part ii: poly (ethylene sebacate) nanoparticles of anti...

Part II: Poly (ethylene sebacate)

Nanoparticles of Anti HIV-Anti tubercular drug

combinations

Poly (ethylene sebacate) Nanoparticles of Anti HIV-Anti tubercular drug combinations

106 Particulate Carriers as Drug Delivery Systems for Anti-Tubercular and Anti-Cancer Agents

6.1. INTRODUCTION

Development of AIDS from acquisition of HIV infection to disease progression

represents a tremendous social, economical and political challenge in the 21st Century

[Strawford and Hellerstein, 1998]. Further existence of tuberculosis in HIV patients

results in significantly higher mortality. Treatment failures in HIV-infected patients

have been associated with reduced drug concentrations due to malabsorption of anti-

mycobacterials. Also the M. avium–M. intracellulare (MAC) complex is the main

cause of complications in immunodepressed patients. Patients with severe

immunodeficiency benefit from co-administration of anti-HIV and anti-tubercular

drug.

Highly active antiretroviral therapy (HAART), a combination therapy of at least three

antiretroviral drugs was a major step forward in the treatment of AIDS and has led to

a significant reduction in the mortality [Frezzini et al., 2005; Holtgrave, 2005; Piliero,

2004]. Lopinavir (LOPI) a potent protease inhibitor is an integral drug in Highly

Active Anti-Retroviral Therapy (HAART) and rifampicin is a first line anti-tubercular

drug. Administration of Lopinavir alone has insufficient bioavailability due to

extensive first pass effect and limited intestinal uptake due to p-glycoprotein efflux

however, its blood levels are greatly increased by subtherapeutic doses of ritonavir, a

potent inhibitor of cytochrome P450 3A4. Ritonavir acts as a pharmacokinetic booster

for lopinavir but at the same time it increases the cost and exposure associated toxicity

in the patients.

The therapeutic strategy for the treatment of AIDS and tuberculosis has undergone a

paradigm shift in the past decade, wherein targeted delivery of anti-HIV and anti-

tubercular drugs is emerging as a new dimension. Nanoparticulate based drug delivery

system represents an attractive carrier for targeted drug delivery to viral and bacterial

reservoirs [Vyas et al., 2006; Chellat et al., 2005; Gunaseelan et al., 2010; Lanao et

al., 2007; Gupta and Jain, 2010; Neves et al., 2010; Wong et al., 2010].

Our group has recently reported a new biodegradable polymer, polyethylene sebacate

(PES) which offers some unique advantages including ease of synthesis, hydrophobic

nature, good hydrolytic stability and low cost. Enzymatic degradation studies with

lipase revealed PES as biodegradable and toxicity studies including genotoxicity and

mutagenicity have confirmed safety of PES for biomedical and pharmaceutical

applications [Malshe et al., 2006; More et al., 2009]. FDA approved polymer PLGA

and PLA were selected for comparative evaluation. Although rifampicin is an inducer



of cytochrome P450 we hypothesized that nanoparticulate drug delivery systems

(NPDDS) would enable improved bioavailability of lopinavir by providing protection.

The objective of the study is to evaluate the role of the NPDDS of rifampicin-

lopinavir combinations on enhanced bioavailability of the drugs following oral

administration. Entrapping both drugs in the ratio 1:1 is an additional objective.

6.2. LOPINAVIR

Lopinavir is a peptidomimetic HIV protease inhibitor that is structurally similar to

ritonavir but is three- to tenfold more potent against HIV-1 in vitro. Lopinavir is

active against both HIV-1 and HIV-2.

6.2.1. Chemical structure:

Formula - C37H48N4O5 Molecular weight – 628.80 g/mol

6.2.2. Chemical name- (2S)-N-[(2S,4S,5S)-5-[2-(2,6-dimethylphenoxy) acetamido]-

4-hydroxy-1,6-diphenylhexan-2-yl]-3-methyl-2-(2- oxo-1,3- diazinan-1-l)butanamide

6.2.3. CAS number - 192725-17-0

6.2.4. Physicochemical properties

Lopinavir is a white to light tan powder. It is freely soluble in methanol and ethanol,

soluble in isopropanol and practically insoluble in water. Lopinavir has melting point

of 120-124°C.

6.2.5. Analysis

Several chromatographic methods have been reported for analysis of lopinavir as a

bulk drug, in formulations and from the biological fluids. Reported methods include

those based on HPLC separation with ultra-violet detection [Kuschak et al., 2001;

Choi et al., 2007; Weller et al., 2007; Hirano et al., 2010] and liquid

chromatography/tandem mass spectroscopy [Rezk et al., 2008].

6.2.6. Indications (Qazi et al., 2002)

Lopinavir is indicated in combination with other antiretroviral agents for the treatment

of HIV-infection.



6.2.7. Mechanism of action (Qazi et al., 2002)

Lopinavir inhibits the HIV viral protease enzyme. This prevents cleavage of the gag-

pol polyprotein and, therefore, improper viral assembly results. This subsequently

results in non-infectious, immature viral particles.

6.2.8. Pharmacokinetic properties (Cvetkovic and Goa, 2003)

Lopinavir is only available as a coformulation with low doses of ritonavir. When

administered orally without ritonavir, lopinavir plasma concentrations were

exceedingly low mainly owing to first-pass metabolism. Lopinavir is absorbed rapidly

after oral administration. A moderate-to high-fat meal increases oral bioavailability by

up to 50%, and it is therefore recommended that the drug be taken with food.

Although the capsules contain lopinavir–ritonavir in a fixed 4:1 ratio, the observed

plasma concentration ratio for these two drugs following oral administration is nearly

20:1, reflecting the sensitivity of lopinavir to the inhibitory effect of ritonavir on

CYP3A4. Lopinavir undergoes extensive hepatic oxidative metabolism by CYP3A4.

Approximately 90% of total drug in plasma is the parent compound, and less than 3%

of a dose is eliminated unchanged in the urine. Both lopinavir and ritonavir are highly

bound to plasma proteins, mainly to α1-acid glycoprotein, and therefore have a low

fractional penetration into cerebrospinal fluid (CSF) and semen.

6.2.9. Adverse drug reaction (Cvetkovic and Goa, 2003)

The most common adverse events reported with the lopinavir–ritonavir coformulation

have been gastrointestinal, including loose stools, diarrhea, nausea, and vomiting. The

most common laboratory abnormalities include elevated total cholesterol and

triglycerides. Because the same adverse effects occur with ritonavir, it is unclear

whether the side effects are due to ritonavir, lopinavir, or both.

6.2.10. Contraindications (Cvetkovic and Goa, 2003)

Numerous dosing schedules exist for the treatment of DOX depending on disease,

response and concomitant therapy. Guidelines for dosing also include consideration of

white blood cell count. Dosage may be reduced and/or delayed in patients with bone

marrow depression due to cytotoxic/radiation therapy.

