Alternative Pharmaceutical Formulation for Oral Administration of Rifampicin

Abstract

Tuberculosis (TB) is considered an emergency global public health, mainly due to the TB-HIV co-infection, bacillus dormancy stage, and emergence of resistant strains. In addition, the therapeutic toxicity and its pharmacokinetic interactions with other drugs may influence treatment non-compliance, low serum concentration of drugs, and, consequently, treatment failure. Strategies using nanotechnology represent a new tool for the therapy, since they are effective delivery systems due to the possibility of solubilization of hydrophobic compounds, enable the production of formulations for oral use, and, in addition, increase bioavailability of drugs. This study aimed to develop a nanoemulsion (NE) containing rifampicin (RIF-NE) and evaluate its in vitro antimycobacterial activity using Resazurin Microtiter Assay against three Mycobacterium tuberculosis strains: two susceptible and a multidrug-resistant. Using the hot solvent diffusion method associated with phase inversion technique was possible to develop a liquid formulation containing 500 μg/mL rifampicin (RIF), which is a hydrophobic compound, of average size 25 nm. The results showed that the minimum inhibitory concentration of the encapsulated RIF was equal to the free form of RIF, indicating that the process of production of NEs did not affect the activity of the compound. Thus, RIF-NE could be a promising alternative for oral administration of RIF, being considered a child-friendly pharmaceutical formulation. Its application could avoid the administration of unknown and/or non-ideal concentrations, being functional in the regimes of prevention and treatment of TB and, in addition, in the fight against drug resistance.

Introduction

Although a curable disease, since 1993 the tuberculosis (TB) has been considered an emergency global public health, and some factors contribute to this scenario: TB-HIV co-infection, bacillus dormancy stage, emergence of resistant strains, toxicity of the treatment, and its pharmacokinetic interactions with other drugs.¹ Therefore, there is also an incentive to develop new strategies for the treatment of TB, especially those that can facilitate the administration and enhance the activity of drugs already used in therapy.² Considering these purposes, several types of carriers have been developed to carry anti-TB drugs, such as liposomes, nanopolymersomes, and lipospheres.^3
–5

Among the technological tools, we highlight a variety of nanostructures, such as lipid nanocarriers, that are good candidates for the administration of drugs that are biocompatible and biodegradable. These delivery systems, in general, can increase the solubility of hydrophobic drugs and consequently their bioavailability in vivo.⁶ Also, the delivery systems may lead to dose and treatment time reduction and, therefore, reduction of side effects and systemic toxicity, increasing patient compliance to therapy.^6
–8

Some studies have proposed the use of nanoemulsions (NEs) in the treatment of TB, but through differentiated routes of administration, including transdermal⁹ and pulmonary routes.¹⁰ However, NEs have been considered promising, especially for oral drug administration, increasing bioavailability and improving therapeutic efficacy.¹¹

Specially in cases of childhood TB, the lack of child-friendly dosage forms leads to the use of crushed tablets or opening capsules for the preparation of solutions to facilitate drug administration.^12,13 However, some drugs used in the treatment of TB are hydrophobic, such as rifampicin (RIF), and so may hinder this process and can result in low serum concentration of drugs and, consequently, interfere with the effectiveness of the treatment.^12,14 Thus, the development of a liquid formulation of anti-TB drugs that could be administered by oral route and that are effective against Mycobacterium tuberculosis could be an interesting alternative for children.

In this context, this study aimed to prepare a NE containing RIF (RIF-NE) and evaluate its in vitro antimycobacterial activity to contribute to the development of alternative therapies for TB.

Materials and Methods

Preparation of NE

Castor oil (CO), 12-hydroxystearic acid-polyethylene glycol copolymer (PEG-660 stearate/Solutol HS15^®), and RIF were purchased from Sigma-Aldrich (St. Louis, EUA), and hydrogenated soybean lecithin (LEC; Phospholipon 80^®) was obtained from Lipoid (Steinhausen, Switzerland). Unloaded-NE (UN-NE) was prepared by hot solvent diffusion method associated with phase inversion temperature technique, previously described by Hädrich et al., using CO as the oil phase and PEG-660 stearate and LEC as surfactant and co-surfactant,¹⁵ respectively. A mixture containing CO and LEC diluted in 5 mL acetone/ethanol (60:40, v/v) at 60°C was added to an aqueous phase containing PEG-660 stearate (50 mL) at 82°C under magnetic stirring (700 rpm). Subsequently, the formulations were kept under magnetic stirring at room temperature and allowed to cool. The organic solvents were evaporated under low pressure (23 mbar) to give a final volume of 20 mL. The final formulation was filtered using a 8 μm filter. The formulations were prepared in triplicates in the following concentrations: 15 mg/mL PEG-660 stearate, 7.5 mg/mL CO, and 1 mg/mL LEC. For the preparation of RIF-NE, we used the same conditions and concentrations described above, with the addition of RIF to the organic phase of formulation, to obtain a final concentration of 500 μg/mL. After 7 days of nanocarrier production it was given a visual inspection to determine if the formulations were not destabilized during this period, separating phases or precipitating RIF.

