Stability of extemporaneously prepared clofazimine oral suspensions

Clofazimine is recommended as a treatment for tuberculosis in infants. Extemporaneously prepared clofazimine oral suspensions are more appropriate dosage forms for infants than tablets, however no data exist to inform appropriate storage conditions.


INTRODUCTION
Clofazimine is a riminophenazine antibiotic marketed for the treatment of leprosy in combination with dapsone and rifampicin.The mode of action of clofazimine as an anti-mycobacterial agent remains unclear, but it may involve multiple mechanisms of antimicrobial activity. 1 Clofazimine has been shown to increase the effect of bacterial phospholipase A2 and releases lysophospholipids, which are toxic to mycobacteria. 2 Some studies also suggest that clofazimine may interfere with bacterial adenosine 5 0 -triphosphate (ATP) production via membrane interaction with the respiratory chain, resulting in the production of reactive oxygen species. 2 Clofazimine is now also recommended by the World Health Organization as second-line therapy for the treatment of multi-drug-resistant tuberculosis (MDR-TB). 3It is estimated that between 25 000 and 32 000 children develop MDR-TB every year, with infants and children at a high risk of developing disseminated diseases that are associated with a high mortality rate. 3he lack of child-friendly formulations for second-line anti-TB medicines was previously highlighted as a challenge for the treatment of MDR-TB in children. 4,5lthough child-friendly formulations have now been developed for all oral medicines recommended for the treatment of MDR-TB, 6 these products may not be *Address for correspondence: Ben J. Boyd, Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia.E-mail: ben.boyd@monash.edureadily available in all countries or may need to be sourced via special access programs.Additionally, these products may not be suitable for administration to critically ill or preterm neonates, who may require part doses from a tablet or capsule and administration via enteral feeding tube.
Clofazimine is commercially available as a 50 mg tablet and capsule, as well as a 100 mg tablet and capsule.Clofazimine is lipophilic and has a low water solubility.For this reason, it has been formulated as a microcrystalline suspension in an oil-wax base, encapsulated within a soft gelatine capsule. 7Although clofazimine tablets are technically not dispersible, they have been shown to dissolve in water over time. 3However, this is not an ideal method of administration for doses less than a full tablet as the medicine will not disperse evenly due to the poor aqueous solubility of clofazimine.This makes the accurate administration of a part dose for neonates and young children challenging.Compounding a clofazimine suspension from tablets would allow for easier and more accurate dosing for the neonatal and paediatric populations, particularly when doses less than a full tablet are required.Due to the poor aqueous solubility of clofazimine, use of a suspending agent is necessary to produce a formulation with a uniform concentration.Ora-Blend SF (sugar-free) (Perrigo Company, PLC, Belrose, NSW, Australia) is a commonly used suspending vehicle designed for extemporaneous compounding of oral suspensions.Medicated powder (obtained either as active pharmaceutical ingredient or from crushed tablets) can be incorporated into Ora-Blend SF to produce a suspension for oral or enteral administration.
The aim of this study was to determine the physical and chemical stability of extemporaneously prepared clofazimine suspensions in Ora-Blend SF over 60 days under both refrigerated and room temperature storage conditions.

METHOD Ethics Statement
Ethics approval was not required for this research article as it was a stability study and did not involve human subjects.

Extemporaneous Formulation of Clofazimine Oral Suspension
An oral suspension of clofazimine (10 mg/mL) was prepared by thoroughly crushing and grinding six 100 mg clofazimine tablets with a mortar and pestle to produce a fine powder.A small amount of Ora-Blend SF was added to the clofazimine powder to form a thick, smooth paste.Additional Ora-Blend SF was incrementally added until a smooth mixture was formed, which was then transferred into a graduated conical measuring cylinder.Ora-Blend SF was used to rinse the residual contents of the mortar into the measuring cylinder and make up a final volume of 60 mL.The suspension was mixed well using a glass stirring rod; then 10 mL aliquots were transferred to six amber plastic bottles (Cospak, Knoxfield, VIC, Australia).The bottles were capped using NeoConnect pharmacy plastic bottle caps (size D) (Woodstock, GA, USA) and shaken well before use.The samples were prepared by Western Health Pharmacy Department, Victoria, Australia and sent to a laboratory at Monash University, Victoria, for sampling on the same day.During transport, three bottles were transported in a polystyrene esky at ambient temperature, and three bottles were transported in a polystyrene esky with an ice pack.On arrival at the laboratory, three samples were stored at room temperature (19°C AE 2°C) and three were refrigerated at 4°C.All samples were stored out of direct sunlight.

Sample Analysis
Each bottle was shaken thoroughly by hand and a 1 mL aliquot was taken on days 0, 7, 14, 28, and 60.Samples were stored at À20°C in 1.5 mL Eppendorf (Macquarie Park, NSW, Australia) tubes then thawed at room temperature before preparation for HPLC analysis.Stability samples taken on days 0-28 were initially analysed together on day 28, followed by samples taken on day 60.
The samples were visually inspected for colour change and separation at each time point by qualitative analysis.As the commercial suspending vehicle contained effective preservatives, microbiological testing was not performed.