6.2.11. Marketed formulations

Several marketed dosage forms of lopinavir as fixed dose combination with ritonavir

are available in the US and Indian market including: Kaletra® capsules (Abbott),

Kaletra® oral solution (Abbott), Kaletra® tablets (Abbott), Emletra tablets (Emcure),



Lopimune tablets (Cipla), Ritocom tablets (Hetero HC) and V-Letra tablets

(Ranbaxy).

6.3. ANALYTICAL METHOD DEVELOPMENT

The following methods for the simultaneous analysis of rifampicin and lopinavir were

developed:

Stability indicating HPLC method

HPLC method for the simultaneous analysis of rifampicin and

lopinavir in plasma and organ homogenate

6.3.1. Stability indicating HPLC method

Instrumentation:

The HPLC system used was JASCO LC900 Intelligent pump coupled with UV

detector (Jasco UV/VIS 1570/1575) and Rheodyne injector model (7725) fitted with

100μl sample loop. Data integration was done by Borwin chromatography software

version 1.21.

Chromatography

Chromatography was performed on a Waters Spherisorb® S5 ODS2 (250 × 4 mm i.d.,

5 μm particle size) column. The mobile phase comprised of tetrahydrofuran:methanol:

phosphate buffer pH 5.2 in the ratio 30:30:40 v/v was used. The mobile phase was

filtered through a nylon membrane (0.22 μm, Pall Gelman) and degassed by

sonication prior to use. Chromatography was performed at room temperature under

isocratic conditions at a flow rate of 1 mL/min. UV detection was done at a λmax of

212 nm.

Preparation of standard solutions

Rifampicin (10mg) and lopinavir (10mg) was accurately weighed and transferred to a

10 mL volumetric flask. The volume was made up to 10 mL with methanol to obtain

a stock solution (1000μg/mL). From the above solution, 0.05 and 0.1 ml was diluted

upto 10 ml with mobile phase to get concentration of 5 and 10μg/mL. Aliquots of

10μg/mL solution corresponding to 0.01, 0.05, 0.1, 0.5, and 1.0mL were diluted to

10mL with mobile phase to get concentration range from 10 to 1000ng/mL. Each

solution was injected in triplicate. Average of the peak areas was considered for

calculation purposes.



Validation

a) Stability of analyte in solution:

The stability of rifampicin and lopinavir combination in mobile phase was assessed by

injecting the standard solution (10μg/ml) at interval of 0, 3 and 6 hrs post preparation

kept in amber colored volumetric flasks at room temperature. The chromatograms

were checked for presence of peaks corresponding to degraded product.

b) Linearity

Standard solutions (10, 50, 100, 500, 1000 ng/mL, 5 and 10μg/ml), each in three

replicates, were injected into the system. The method of linear regression was used for

data evaluation. Peak areas were plotted against theoretical concentrations of

standards. Linearity was expressed as a correlation coefficient.

c) Precision

System precision (repeatability) was determined by performing five consecutive

injections of the 10 μg/ml standard solution. Method precision was determined by

injecting three different samples (10 μg/ml) prepared individually.

Forced Degradation Studies

It was necessary to perform forced degradation studies to verify and prove the

stability-indicating feature of the proposed method. Intentional degradation was

attempted by heating the drug in the presence of base, acid and hydrogen peroxide

and exposing to sunlight.

i) Acid degradation

To 1mL of standard stock solution (100μg/mL) of rifampicin and lopinavir

combination, 1 mL of 0.1N HCl was added and the solution was placed in a boiling

water bath for 2h. The sample was allowed to cool to room temperature and

neutralized using 0.1N NaOH. The volume was adjusted to 10 ml with mobile phase,

and this solution was injected in the HPLC column.

ii) Base degradation


combination, 1 mL of 0.1N NaOH was added and the solution was placed in a boiling

water bath for 2h. The sample was allowed to cool to room temperature and

neutralized using 0.1N HCl. The volume was adjusted to 10 ml with mobile phase,

and this solution was injected onto the HPLC column.



iii) Oxidation


combination, 1 mL of 3% H2O2 was added and the solution was placed in a boiling

water bath for 2h. The sample was allowed to cool to room temperature. The volume

was adjusted to 10 ml with mobile phase, and this solution was injected onto the

HPLC column.

iv) Photodegradation

Drug solution (10μg/mL) in mobile phase was exposed to sunlight for 2h and this

solution was injected onto the HPLC column.

The degraded samples were analyzed against an untreated control sample.

Results and Discussion

a) Stability of analyte in solution

Rifampicin and lopinavir was found to be stable in the mobile phase, when the

standard solution of strength 10μg/mL was analyzed at 0-6h post preparation. No

peaks corresponding to the degradation products were observed. A low RSD value

indicated that there was no significant change in the drug peak area (table 6.1).

Table 6.1: Stability of rifampicin and lopinavir in mobile phase

Rifampicin Area 1 Area 2 Average %RSD

0 hr 1373917 1363530 1368724 0.5366 3 hr 1367295 1378098 1372697 0.5564 6 hr 1358894 1368932 1363913 0.5205

AVG. 0.5378 Lopinavir Area 1 Area 2 Average %RSD

0 hr 2390291 2410153 2400222 0.5851 3 hr 2367945 2418125 2393035 1.4827 6 hr 2395331 2429923 2412627 1.0138

AVG. 1.0272

b) Linearity

Graph of the peak area vs concentration was plotted in order to check the linearity.

The developed method was found to be linear between concentration range of 0.01-

10μg/mL (figure 6.1 and 6.2). The regression coefficient was found to be 1 for both

RIF and LOPI.



Standard Curve of Rifampicin

y = 136230x + 3546.4R2 = 1

0200000400000600000800000

1000000120000014000001600000

0 2 4 6 8 10 12Concentration mcg/ml

Are

a un

der

curv

e

Figure 6.1: Standard curve of rifampicin by HPLC

Standard Curve Lopinavir

y = 239530x - 3320R2 = 1

0

500000

1000000

1500000

2000000

2500000

3000000

0 2 4 6 8 10 12Concentration mcg/ml

Are

a un

der

curv

e

Figure 6.2: Standard curve of lopinavir by HPLC

c) Precision

Low RSD values of 0.35% for RIF and 0.72% for LOPI for system precision and for

method precision 0.72% for RIF and 0.50% for LOPI were obtained (table 6.2).

Table 6.2: Precision study of the assay

Sample No.

