Nano-formulation of rifampicin with enhanced bioavailability: development, characterization and in-vivo safety.

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Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

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Pharmaceutical nanotechnology

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Nano-formulation of rifampicin with enhanced bioavailability: Development, characterization and in-vivo safety

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Harinder Singh, Sahil Jindal, Mandeep Singh, Gaurav Sharma, Indu Pal Kaur *

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University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India

A R T I C L E I N F O

A B S T R A C T

Article history: Received 27 November 2014 Received in revised form 17 February 2015 Accepted 18 February 2015 Available online xxx

Rifampicin (RIF) was encapsulated into solid lipid nanoparticles (SLNs) to overcome its poor and unreliable oral bioavailability. Novel microemulsification method with high drug loading (50%) and entrapment efficiency (67%) was developed (Indian Patent Application 3356/DEL/2013). RIF-SLNs were characterized using TEM, AFM, DSC and XRD. Near neutral SLNs (zeta 3.5 0.8), with average particle size of 130.0 22.6 nm showed 70.12% release in phosphate buffer pH 6.8 in 9 days. Single oral dose (50 mg/kg) pharmacokinetic studies in Wistar rats indicated 8.14 times higher (in comparison to free RIF) plasma bioavailability with sustained levels for 5 days. Pharmacodynamic parameters viz. TMIC (120 h; time for which plasma levels were above MIC of 0.2 mg/ml), AUC0–1/MIC (1868.9 h) and Cmax/MIC (75.6) for RIF-SLNs were greater than free RIF by 2.5, 8.2 and 6.6 times, respectively. Similar LD50 (1570 mg/kg) and absence (or reversal in satellite group) of adverse events in repeat dose (three doses; highest dose was up to 50 times the human therapeutic dose) toxicity studies confirmed safety of RIF-SLNs. Improved pharmacokinetic profile of RIF-SLNs can be translated to a reduced dose and dosage frequency of RIF, thus resulting in lower or no hepatotoxicity commonly associated with its use. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Lipidic nanoparticles Rifampicin Acute toxicity Repeat dose toxicity Pharmacokinetics

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1. Introduction

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Rifampicin (RIF) is one of the first-line antitubercular drug (ATD) recommended by World Health Organization in the treatment of tuberculosis (TB) due to its unique ability to rapidly kill tubercular bacilli (WHO, 2008). However, poor and unreliable bioavailability, short biological half-life and severe hepatotoxicity are associated with its use (Shishoo et al., 2001; Singh et al., 2001). To overcome the problems of poor absorption, acid and other ATDs, viz. INH, induced gastric degradation, high hepatic clearance due to auto induction, intestinal metabolism, drug interactions (Zhang et al., 1998) and adverse side effects, controlled-release micro- and nanoparticulate formulations of RIF have been explored in the past decade (Dutt and Khuller, 2001; Khuller et al., 2004; O’Hara and Hickey, 2000; Qurrat-ul et al., 2003). Earlier we explored solid lipid nanoparticles (SLNs) as carrier systems for a wide variety of water soluble and insoluble drug molecules (Kakkar et al., 2013, 2011; Singh et al., 2013) like curcumin (prone to intestinal metabolism and high hepatic degradation and clearance), streptomycin (prone to acid degradation) and ketoconazole (completely insoluble lipophilic molecule;

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* Corresponding author. Tel.: +91 172 2534191; fax: +91 172 2543101. E-mail address: [email protected] (I.P. Kaur).

unpublished work) with great success (Bhandari and Kaur, 2013a; Kumar et al., 2013). Further SLNs can have a special application as carriers for ATDs including RIF, since the mycobacterium possess a special range of lipases to act on lipidic substrates (Smith, 2003) and have a special affinity for the same, hence lipid matrix of SLNs may attract mycobacterium. Action of lipases on RIF loaded SLNs will release RIF in a close vicinity of the mycobacterium resulting in a much faster and higher antimicrobial effect (Kaur and Singh, 2014). Furthermore, these SLNs being nano in size may also intercalate or enter more freely within and across the macrophage wall so that high local concentration of drug would be present at the seat of high occupancy of the mycobacterium. Intent of the present study was to develop concentrated SLN dispersion both in terms of high payload of RIF (50% with respect to the lipid used) and a minimal aqueous volume. Former was achieved by suitably modifying (Kaur and Singh, 2013a) the microemulsification method of preparing SLNs. Latter was also optimized using the ternary phase diagram studies. Low aqueous volumes were achieved by diluting the microemulsion with a very small (equal) volume of cold water than normally proposed 1:10 or 1:25 dilution with cold water. Former concept has been developed and reported by us for the first time (Kaur and Bhandari, 2012; Kaur and Singh, 2013b). Low aqueous volume is expected to prevent drug expulsion while the (Kumar et al., 2013), high loading will

http://dx.doi.org/10.1016/j.ijpharm.2015.02.050 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Singh, H., et al., Nano-formulation of rifampicin with enhanced bioavailability: Development, characterization and in-vivo safety. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.02.050