HPLC Method
A HPLC assay was used to quantify the concentration of clofazimine within the prepared oral suspensions.The HPLC method used was adapted from a clofazimine quantification method previously described by Salim et al. 8 Multiple bottles of the prepared suspension were kept in each storage condition (n = 3) and sampled individually at each time point to represent any potential variability in the suspension.Each sample was analysed by single injection using the detailed method.
From the original 1 mL stability sample taken, a 25 mg aliquot was weighed into a 1.5 mL Eppendorf tube.To the weighed sample, 1 mL of methanol containing salicylic acid internal standard was added.The sample was vortexed for 5 min to extract the clofazimine.Following extraction, the sample was centrifuged at 16 160 g for 7 min to sediment the Ora-Blend SF.The supernatant was collected and diluted 1 in 20 volume per volume (v/v) in mobile phase (26:74 v/v 0.25 M sodium acetate trihydrate, pH 4.5/methanol) prior to measurement using the HPLC method.Separation of the clofazimine was performed using a reversed-phase Phenomenex C8 column (150 mm length 9 5 lm particle size 9 100 A pore size 9 4.6 mm inner diameter) on a Shimadzu (Shimadzu Scientific Instruments [Oceania] Pty Ltd, Rowville, VIC, Australia) Nexera instrument.The oven temperature of the HPLC instrument was set to 35°C and a ultraviolet detection wavelength of 286 nm was used.The injection volume of the samples was 10 lL and the flow rate was 1 mL/min, which gave a retention time of 4.5 min for clofazimine and 1.7 min for the internal standard.A standard curve was prepared from 0.5 to 40.0 lg/mL of clofazimine made up in methanol and spiked with Ora-Blend SF.An acceptable linearity was obtained for the standards across the concentration range of 0.5 to 40.0 lg/mL with a correlation coefficient (r 2 ) of 0.9998 for clofazimine.The HPLC method was validated and found to be precise and accurate (Table S1).The final concentration of clofazimine in each sample was then determined by normalising the area of the clofazimine peak against the area of the internal standard to give a concentration area ratio and comparing it to the determined standard curve.

Forced Degradation Study
A forced degradation study was performed to verify that the HPLC method used in this study was stability indicating and able to resolve degradation products from the intact drug.The forced degradation conditions consisted of accelerated thermal conditions of 80°C for 7 days.Samples (3 mL) of the clofazimine oral suspension were aliquoted and centrifuged for 10 min at 4444 g to separate out the solubilised clofazimine present in the suspension.Following centrifugation, the supernatant containing the solubilised clofazimine was collected and transferred to a sealed glass vial.Glass vials containing the suspension were stored in an 80°C oven, and a 200 lL sample was taken daily for 7 days.All forced degradation samples were stored at À20°C in 1.5 mL Eppendorf tubes following collection and thawed on the day of HPLC analysis.

Data Analysis
All experiments were performed using batch samples from each storage condition (n = 3).The stability of clofazimine was assessed by calculating the percentage of the initial concentration remaining at each time point and was reported as the mean AE standard deviation of remaining drug concentration (%).Stability was defined as the retention of at least 90% of the initial drug concentration.GraphPad Prism software version 9.0.1 for Windows (GraphPad Software, San Diego, CA, USA) was used to perform t-test and one-way ANOVA statistical tests.

Physical Stability
Visual observations of the suspensions at 60 days of storage showed there were no apparent changes in physical stability, with no separation noted under either storage condition.

Forced Degradation Studies
Forced degradation studies were carried out to ensure the HPLC method used to quantify the final concentration of clofazimine could distinguish between the parent drug and any potential clofazimine degradation products.Samples of the oral suspension were stored at 80°C and sampled daily for 7 days.Analysis of the samples on HPLC showed the presence of a potential degradation product (Figure S2).Quantification of the clofazimine drug peak (Figure 1a) across the 7 days showed there was a small, but not significant, decrease in the area of the drug peak, indicating the drug and suspension were quite stable.Samples taken on days 3-7 showed an additional peak following analysis on HPLC indicating potential degradation of the parent drug (Figure 1b).Quantification of the potential degradation product showed a steady increase in the area of the degradation product after day 3.