Area for Rifampicin Area for Lopinavir System

precision (10 µg/ml)

Method precision (10 µg/ml)

System precision (10 µg/ml)

Method precision (10 µg/ml)

1 1363530 1373917 2388912 2390291 2 1364972 1363530 2430674 2410153 3 1369832 1354160 2391234 2387991 4 1359454 - 2415397 - 5 1371298 - 2410164 -

% RSD 0.3522 0.7246 0.7243 0.5085

Forced Degradation Studies

The HPLC procedure was optimized with view to develop a stability indicating

method so as to resolve the degraded products from the drugs. Various mobile phase



compositions were tried so as to obtain a sharp peak and also resolve the peaks of

degraded product from the peak of drug. The mobile phase consisting of

tetrahydrofuran:methanol: phosphate buffer pH 5.2 in the ratio 30:30:40 v/v resulted

in a retention time of 3.7 min for rifampicin and 9.5 min for LOPI. The

chromatograms of rifampicin and LOPI (undegraded) and rifampicin and LOPI

degraded in the presence of acid, base, hydrogen peroxide (H2O2) (oxidative

degradation) and light (photo degradation) are shown in figure 6.3 and indicated a

good separation of the undegraded drug from the degradation products. The peaks of

all the degraded products were resolved from the rifampicin and LOPI peak. The

chromatograms after forced degradation are shown in figure 6.3.

a) Standard rifampicin and LOPI

b) Acid degraded rifampicin and LOPI

c) Base degraded rifampicin and LOPI



d) H2O2 degraded rifampicin and LOPI

e) Photo degraded rifampicin and LOPI

Figure 6.3: Forced degradation studies of RIF and LOPI

Conclusion: The RP-HPLC method developed for rifampicin and Lopinavir

combination was found to be precise, rapid, accurate, and stability indicating. Thus

the method could be used for determining the stability of rifampicin and lopinavir.

6.3.2. HPLC method for plasma and organ homogenate

Instrumentation

The HPLC system used was JASCO LC900 Intelligent pump coupled with UV

detector (Jasco UV/VIS 1570/1575) and Rheodyne injector model (7725) fitted with

100μl sample loop. Data integration was done by Borwin chromatography software

version 1.21.

Chromatography

Chromatography was performed on a Agilent ZORBEX SB-C18 (250 × 4 mm i.d.,

5μm particle size) column. The mobile phase comprised of Acetonitrile: Ammonium

formate buffer pH 3.8 in the ratio 48:52 v/v was used. The mobile phase was filtered

through a nylon membrane (0.22 μm, Pall Gelman) and degassed by sonication prior

to use. Chromatography was performed at room temperature under isocratic

conditions at a flow rate of 0.8 mL/min. UV detection was done at a λmax of 212 nm.



Preparation of standard solutions

Rifampicin (10mg) and lopinavir (10mg) was accurately weighed and transferred to a

10 mL volumetric flask. The volume was made up to 10 mL with methanol to obtain

a stock solution (1000μg/mL). From the above solution, 0.1 ml was diluted upto 10 ml

with mobile phase to get concentration of 10μg/mL. Aliquots of 10μg/mL solution

corresponding to 0.1, 0.25, 0.5, 1.0 and 5.0mL were diluted to 10mL with mobile

phase to get concentration range from 100 to 5000ng/mL in the presence of plasma

and various organ homogenate (lung, liver, spleen and kidney). Each solution was

injected in triplicate. Average of the peak areas were considered for calculation

purposes.

Recovery/extraction from plasma and organ homogenate

The recovery of RIF and LOPI from plasma and various organ homogenate (lung,

liver, spleen and kidney) was determined at five different concentrations namely 100,

250, 500, 1000 and 5000 ng/mL (n=3). To determine drug extraction efficiency of

method from plasma and various organ homogenate (lung, liver, spleen and kidney),

RIF-LOPI solution (100µL) (1–50µg/mL) was spiked to drug-free plasma and various

organ homogenate (lung, liver, spleen and kidney) (400µL) and vortexed vigorously

for 2 min followed by addition of equal volume (500µL) of methanol. The resulting

mixture was vortexed vigorously for 2 min and centrifuged at 20,000 rpm for 20 min

at 25 ºC. The supernatant was injected into the HPLC system. Extraction efficiency of

RIF-LOPI from plasma and various organ homogenate (lung, liver, spleen and

kidney) was calculated by comparing the peak heights of standard RIF-LOPI solution.

Validation

The chromatographic method was validated for linearity, specificity, sensitivity,

precision, and accuracy.

i) Linearity: All validation runs were performed in triplicate to assess variation.

Calibration curves were constructed in presence of plasma and various organ

homogenate (lung, liver, spleen and kidney) over the concentration range 100–

5000ng/mL for RIF-LOPI combination.

ii) Sensitivity: Limit of quantification (LOQ) of standard drug and spiked plasma and

various organ homogenate (lung, liver, spleen and kidney) was determined at a signal

to noise ratio of 1:10.

iii) Precision: System precision (repeatability) was determined by performing five

consecutive injections of the 5μg/mL for RIF-LOPI combination. Method precision



was determined by injecting three different samples of 5μg/mL for RIF-LOPI

combination prepared individually.

Results and Discussion

The developed method showed good resolution of the drug with retention time (RT)

of 5.8 mins (RIF) and 26.5 mins (LOPI) for plasma and various organ homogenate

(lung, liver, spleen and kidney) with no interference. The chromatograms indicate that

RIF and LOPI peaks are well separated from other peaks in plasma and various organ

homogenate (figure 6.4). The HPLC analytical method was found to be linear

between concentration range of 100-5000ng/mL in the presence of plasma and organ

homogenate with high correlation coefficient of (table 6.3 and 6.4).

Blank plasma RIF-LOPI extracted from Plasma

Blank lung homogenate RIF-LOPI extracted from lung homogenate

Blank spleen homogenate RIF-LOPI extracted from spleen homogenate

Blank kidney homogenate RIF-LOPI extracted from kidney homogenate

Blank liver homogenate RIF-LOPI extracted from liver homogenate

Figure 6.4: Chromatogram of RIF and LOPI from biological samples



Extraction efficiency/recovery was greater than 85% (table 6.3 and 6.4) for both RIF

and LOPI and low RSD values (<5%) were obtained for system precision (table 6.5

and 6.6).