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allow administration of the required dose in smaller volumes. Ensuring a small particle size (500 nm (Supplementary Fig. 1) confirming the absence of any microparticulate structures or

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Table 1 Characterisation parameters of three SLN batches of final formulation ‘E’. Formulation code

TDC (%)

EE (%)

PS (nm)

PDI

EI E II E III

94 90 93.4

65 61 67

154 127 109

0.322 0.287 0.247


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probable aggregates. Further to this, a fair distribution of particles of different sizes are recommended for oral administration (Hu et al., 2005). The near neutral zeta potential indirectly implies a stable dispersion because neutral particles may be considered intrinsically stable due to absence of any inter-particulate attractive and repulsive interactions (Bhandari and Kaur, 2013a). Charge on the nanoparticles also influences their circulation time, metabolism, clearance and immune response as the proteins circulating in the plasma instantaneously bind on the surface of nanoparticles and tag them by forming a soft corona. This corona can initiate elimination of solid particulates from the body within few minutes by presenting them to the mononuclear phagocyte system inside the liver and spleen. 3.2.2. Total drug content (TDC) and percent entrapment efficiency (%EE) A high drug load of 50% with respect to the lipid phase was obtained when RIF was added to the hot lipid-surfactant phase (formulation E, Supplementary data, Table 1). The dispersion included both entrapped (with in SLNs; 67%) and free RIF with a total drug content (Table 1) of 24.5 2.5 mg/ml (formulation A, Supplementary data – Table 1 and 2 data). The free RIF present in small volume of water in the SLN dispersion ensures significant partition of drug with in the lipid matrix of the SLNs resulting in high EE (Burman et al., 1997). Both high drug loading and small dilution of 1:1 (usually a dilution ranging from 1:10 to 1:100 is reported) of the microemulsion with cold water to form SLNs resulted in high drug concentration/ml of the SLN dispersion. 3.2.3. Transmission electron microscopy Particle size distribution (Supplementary Fig. 1) indicated that 6–35% particles of RIF-SLNs were in the range of 46–100 nm. The nanoparticles were found to occur singly and were spherical in shape (Fig. 4a) when observed under TEM. High magnifications (1.5 lac) indicated a solid lipidic core surrounded by an outer surfactant layer (Fig. 4b). 3.2.4. Atomic force microscopy (AFM) The AFM images of formulated SLNs (Fig. 5a and b) demonstrated spherical particles (as indicated by topographic images) of 50–200 nm in diameter, and 75 nm in height. The

outcome is in agreement with particle size measurements using DLS where the majority of nanoparticles were found to lie between 100 and 200 nm. SLNs of soft matter tend to flatten due to gravitational force or the pressure of tip (Peloquin) of the AFM, hence, the height of these particles was lower than the other two dimentions. In addition the tip may be submerged into the outer soft surfactant coat of SLNs (Fig. 4b) and thus result in an underestimation of the particle height.

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3.2.5. Powder X-ray diffraction study Pure RIF exhibited characteristic peaks between 2u of 10 and 25 (Fig. 6). It was observed that the characteristic drug peaks disappeared in the XRD pattern of lyophilized RIF-SLN formulation and undefined, broad and diffuse peaks with low intensities were observed. However, some peaks corresponding to pure drug were still present and may be attributed to the presence of free drug (33%) in the formulation (Fig. 6).