Size, Polydispersity Index, and Zeta Potential

The average diameter and polydispersity index (PDI) of the formulations were determined by dynamic light scattering using Zetasizer Nano Series (Malvern Instruments, Worcestershire, United Kingdom). The light-scattering measurements at 90° were performed at 25°C. The hydrodynamic radius was determined using the Stokes-Einstein equation, R = (κ_B T/6πηD), where κ_B is Boltzmann's constant (J/K), T is temperature (in K), D is diffusion coefficient, and η is viscosity of the medium—water in this case (η = 0.89 cP at 25°C). For zeta potential measurements, the samples were diluted in 1 mM NaCl and placed in an electrophoretic cell where an alternating voltage of ±150 mV was applied. The zeta potential values were calculated as mean electrophoretic mobility values using Smoluchowski's equation.

Determination of RIF Concentration in the Nanocarriers

The RIF content was determined by UV/VIS analysis. The measurements were performed with a spectrophotometer (UV/VIS Spectrophotometer Perkin-Elmer) using a quartz cuvette of optical path 1 cm and detection at 337 nm. The content was compared with a standard of RIF in methanol, and methanol was used for background correction.¹⁶ The analytical methodology was validated for parameters such as specificity, linearity, limit of quantification (LQ) and detection (LD), precision, and accuracy, as described in the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (2005).^17,18 For UV analysis, an aliquot of nanocarrier was completely dissolved in methanol. The RIF content (total concentration) in the nanocarrier was calculated after determining the drug concentration in methanolic solutions and was expressed in micrograms of RIF/mL of nanocarrier. The RIF recovery was calculated as being the percentage of total drug concentration found in the nanocarrier in relation to the initially added amount.

Strains and Inoculum

Antimycobacterial activity of UN-NE, RIF-NE, and free RIF was evaluated against two susceptible M. tuberculosis strains (strain 1 and H37Rv-ATCC 27294) and a multidrug-resistant (MDR) strain with mutations in katG (S315T) and rpoB (S531L) genes (Strain 2). The mycobacterias were from the strains bank of the Medical Microbiology Research Center (Federal University of Rio Grande) and were cultured in Ogawa-Kudoh for up to 14 days at 37°C. For the evaluation of antimicrobial activity, a bacterial suspension was prepared in sterile distilled water according to 1.0 McFarland scale (3 × 10⁸ UFC/mL). The inoculum was prepared by diluting the bacterial suspension at a ratio of 1:20 in Middlebrook 7H9 Broth.¹⁹

Resazurin Microtiter Assay

The antimycobacterial activity of the compounds—free RIF, RIF-NE and UN-NE—was determined by microdilution broth method, using resazurin as an indicator of cell viability.¹⁹ RIF was diluted in dimethyl sulfoxide at a concentration of 20 mg/mL and was stored until use. Briefly, the wells of a 96-well microplate received 100 μL of Middlebrook 7H9 medium supplemented with 10% OADC (oleic acid, albumin, dextrose, and catalase) and 100 μL of the compound, and next, a serial microdilution (1:2) was performed. The concentrations evaluated ranged from 5,875 to 5.7 μg/mL for UN-NE and from 6,000 to 5.9 μg/mL for RIF-NE. In addition, free RIF was also evaluated from 2,048 to 0.06 μg/mL. At the end of microdilution, 100 μL of the inoculum was added in each well. Controls of bacterial growth and sterility were included. The plate was incubated at 37°C for 7 days; after this period, 30 μL of resazurin (Sigma-Aldrich) 0.02% was added in each well, the plate was reincubated for 24 h, and then the MIC was determined.¹⁹ The MIC was defined as the lowest concentration of the compound capable of inhibiting bacterial growth.