Table 6.3: Linearity and recovery data of RIF from plasma and organ homogenate

Medium Linearity range Slope Intercept r2

Extraction efficiency/ recovery

Standard RIF 100-5000 ng/ml 201.5 26139 0.9990 -

Plasma 100-5000 ng/ml 224.5 39973 0.9980 97.32±7.73

Lung Homogenate

100-5000 ng/ml 160.6 14022 0.9990 89.96±6.78

Spleen Homogenate

100-5000 ng/ml 174.2 22172 0.9990 91.23±4.81

Liver Homogenate

100-5000 ng/ml 183.7 25876 0.9994 90.26±4.15

Kidney Homogenate

100-5000 ng/ml 195.2 28954 0.9995 91.12±3.68

Table 6.4: Linearity and recovery data of LOPI from plasma and organ homogenate

Medium Linearity range Slope Intercept r2

Extraction efficiency/ recovery

Standard LOPI 100-5000 ng/ml 300.8 5297 1.0000 -

Plasma 100-5000 ng/ml 358.2 21226 0.9990 98.71±3.10

Lung Homogenate

100-5000 ng/ml 294.4 19748 0.9970 98.61±1.37

Spleen Homogenate

100-5000 ng/ml 322.5 4310 1.000 100.28±3.84

Liver Homogenate

100-5000 ng/ml 312.3 10823 0.9994 89.86±5.59

Kidney Homogenate

100-5000 ng/ml 302.5 14352 0.9995 92.52±6.83



Table 6.5: System precision data of RIF from plasma and organ homogenate

Sample No. Area (5000 ng/mL)

Plasma Lung Spleen Kidney Liver 1 1055963 985394 932481 1006862 980579 2 1096205 952783 962086 1010868 1006885 3 1110935 990182 955479 1019832 1012139 4 1052762 1009823 910862 1010024 1009856 5 1058235 973422 920973 1010023 1016732

Average 1074820 982320.8 936376.2 1011522 1005238 RSD 2.49 2.14 2.34 0.48 1.41

Table 6.6: System precision data of LOPI from plasma and organ homogenate

Sample No. Area (5000 ng/mL)

Plasma Lung Spleen Kidney Liver 1 1749988 1629056 1637075 1609423 1600352 2 1775248 1593497 1614940 1597271 1609842 3 1801976 1528615 1577209 1560862 1610239 4 1775737 1583723 1609741 1600487 1619581 5 1779823 1612986 1608627 1509892 1611929

Average 1776554 1589575 1609518 1575587 1610389 RSD 1.03 2.41 1.33 2.61 0.42

HPLC method was validated for linearity, specificity, sensitivity & precision thus

establishing that the method can be efficiently used for RIF-LOPI analysis in plasma

and various organ homogenate (lung, liver, spleen and kidney).

Conclusion: The RP-HPLC method developed for RIF-LOPI was found to be

specific, precise, rapid and accurate. Thus the method could be efficiently used for

RIF-LOPI analysis in plasma and various organ homogenate (lung, liver, spleen and

kidney).

6.4. EXPERIMENTAL METHODS

6.4.1. Materials

Poly (ethylene sebacate) [PES] was synthesized in our laboratory (Mw= 11300),

rifampicin (RIF), lopinavir (LOPI), poly vinyl alcohol (PVA), and trehalose 100

(Hayashibara Co. Ltd., Japan) were kindly gifted by Maneesh Pharma (Mumbai,

India), Hetero Drugs Pvt. Ltd.(Hyderabad, India), Colorcon Asia Pvt ltd and Gangwal

Chemicals Pvt. Ltd. (Mumbai, India) respectively. PLGA 50:50 (PDLG 5010;

inherent viscosity midpoint of 1 dl/g) was purchased from PURASORB®. Lutrol-F-68



(polyoxyethylene polyoxypropylene block co-polymer) was a gift sample from BASF.

Dichloromethane AR, methanol AR, dioctyl sodium sulphosuccinate AR (Aerosol

OT®, AOT), tetrahydrofuran AR (THF), ammonium format AR, disodium hydrogen

phosphate AR and sodium chloride AR were purchased from s. d. fine-chem limited

(Mumbai, India). Ethyl alcohol AR (99.9% pure) was purchased from Changshu

Yangyuan Chemical (China). Filtered (0.45 µ membrane filter) doubled distilled

water was used for preparation of nanoparticles. All other chemicals and solvents

were either spectroscopic or analytical grade.

6.4.2. Preparation and optimization of nanoparticles

RIF-LOPI loaded PES nanoparticles were prepared by emulsion solvent evaporation

method. Briefly, RIF (25-35mg), LOPI (15-25mg) and PES (100mg) were dissolved

in dichloromethane (5ml). The non solvent phases comprised an aqueous solution of

poly vinyl alcohol (75mg) in 20ml water. The organic phase was added to the non

solvent phase by probe sonication for 5minutes (10sec on/10sec off cycle) to form a

stable emulsion. The dispersion was kept under continuous stirring on a magnetic

stirrer at room temperature till complete evaporation of organic solvent (approx. 2-

3h). The nanoparticle suspension was centrifuged at 15000 rpm for 30 min and the

supernatant analyzed for drug to determine entrapment efficiency. Similarly by

replacing PES with PLGA and PLA, RIF-LOPI loaded nanoparticles of PLGA and

PLA were prepared. Parameters evaluated included concentration of polymer,

concentration of surfactants and rifampicin to lopinavir ratio, to optimize particle size

and entrapment of both drugs in 1:1 ratio.

a) Effect of PVA concentration

Table 6.7: Effect of PVA concentration on %EE and particle size

RLPES/1 RLPES/2 RLPES/3

Rifampicin (mg) 25 25 25

Lopinavir (mg 25 25 25

PES (mg) 100 100 100

Dichloromethane (ml) 5 5 5

Water (ml) 20 20 20

Poly vinyl alcohol (mg) 50 (0.25% w/v)

75 (0.375% w/v)

100 (0.5% w/v)



The surfactant concentration was varied as 0.25, 0.375 and 0.5% in order to find the

ratio that gives optimum entrapment efficiency table 6.7.

b) Effect of rifampicin to lopinavir ratio

The rifampicin to lopinavir ratio was varied to find the ratio that gives optimum

entrapment efficiency of both drugs in 1:1 ratio (table 6.8).

Table 6.8: Effect of RIF:LOPI ratio on %EE and particle size

RLPES/4 (1:0)

RLPES/5 (7:3)

RLPES/6 (6:4)

RLPES/2 (1:1)

Rifampicin (mg) 50 35 30 25 Lopinavir (mg - 15 20 25 PES (mg) 100 100 100 100 Dichloromethane (ml) 5 5 5 5 Water (ml) 20 20 20 20 Poly vinyl alcohol (mg) 75 75 75 75

c) Effect of drug: polymer ratio

Effect of RIF-LOPI:PES ratio (drug:polymer) on entrapment efficiency and particle

size was evaluated. Ratio of RIF-LOPI:PES evaluated include 1:1, 1:1.5, 1:2 and 1:3

(table 6.9).