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3.2.6. Differential scanning calorimetry (DSC) studies In case of freeze dried SLNs, endothermic peak corresponding to RIF (183 C) shifts to a much lower temperature of 107.72 C. RIF-SLN dispersion however did show a small endothermic peak at 188 C probably representing the small amount of free drug present in the sample. The heat change for the peak was 0.25 J/g as compared to 11.23 J/g for pure drug. A small sharp peak at 67.68 C corresponding to Compritol1 888 ATO along with a shoulder at 50 C was also observed while bulk lipid showed a peak at 73.06 C. Reduction of peak height and/or disappearance of peaks corresponding to free RIF and Compritol1 888 ATO coupled with a decrease in respective enthalpy were observed in general (Fig. 7). These factors indicate formation of a molecular dispersion of RIF in the matrix of lipidic nanoparticles and also confirm its existence in an amorphous form. Decrease in the melting point peak of lipid in nanoparticles is also accredited to the small size (nanometer range), high specific surface area, and the presence of a surfactant (Burman et al., 1997). In case of freeze dried SLNs, shift of RIF peak to a much lower temperature confirmed its incorporation within SLNs. All of the above indicate successful entrapment of RIF in SLN core, with little chance of its expulsion and a better controlled release with minimal burst effect (as observed subsequently in the in vitro release studies).

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Fig. 4. TEM micrographs of RIF-SLNs at (a) 150,000, (b) 20,000 magnification.


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Fig. 5. AFM of RIF-SLNs (a) D image of RIF-SLNs in non-contact mode, (b) topographic image of RIF-SLNs.

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3.2.7. In vitro release Release of RIF from RIF-SLNs showed a triphasic behavior comprising an initial slow release (7%) phase (upto 24 h) being attributed to the passage of very small SLNs (10 nm comprising 2% of particles) into the dialysate. Release (18%) and dissolution of free drug (the entrapped drug 35% was not removed from the prepared SLN dispersion loaded into the dialysis bag) through the dialyzing membrane is indicated by an exponential phase extending from 24 to 48 h. A lag phase seems to set in (upto

72 h) after this, probably due to slow release of drug entrapped in the solid core of developed SLN system. An extended and controlled release (Peppas model release kinetics) of RIF was observed post 96 h continuing over several days. RIF-SLNs showed a release of 70% RIF in phosphate buffer pH 6.8 after 9 days while more than 90% free RIF was released from the dialysis bag with in 24 h (Fig. 8). The prolonged and slow release of RIF from RIF-SLNs can be explained in terms of a very slow diffusion of RIF from the inner


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Fig. 6. Powder X-ray diffraction graph of RIF and freeze dried RIF-SLNs (RFD).

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solid lipidic core matrix of the SLNs, where it is probably entrapped in its solubilised form, to the surface of SLNs, from where it is released and passes across the dialysis membrane. During preparation of SLNs, RIF was added to the lipid-surfactant phase and it was found to dissolve completely in this phase. Various release models viz. Korsmeyer–Peppas, HixsonñCrowell, Higuchi, Zero-order model and First-order were applied on the release data with the release kinetics indicating Hixon–Crowell release upto 24 h signifying a surface dependent release as postulated by us. This is probably due to passage of very small SLNs into the dialysate and release of drug therein (Bhandari and Kaur, 2013 a,b). Further to it, a zero order slow release at constant rate was indicated for data from 0 to 96 h and Peppas controlled release was observed from 96 h onwards till the end of the study. All this points toward the incorporation of drug in the core of lipid matrix as expected for a lipophilic drug and confirm a highly controlled release system. It may also be concluded that composition of Compritol1 888 ATO is suitable to not only entrap large quantities of RIF but also to release the latter slowly. Former being rich in diglycerides is known to form less perfect crystals with many imperfections and thus may offer space to accommodate more RIF inside its matrix. 3.2.8. Stability of developed SLNs RIF-SLNs were found to be stable for 1.5 years with respect to TDC (not more than 5% change), %EE (not more than 10% change), particle size and PDI (Table 2). The high stability inspite of a highly concentrated dispersion (solid content of 9%) is attributable to the optimized formula with suitable concentration of surfactant– cosurfactant molecules which surround the nanoparticles and are also present in the aqueous dispersing medium so as to ensure a stable deflocculated dispersion. It is postulated that free RIF is present in equilibrium with small volume of water in the SLN dispersion so that leakiness from RIF-SLNs is minimal and no significant change in %EE is thus observed. 3.3. Pharmacokinetic study RIF though well absorbed in humans from the gastrointestinal tract suffers compromised bioavailability due to the influence of delayed gastric emptying and alterations in gastric pH on RIF bioavailability. Following a single oral dose (50 mg/kg) of RIF-SLNs to rats, a significant drug concentration above MIC90 (0.2 mg/ml)