Chemical Stability of Clofazimine under Different Storage Conditions
The final stability of the clofazimine suspension was assessed by evaluating the percentage of clofazimine remaining in intervals across 60 days.As per the United States Pharmacopeia (USP) guidelines, 9 more than a 10% decrease in the initial clofazimine concentration represented an unacceptable decrease in the potency of the suspension.As no consistent guidelines exist for paediatric patients, greater than 10% variability in the final concentration was set as the stability threshold.After 28 days, at least 90% of the clofazimine concentration remained when stored under either storage condition (Table 1).Samples stored at room temperature for 60 days showed similar concentrations, with greater than 90% of the initial clofazimine remaining.Comparatively, samples stored under refrigerated conditions (4°C) were found to have slightly less than 90% (87.4%) of the initial clofazimine concentration remaining after 60 days.Some deviation in the average concentration of clofazimine was observed across the sampled time points.This variation was predominantly observed in samples taken on days 7 and 28 from both storage conditions.Despite the care taken when compounding, deviation at these time points may have been a result of slight variations in the initial mass of clofazimine compounded in the suspension.As stated in the USP guidelines, 9 there must be no more than a AE10% difference in the mass compounded to the labelled mass, which would not be exceeded as a result of the determined deviations.Additionally, a slight difficulty with sampling of the viscous suspension during HPLC preparation may have resulted in minor variation in the mass of suspension analysed.As no additional degradation peaks were observed in the chromatogram (Figure S3) or large changes in the subsequent clofazimine concentrations present, it was concluded that degradation of the clofazimine was most likely not occurring at these points.
Following these results, statistical analyses were performed to compare the effect of both storage conditions and time on the final clofazimine concentrations.An unpaired t-test demonstrated no significant difference in the concentration of clofazimine when stored under either condition (p > 0.05).A one-way ANOVA showed there was no significant difference in the concentration of clofazimine in the suspensions stored at room temperature.A significant difference was found in the concentration of clofazimine stored under refrigeration for  60 days compared to the initial concentration at day 0 (p < 0.05), suggesting the API appeared to have undergone more rapid degradation over time when stored at 4°C compared to room temperature.These results show that the suspension may be stored at room temperature for up to 60 days and does not require refrigeration.

DISCUSSION
In the treatment of MDR-TB, the lack of suitable oral clofazimine formulation poses a problem in neonates and children who cannot swallow tablets or capsules.
As an alternative, an extemporaneous compounding method is commonly used in hospital pharmacy departments to provide care to children and neonates in cases where medicine is not commercially available a childfriendly formulation.
The commercial suspending vehicle used in the study, Ora-Blend SF, was designed to be used in the extemporaneous preparation of oral suspensions and was not expected to adversely impact absorption of clofazimine in the gastrointestinal tract.Clofazimine is a highly lipophilic drug and administration with food is recommended to increase absorption.For use in the neonatal population, administration of the extemporaneous suspension with or after a breastfeed or mixed with a small volume of expressed breast milk or infant formula is also suggested to improve absorption in suitable situations.Although the release and absorption of clofazimine from the suspension in vivo is unknown, it would not be expected to negatively impact this process.
An initial forced degradation study was performed to determine the major degradation products present as a result of thermal stress.As the lack of a temperaturecontrolled environment was the main concern for the stability of the formulation, the presence of degradation peaks was observed across 7 days at 80°C to represent a worst-case scenario.An additional peak was found to elute at 9.7 min following 3 days at elevated temperatures, indicating some thermal-based degradation was occurring to the clofazimine API.The presence and elution time of this degradation peak was found to be comparable to other studies investigating the thermal degradation of clofazimine in orally delivered formulations. 10verall, the HPLC method was deemed suitable for accurately separating the parent clofazimine drug from the only observed degradation product.
Results from the study showed that grinding of clofazimine tablets to source the API and suspending the powder in the aqueous suspending agent, Ora-Blend SF, did not negatively impact the stability of clofazimine when stored at room temperature for 60 days.These results are consistent with a recently published study by Taneja et al., where the stability of a 10 mg/mL clofazimine oral suspension was investigated over a shorter time period of 30 days. 11That study demonstrated that a clofazimine suspension prepared in simple syrup retained at least 90% of the original concentration at 30 days when stored both at room temperature and at 30°C.Additionally, a sugar-free suspension was reported as stable at 15 days in both storage conditions.To our knowledge, our study is the first to show the stability of an extemporaneously compounded clofazimine suspension when kept at room temperature for 60 days.The oral suspension of clofazimine was shown to have higher stability when stored at room temperature.Although the reasons for this have yet to be determined, the implications for this research are clear as an oral suspension of clofazimine may be stored at room temperature without the need for refrigeration.This result could facilitate the use of oral suspensions of clofazimine for neonatal and paediatric patients in areas where TB is endemic and refrigerated storage may be difficult.The suspension will allow for more accurate dosing as well as easier administration, particularly when doses less than a full tablet or capsule are required, as may be necessary for critically ill or preterm neonates.
Extemporaneously compounded clofazimine (10 mg/mL) oral suspensions packaged in amber plastic bottles were chemically stable for 60 days when stored at room temperature and 28 days when stored under refrigerated conditions (4°C).

Figure 1
Figure 1 Area of (a) initial clofazimine parent drug and (b) resulting degradation product following 7 days under forced degradation conditions of 80°C.Data are represented as mean (n = 3) and error as standard deviation.

Table 1
Stability of clofazimine oral suspension when stored under room temperature and refrigerated (4°C) conditions over 60 days.Results are presented as mean AE standard deviation (n = 3) at each storage temperature.