Table 6.9: Effect of drug:polymer ratio on %EE and particle size

RLPES/8 (1:1)

RLPES/9 (1:1.5)

RLPES/7 (1:2)

RLPES/10 (1:3)

Rifampicin (mg) 32.5 32.5 32.5 32.5 Lopinavir (mg 17.5 17.5 17.5 17.5 PES (mg) 50 75 100 150 Dichloromethane (ml) 5 5 5 5 Water (ml) 20 20 20 20 Poly vinyl alcohol (mg) 75 75 75 75

6.4.3. Freeze-drying of nanoparticles

Freeze-drying of various nanoparticle batches were carried out using trehalose (10:1

by weight of nanoparticles) and lutrol-F-68 (0.1:1 by weight of nanoparticles).



Samples of 10 mL dispersion of nanoparticles were dispensed in 250mL freeze drying

glass vessels, frozen at -70 °C for 12 h and then subjected to freeze-drying using

Labconco freeze-drying system (FreeZone 4.5, USA). Sublimation lasted for 36-48 h

at a vacuum pressure of 10-50×10-3 bar, with the condenser surface temperature

maintained at less than -50 °C. Lyophilized samples were collected under anhydrous

conditions and stored in a dessicator until re-hydrated. Re-hydration of lyophilized

nanoparticles was carried with 0.2 µm filtered water by simple manual shaking.

Particle size and PI of the re-hydrated samples were determined by PCS to assess the

cryoprotection provided by the cryoprotectant.

6.4.3. Evaluation and physical characterization

a) Entrapment Efficiency

Nanoparticle dispersion was centrifuged at 15,000 rpm for 30 min at 20 ºC. The

resultant supernatant was analyses for free drug. The concentration of RIF and LOPI

in the supernatant was determined using stability indicating HPLC method reported in

section 6.3.1. Entrapment efficiency was calculated using equation 6.

%EE = (RIF/LOPIinitial – RIF/LOPIsupernatant) / RIF/LOPIinitial ×100 (6)

b) Particle Size

Particle size was determined by Photon Correlation Spectroscopy using N4 plus

submicron particle size analyzer (Beckman Coulter, USA). The analysis was

performed at a scattering angle of 90º at a temperature of 25 ºC. All the

nanoparticulate dispersions were sonicated using ultrasonic probe system (DP120,

Dakshin, Mumbai, India) for 5 min with 10 sec pulse at 200 voltages over an ice bath.

Dispersions were then appropriately diluted with filtered water (0.2 µm filter,

Millipore India Pvt. Ltd.) to obtain 5 ×104 to 1×106 counts per second. Each sample

was analyzed in triplicate and average particle size and polydispersity index (PI)

measured.

c) Drug loading

Measured quantity of freeze dried nanoparticles were dissolved in THF:water (1:1) by

sonication for 5 mins and assayed for drug content by developed HPLC method.

Percent Drug loading (DL%) was calculated using the equation:

DL (%) = WDL/WNP×100 ………………………………………….. (7)

where WDL = weight of Drug in Np and WNP = weight of Np



d) Zeta Potential

Zeta potential of nanoparticle dispersion was measured using Malvern Zetasizer

Nanoseries using DTS Nano software. Nanoparticle dispersion was centrifuged at

15,000 rpm for 30 min at 20ºC. The resultant pellet was washed and redispersed with

distilled deionized water (nanoparticles 100µg/ml) by sonication. Samples were filled

in to the folded capillary cell and zeta potential was measured. Each sample was

analyzed in triplicate.

e) Scanning Electron Microscopy (SEM)

The morphology/shape of nanoparticles was determined by SEM (JSM-6380-LA,

JEOL, Tokyo, Japan). A drop of colloidal dispersion was deposited onto a carbon tape

and dried under vacuum. The samples were sputtered with platinum using an auto fine

coater prior to analysis (JFC-1600, JEOL, Tokyo, Japan).

f) Fourier Transform Infrared (FTIR) Spectroscopy

FT-IR for rifampicin, lopinavir, PES, PLGA, PLA, the nanoparticles and excipients

were recorded on a Perkin-Elmer FTIR spectrophotometer by the KBr disk method

from 4000 to 500 cm-1. Samples were crushed to a fine powder, mulled with

anhydrous potassium bromide, compressed to form a thin transparent pellet and

subjected to FTIR.

g) Differential Scanning Calorimetry (DSC)

DSC thermograms of rifampicin, lopinavir, PES, PLGA, PLA, the nanoparticles and

excipients were recorded on a Perkin Elmer Pyris 6 DSC (PerkinElmer, Netherlands)

system in the temperature range 40 -300°C at a heating rate of 10°C /min in a

dynamic nitrogen atmosphere (20 mL/min). A sample of 5-6 mg was sealed in an

aluminum pan and an empty sealed aluminum pan was used as the reference.

h) Powder X-Ray Diffraction (PXRD)

Powder XRD patterns were obtained for rifampicin, lopinavir, PES, PLGA, PLA and

the nanoparticles were recorded using a Rigaku Miniflex diffractometer, with Cu Kα

target tube, NaI detector, variable slits, a 0.050 step size, operated at a voltage of 30

kV, 15mA current, at 2θ/min scanning speed, and scanning angles ranged from 8-

60º(2θ).

i) Hydrophobicity evaluation of nanoparticles (contact angle measurement)

Hydrophobicity of rifampicin, lopinavir, PES, PLGA, PLA and the nanoparticles was

evaluated by measuring the static contact angle of nanoparticles/drug pellet. Briefly,

nanoparticle pellets were prepared in KBr press using 25 mg freeze dried



nanoparticles and press it at 10 tone pressure for 1 minute. Drop water contact angles

were measured (Kruss contact angle measuring instrument G10, Germany) using

approximately 5µl drop of Milli-Q water. Results are presented as an average of 3

measurements.

6.4.4. Stability study

The nanoparticles were freeze dried packed and sealed in amber glass vials and

subjected to stability studies as per the ICH guidelines at 300C/65RH and 400C/75RH.

6.4.5. Pharmacokinetic and biodistribution of nanoparticles

a) Pharmacokinetic study

Male Wistar rats (200-250gms) (n=6) were fasted 12-18h prior to dosing. Rats were

divided into four groups, one group received plain drug dispersion, the second group

received RIF-LOPI PES Np’s, third group received RIF-LOPI PLGA Np’s and fourth

group received RIF-LOPI PLA Np’s (equivalent to 10mg/kg body weight of each RIF

and LOPI) administered by oral gavage. Blood samples were withdrawn from the

retro-orbital plexus prior to dosing and at 1, 2, 4, 6, 12 and 24h post dosing and

collected in tubes containing 4.1% EDTA. Plasma was separated and extracted and

evaluated for drug content by HPLC. The peak plasma concentration (Cmax) and

peak plasma time (Tmax) were obtained by visual data inspection. The area under

plasma drug concentration over time curve (AUC0–t) and t1/2 were calculated using

BASICA software.

b) Biodistribution study

Rats used in the pharmacokinetic study were euthanized at 24h by excessive carbon

dioxide, lungs, liver, spleen and kidney were isolated, placed in phosphate buffered

saline (PBS) pH 7.4 and homogenized using a tissue homogenizer. RIF and LOPI was

extracted and evaluated for drug content by HPLC.