was maintained in the plasma for 5 days (Fig. 9). The nanoencapsulation of RIF served to ensure a faster and larger uptake and slow sustained clearance of drug, a feature that was not demonstrated with encapsulated/free RIF (Fig. 9). Values for Cmax, Tmax, t1/2, AUC0–1 and relative bioavailability of encapsulated drug were significantly higher than those obtained for the free drug (P = 0.05) (Table 3). The maximum concentration (Cmax) of RIF observed in plasma was 15.12 mg/ml and 2.27 mg/ml, respectively for RIF-SLNs and free drug (Fig. 9; Table 2). Results show that the bioavailability of RIF achieved after administration of RIF-SLNs was 8.14 times higher than that obtained for free RIF. Furthermore, significant plasma concentration levels were observed for upto 5 days as compared to free RIF which showed detectable levels only till 48 h. (Supplementary Fig. 2 shows comparison of predicted and observed pharmacokinetic profile of RIF after oral administration of free RIF and RIF-SLNs using PK solver 2.0). The pharmacodynamic parameters indicated that the plasma drug concentration remained above MIC of 0.2 mg/ml (Chanwong et al., 2007) for 120 h (TMIC). The Cmax/MIC and AUC0–1/MIC values for RIF-SLNs were 75.6 h and 1868.9 h while the values for free drug were 11.35 h and 229 h, respectively. Another highlight of the study was that the time taken to achieve maximum concentration (Tmax) for RIF-SLNs (4.67 h) was the same as that observed for free RIF (4.43 h), although the Cmax achieved in plasma was 6.7 times higher. This is attributed to (i) a fast uptake from stomach and parts of upper intestine (Bhandari and Kaur, 2013a; Kakkar et al., 2011) and (ii) lymphatic uptake of SLNs pouring the drug directly into blood circulation (Khan et al., 2013). Achieving higher Cmax of 6.6 times with respect to free drug at the same tmax in contrast to a much longer tmax observed by other groups with alginate or PLG-NPs and SLNs; (Ahmad et al., 2006; Pandey et al., 2005) is presently an important achievement which can be translated to treatment success of RIF as RIF-SLNs. The target range of peak plasma concentrations of RIF at 2 h with significant antimycobacterial activity has been reported to be 8–24 mg/ml. RIF concentrations between 4 and 8 mg/ml are considered low, and concentrations below 4 mg/ml are considered subtherapeutic (Peloquin, 1997). Hence, a high Cmax of 15.12 mg/ml and Cmax/MIC of 75.6 achieved by us is highly significant. Studies demonstrate that early bactericidal effect, suppression of resistance, and post-antibiotic effect of RIF are strongly linked with its Cmax/MIC ratio (Jayaram et al., 2003; Verbist, 1969). Considering a MIC of 0.05 mg/ml in vitro, which translates to 0.25 mg/ml after adjusting for 80% protein binding in-vivo, the optimal Cmax/MIC ratio is indicated to be 32. Presently observed Cmax/MIC is 2 times of the optimal value. Other workers have reported Cmax/MIC ratio of 5.3 and 8 which is well below the required value (Pandey and Khuller, 2006; Pandey et al., 2005). Another general observation for all the previously reported pharmacokinetic studies on RIF loaded polymeric (PLG/alginate) and lipidic nanoparticles indicate a highly delayed Tmax varying from 24 to 48 h or even more while we achieved an almost similar Tmax as that of free RIF. This will reduce the exposure to suboptimal concentrations which though not effective may cause toxic reactions especially in combination with other ATDs and may also result in induction of resistance. A significant reduction in clearance (10 times), Kel (2 times) and Vd (3.8 times) indicate a prolonged circulation and major distribution in plasma. This may be attributed to small size and hydrophilic coat of Tween 80 on these particles such that they can overwhelm pick up by RES organs. Since liver is also a site of metabolism and since RIF is known to induce its own metabolism (Zhang et al., 1998), so the RIF entrapped within SLNs is protected form metabolic conversion and only free drug released from the


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Fig. 7. DSC of RIF-SLNs dispersion and other and constituents.



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Fig. 8. Percent cumulative release of RIF-SLNs and drug (n = 3) in phosphate buffer pH 6.8 upto 24 h.