Cytotoxicity Assay

In a 96-well plate, 200 μL of a suspension of adhered cells of J774A.1 lineage (ATCC TIB-67) at a concentration of 1 × 10⁵ cells/mL was cultivated in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum. The cells were maintained for 24 h at 37°C in a humid atmosphere (5% CO₂), and after this period, the adherent cells were exposed to UN-NE and RIF-NE at the same concentrations evaluated in the antimycobacterial activity assay. Controls of sterility and growth were included. After 24 h of exposure, the contents of the wells were removed, 30 μL resazurin (0.01%) was added to it, and the plate was reincubated overnight until measurement of optical density to determine the half maximal inhibitory concentration (IC₅₀), that is, concentration of the compound capable of maintaining viability of 50% of cells.²⁰

Results and Discussion

Among the priorities in the scenario of TB there are issues related to the development of new interventions of drug administration aiming to control drug resistance, increase cure rates, and reduce mortality, including for children.²¹ Using hot solvent diffusion method associated with phase inversion temperature technique, it was possible to obtain a liquid pharmaceutical formulation in which approximately 500 μg/mL RIF, which was completely soluble, could be administered by oral route, with the following physical-chemical characteristics: size ∼25 nm; PDI = 0.18; and zeta potential = −8.22 mV (Table 1).

Table 1.

Physical and Chemical Characteristics of RIF-NE

Formulation	Size (nm)	PDI	Zeta potential (mV)	RIF amount (μg/mL)	Recovery (%)
RIF-NE	25.75 ± 0.01	0.18 ± 0.01	−8.22 ± 4.59	488.8 ± 22.7	97.7% ± 4.51

The results are expressed as mean and standard deviation.

PDI, polydispersity index; RIF, rifampicin; RIF-NE, nanoemulsion containing rifampicin.

Regarding analytical validation parameters, the calibration curve was linear in the ranges of evaluated concentrations, and LQ and LD were 2.75 and 0.91 μg/mL, respectively (Table 2). These data indicate that the method is sufficiently sensitive for determining the total content of RIF in the formulation; and in relation to accuracy, the recovery values varied between 71% and 116% (Table 3). After 7 days, from the day of production, a visual inspection of UN-NE and RIF-NE was performed, and no phase separation or precipitation of RIF was observed. Thus, in vitro experiments were performed.

Table 2.

Linear Regression Analysis of Calibration Curve Data

Parameters	Spectrophotometry (UV/Vis)
Concentration (μg/mL)	2.5–15
Equation of straight line	y = 0.0289x + 0.0392
r ²	0.99
LD (μg/mL)	0.91
LQ (μg/mL)	2.75

LD, limit of detection; LQ, limit of quantification; UV/Vis, ultraviolet–visible.

Table 3.

Recovery Values

Concentration level	Presumed content of RIF (μg/mL)	Average concentration of RIF (μg/mL)	Recovery
Low	7.0	7.1	71%
Medium	10.0	9.0	90%
High	13.0	11.6	116%

Considering the several nanocarriers developed for the delivery of anti-TB drugs, NEs could be a promising alternative to overcome some barriers: they allow encapsulation of lipophilic compounds (such as RIF), are low-viscosity liquids sterically stable, can be produced on a large scale, and can increase the bioavailability of drugs.^15,22
–24

LEC used in the organic phase is a co-surfactant of natural origin with amphoteric character, and ∼70% of its composition is phosphatidylcholine. Due to the lipophilic character of lecithin, the use of surfactants in the formulation is recommended, such as PEG-660 stearate, to promote stabilization of the system. PEG-660 stearate is a nonionic surfactant, having a lipophilic region consisting of mono- and diesters of polyglycol chains of 12-hydroxystearic acid and a hydrophilic region consisting of ∼30% free polyethylene glycol. Indeed, the presence of polyethylene glycol chains of PEG-660 stearate at droplet surface of NEs allows stabilization by the steric effect, which is confirmed by a zeta potential close to neutrality in the formulations. This excipient is widely utilized in drug delivery and nanotechnology that was firstly described to have “stealth” properties. The stealth effect is due to the formation of a dense hydrophilic barrier of PEG-660 stearate chains on the surface of the carrier, thereby reducing interactions with the reticular-endothelial system. In addition, pegylation increases the hydrodynamic size of drug delivery systems and consequently decreases their clearance from the body.^25,26

