Highly Increasing Solubility of Clofazimine, an Extremely Water-insoluble Basic Drug, in Lipid-based SEDDS Using Digestion Products of Long-chain Lipids

Abstract

Clofazimine (CFZ) is a highly effective antibiotic against leprosy and drug-resistant tuberculosis and is on the WHO List of Essential Drugs. However, no CFZ product with optimal bioavailability is available worldwide. The manufacturer withdrew its only marketed product, presumably due to poor and erratic bioavailability because of extremely low aqueous solubility in the gastrointestinal pH range. We developed a self-emulsifying drug delivery system (SEDDS) using a lipid digestion product (LDP) containing glyceryl monooleate and oleic acid at ∼1:2 molar ratio to increase drug solubility and ensure rapid dispersion into microemulsion. While solubilities of CFZ in glyceryl monooleate, glyceryl trioleate, and two common surfactants (Tween 80 and Kolliphor EL) were comparatively low (<15 mg/g), oleic acid provided a very high solubility of ∼500 mg/g. Because of the presence of oleic acid, the clofazimine solubility in SEDDS containing a 50:50 w/w mixture of LDP and surfactants increased to 130 mg/g. Two formulations having 50 or 100 mg CFZ in one gram of SEDDS were developed. They dispersed rapidly and almost completely in simulated intestinal fluid and in the USP pH 6.8 phosphate buffer containing 3 mM sodium taurocholate. There was some precipitation of CFZ as the HCl salt at low gastric pH during dispersion testing, but the effect could be avoided using enteric-coated capsules. Thus, an enteric-coated lipid-based formulation for CFZ with as high as 100 mg/g drug loading was developed, providing complete drug release and producing microemulsions under intestinal pH conditions.

Introduction

Clofazimine (CFZ) is an old drug first described in 1957 as an antibiotic for potential use against Mycobacterium tuberculosis responsible for tuberculosis (TB).1, 2, 3, 4 However, after some initial studies, it was also found to be effective against Mycobacterium leprae, which causes leprosy. Since there were other anti-TB drugs available in the market, the focus of the clinical research for CFZ shifted to leprosy.3,5 A CFZ product, Lamprene, a suspension of the micronized drug in an oily base, was described in 1979 and marketed in the USA in 1986 for treating leprosy.6 However, its manufacturer, Novartis, withdrew Lamprene from the USA market in 2004, and no generic equivalents or other CFZ products are currently available in the USA. There are also no other viable CFZ products available outside the USA. Though the reason for the discontinuation of Lamprene in the market has not been explicitly given, the possible cause could be its low and erratic bioavailability, high dependency on food for bioavailability, and high inter- and intra-subject variability, all related to the low solubility of the compound.7

There is currently a renewed interest in CFZ for treating TB, as it has been found to be effective against multidrug-resistant tuberculosis (MDR-TB).4,8,9 According to the 2023 World Health Organization (WHO) Global Tuberculosis Report,10 TB is one of the deadliest infectious diseases in the world, only after Covid-19, with over 10 million people infected with TB in 2022, out of which 7.5 million were newly infected. There were 1.3 million deaths from TB in the same year. However, despite the availability of multiple drugs against TB, the treatment of TB is difficult due to the prevalence of two different types of TB, one susceptible to common anti-TB drugs and the other multi-drug resistant or rifampicin-resistant TB (MDR/RR-TB). While the success rate for the treatment of drug-susceptible TB is 88 percent, it is only 63 percent among drug-resistant. Since the proportion of people with MDR/RR-TB has been estimated to be 3.3% among new cases and 17% among those previously treated with other medications, there may not be effective treatment available for a considerably large number of TB patients, thus defeating the United Nations (UN) goal of eradicating the disease by 2030. For these reasons, there is an all-out effort by WHO to find treatment for MDR/RR-TB. In addition to searching for new drugs, there is a great interest in repurposing old drugs like CFZ for this indication. The effect and benefit of CFZ in drug-resistant TB have been reviewed in the literature, and reports suggest that CFZ could provide better therapeutic outcomes and lower the risk of failure in the treatment of drug-resistant TB.11
It is evident from the above brief review of literature that CFZ is an essential drug for the treatment of leprosy and MDR/RR-TB.6,8,9,12 In addition to these diseases, it showed impressive inhibitory activity against various coronaviruses, including SARS-CoV-2 and MERS-CoV, in various in vitro systems.13 Indeed, CFZ belongs to the World Health Organization’s (WHO) List of Essential Medicines despite having no product in the market due to its unfavorable physicochemical properties.