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SLNs will undergo elimination. Furthermore, since RIF is highly bound to plasma proteins, so only a very small fraction (20–40%) out of the available free RIF is metabolized (Jayaram et al., 2003). Approximately 18 fold increase in relative bioavailability of RIF when compared to the free drug, with plasma levels being maintained for 5 days have been reported (Pandey and Khuller, 2006) in a study. Nanoparticles reported by these authors are polymeric (PLG, a synthetic polymer) in nature prepared using organic solvents. This is in addition to the concern for biodegradable nature of the polymer used and the toxicity/safety of the monomers formed therein. In addition to the listed benefits of lipidic nanoparticles, latter would have a specific advantage for treatment of mycobacterium infections as described earlier. Presently, an 8 fold increase in bioavailability was achieved with RIF-SLNs when administered orally in comparison to RIF suspension. Pandey et al. (2005) also report a relative bioavailability of 11 with RIF-SLNs but the Tmax reported by them is 24 h (Cmax 2 mg/ml) and they prepared SLNs by emulsion solvent diffusion involving the use of an organic solvent. They achieve an entrapment efficiency of 51%, whilst we achieved entrapment efficiency of 67%. The drug loading reported by them is 33% versus 50% reported by us presently. Though the basic mechanisms of improved oral bioavailability yet remain to be elucidated for SLNs, however reports including our studies indicate that small particle size and surface characteristics are largely responsible for the improved oral absorption and

Table 2 Pharmacokinetic parameters of RIF following oral administration of RIF (as free or SLN) in male Wistar rats at a dose of 10 mg/kg. Drug

Free RIF

RIF-SLNs

Dose (mg/kg) Tmax(h) Cmax(mg/ml) Cl (mg/ml/h) Kel(h1) T1/2(h) Vd(L) AUClast(mg h/ml) AUCinf(mg h/ml) AUMC(mg/ml h2) AUC/MIC Relative bioavailalability

10 4.43 2.27 0.22 0.121 2.9 1.79 37.43 45.8 1268.55 229 1

10 4.67 15.12 0.027 0.057 7.6 0.47 363.92 373.78 10398.4 1868.9 8.16

Fig. 9. Plasma-concentration profile of RIF after oral administration of free RIF and RIF SLNs (n = 6).

availability of drugs entrapped with in SLNs (Hanafy et al., 2007; Hu et al., 2004; Muller et al., 2006; Niemi et al., 2003; Paliwal et al., 2009 Zhuang et al., 2010).

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3.4. Toxicity study

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3.4.1. Acute toxicity study (OECD TG 425) Pre-clinical phase of drug development includes toxicity studies in animals. The principle behind these experiments is that the use of high doses of a compound in a group of animals should reveal intrinsic potential toxicity (of a compound) that would occur with a low frequency in patients receiving much smaller therapeutic doses. Nanoparticles possess different physicochemical properties than the free drug particles due to their extremely small size and large surface area, so they are expected to interact intimately with the biological systems. Further to this, according to US-FDA, if plasma levels of a pharmaceutical product increases substantially (8.16 times presently) as compared to the reference product (free RIF), then major concern is not therapeutic failure but the safety of the product. Assuming that the toxic potential of RIF-SLNs may be different from free RIF; it was with this perception that the toxicity studies were conducted. LD50 for both free RIF and RIF-SLN was 1570 mg/kg body weight (BW) (Supplementary data, AOT software sheet A and B) as calculated using the official software AOT 425 StatPgm. RIF-SLNs thus fall in the class III (LD50 from 300 to 2000 mg/kg) of Globally Harmonized System for classification of chemicals which cause acute toxicity (OECD, November 1998). No significant change was observed in BW of animals of either group administered the determined LD50. Food and water consumption was reduced on the first day of administration of RIF-SLNs but returned to normal by second to fourth day. All other observable parameters and activities of animals were normal except for a diminished activity at the highest dose of 5000 mg/kg BW for both free RIF and RIFSLNs, in which case death occurred. No mortality was observed in either case at the lowest dose of 500 mg/kg BW. Even though RIF is majorly reported to induce hepatotoxicity but pathological changes in brain (Fig. 10 1A) and kidney (Fig. 10 1B) were also monitored, in addition to liver (Fig. 10 1C), as prescribed under OECD 425 guidelines. As SLNs are reported to cause hyperplasia in spleen, therefore spleen (Muller et al., 1997) was also monitored for necropsy (Fig. 10 1D). Histology of liver (Fig. 10 1C) did not show any architectural alterations in either case. However, mild increase in lymphoid

