Stable sugar and sugar-free suspensions of pretomanid

Abstract

BACKGROUND:

Pretomanid (PMD) tablets are indicated as part of a combination regimen for the treatment of adults with pulmonary extensively drug-resistant, treatment-intolerant or non-responsive multidrug-resistant TB. No commercial liquid formulation is currently available for patients unable to swallow these tablets.

OBJECTIVE:

To develop stable extemporaneous liquid formulations of PMD that can be stored at room temperature or 30°C for at least 4 weeks.

METHODS:

Crushed PMD tablets were formulated into 20 mg/mL suspensions in a simple syrup and sugar-free formulation. The PMD formulations were stored at room temperature and at 30°C for 30 days in dispensing bottles. Appearance, pH, potency and microbial counts of the suspensions were determined on Days 0, 15 and 30.

RESULTS:

The potency of PMD remained at 99.7–103.4% of the theoretical concentration in each formulation. The appearance, pH and microbial count did not change during the 30-day storage period. Simple syrup formulations did not require preservatives for microbial stability.

CONCLUSIONS:

PMD oral suspension formulations in simple syrup or in sugar-free vehicle were easily prepared by utilising commonly available equipment and ingredients and were stable for 30 days. These formulations are appropriate alternatives for patients with swallowing difficulties.

The 2020 WHO Global Tuberculosis Report highlighted TB as a major cause of illness. TB remains among the top 10 causes of death worldwide and the leading cause of death from a single infectious agent. An estimated 10 million people experienced TB disease, and there were an estimated 1.4 million TB associated deaths worldwide in 2019. Over 500,000 of the cases had rifampicin-resistant TB (RR-TB), 78% of which were multidrug-resistant TB (MDRTB) cases. Only 38% of the estimated number of people who developed MDR/RR-TB in 2019 were enrolled in a treatment programme, emphasising the need to improve diagnosis and treatment.¹

Pretomanid (PMD) was approved by the US Food and Drug Administration as a part of a combination regimen with bedaquiline and linezolid for the treatment of adults with pulmonary extensively drug-resistant (XDR-), treatment-intolerant or non-responsive MDR-TB in 2019.^2,3 The tablet contains 200 mg PMD as the active ingredient. It may be challenging to administer these tablets to patients who have trouble swallowing tablets. As no commercial liquid dosage form of PMD is currently available, the objective of this work was to develop stable extemporaneous liquid (suspension) formulations of PMD that could be compounded in a pharmacy or dispensary. The vast majority of the patient population resides in low- and middle-income countries (LMICs). Our intent was to conduct this study by utilising easily accessible and affordable ingredients and minimal equipment under practical storage conditions. Therefore, these formulations were designed to be stable at 30°C for at least 4 weeks.

METHODS

Preparation of pretomanid formulation in simple syrup

Simple syrup was prepared from food grade cane sugar. A total of 85 g of sugar was transferred to a container and mixed with 45 mL of hot distilled water until completely dissolved. The mixture was allowed to cool to ambient room temperature as a 65% w/w solution of sucrose. Ten PMD 200 mg tablets were placed into a 4-ounce glass mortar and ground to a fine powder using a glass pestle. Using an oral syringe, 15 mL of simple syrup vehicle was added and the contents in the mortar were mixed with the pestle to obtain a smooth mixture. An additional 23 mL of simple syrup was added to the mortar and mixed well. Finally, an additional 54 mL of syrup was added to bring the volume in the mortar to 100 mL. The contents were mixed well to obtain a uniformly dispersed suspension of PMD 20 mg/mL. This well-mixed formulation was transferred into an amber 120 mL plastic prescription bottle made of polyethylene terephthalate with a syringe adapter and child-resistant cap for storage. The instructions for preparation are described in Table 1.

Table 1.

Instructions for preparation of pretomanid 20 mg/mL formulation in simple syrup

Instructions for preparing simple syrup (65% w/w) vehicle: Weigh 85 g of food-grade cane sugar into a suitable container Add 45 mL of hot distilled water and mix well until sugar is dissolved Cool syrup to ambient room temperature Instructions for pretomanid formulation in simple syrup: Grind 10 tablets of pretomanid (200 mg each) to a fine powder in a mortar and pestle Tare out the combined weight of mortar, pestle and triturated tablets Mix powder with a small, measured amount (15 mL) of vehicle to form a uniform paste Add an additional 77 mL of syrup vehicle in increasing measured amounts while mixing thoroughly to bring volume in mortar to 100 mL Transfer the final contents from the mortar and pestle into an appropriately sized amber bottle

Preparation of pretomanid sugar-free formulation

Thick & Easy^® Instant Food and Beverage Thickening Powder (Hormel Foods Sales, Austin, MN, USA) is a widely available modified food starch that is used as a thickening agent in foods and liquids for patients with difficulty swallowing. Thick & Easy powder 11.5 g was mixed for 30 sec with 8 ounces (236 mL) of distilled water with a spoon or spatula in a suitable container. The mixture was held for 5 min to ensure complete hydration before use as a vehicle.