The structure of CFZ is shown in Fig. 1. It is a basic drug with a molecular weight of 473.4 g/mol, melting point of 224°C, high lipophilicity (log P = 7.66), and extremely low aqueous solubility.14 Verbić et al.15 determined the pKa value of the compound by potentiometric and spectrophotometric analyses of its solutions in the methanol-water mixture and then extrapolating values to zero methanol concentration and observed pKa values of 9.43 and 9.61, respectively, by the two methods (avg. 9.52 ±0.13). Despite high pKa values indicating protonation throughout the gastrointestinal pH range of 1 to 7.4, CFZ is extremely insoluble in aqueous media. In their attempts to determine the compound’s solubility in aqueous media, Bannigan et al.16 could not determine its solubility in water alone as the concentration was below the UV/Vis spectrophotometric detection limit. Using the citrate salt as the starting material, they observed that its saturation solubility was ∼25 µg/mL at pH 3.75, which decreased to <1 µg/mL when the solution pH was raised above 5 by adding NaOH. The solubility also decreased when the pH was lowered below 3.75 by adding HCl due to the conversion of the citrate salt to the hydrochloride salt and the common ion effect in the solution. The solubility was only about 2 µg/mL or less under the gastric pH of 1 to 2. Recently, Topalović et al. 17 reported that CFZ was extremely insoluble in the pH range of 1 to 7 when the pH was adjusted by using such weak acids as adipic, citric, maleic, malic, succinic, and tartaric acids; only with glutaric acid, there was an increase in solubility up to 10 mg/mL at pH below 2 that was attributed to acid-base supersolubilization (ABS). Even in the fasted-state simulated gastric fluid (FaSSGF), fasted-state simulated intestinal fluid (FaSSIF), and fed-state intestinal fluid (FeSSIF), the solubility of CFZ was reported to be ∼0.36, ∼6.20, and 29.60 µg/mL, respectively.18 In contrast, the recommended CFZ dose in humans for leprosy is 100-200 mg once a day.6 Considering the extremely low solubility of the compound mentioned above, it could be possible that the dissolution rate of the drug in the gastrointestinal tract and, therefore, the bioavailability of the drug would be extremely low. A recent Phase IIa clinical trial in HIV-infected adults for treating cryptosporidiosis, a severe diarrheal disease caused by Cryptosporidiosis, using the previously marketed product, Lamprene, showed no significant efficacy, likely due to subtherapeutic CFZ concentrations in the body.19 No definitive reports on the bioavailability of any CFZ product in humans have been published in the literature. Various strategies like salt formation,14,20, 21, 22 cyclodextrin complexation,23,24 amorphous solid dispersion (ASD),25, 26, 27 liposomal formulation,28 PLGA nanoparticles29, etc., have been unsuccessful in producing an oral product. This is because all salt forms of the drug tested were also very poorly water-soluble, the drug loading and dissolution rates of cyclodextrin complexes, ASDs, and liposomes were low, and the dissolution rates of nanoparticles were slow. For example, Narang et al.26 developed an ASD of CFZ in PVP K14 with 10% drug loading and obtained almost complete (90%) drug release only when a low dose of 2.5 mg equivalent of the drug was added to 900 mL of pH 1.0 medium (0.1N HCl). An ASD would not be feasible for a higher dose, especially for the normal CFZ doses of 100 to 200 mg. These results show that there is a need for alternative formulations with improved oral absorption to achieve effective treatment levels with CFZ.

The present investigation aims to develop a lipid-based formulation (LBF) for CFZ that would disperse in aqueous media rapidly as microemulsion, keep the drug solubilized in simulated gastrointestinal fluid, and potentially provide higher bioavailability in humans. The LBFs use lipids, surfactants and often cosolvents as excipients to create homogenous mixtures where the drug is dissolved.30 Pouton et al.31,32 introduced a lipid formulation classification system (LFCS) that categorizes various LBFs into four classes, namely, Types I, II, III, and IV, where Type III formulations lead to the formation of microemulsions. As explained previously, the prefix ‘micro’ in microemulsion usually refers to “small” and not necessarily any particle in the micrometer range (>1000 nm).33,34 In aqueous media, a dispersion with average particle sizes usually up to 300 nm is often called a microemulsion. For practical purposes, any dispersion that passes through the 0.45 µm (450 nm) filter is also called a microemulsion. Since differentiating microemulsions from nanoemulsions is experimentally tedious and often unclear, the exact definition of microemulsion is mostly of academic interest.35 In the present investigation, we call all lipid particles that pass through a 0.45 µm filter microemulsion. Type III LBFs are again divided into IIIA and IIIB based on lipid or glyceride contents of the formulations, where lipid contents in IIIA and IIIB are 40–80 and < 20% w/w, respectively, and the remaining components are surfactants, organic solvents, or both.31,32,36 Various other terms are also used in the literature to describe Type III LBFs. The general term self-emulsifying drug delivery system (SEDDS) refers to a clear, isotropic mixture of oil, surfactant, co-surfactant, co-solvent, etc., that spontaneously forms an oil-in-water microemulsion upon dilution with aqueous media with gentle agitation.37 Because of their small particle size and spontaneous emulsification, they are also called self-microemulsifying drug delivery systems (SMEDDS) or self-nanoemulsifying drug delivery systems (SNEDDS). The term microemulsion preconcentrate is also used to describe SEDDS or Type III LBF since they form a microemulsion upon dilution with water. The superior clinical effects and commercial success of Neoral® (Novartis), a SEDDS formulation for cyclosporine A, is a great testimonial to the benefit of the LBF in increasing bioavailability, reducing inter-subject variation, and reducing food effects of poorly water-soluble drugs.38,39