Regarding the antimicrobial assay, both UN-NE and RIF-NE were effective against the three M. tuberculosis strains, with different MIC for susceptible strains. However, for the MDR strain, UN-NE and RIF-NE showed the same MIC (Table 4), and RIF-NE was effective at a drug concentration (MIC_RIF = 7.8 μg/mL) less than that of free RIF (MIC_RIF = 1024 μg/mL). Although RIF-NE increased the susceptibility of strain 2, considering just RIF concentration, the inhibition of growth may not be due to the activity of RIF, but may be due to the activity of some excipients from NEs and/or from the interaction between excipients and RIF, since RIF-NE and UN-NE showed similar MIC. In a formulation, the excipients are as important as the drug, since they may influence the absorption rate and, consequently, the bioavailability of the encapsulated compound. They cannot be considered inert even though their internal use was approved by the Food and Drug Administration, facilitates the preparation and use of certain substances, and protects them from degradation.²⁷

Table 4.

Minimum Inhibitory Concentration and IC₅₀ of UN-NE and RIF-NE

MIC (μg/mL)				IC₅₀ (μg/mL)
	Strain 1	Strain 2	H37Rv	J774A.1
UN-NE total	183.1	367.2	367.2	14.09
RIF-NE total	11.7 (0.25 RIF)	375 (7.8 RIF)	23.4 (0.5 RIF)	25.71

UN-NE total = CO + LEC + PEG-660 stearate; RIF-NE total = CO + LEC + PEG-660 stearate + RIF.

IC₅₀, half maximal inhibitory concentration; MIC, minimum inhibitory concentration; UN-NE, unloaded nanoemulsion.

RIF-NE and free RIF showed no difference in antimicrobial activity against M. tuberculosis strains evaluated in vitro, and the potential of RIF-NE in vivo should not be ruled out. It is important to note that even after the procedure used in the preparation of RIF-NE—which contains heating steps above 80°C and evaporation of solvents under pressure—the RIF remained with its antimycobacterial activity, and this fact evidences a success in the preparation of the proposed formulation. In addition, these liquid formulations are stable, and they can be administrated orally and stored easily, which are characteristics that should be considered when developing new formulas for anti-TB drugs.^12,14

Furthermore, UN-NE and RIF-NE showed IC₅₀ of 14.09 and 25.71, respectively, for macrophage lineage. Also, PEG-660 stearate may have contributed to the IC₅₀ values. The cytotoxicity assay was performed with adherent cells, and considering the surfactant property of this excipient, PEG-660 stearate as well as other surfactants can interfere with cell adhesion to the surface and promote changes in membrane permeability.²⁸ Therefore, these characteristics may have led to the cellular viability levels found.

In spite of this, our UN-NE showed similar chemical-physical characteristics from Hädrich et al.,¹⁵ and considering those reported in vivo results, these NEs can be considered safe for oral administration. In addition, Hädrich et al. ²⁹ also evaluated the anti-inflammatory activity of a NE containing quercetin, with the same basic constitution of NEs evaluated in our study. Interestingly, in the control group, the rats treated with UN-NE showed no damage to the liver and kidneys, or to the neutrophils, lymphocytes, and monocytes. Moreover, Sanzhakov et al. ³⁰ have reported an increased peak concentration of RIF in rat plasma after its packaging in lipid-based nanoparticles for oral administration, and it is known that encapsulation of drugs is able to prevent their degradation and enhances their pharmacological activity.³¹

With regard to childhood TB, one of the strategies is the preventive treatment of latent TB, especially in those under 5 years who maintain household contact with confirmed cases of pulmonary TB, and among the proposed treatments is the administration of RIF daily for 3–4 months.¹ Thus, in this context, RIF-NE could be a viable, alternative pharmaceutical formulation for the treatment of childhood TB and a possible solution for low serum concentrations of antibiotics.

Conclusion

The encapsulated RIF has shown similar activity to free RIF against strains of M. tuberculosis in vitro, and we cannot ignore its potentiation in biological systems if NEs can be administered as liquid formulations that can be considered safe for oral administration and are able to reduce drug degradation and increase its bioavailability in vivo. Thus, RIF-NE can be a promising, alternative pharmaceutical formulation of RIF, including for children, allowing its application in the regimes of prevention and treatment of TB and, in addition, in the fight against drug resistance.

Footnotes

Authors and Contributors

P.C., G.H., D.F., C.L., and P.E. designed the study. P.C., G.H., and L.A. performed the experiments. P.C., G.H., D.F., C.L., and P.E. analyzed the data and wrote the article. All authors approved the version to be published.

Acknowledgments

This work was supported by the National Council for Technological and Scientific Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES). This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Disclosure Statement

This research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All authors declare that they have no conflict of interest.

Abbreviations Used

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