Ten PMD 200 mg tablets were placed into a 4-ounce glass mortar and ground into a fine powder using a glass pestle. Sodium saccharin 125 mg, citric acid 125 mg, potassium sorbate 100 mg and methylparaben 100 mg were added to the mortar and all powders were mixed well with the pestle. Distilled water 20 mL was transferred using an oral syringe to the mortar. The contents were mixed with the pestle to form a smooth paste. The contents were transferred quantitatively into a Class-A 100 mL graduated cylinder by using an additional 30 mL of distilled water in 5–10 mL increments to rinse the mortar and pestle. About 30 mL of the Thick & Easy vehicle described above was then added to the graduated cylinder and mixed using a long plastic spatula or glass rod. Thick & Easy vehicle was added to make a total volume of 100 mL and mixed well to obtain a uniform suspension of 20 mg/mL PMD. The suspension was transferred into an amber 120 mL plastic prescription bottle with a syringe adapter and child-resistant cap. If a graduated cylinder is unavailable, the powders could be mixed in the mortar with 50 mL of distilled water and 46 mL of Thick & Easy vehicle to prepare 100 mL of this formulation. These instructions for preparation are described in Table 2. The general technique for preparing the suspension and withdrawing doses is shown in the Figure.

Table 2.

Instructions for preparation of pretomanid 20 mg/mL sugar-free formulation

Instructions for preparing Thick & EasyW Sugar-free vehicle: Measure 236 mL (8 oz) of distilled water into a suitable container Add 11.5 g of Thick & Easy powder Mix well with a spoon for 30 seconds and let the mixture sit for at least 5 min before use Mix again just prior to use Instructions for preparing pretomanid sugar-free formulation: Grind 10 tablets of pretomanid (200 mg each) to a fine powder in a mortar and pestle Add 100 mg of methyl paraben, 100 mg of potassium sorbate, 125 mg of citric acid and 125 mg of sodium saccharin to the ground tablets in the mortar and mix all the powders well with the pestle Add 20 mL of distilled water into the mortar using an oral syringe and mix the powder with a pestle to form a uniform suspension Transfer contents of the mortar to a 100-mL graduated cylinder Rinse the mortar and pestle with additional 30 mL of distilled water in 5–10 mL amounts and pour into graduated cylinder Add 30 mL of Thick & Easy vehicle to the graduated cylinder and mix with a long plastic spatula or glass rod Add Thick & Easy vehicle to the graduated cylinder to achieve the total volume of 100 mL and mix well to obtain a uniform suspension Transfer the final contents from the graduated cylinder into an appropriately sized amber bottle

Figure.

Preparation of pretomanid suspensions.

Storage stability of pretomanid sugar and sugar-free formulations

The methods described above were used to prepare a total of 12 bottles of each PMD formulation. Six bottles were stored at room temperature and the other six bottles at 30°C for 30 days. For each formulation, 3 bottles were designated for potency testing, 1 bottle for appearance and pH testing, 1 bottle for microbial testing, and 1 bottle was extra as a backup, if needed. Aliquots were withdrawn from each bottle at the designated time-point for performing the different tests.

Appearance and pH

From each bottle of PMD suspension, 25 mL of the well-mixed suspension was transferred to a 30-mL beaker and examined by visual inspection to observe any changes in appearance during the 30-day study period. A sample of each suspension was also placed on a glass microscope slide to observe and photograph any changes in appearance under a light microscope at both 40X and 100X magnifications. Appearance was assessed on Days 0, 15 and 30.

The pH of the two PMD formulations was measured using a digital pH meter calibrated with pH 4 and 7 buffers. The pH was determined on Days 0, 15 and 30.

Measurement of pretomanid potency

A stability-indicating high-performance liquid chromatographic (HPLC) method was developed and verified by conducting forced degradation studies on PMD. Forced degradation studies included exposure to light, heat, acid, base and oxidation. This method was used to determine potency of PMD in the two formulations on Days 0, 15 and 30. Three 5-mL aliquots were withdrawn from each of the three bottles designated for potency determination for a total of n = 9 at each time-point. A syringe adaptor was inserted in each bottle and an oral syringe was used to withdraw the aliquots after shaking the bottle (Figure).

