in plasma,lung and bronchi tissue fluids has sufficient precision and accuracy.Its sensitivity,specificity and stability is appropriate to determine the drug concentration in plasma,BALF,sputum,lung and bronchia membrane samples.Part 2:The penetration of LVFX into lung and bronchi tissuesThis study was conducted under approval of The Ethic Committee of Huashan Hospital affiliated to FuDan University with corresponding informed consent.The potential candidates were screened based on the pre-specified inclusion and exclusion criteria of the protocol.A written informed consent sheet must be provided by each subject before any procedure of protocol.The study population was defined as:(1) the patients with lower respiration infection who needed to receive fiberoptic bronchoscope examination and bronchoalveolar lavage procedure;(2) the patients receiving lung surgery.All subjects received orally a single 500-rag tablet of LVFX in no-fasting status.The time of oral drug depended on the time-point of sampling. The patients with lower respiratory tract were randomized into 5 time-point groups of each 8～10subjects.Prior to the examination with bronchoscope,the patient administrated orally one 500-mg tablet of LVFX.The blood samples and simultaneous BALF,as well as sputum were collected at pre-specified time-point before and after dosing.The patients who required lung surgical procedure were assigned randomly into 4 time-point groups of each 8～10 subjects. These patients each received a single 500-mg tablet of LVFX before operation.The blood samples were collected at the pre-specified time-point before and after dosing,but the lung and bronchia samples only at the time-point post dosing.The verified HPLC mentioned in the above was conducted to detect the concentration of LVFX in plasma,BALF,sputum and tissue-extracted fluids from lung and bronchia membrane samples.The demographic and baseline data of the patients were analyzed statistically.The average concentration in plasma,BALF, sputum and tissue-extracted fluids from lung and bronchia membrane samples at each time-point was calculated and the highest concentration among these average values was served as the C_max. The AUC_0-24h were calculated.Based on the above PK variables and pharmacodynamic(PD) parameters of LVFX on the pathogen infecting lower respiratory tract,the PK/PD values, including C_max/MICs and AUC_0-24h/MICs for plasma and other biologic tissue extractive fluids were calculated for evaluation of 500 mg dose regime of LVFX in lower respiratory infections. This study recruited 72 patients in total.Among them,the patients with the lower respiratory infection and bronchoalveolar lavage procedure were 40,and other 32 patients needed to receive lung surgical procedure.In 40 patients with bronchoalveolar lavage procedure,male was 27,and female was 13.The average age in these patients was 56±17(21～82) years old,and average Creatinine Clearance(Ccr) was 92.47±38.64(39.33～195.8) mL/min.The major diagnoses of the lung infections were pneumonia and pleural effusion associated with the original disease of lung cancer or chronic bronchitis,etc.No significant difference was found in the gender distribution,ages,weights,Ccr values,simultaneously concomitant drug therapy,and smoking status among 5 time-point groups of lh,4h,8h,12h and 24h post dosing(p＞0.05) exception of the body temperature(p＜0.05).Among the 32 patients receiving lung surgical procedure,20 were male,and 12 were female.The ages were 56±12(23～80) years old,and Ccr values were 92.20±20.89(51.73±141.2) mL/min.The major diseases were lung cancer concomitantly with hypertension or diabetes,etc.No significant difference was found in the 4 time-point groups of 4h,8h,12h and 24h post dosing regarding the gender distribution,ages,weights,Ccr values, concomitant drug therapy and smoking history(p＞0.05).In the 5 time-point groups of 1h,4h,8h,12h and 24h of bronchoalveolar lavage procedure patients,LVFX concentrations(mean±SD,n=8 for each group) were 3.339±3.004μg/mL at 1h time-point,4.057±1.485μg/mL at 4h,2.124±1.106μg/mL at 8h,1.899±0.637μg/mL at 12h and 0.926±0.606μg/mL at 24h,respectively in plasma;3.438±3.694μg/mL,2.345±1.970μg/mL, 1.641±1.506μg/mL,0.953±0.928μg/mL and 0.867±0.723μg/mL,respectively in ELF, 0.843±1.438μg/g,1.377±2.948μg/g,0.394±0.427μg/g,0.118±0.160μg/g and 0.078±0.035μg/g, respectively in sputum.The relative values of LVFX concentrations in ELF and sputum to simultaneous those in plasma were 0.788±0.432 for 1h,0.583±0.455 for 4h,0.725±0.322 for 8h, 0.572±0.596 for 12h and 1.043±0.898 for 24h,respectively,in ELF,and 0.614±0.846 for 1h, 0.223±0.384 for 4h,0.203±0.236 for 8h,0.074±0.116 for 12h and 0.149±0.149 for 24h, respectively,in sputum.The C_max,values were 4.066μg/mL for plasma,3.438μg/mL for ELF