One major challenge in developing SEDDS is the low solubility of drugs in commonly available and pharmaceutically acceptable lipids and surfactants. High amounts of organic cosolvents are added to the formulation to address the low solubility issue. Additionally, high amounts of surfactants are added to enable the emulsification or dispersion of formulations into microemulsions in aqueous media. In many reports in the literature, Type IIIB formulations contain as little as 10% w/w lipids (triglycerides, diglycerides, monoglycerides, etc.).40, 41, 42 Because of such low amounts of lipids used, it has been suggested that these formulations should rather be called ‘lipid-flavored’ than ‘lipid-based’ since not enough lipids are present to hold lipophilic, water-insoluble drugs in the solubilized form.34 Consequently, drugs may precipitate in aqueous media or gastrointestinal (GI) fluid during dispersion testing or after oral intake.36,40,43 Ideally, the formulation should contain enough lipids, only minimal amounts of surfactants as necessary, and no organic solvents, and yet it should enable sufficient solubilization and sustained drug supersaturation of the entire drug dose during intestinal dispersion and digestion of LBFs to prevent drug precipitation.44

Lipids used in LBFs undergo digestion in the GI tract. We previously reported the formulation of LBFs with much higher solubility of weakly basic drugs like cinnarizine and ritonavir by using lipid digestion products of long-chain lipids (oleic acid + glyceryl monooleate) instead of the parent lipid glyceryl trioleate.33,34 The long-chain fatty acid, oleic acid, greatly increased drug solubility in the formulation. The LBFs used contained Kolliphor EL, Tween 80, or their mixtures as surfactants. They dispersed readily in aqueous media, and in the presence of sodium taurocholate, mixed micelles were formed. The present study aimed to determine whether a similar approach would apply to CFZ. The following are the specific objectives of the investigation:

(a) Determine the solubility of CFZ in long-chain fatty acid, monoglyceride, and triglyceride.

(b) Apply quantum-chemical calculations of surface charges for an improved mechanistic understanding of possible molecular interactions between CFZ and excipients when high solubility was observed.

(c) Construct pseudo-ternary phase diagrams of lipid digestion product (oleic acid + glyceryl monooleate), surfactant (or mixtures of surfactants), and water to identify optimal formulations for the formation of microemulsion upon dilution with aqueous media.

(d) Conduct dispersion tests of the selected LBF in aqueous media at low and high pH, in the presence of sodium taurocholate, an endogenous surfactant, and in simulated intestinal fluid. Step dissolution at low gastric pH conditions followed by the change in pH to higher intestinal pH condition is also studied.

(e) Conduct dispersion testing of selected LBFs using enteric-coated capsule by step dissolution at low gastric pH conditions, followed by the change in pH to a higher intestinal pH condition, to determine whether any potential for the crystallization of CFZ at low pH could be avoided.

Materials

Clofazimine (CFZ) was purchased from Chemshuttle (California, USA). Long-chain lipid digestion products (oleic acid and glyceryl monooleate) and surfactants (Kolliphor EL and Tween 80) used in the present investigation are listed in Fig. 2, along with their trade names, manufacturers, chemical structures, and compositions. All data in Fig. 2 are taken from the manufacturers’ brochures.

Hari P. Kandagatla, Mufaddal H. Kathawala, Amber Syed, Tatjana Ž. Verbić, Alex Avdeef, Martin Kuentz, Abu T.M. Serajuddin, Highly Increasing Solubility of Clofazimine, an Extremely Water-insoluble Basic Drug, in Lipid-based SEDDS Using Digestion Products of Long-chain Lipids, Journal of Pharmaceutical Sciences, 2025, 103782, ISSN 0022-3549, https://doi.org/10.1016/j.xphs.2025.103782.

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