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Fig. 10. 1(A) Brain (200); (B) kidney (200); (C) liver (100); (D) mildly reactive spleen (100) sections of animals administered LD50 dose (1570 mg/kg) of RIF SLNs in acute Q13 toxicity study. (E) Indicates normal spleen section (100) for corresponding LD50 for free RIF. 2(A) (i–ii) Brain sections (100, 200), (iii–iv) kidney sections (100, 200) of animals administered 500 mg/kg RIF SLNs in repeated dose 28 days toxicity. (B) (i) Normal liver of naive control, (ii–iii) mild portal triaditis in liver tissue at 250 mg/kg dose observed for both males and females (200, 100), (iv) micro vesicular fatty changes in liver tissue at 500 mg/kg dose of males (200). (C) (i) Spleen section (40) showing normal histology in naive control animals; (ii–iv) showing reactive spleen (40, 100 and 200) observations made both for vehicle control and 500 mg/kg dose group.

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follicles (Fig. 10 1D) was observed in spleen at the highest tested dose of RIF-SLNs. Latter may be indicative of mild reactive hyperplasia of spleen. Spleen of animals administered free RIF was found to be normal (Fig. 10 1E). Rifampicin is associated with hepatocellular pattern of drug induced liver injury (Nakajima et al., 2011) and more often it potentiates the hepatotoxicity of other anti-TB drugs (Liu et al., 2008). It was indicated that RIF induces hepatocellular dysfunction early in the treatment, which tends to resolve without a need to discontinue the drug (Girling, 1977). The use of phosphatidylcholine presently in the preparation RIF-SLNs, is expected to protect against RIF induced liver injury because of its established hepatoprotective nature (Kidd, 1996). The decrease in food and water uptake by animals administered RIF-SLNs or blank SLNs was observed and may be attributed to the fulfillment of energy needs of animals from lipids present in SLNs and water requirements from the aqueous nature of the SLN dispersion administered at large volumes of 4 ml, 12 ml and 30 ml on an average for 500, 1570 and 5000 mg/kg dose. Similar loss of appetite and decreased water intake is also reported for SLNs by other workers (Xie et al., 2011). The changes observed in spleen were mild and similar changes have also been reported for SLNs prepared from Compritol1 ATO 888 showing an altered architecture of spleen. However, such a change was found to be reverse in 6 weeks (Blasi et al., 2007). Histological observations made on all animals (free RIF and RIFSLNs) did not indicate any gross pathological change in the major organs viz. liver, brain, kidney and spleen. Since LD50 of both RIF SLNs as well as free RIF was found to be same, thus it may be predicted that the nano nature of the formulation did not enhance the intrinsic toxicity of RIF.

3.4.2. Repeated dose 28 days oral toxicity (OECD TG 407) The general observations including wellness parameters and long term effects of the treated as well as the control animals were found to be normal. However, after administration of the high dose (500 mg/kg) of RIF-SLNs, a decrease in food and water consumption was observed. A reduction, though of a lower intensity was observed for lower dose group too. Daily food and water consumption (Supplementary Table 3) in RIF-SLNs dose groups was similar to that in blank SLNs group (i.e., vehicle control). Similar observations were also made during acute toxicity studies and are also reported by other workers (Xie et al., 2011). No significant difference in absolute weights as well as in relative organ weights (liver, kidney and brain) was observed for any treatment group with respect to the control at any of the tested dose (Supplementary Table 4), except for a significant increase in relative spleen weight in male animals of the highest dose and vehicle control group (blank SLNs administered in a volume corresponding to the highest dose). No mortality was observed in male and female rats at the low (125 mg/kg; 12.5 times human therapeutic dose) and median dose (250 m/kg; 25 times human therapeutic dose) as well as in vehicle control group. However, at the highest dose levels (500 mg/kg) 1 male rat (out of 10) died on 9th day of dosing, while 1 female rat died on 18th day of dosing. No behavioral changes viz. change in muscle strength and pain threshold (nociception) were observed at any of the tested doses or in the vehicle control group (Supplementary Figs. 3–5). A significant decrease in hemoglobin of males at doses higher than 125 mg/kg dose group and in the vehicle control group was observed. However, no significant change in any other hematological parameter was observed in animals of either sex at all the tested doses (Supplementary Table 5).