and 1.377μg/g for sputum.The AUC_0-24h were 50.12μg·h/mL in plasma,34.51μg·h/mL in ELF and 9.49μg·h/g in sputum.In 4 time-point groups of 4h,8h,12h and 24h of lung-surgical procedure patients,the LVFX concentration(mean±SD,n=8 for each group) were 3.621±1.141μg/mL for 4h, 3.400±1.288μg/mL for 8h,1.809±0.761μg/mL for 12h and 0.469±0.153μg/mL for 24h, respectively,in plasma;6.413±4.652μg/g for 4h,6.080±3.678μg/g for 8h,3.723±1.929μg/g for 12h and 1.591±0.551μg/g for 24h,respectively,in lung samples;8.742±7.574μg/g for 4h, 6.796±3.461μg/g for 8h,7.073±4.887μg/g for 12h and 4.067±3.334μg/g for 24 h, respectively,in bronchia membrane samples.The relative concentration in lung and bronchia membrane samples to those simultaneously in plasma were 1.709±0.981 for 4h,1.797±0.879 for 8h,2.279±1.473 for 12 h and 3.573±2.001 for 24h,respective,in lung samples;and 2.161±1.095 for 4h,2.093±0.829 for 8h,4.168±2.485 for 12h and 9.018±8.045 for 24h, respectively,in bronchia membrane samples.The Cmax values were 6.413μg/g in lung samples and 8.742μg/g in bronchia membrane samples,respectively.The AUC_0-24h were 89.30μg·h/g in lung samples and 143.1μg·h/g in bronchia membrane samples,respectively.The above results indicated that following a single oral administration of 500-mg tablet of LVFX,drug concentration achieved the highest in bronchia membrane samples,and next,as descending order,was in lung tissue samples,plasma,ELF and sputum.Based on the relative values to the simultaneous concentration in plasma,the extent of LVFX penetration was,as descending order,in bronchia membrane,lung,ELF and sputum.In general,the clinical and bacteriologic efficacy can be achieved only when the drug concentrations are up to the bacteria-inhibitory or bactericidal levels on the infected sites. Commonly,the drug concentrations on the infected sites are far lower than those in plasma, approximately 1/2 to 1/5 of the later.Only on local sites can 2-fold or more of drug concentration over MICs produce bacterial inhibition,and up to 5-flod or higher will not only produce bactericidal effects but also prevent from drug resistance.Strepotococcus pneumoniae, Hemophilus influenza,Moraxelle catarrhalis,Meticillin-Sensitive Staphylococcus aureus and Klebsiella pneumoniae are the most seen pathogens in the community-acquired lower respiratory infections,such as the community-acquired pneumonia,acute exacerbation of chronic bronchitis (AECB).LVFX C_max following a single oral dosing of 500-rag tablet can achieve 5 multiples more over its MIC₉₀ against Moraxelle catarrhalis in bronchia,lung tissue,ELF and sputum,and its effective bactericidal concentration will sustain for 24 hours post dosing except in sputum.For Hemophilus influenza,the drug C_max can also attain 5 multiples over its MIC₉₀ in bronchia membrane,ELF and lung tissues,and bactericidal concentrations in bronchia membrane and lung tissues will last until 24 hours post dosing,and in ELF,the effective level for inhibition or sterilization of Hemophilis influenza will maintain for 8 hours.In sputum,the C_max was 3 multiples higher than its MIC₉₀ values,but later the concentration declined down to the level lower than MIC₉₀ at 8 hours post dosing.This investigation also reveals that in all 4 kinds of biological fluids,LVFX C_max following the testing dose regime was up to 5-folds more over its MIC₉₀ on Meticillin-Sensitive Staphylococcus aureus,and its bactericidal level will sustain until 24 hours following dosing.Meanwhile,this C_max was also higher than its MIC₉₀(1μg/mL) on Strepotococcus pneumoniae or Klebsiella pneumoniae,up to 5-flods more.Its effective bactericidal concentration will last until 13 hours following oral administration,even though in ELF the ratio of C_max to MIC₉₀ was only 3 and in sputum the concentration was approximate to MIC₉₀ values.Klebsiella pneumoniae,Enterobacteriaceae,Acinetobacter and Pseudomonas aeruginosa are the major pathogens for the hospital-acquired lower respiratory tract infections following a single oral dosing of 500-mg tablets of LVFX,the Cmax in bronchia membrane,lung tissues,ELF and sputum achieved 5-folds more than its MIC90 against Acinetobacter.However,these values in the above biological samples are great lower than 16μg/mL of its MIC₉₀ for Pseudomonas aeruginosa,although they could higher than MIC against 50%of this pathogen according to the 0.5μg/mL of its MIC₅₀ values.This investigation on PK/PD predicts a satisfactory outcome in the treatment of Strepotococcus pneumoniae-,Hemophilusinfluenza-,Moraxelle catarrhalis-,Meticillin-Sensitive Staphylococcus aureus- orKlebsiella pneumoniae-induced community-acquired lower respiratory infections,as well as Klebsiella pneumoniae-induced hospital-acquired pneumonia with the therapeutic regime of LVFX 500 mg,once daily.