All experimental procedures were reviewed and approved by the Institutional Animal

Ethics Committee (IAEC) of Institute of Chemical Technology, Department of

Pharmaceutical Sciences and Technology, Mumbai, India (ICT/IAEC/2011/P71).

6.5. RESULTS AND DISCUSSION

6.5.1. Preparation and optimization of nanoparticles

Emulsion solvent evaporation method was selected for the preparation of RIF-LOPI

loaded PES nanoparticles. Method employs a solution of drug(s) and polymer in a

water immiscible solvent is emulsified in water with the aid of surfactant/emulsifying

agent. Method is advantageous for good entrapment efficiency for combination of



hydrophobic agents (Jeffery et al., 1991, Okochi et al., 2000) compared to

nanoprecipitation method. In nanoprecipitation method when combinations of two

drugs are used, one drug which has more affinity towards organic phase displaces the

other drugs in an aqueous phase, whereas due to water immiscibility this phenomenon

is very low with emulsion solvent evaporation method.

For obtaining nanoparticles, studies were started with a prototype formula consisting

of the 100mg PES, 20mL aqueous phase, 10mL organic phase (dichloromethane),

25mg RIF, 25mg LOPI and 50mg PVA. The parameters were varied with this

prototype formula are as below

a) Effect of PVA concentration

Table 6.10 : Effect of PVA concentration on %EE and particle size

RLPES/1 RLPES/2 RLPES/3 Particle size (nm) 401.0 ± 25 302.5 ± 30 243.9 ± 22

PI 0.246 ± 0.103 0.293 ± 0.098 0.302 ± 0.102 %EE RIF 28.22 ± 1.67 % 27.37 ± 1.13 23.75 ± 3.04

% EE LOPI 93.7 ± 0.98 93.36 ± 1.01 92.76 ± 1.14

Figure 6.5: Effect of RIF-LOPI:PVA ratio on %EE and particle size

As seen from figure 6.5 while PVA concentration did not significantly influence

entrapment efficiency (P>0.05). A significant reduction in particle size (P<0.05) was

seen with increase in PVA concentration (table 6.10). PVA exerts its stabilizing effect

by adsorbing at the droplet interface thus reducing surface tension and promoting

mechanical and steric stabilization. Similar data is reported in literature for other

drugs (Lamprecht et al., 2001, Patil et al., 2008).



b) Effect of rifampicin to lopinavir ratio

Figure 6.6: Effect of RIF to LOPI ratio on %EE and particle size

As shown in figure 6.6 RIF:LOPI ratio did not significantly influence particle size

(P>0.05). RIF showed maximum entrapment efficiency of ~60% when no LOPI

added during the preparation of nanoparticles (table 6.11). Addition of LOPI together

with RIF significantly (P<0.05) decrease the entrapment efficiency of RIF while

entrapment efficiency of LOPI was not affected. LOPI and RIF compete for the

organic phase however being hydrophobic LOPI (aqueous solubility 0.0019g/L)

preferably dissolves in the organic phase, as a consequence RIF (aqueous solubility

1.4g/L) partitions in to the aqueous phase. This is observed as a decrease in

entrapment efficiency of RIF with increase in LOPI concentration.

Table 6.11: Effect of RIF to LOPI ratio on %EE and particle size

RLPES/4 RLPES/5 RLPES/6 RLPES/2 Particle size

(nm) 299.3 ± 20 283.1 ± 25 273.6 ± 28 302.5 ± 30

PI 0.311 ± 0.088 0.362 ± 0.091 0.336 ± 0.110 0.293 ± 0.098 %EE RIF 60.15 ± 2.27 49.81 ± 1.08 47.24 ± 1.19 27.37 ± 1.13

% EE LOPI - 91.84 ± 2.01 92.12 ± 1.15 93.36 ± 1.01

c) Effect of drug: polymer ratio

Effect of different drug:PES ratio (RIF:PES-1:0.53) on entrapment efficiency and

particle size is shown in figure 6.7. Varying the drug:PES ratio from 1:1 to 1:3 did not

significantly (P>0.05) affect entrapment efficiency of RIF and LOPI (table 6.12). A

significant (P<0.05) increase in particle size was observed with increase in drug:PES

ratio from 1:1 to 1:3. The increase in particle size with increasing polymer

concentration was discussed in section 5.1.1.



Table 6.12: Effect of drug to polymer ratio on %EE and particle size

RLPES/8 RLPES/9 RLPES/7 RLPES/10 Particle size

(nm) 239.9 ± 15 258.4 ± 18 287.6 ± 8.27 319.89 ± 9

PI 0.322 ± 0.108 0.278 ± 0.10 0.297 ± 0.104 0.390 ± 0.098 %EE RIF 39.03 ± 1.98 41.71 ± 2.15 48.1 ± 2.07 45.73 ± 1.13

% EE LOPI 94.93 ± 2.62 94.65 ± 2.35 95.3 ± 0.81 96.52 ± 2.11

Figure 6.7: Effect of Drug to Polymer ratio on %EE and particle size

Table 6.13: Optimized batch for RIF-LOPI PES, PLGA and PLA nanoparticles

RLPES/7 RIF:LOPI PES Np’s

RLPLGA/1 RIF:LOPI

PLGA Np’s

RLPLA/1 RIF:LOPI PLA Np’s

Average particle size 287.6 ± 8.27 nm 274.2 ± 14.9 nm 288.4 ± 13.2 nm %EE For RIF 48.1 ± 2.07 % 58.06 ± 0.78 % 58.07 ± 0.12 % %EE For LOPI 95.3 ± 0.81 % 95.1 ± 0.8 % 93.95 ± 0.22 % Drug Entrapped For RIF 16.63 ± 0.63 mg 17.89 ± 0.07 mg 18.0 ± 0.04 mg For LOPI 16.67 ± 0.14 mg 18.02 ± 0.08 mg 17.85 ± 0.04 mg RIF:LOPI ratio ~1:1 ~1:0.99 ~1:1.008 Total drug loading 24.98 ± 1.3 % 26.47 ± 1.2 % 26.2 ± 0.9 %

For comparative evaluation in vivo RIF-LOPI nanoparticles were also prepared using

FDA approved polymer PLGA (intermediate hydrophobicity) and PLA

(hydrophobic). PES was replaced with PLGA and PLA in RLPES/7 to get RIF-LOPI

loaded PLGA (RLPLGA/1) and PLA (RLPLA/1) nanoparticles respectively. Table

6.13 shows optimized batch for RIF-LOPI loaded PES, PLGA and PLA nanoparticles.