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The phospholipid levels were significantly decreased in males at the dose of 125 mg/kg and 250 mg/kg (dose dependent) but there was no significant effect in cholesterol and triglycerides level. Further, a significant decrease was also observed in total lipids at 250 mg/kg and 500 mg/kg doses in males, at 500 mg/kg in females and in vehicle control group. In terms of the liver function tests (Supplementary Table 6), an increase in hepatic enzymes was observed at some of the tested doses. However, no clear correlation could be drawn with the level of enzymes induced and the administered dose. SGOT (AST) was significantly increased at the highest tested dose of 500 mg/kg in females and increase in SGPT (ALT) was observed only at 125 mg/kg in both males and females. Similarly a significant increase was observed in case of ALKP in males at 125 mg/kg and 500 mg/kg as well as in satellite group. However, an arithmetic reversal in the value was observed (from 762 to 533) in the satellite group in comparison to the corresponding high dose group (group 3) of 500 mg/kg. Significantly increased bilirubin levels (almost three times) in respect to the control values were observed at all the tested doses in both males and females. A reversal of values for SGOT, SGPT and bilirubin in the satellite group established the safety of RIF-SLNs upon withdrawal of drug administration which, is otherwise a hepatotoxin and is expected to disturb the levels of these hepatic enzymes. None of the animals except 1 female rat (taken as outlier) of the satellite group showed exceptionally high plasma sugar levels of 233 mg/dl versus 73 8.83 mg/dl for naive control animals. Histological investigations revealed significant though mild alterations in the structural architecture of liver (Fig. 10 2B (ii–iv)) and spleen in groups administered RIF-SLNs. No animal showed any significant change in kidney function (creatinine and plasma sugar levels; Supplementary Table 6). Absence of any adverse histological event in any organ of the satellite group and in brain and kidney (Fig. 10 2A (i–iv)) of all animals at any of the tested doses establish the safety of RIF-SLNs in these organs. Histology of liver revealed that at median dose, animals showed mild lymphocytic collection (Fig. 10 2B (ii)) in comparison to the normal liver of naive control (Fig. 10 2B (i)). Lymphocytes were collected in portal triads showing their inflammation also referred to as mild portal triaditis (Fig. 10 2B (iii)). The collection of lymphocytes is always indicative of liver damage. Microvesicular changes (Fig. 10 2B (iv)) were also observed in the liver of male rats, liver at the highest tested dose of RIF-SLNs. However, those histological alterations were absent in the satellite group, indicating complete reversal of the induced changes. Mild follicular hypertrophy (Fig. 10 2C (ii–iv)) was observed in spleen of 2 out of 4 female rats at the highest dose of 500 mg/kg and in 1 each male and female animal of vehicle control group. The photographs show reactive enlargement of the white pulp i.e., lymphoid tissue. Each follicle shows a reactive center or follicular center (FC) and an enlarged or expanded marginal zone (MZ). However, no such changes were observed in any of the animal at lower doses as well as in the satellite group and histology compared well with the naive control animals (Fig. 10 2D (i)). To our knowledge, no repeated dose 28 days oral toxicity study is reported for RIF although an (Yun et al., 1991) intraperitoneal administration of RIF (30 mg/kg) for 10 days has been reported to show no adverse effects. We however did observe some adverse events like an increase in SGOT, SGPT and bilirubin, on oral administration of much higher doses of 125, 250, 500 mg/(50 times human recommended dose and 10 times applying conversion factor of 5) of RIF as RIF-SLNs. However, the effects were reversible as indicated by disappearance of most of these effects in the satellite group. The observed increase in relative weight of spleen (reversible in satellite group) was attributable to the deposition of Compritol1

