3.3 Application
The validated method was utilized to quantify the steady-state trough concentration of TGC in serum and CSF samples from 12 MDRAB CNSI patients treated with TGC. The quantification results were listed in Table 5. The concentrations of TGC in CSF and serum were quantified to be 16.35-53.56 ng/mL and 67.76-211.9 ng/mL respectively. The CSF-to-serum ratio was calculated according to the TGC concentration. For these 12 MDRAB CNSI patients, the CSF-to-serum ratio of TGC at steady-state trough concentration was ranged from 21.46% to 44.46%, and the mean value was 31.61 ± 8.13%. To the best of our knowledge, this is the first study to report the CSF-to-serum ratio of TGC at steady-state trough concentration based on samples from 12 MDRAB CNSI patients. This study evaluated the penetration of TGC into CSF for CNSI patient at the steady-state trough concentration. Correlation of TGC in CSF and serum samples from these patients was also evaluated in this study. The correlation curve was showed in Fig.2, and the correlation coefficient (r2) was 0.5065.
Disscussion
Generally, only small molecular (under the threshold 400-500Da) with high lipid solubility have potential to cross the BBB easily[12, 13]. Lots of effective drugs are restricted to treat neurological diseases because of their inability to penetrate the BBB [14]. CNSI caused by MDRAB has an increasing trend and high mortality. Owing to the drug-resistant ability of MDRAB and barrier function of BBB, few antibiotics are available for the treatment. TGC is known as an antibiotic that has activity against acinetobacter baumannii [15]. The ability of TGC to penetrate BBB is important for the possibility to treat MDRAB CNSI by TGC [16]. TGC is a compound with high water solubility (>100mg/mL) and protein binding rate (71-89%). These decide that TGC has little potential to cross the BBB. However, intracranial diseases such as cerebritis might facilitate the permeability of the BBB. The data about the CSF penetration of TGC is definitely deficient, especially for the patients diagnosed with CNSI. Rodvold et al . firstly reported CSF penetration of TGC in humans in 2006. CSF and serum samples were collected from 17 subjects without inflamed meninges within hours after a single 100mg dose of TGC. 11 samples were obtained between 0.92 and 2.1 h, and 6 samples were obtained between 18-24h. CSF-to-serum ratio obtained between 18 and 24 h was ranged from 0.33 to 0.52, which was much higher than those obtained from 0.92 and 2.1 h (0.016–0.095), and the CSF-to-serum AUC0-24 ratio was 0.11. The differences were caused by longer Tmax and slower clearance velocity of TGC in CSF than that in serum [8]. This study observed poor penetration of TGC in subjects without inflamed meninges. In 2007, Chen et al. used 100mg TGC every 12h to treat a patient with sepsis and complicated ventriculitis caused by MDR K. pneumoniae. CSF and blood samples were collected on the 12th day of therapy immediately prior to the scheduled dose of TGC (trough) and 30 minutes after completion of the infusion (peak) to evaluate the BBB penetration ratio of TGC at stay-state. Unfortunately, the analytical method with detectable limit of 0.05 μg/mL, was not able to determine the concentration of TGC in CSF [17]. Ray et al . also established an HPLC method with a detectable limit of 0.05 μg/mL to evaluate the CSF-to-serum ratio based on samples from a female patient with meningitis. However, all of the CSF concentrations in this study were below the lower limit of detection. Depending on observed peak area, they estimated the concentration of TGC in CSF was 0.035-0.042 μg/mL, and the penetration ratio of this patient was ranged from 0 to 0.52 [18]. Another case was reported by Pallotto et al . in 2014. In this report, a patient with CNSI caused by multidrug-resistant Enterococcus faecium was treated by TGC 100 mg intravenous as a loading dose, and followed by 50 mg intravenous every 12 h thereafter. For this patient, CSF-to-serum ratio at peak and trough concentration were 0.066 and 0.106 respectively, and CSF-to-serum AUC0-24 ratio was 0.067. The BBB penetration ratio was significantly lower than that in previous studies[6].
To our knowledge, these three case reports mentioned above were the only studies to describe BBB penetration ratio of CNSI patients. Two of these reports didn’t provide accurate data due to the limit of analytical methods. Moreover, the results of these reports were also quite different. Ray et al . showed that the TGC CSF concentrations of the patient with CNSI were higher than those of subjects without inflamed meninges in Rodvold’s study owing to compromised BBB. On the contrary, the CSF penetration ratio of patient with CNSI in the study of Pallotto were much lower than those of subjects without inflamed meninges in Rodvold’s study. Heterogeneity of these cases, such as dosage scheme, sample time and analytical methods, makes it difficult to compare and evaluate these results (Table 6). Consequently, it is important to establish a sensitive method to analyze TGC in CSF of CNSI patients accurately. Moreover, multiple subjects with same dosage scheme and sample time were also important to evaluate the penetration of TGC in CSF of patients with CNSI.
According to our study, the trough concentration of TGC in the CSF at stay-state was ranged from 16.35 to 53.56 ng/mL. The method we established with the LLOQ of 5 ng/mL was sensitive enough to quantify TGC in CSF. This mainly benefited from the high sensitivity of LC-MS/MS. And appropriate chromatographic condition of this method perform a narrow and sharp peak of TGC in 1.47 min, which made the method more sensitive and efficient.
For most results of previous studies, concentration of TGC in CSF was under 50 ng/mL, some researchers proposed that there might be a saturability of TGC to penetrate the BBB. In our study, the positive correlation between the concentrations of TGC in CSF and serum at trough concentration was observed obviously. This indicated that the concentration of TGC in CSF could be affected significantly by the concentration of TGC in serum, and the saturability of TGC in CSF was not observed at common dosage. The CSF-to-serum ratio of TGC for the patient with CNSI at trough concentration was ranged from 0.21 to 0.44. This result was consisted with the data (0.33-0.52) obtained from adults with non-inflamed meninges in the study of Rodvold et al . in 2006. Depending on present study, CNSI did not show the potential to increase the penetration ability of TGC to CSF. Though pathologic states caused by inflammation plays an important role to increase the permeability of BBB, other factors, such as up regulation of efflux transporters at minor damage and high protein binding rate of some drugs, also might restrict the permeation of drugs[13].