Despite the change in polymer we obtained comparable drug entrapment and



RIF:LOPI ratio ~1:1 as seen in table 5.1. Average particle size was comparable with

the RIF-LOPI PES nanoparticles (RLPES/7).

6.5.2. Freeze-drying of nanoparticles

Freeze-drying of RIF-LOPI loaded PES, PLGA and PLA nanoparticle batches were

carried out using trehalose (10:1 by weight of nanoparticles) and lutrol-F-68 (0.1:1 by

weight of nanoparticles) revealed the best cryo-protection with a Sf/Si (Sf- final

particle size after freeze thaw, Si- initial particle size) ratio of <1.3 as optimized in

section 5.2.2. The data is shown in table 6.14.

Table 6.14: Freeze drying of RIF-LOPI loaded PES, PLGA and PLA nanoparticle

Sr. No Freeze dried NPDDS Size before

FD (Si) Size after FD (Sf)

Sf/Si ratio

1 RIF-LOPI PES Np (RLPES/7) 289.1 335.9 1.16 2 RIF-LOPI PLGA Np (RLPLGA/1) 279.5 332.6 1.19 3 RIF-LOPI PLA Np (RLPLA/1) 297.2 350.7 1.18

6.5.3. Evaluation and physical characterization

a) Zeta Potential

Nanoparticles exhibited a negative zeta potential due to free terminal hydroxyl group

of PES and carboxylic group of PLGA and PLA (table 6.15). Zeta potential values

ranged from -25 to – 35 mV which is an indicator of good colloidal stability (Sugrueet

al., 1992).

Table 6.15: Zeta potential of RIF-LOPI loaded PES, PLGA and PLA nanoparticle

NPDDS Zeta Potential mV RIF-LOPI PES Np (RLPES/7) -21.2 ± 3.4 mV RIF-LOPI PLGA Np (RLPLGA/1) -19.5 ± 2.9 mV RIF-LOPI PLA Np (RLPLA/1) -24.1 ± 4.1 mV

b) Scanning Electron Microscopy (SEM)

a) b) c)

Figure 6.8: SEM images of a) RIF-LOPI PES Nps b) RIF-LOPI PLGA Nps c) RIF-LOPI PLA Nps

Scanning Electron Microscopy (SEM) revealed polydispersed nanoparticles with

spherical morphology (figure 6.8).



c) Fourier Transform Infrared (FTIR) Spectroscopy

FTIR studies are rapid method of accessing drug: excipients interaction in the

formulation. FTIR of RIF, LOPI, PES, PLGA, PLA and their respective nanoparticles

along with excipients revealed all the characteristic peaks of RIF and LOPI. RIF has

principal peaks at wavenumbers 1250, 1567, 976, 1098, 1064, 1650 cm−1which are

also found in RIF PES, PLGA and PLA nanoparticles. LOPI has principal peaks at

wavenumbers 1090, 1061, 1142, 987, 1050, 1009 cm−1. No drug: excipient

interaction was evident in the spectra (figure 6.9).

Rifampicin Lopinavir

Poly vinyl alcohol Poly (ethylene sebacate)

PLGA PLA



RIF-LOPI PES Nps RIF-LOPI PLGA Nps

RIF-LOPI PLA Nps

Figure 6.9: FTIR of drugs, excipients and nanoparticles

d) Differential Scanning Calorimetry (DSC)

a) b)

c)

Figure 6.10: DSC thermogram of a)RIF-LOPI PES Nps b) RIF-LOPI PLGA Nps c) RIF-LOPI PLA Nps



DSC enables detection of all the processes in which energy is required or produced

(i.e. endothermic and exothermic phase transformations). The thermograms of RIF,

LOPI, PES, PLGA, PLA and their respective nanoparticles along with excipients are

shown in figure 6.10. Pure RIF and LOPI revealed a sharp melting endotherm

corresponding to their melting point indicating crystalline nature. The disappearance

of RIF and LOPI melting endotherm in their respective nanoparticle suggests

remarkable decrease in crystallinity.

e) Powder X-Ray Diffraction (PXRD)

Crystallinity in the sample is reflected by a characteristic fingerprint region in the

diffraction pattern. The XRD spectra of RIF, LOPI, PES, PLGA, PLA and their

nanoparticles are shown in figure 6.11. RIF, LOPI, PES, PLGA and PLA are highly

crystalline powders showing characteristic sharp diffraction peaks. These sharp

diffraction peaks disappeared in the respective nanoparticles indicating

amorphization/remarkable decrease in crystallinity of the drug.

Rifampicin Lopinavir

Poly (ethylene sebacate) PLGA

PLA RIF-LOPI PES Nps

RIF-LOPI PLGA Nps RIF-LOPI PLA Nps

Figure 6.11: pXRD spectra of RIF, LOPI, PES, PLGA, PLA and their respective nanoparticles



f) Hydrophobicity evaluation of nanoparticles (contact angle measurement)

As shown in table 6.16 hydrophobicity in term of contact angle of lopinavir was high

(91.45 ± 1.44) followed by rifampicin (74.33 ± 1.15), PLA (71.46 ± 1.47), PLGA

(61.1 ± 2.81) and PES (60.0±1.0). Hydrophobicity of LOPI and RIF was significantly

decreased when formulated in polymeric nanoparticles. PLA showing higher contact

angle was relatively more hydrophobic than PES and PLGA. PES and PLGA

exhibited comparable hydrophobicity.

Table 6.16: Hydrophobicity in terms of water contact angle of nanoparticles

Sr. No. Formulations Contact angle (n=3) 1. Rifampicin 74.33±1.15 2. Lopinavir 91.45±1.44 2. PES 60.0 ± 1 3. PLGA 61.1 ± 2.81 4. PLA 71.46 ± 1.47 5. RIF-LOPI PES Np (RLPES/7) 48.93 ± 2.03 4. RIF-LOPI PLGA Np (RLPLGA/1) 47.28 ± 1.32 7. RIF-LOPI PLA Np (RLPLA/1) 58.98 ± 2.57

6.5.4. Stability study

Freeze dried NPDDS were stable for 6 months at 30°C/60%RH and 40°C/75%RH.

All the nanoparticles revealed good redispersibility, no significant change in particle

size (as indicated by Sf/Si ratio<1.3) and drug content >90% suggest stability (table

6.17-6.19).

