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].