888 ATO. Latter is reported to provoke a reversible increase in the liver and spleen weight increase (Weyhers et al., 1995), though presently we did not observe any significant change in the liver weights. There was change in the number of leucocytes or platelets. However, a decrease in Hb was observed in male rats administered RIF-SLNs at all the tested doses and also the vehicle control group. This may be attributed to an enhanced oxidation of Hb due to the lipids (Stratford and Murthy, 1997) used in the production of SLNs. A significant decrease in Hb levels in a vehicle control group has been reported earlier (Xie et al., 2011). The safety of RIF-SLNs may, however, be highlighted in terms of no significant change in platelet count at any of the tested doses which is a major concern for free RIF. RIF is an effective liver enzyme-inducer, promoting the upregulation of hepatic cytochrome P450 enzymes (such as CYP2C9 and CYP3A4) (Backman et al., 1998) and the breakdown of fats and lipids. Since in the present study no such changes were observed even at a dose as high as 500 mg/kg, so it may be concluded that RIF-SLN administration in no way influenced lipid metabolism even at a dose 10 times higher than that recommended usually. The cytochrome P450 enhancing property of RIF tends to alter the liver enzymes like serum glutamic-oxaloacetic transaminase/ aspartate transaminase (SGOT/AST), serum glutamic-pyruvic transaminase/alanine transaminase (SGPT/ALT) and alkaline phosphatase (ALKP). Increase of any of these three enzymes in serum shows hepatocellular damage. When liver cells get damaged, they release these enzymes into the blood. RIF is a potent and established hepatotoxicant as discussed earlier, hence an increase in the serum levels of all these enzymes is expected upon its administration, however, RIF-SLNs were found to be generally safe (except SGOT levels). Furthermore, the changes observed at the highest tested dose of 500 mg/kg showed a reversal of these effects in the satellite group (Supplementary Table 6). A significant hyperbilirubinemia is also reported with the use of free RIF (Schiff et al., 2002) producing jaundice like symptoms. Same (2–3 times increase) was also observed with RIF-SLNs especially at 250 and 500 mg/kg dose. However, the alterations observed in liver function test (LFT) for RIF-SLNs may be ignored as not of much clinical significance. According to TB manual-NTP guidelines, rise of any LFT up to 5 times of the control value is documented to be safe and it is indicated that the therapy does not need to be discontinued. In the present study an increase of not more than 2 times was observed for any of the determined LFT parameter (except for bilirubin which was about 3 times). Furthermore, the bilirubin levels also showed reversal to baseline (control) after discontinuation of the therapy in the satellite group. This clearly establishes the safety of RIF-SLNs in terms of induced hepatic alterations. To the best of our knowledge, literature does not show any nephrotoxicity with RIF but it may raise blood sugar levels. This rise in blood sugar levels is linked to impairment of hepatic cells. As liver cells store sugars, so any damage to them can lead to release of stored sugars in to the blood resulting in increased blood sugar levels (Waterhouse et al., 2005). However, no such changes were observed in plasma sugar levels in the repeated dose 28 day toxicity study conducted by us. The pattern of change in histology slides was almost the same in all animals and is hence displayed only for a single animal group to avoid duplication of data. In a previously reported study in patients, portal triaditis was seen at a significantly lower dose of free RIF (50 mg/kg) (Rana et al., 2006). The changes in spleen of vehicle control group clearly shows that it was because of the deposition of SLNs as is also documented in the literature (Blasi et al., 2007). The changes observed presently in liver may be attributed to mild damage of hepatocytes and thus lymphocytic infiltration


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which caused the inflammation of portal triads. Microvesicular fatty changes occur because of the altered lipid metabolism. Generally, altered lipid metabolism leads to deposition of fat molecules in cytoplasm of hepatocytes on the account of which they become swollen. However, the reversal of these alterations in 2 weeks after the therapy was terminated, confirms the safety of RIF-SLNs. Literature also showed such alterations to be reversible but in 6 weeks (Weyhers et al., 1995). Observation of very mild changes at such high doses which are reversible on discontinuation of therapy reestablish the safety of RIF-SLNs.

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4. Conclusion

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A novel nanoparticulate delivery system of RIF with a significantly high drug loading of 50% with respect to lipid used (1:2) was presently developed for enhancing bioavailability and prolonged release. Pharmacokinetic studies validated the design concept and signify the potential of the developed system for improved bioavailability. Part of the latter, may be due to resistance to degradation at acidic pH of stomach upon oral administration as established and reported earlier by us (Singh et al., 2013). On one side, high RIF doses are indicated for treatment success and on the other side RIF tends to autoinduce its metabolism or may enhance metabolism of INH to more toxic metabolites (Nannelli et al., 2008) by activating cytochrome P450 (3A4). These issues can however be overcome if low free drug plasma concentration is maintained as demonstrated presently with RIF-SLNs. The study established that RIF-SLNs produced no treatment related toxicity in rats following oral administration, thus the latter can be exploited for potential therapeutic applications in humans. A more than 8 times higher may thus be manifested in clinics in terms of using a lower dose which will ensure low or no toxicity with RIF-SLNs at the administered dose. The manufacturing technology used for the preparation of novel RIF-SLNs is relatively simple as compared to other costly techniques and can be easily adapted to an industrial setup on commercial scale.

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Acknowledgements

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Authors are thankful to the Department of Biotechnology, Government of India for providing financial help to carry out the research work. SAIF facility of Panjab University, Chandigarh, India for TEM and XRD studies is also acknowledged.

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Appendix A. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2015.02.050.

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