Table 6.17: Stability results of RLPES/7 at 300C/65%RH and 400C/75%RH

30°C/65%RH 40°C/75%RH

Drug content Sf/Si ratio

Drug content Sf/Si ratio RIF LOPI RIF LOPI

Initial 99.42 ±3.12

99.48 ±2.29 1.160 99.42

±3.12 99.48 ±2.29 1.160

1 month 97.72 ±1.46

98.85 ±2.65 1.171 98.14

±1.86 98.29 ±1.36 1.172

3 month 97.45 ±1.92

97.96 ±1.48 1.169 97.87

±2.01 97.54 ±1.93 1.171

6 month 96.99 ±1.28

97.23 ±2.02 1.178 97.12

±1.37 97.44 ±1.26 1.181



Table 6.18: Stability results of RLPLGA/1 at 300C/65%RH and 400C/75%RH

30°C/65%RH 40°C/75%RH Drug content Sf/Si

ratio Drug content Sf/Si

ratio RIF LOPI RIF LOPI

Initial 98.99 ±2.49

99.95 ±3.05 1.190 98.99

±2.49 99.95 ±3.05 1.190

1 month 99.12 ±2.92

98.92 ±2.27 1.187 99.04

±2.12 99.19 ±2.33 1.192

3 month 98.59 ±1.78

98.63 ±1.86 1.191 98.95

±2.10 98.43 ±1.98 1.190

6 month 97.43 ±1.89

97.90 ±1.02 1.195 96.42

±1.94 97.04 ±1.79 1.197

Table 6.19: Stability results of RLPLA/1 at 300C/65%RH and 400C/75%RH

30°C/65%RH 40°C/75%RH Drug content Sf/Si

ratio Drug content Sf/Si

ratio RIF LOPI RIF LOPI

Initial 99.69 ±2.82

100.5 ±3.25 1.180 99.69

±2.82 100.5 ±3.25 1.180

1 month 99.66 ±2.22

99.59 ±2.93 1.178 99.79

±2.36 99.84 ±2.23 1.175

3 month 98.25 ±1.03

98.43 ±1.39 1.179 98.36

±2.25 98.84 ±1.35 1.181

6 month 97.66 ±1.90

97.41 ±1.87 1.181 97.93

±1.83 97.32 ±1.74 1.187

6.5.5. Pharmacokinetic and biodistribution of nanoparticles

Figure 6.12: Pharmacokinetic profile of RIF-LOPI nanoparticles

Pharmacokinetic evaluation of RIF-LOPI nanoparticles revealed significantly higher

and sustained plasma drug concentration, delayed Tmax and enhanced oral

bioavailability for both RIF and LOPI compared to plain drugs (figure 6.12). T1/2

values were significantly higher with the nanoparticles for both RIF and LOPI (table



6.20). Relative oral bioavailability increased upto ~150% for RIF and ~200% for

LOPI with RIF-LOPI PES NPs and RIF-LOPI PLGA NPs respectively. RIF-LOPI

PLA NPs revealed even greater increase in relative oral bioavailability of ~200% for

RIF and ~255% for LOPI compared with plain drug. Hydrophobic particles are

readily taken up M-cells of by Peyer’s patches and lymphoid tissues. The contact

angle value intable 6.15 revealed PLA as the most hydrophobic polymer while PES

and PLGA exhibit comparable hydrophobicity. The hydrophobic nature enables

higher uptake through the Peyer’s patches and lymphoid tissues in the GI tract thereby

exhibiting enhanced bioavailability.

Table 6.20: Pharmacokinetic parameters of RIF-LOPI nanoparticles

PK Parameters

RIF-LOPI Dispersion RLPES/7 RLPLGA/1 RLPLA/1

RIF LOPI RIF LOPI RIF LOPI RIF LOPI

C max (μg/ml) 6.42 ±0.85

2.05 ±0.23

7.47 ±1.64

4.20 ±0.3

8.07 ±0.67

4.78 ±0.32

10.26 ±0.83

6.22 ±0.37

T max (h) 1 2 4.4 ± 0.89 4 4 4 6 6

Slope -0.014 ±0.002

-0.018 ±0.005

-0.023 ±0.003

-0.026 ±0.005

-0.021 ±0.001

-0.025 ±0.001

-0.028 ±0.001

-0.035 ±0.001

Kel(h-1) 0.034 ±0.005

0.042 ±0.012

0.053 ±0.008

0.061 ±0.013

0.048 ±0.003

0.058 ±0.004

0.065 ±0.004

0.081 ±0.003

T½(h) 6.68 ±2.89

7.33 ±4.02

13.23 ±2.09

11.64 ±2.06

14.25 ±1.11

11.91 ±0.81

10.62 ±0.74

8.51 ±0.35

AUC(μg/ml*h) 60.65 ±3.87

24.23 ±0.94

90.37 ±6.62

45.73 ±1.27

93.91 ±3.73

50.49 ±1.89

122.95 ±4.87

61.87 ±1.86

AUC infinity (μg/ml*h)

105.2 ±5.28

39.06 ±5.72

132.92 ±16.64

62.91 ±5.43

142.67 ±4.91

69.97 ±2.41

167.09 ±4.68

76.65 ±1.74

Bioavailability enhancement Ref. Ref. 149% 189% 155% 209% 203% 255%

Figure 6.13: Biodistribution profile of RIF-LOPI nanoparticles



Following oral administration of RIF-LOPI PES and PLGA nanoparticles revealed

significantly higher lung concentration compared to RIF-LOPI solution. However, RIF-

LOPI PLA nanoparticles revealed maximum lung uptake (figure 6.13). In other organs

(liver, kidney and spleen) no significant difference were seen with the nanoparticles.

Enhanced bioavailability and lung concentration is attributed to rapid and high uptake via

M cells of the Peyer’s patches. High bioavailability of LOPI confirmed that entrapping

the drug in a nanoparticulate carrier could provide adequate protection from cytochrome

P450 even in the presence of a drug like RIF a known inducer of cytochrome P450 to

enable significantly enhanced bioavailability. This protective effect of the nanoparticles

could have enabled high bioavailability of LOPI despite being in combination with RIF

an inducer of cytochrome P450. Similar observation on the metabolism of PLA

nanoparticles of isoniazid in vivo is demonstrated (Zhou et al., 2005).

In conclusion, design of RIF-LOPI nanoparticles provides a promising approach for

combining the two drugs for improved therapy of tuberculosis in HIV patients.

6.5.6. Highlights

Emulsion solvent evaporation technique represents a simple approach for

simultaneous entrapment of two drugs in a ratio 1:1 with high drug loading

RIF-LOPI PLA nanoparticles revealed enhancement in bioavailability 205%

for RIF and 255% for LOPI

The high bioavailability of LOPI observed confirm the entrapping is a suitable

strategy of bioenhancement despite combination with RIF an inducer of

cytochrome P450.

part ii: poly (ethylene sebacate) nanoparticles of anti...

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