Efficacy of Biodegradable Lipid Glyceryl Monostearate Lipomer of Dosyclycline Against Brucella Abortus
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Date
2012
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MAFSU, Nagpur
Abstract
The efficacy of doxycycline hydrochloride (DH), doxycycline hydrochloride
lipomer (DH LNP), rifampicin (RIF), rifampicin entrapped in polymer Gantrez 119
(NP RIF) and the combinations of these drug formulations against B. abortus was
evaluated in vitro in U937 human macrophage cell line and in vivo in mice. All the
drug formulations were subjected to cytotoxicity assays, uptake studies and
estimation of minimum inhibitory concentration (MIC) before taking up efficacy
studies.
The assessment of cytotoxicity by MTT assay and trypan blue dye
exclusion technique revealed that the maximum non-toxic concentration of DH
was 5 μg/ml whereas that of DH LNP was 2 μg/ml. The maximum non-toxic
concentration of RIF was 10 μg/mL while that of NP RIF 5 μg/mL. The maximum
non-toxic concentration of combinations DH + RIF and DH LNP + NP RIF DH +
RIF was 20 μg/mL Both the methods i.e. MTT assay and trypan blue dye
exclusion technique yielded similar results and were found equally suitable.
The minimum inhibitory concentration (MIC) of the drug formulations
against B. abortus was estimated by resazurin microtitre assay (REMA) and
broth microdilution method (BMM). The MIC of DH, DH LNP, RIF, NP RIF and
DH + RIF was 0.5 μg/ml while that of combination DH LNP + NP RIF was 0.25
μg/ml. The results of MIC produced by both the methods, REMA and BMM were
concordant with each other.
The studies on uptake of the nanoparticulate drugs by the macrophages
by fluorescence microscopy revealed that at higher doses the formulations were
taken up rapidly while the uptake was poor at lower concentrations. All the
formulations attained optimum intracellular concentration at 1 μg/ml between 30
to 60 min.
Evaluation of in vitro efficacy DH and DH LNP against B. abortus in
macrophages revealed that the reduction in log10 cfu/ml values achieved in DH
treated cells was slightly greater that that of DH LNP treated cells. The DH LNP
thus did not prove adequately effective in reducing the intracellular B. abortus. The RIF and NP RIF both caused significant reduction in the intracellular
B. abortus counts at all the concentrations tested. Comparison of log10 reduction
values by unpaired ‘t’ test revealed that the reduction in intracellular B. abortus
counts achieved by NP RIF was significantly greater than that of RIF at all the
doses ranging from 0.250 – 4 μg / ml. The results thus revealed a definite
superiority of NP RIF over RIF in clearance of intracellular B. abortus.
The combinations of DH + RIF and DH LNP + NP RIF were found
effective against B. abortus and caused significant reduction in intracellular
counts as compared to untreated control. The comparison of results of DH + RIF
and DH LNP + NP RIF unpaired ‘t’ test revealed that the log10 reductions
achieved in the cells treated DH LNP + NP RIF were significantly higher than
those of DH + RIF treated cells at all the concentrations tested.
Overall comparison of the values of log10 reductions achieved in response
to treatment with different drug formulations revealed that the greatest log10
reduction in intracellular B. abortus counts was evident in cells treated with NP
RIF followed by those treated with DH LNP + NP RIF. The drug formulations next
in order in terms of efficacy were plain RIF and combination of DH + RIF.
The evaluation of in vivo efficacy of DH and DH LNP in mice revealed that
the reduction in splenic B. abortus counts achieved with both the treatments was
almost equal. The DH LNP treatment thus did not prove superior to DH. The RIF
and NP RIF both caused reduction in mean log10 cfu per spleen values as
compared to untreated control mice. The comparison of log10 reduction between
RIF and NP RIF treated mice revealed that the reduction achieved in NP RIF
treated mice was significantly greater (over two fold) than that of RIF treated
mice. Evaluation of in vivo efficacy of combination RIF + NP RIF revealed that
there was a complete clearance of B. abortus from spleens of infected mice.
Comparison of log10 reductions achieved in spleens of mice treated RIF and RIF
+ NP RIF revealed that the reduction in RIF + NP RIF treated mice was
significantly higher (4.5294+ 0.001) causing complete clearance of B. abortus
from spleens. The combination RIF + NP RIF thus could be of potential value in
therapeutic management of brucellosis in animals and human. The future
research should focus on evaluation of RIF + NP RIF combination in clinical
cases of brucellosis in animals.
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