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Vol. 12 (2) 169-176 April 2018, ISSN 0973-8916 (Print), 2230-7303 (Online)
time curve from time zero to time of last sample Bio-analytical HPLC method : The collected
intake (AUC0b t) and area under the moment curve- serum and tissue samples were analyzed by
time curve from time zero to time of last sample HPLC (Analytical technologies Ltd) comprising
intake (AUMC0b t) were calculated as per the C-18 column & UV detector. The mobile phase
trapezoid rule. The first moment was calculated consists of Triethanolamine acetate: acetonitrile
as concentration times time (Cp x t). The AUMC (97: 3% v/v) eluted by isocratic method and
is the area under the (Cp x t) versus time curve. detected at 262 nm, analysed at 30ºC by injecting
The AUC determines the bioavailability of the drug 20μl by maintaining a flow rate of 0.9ml/min. A
for the given same dose in the formulation. Total wash method program which increased the %
calculations were done by software Phoenix methanol was included at the end of drug
WinNonlin non-compartmental analysis program. elution to ensure washout of all interfering
exciepents. Spectral purity analysis of the
Sample preparation for analysis drugs peak over a range of 200–400 nm was
Blood sample: The blood samples collected at performed. The accuracy and precision of the
respective time intervals during pharmacokinetic developed method for determination of drug
and bio distribution studies were taken in was comparable to the isocratic methods
heparinised micro-centrifuges. Blood samples described in United State Pharmacopoeia (USP).
were centrifuged at 4000 rpm for 10 min to
separate plasma and it was maintained at -20ºC Data Analysis
until analysis. The area under the total plasma
Aliquots of 150μl of plasma were mixed with concentration time curve from zero time to infinity
methanol (300μl) as de-proteinizing agent and the was calculated by equation AUC0–” = area under
obtained dispersal is whirl pooled around 2 min. the plasma concentration-time curve extrapolated
The samples were centrifuged at 15000 RPM for to infinity
10min at 4°C and the supernatant is collected. AUC0–” = AUC0–t +Ct/Ke
Rifampicin was extracted with 3 ml of Where Ct is the rifampicin concentration observed
chloroform-butanol (70:30%v/v) and 3 ml portions at last time and Ke is the apparent elimination
of chloroform-butanol (70:30% v/v) and vortexed rate constant obtained from the terminal slope of
for about 1 min followed by centrifuging at 4000 the individual plasma concentration (12-18).
RPM of 10 min duration. The superficial layer was Durations of serum drug concentration following
decanted and the process was repeated for in inhalational route were analyzed by software
triplicate and superficial liquid was collected. The Phoenix Win Nonlin non-compartmental analysis
collected supernatants were diluted and analyzed program of version 5.1.
by HPLC. Mycobacterial infection study in rat
Tissue sample : Tissue homogenates (20% w/v) MTB H37Rv ATCC 27294, a strain sensitive to all
with aqueous medium were prepared in cold 150M the standard antimycobacterial agents, was used
KCL. Supernatant liquid collected from for all animal infection in the experiments.
homogenates by centrifugation at 15000 RPM for Bacterial cultures were prepared as described
10 min at 4°C was kept aside for further studies. previously published article (8). Wistar rats were
Then, 300μl of the methanol was admixed to 150μl infected via the respiratory route to obtain low-
of the clear homogenates and the dispersal was grade bacillary lung infection (100 bacilli) using a
vortexed for 2 min. The samples were then modified Madison aerosol chamber (9). Bacterial
centrifuged at 15,000 RPM for about 10 min at 4 lung loads were estimated to determine suitable
ºC. An equal volume of water is added to the infection conditions for drug efficacy experiments.
obtained supernatant. The samples were further After infection, the animals were housed for the
filtered using 0.2μm nylon filters and were instilled duration of the study in a bio-safety level 3
to the HPLC system (8-11). facilities. By using microbial enumeration,
dependent variable, the number of animals rifampicin nanospheres slightly more active than
required, per treatment group was three in pure drug rifampicin. Long-term treatment (Table-
experiments for drug evaluation. The course of 3) with rifampicin nanospheres at 25 mg/kg Shown
mycobacterial infection was monitored by statistically very significant when compared to
enumeration of colony forming units (CFU) from negative control in the lungs and spleen (p<0.05).
excised lungs at 1, 2, 3, 4, 6, and 12 weeks of The results elucidated that there was a significant
post infection. decrease in colony forming unit (CFU) of lungs
and spleen in all treated groups during both period
Statistical analysis : All the experiments were
in contrast to negative control group. After 18 days
performed in triplicate. Results were collected as
of treatment, rifampicin nanospheres minimized
mean and standard deviation (mean ± SD).
the bacterial content by 0.54 log 10 CFU. The
Significance in difference was measured with p
activity of rifampicin nanospheres and rifampicin
value as p d” 0.5.
pure drug in the lungs and spleen was statistically
not significant (p = ns) when compared with
Results
negative control after 18 days of treatment. After
The mean bio distribution pharmacokinetic
42days of treatment, rifampicin nanospheres
study parameters of F13 rifampicin nanospheres
minimized the bacterial content 4.62log 10 CFU.
formulation administered through Intravenous (IV)
(9.68 14.30-9.68 log 10 CFU). The efficacy of
and Inhalation are summarized in the Table 1,
rifampicin nanospheres in the lungs and spleen
Figure 1 & 2. The C-max, T-max and clearance of
differs statistically from those of rifampicin pure
F13 through Inhalation route were 42.34 ìg/mL,
drug (P > 0.001) after 42days of treatment. The
14 hr and 0.82(ml/hr) respectively, similarly those
results are shown in (Table 2&3 and Figure no 3).
of IV route were 20.46 ìg/mL,14 hr and 3.44
respectively. Discussion
By comparing the Cmax results between IV
In short term study (Table-3),the potency
and Inhalation administration of Rifampicin
of rifampicin nanospheres at 25 mg/kg body weight
Nanospheres it shows that more concentration of
was compared with those of rifampicin pure drug.
drug was accumulated in the lungs while
Table 1: Bio distribution studies of Rifampicin Nanospheres (F13) – IV & Inhalation administration
Table 2: Growth of M.tuberculosis in culture medium and their control in different conditions
Values are expressed as mean ± SD. n=3; Values are statistically significant at p<0.05;
Significant-p<0.05;** Very significant -p<0.01;***-p<0.001; ns-non-significant;
Values are expressed as mean ± SD. n=3; Values are statistically significant at p<0.05;
Significant-**-p<0.01;Very Significant-***-p<0.001; ns-non-significant; a-Group compared to control;
b-Groups compared to negative control;
administering the formulation through Inhalation. high therapeutic concentration by improving good
From the Tmax comparison data between IV and pulmonary tuberculosis chemotherapy.
Inhalation administration it shows the sustainability In vivo mycobacterium screening studies
time of rifampicin nanospheres in lungs i.e., it show that on long term therapy rifampicin
confirms the sustained action of rifampicin nanospheres shows better control of growth of
nanospheres in lungs. By comparing the AUC0-á microorganism i.e. CFU when compared to short
from Table 1, it concludes that maximum term therapy i.e. for 18 days after administration.
concentration of drug was present in lungs through After 6 weeks of treatment the bacterial counts in
inhalation than any other organ. The organ the lungs were reduced to very low numbers in all
clearance ratio of drug from lungs through treatment groups (range, 14.30 to 9.68 log10 CFU
inhalation was less than the IV administration, Rifampicin Nanospheres Vs untreated controls),
which confirms the sustained release of rifampicin as was the case for the spleens (p<0.001). In
from nanospheres in the lungs. From the above summary, its good activity in vivo models, as well
pharmacokinetic distribution data it shows that as its activity against multidrug-resistant MTBand
rifampicin nanospheres shows more accumulation against MTB isolates in a potentially latent state,
of drug in lungs through inhalation administration makes Rifampicin Nanospheres an attractive drug
than IV, which indicates nanospheres is having dosage form for the therapy of tuberculosis. These
targeted and sustained release of drug results in data indicate that there is significant potential for
lungs. By this it can be confirmed that inhalable effective inhalational delivery of Rifampicin
nanospheres are suitable for targeting with Nanospheres for the treatment of tuberculosis.
negligible toxicity and providing sustained release
of anti-tubercular drugs especially rifampicin in Conclusion
lungs. The result showed the rifampicin In vivo bio distribution studies show that
nanospheres leads to maximum deposition of drug nanospheres form is the best formulation for
in lungs through inhalation which leads to maintain Rifampicin, Nanospheres accumulates maximum
dose in the lungs than other organs over prolonged 7. MaikeLohrmann, Michael Kappl., Hans-
period of time. The plasma levels are more for Juergen Butt., Nora Anne Urbanetz. and
inhalable PLGA Nanospheres and are suitable for Bernhard Christian Lippold. (2007). Adhesion
targeting and providing sustained release of anti- forces in interactive mixtures for dry powder
tubercular drugs to lungs. So inhalation can be inhalers – Evaluation of a new measuring
selected as administration route of Rifampicin method.ýEur. J. Pharm. Biopha, 67: 579 -
PLGA Nanospheres. From the in vivo screening 586.
of M.tuberculosis, it shows good activity in vivo
models, as well as its activity against multidrug- 8. SaniIbnYakubua., Khaled H. Assi. and Henry
resistant MTBand against MTBisolates in a Chrystync. (2013). Aerodynamic dose
potentially latent state, makes Rifampicin PLGA emission characteristics of dry powder
Nanospheres an attractive drug dosage form for inhalers using an Andersen Cascade
the therapy of tuberculosis. These data indicate Impactor with a mixing inlet. ýInt. J. Pharm,
that there is significant potential for effective 455: 213– 218.
intravenous as well as nasal delivery of 9. Francesco Martinelli., Anna Giulia
Nanospheres for the treatment of tuberculosis. Balducci., Alessandra Rossi., Fabio
Sonvico. and Paolo Colombo. (2015). Pierce
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