The diluent was prepared with equal volumes of water and acetonitrile and allowed to equilibrate at room temperature. The stock standard solution was prepared by mixing 50 mg of PMD bulk powder in a 100-mL volumetric flask, and mixed with the diluent to a concentration of 0.5 mg/mL. The working standard solution was prepared by diluting 5 mL of the stock standard solution with sufficient diluent to make a total volume of 100 mL to a concentration of 0.025 mg/mL. The sample solution was prepared by transferring 5 mL aliquot of 20 mg/mL PMD suspension using a 5-mL oral syringe into a 200-mL volumetric flask. This was diluted with the diluent and mixed well with a mechanical stirrer for 10 min. A 5-mL aliquot of this mixture was further diluted with the diluent to make 100 mL and mixed well. A 10-mL aliquot of this mixture was filtered through a 0.45 μm polypropylene membrane syringe filter and the filtrate was analysed by HPLC.

The mobile phase consisted of HPLC grade water, acetonitrile and trifluoroacetic acid (550:450:1). This was allowed to equilibrate at room temperature and degassed by vacuum prior to use. HPLC column C18, 4.6 mm × 100 mm, 3.5 μm was used and maintained at 30°C. The flow rate for the mobile phase was 1 mL/min and the detector was set at the wavelength of 234 nm.

Twenty μL of working standard solution was injected into the chromatographic system and the area under the PMD peak eluting at approximately 7 min was recorded. Twenty μL of the diluent was injected as a blank to ensure no interference occurred with the PMD peak. Five consecutive injections of the working standard were made to show the reproducibility of PMD peak areas. Twenty μL of the sample solution were injected into the chromatographic system and area under the PMD peak was recorded as a percentage of theoretical concentration. Periodically, the working standard solution was injected at the end of the runs to demonstrate HPLC system stability. The relative standard deviation was consistently less than 1%.

Evaluation of microbial growth

Suitability studies were conducted to evaluate and verify the procedures described in the United States Pharmacopeia (USP) <60>, USP<61> and USP<62> were suitable to recover the specific microorganisms in the two formulations. The verified procedures were used to enumerate and determine the absence of microorganisms in the two PMD suspensions at Days 0, 15 and 30. The specific microorganisms tested were Staphylococcus aureus ((American Type Culture Collection [ATCC] 6538), Pseudomonas aeruginosa (ATCC9027), Candida albicans (ATCC10231), Escherichia coli (ATCC8739), Aspergillus brasiliensis (ATCC16404), Burkholderia cepacia (ATCC25416), and Zygosaccharomyces rouxii (ATCC28253), which included those tested for extemporaneous vehicles.⁴ One mL of the sample from the PMD suspension bottle was diluted with 9 mL of neutralising broth, and a separate 10 mL of only the neutralising broth was used for positive and negative controls. The inoculum of each pathogen was prepared using the phosphate buffer and serial dilution made with Tryptic Soy (Alpha Biosciences, Baltimore MD, USA) broth. A volume of 0.1 mL of the selected concentration of 10³ colony-forming unit/mL was spiked into the sample, and the positive control and negative control tubes separately and mixed well. One mL of the spiked sample, and the positive control and negative control were placed on agar plates (bacteria on Tryptic Soy Agar [TSA] plates, and yeast and mould on Sabouraud Dextrose Agar [SDA] plates) (Alpha Biosciences). The TSA plates were incubated at 30–35°C for 3–5 days and SDA plates at 20–25°C for 5–7 days. The colonies for each microorganism were then counted.

For specific microorganisms appropriate plates were streaked and incubated following pre-enrichment per USP<62> at the specified temperatures for specified times. After incubation, the presence or absence of the target organism was verified by comparing against positive controls.

Outcome measures

The criteria for acceptable suspension formulations of PMD included ease of preparation by using commonly accessible and affordable ingredients in high TB prevalence countries. Appropriate storage conditions were defined as the use of easily available plastic prescription bottles to be kept at ambient room temperature and at 30°C. The formulations were considered to be uniformly dispersed and to have acceptable potency if the measured PMD concentrations were within 10% of the theoretical concentration⁵ during the entire study period of 30 days. A smooth, uniform and off-white suspension with no lumps or clumps on visual inspection, and no change in visual or microscopic appearance, or in pH during the study period was considered an acceptable formulation. Conformance with USP<1111> limits for total aerobic microbial counts (TAMC) and total yeast and mould counts (TYMC), and absence of specified microorganisms tested in the formulations during the study period were considered acceptable.

Ethics statement

As this was not a human subjects study, an ethics statement was not applicable to this study.

RESULTS

The two formulations of PMD 20 mg/mL in syrup and sugar-free vehicles were prepared with ease and stored in commonly available amber plastic dispensing bottles at room temperature and 30°C. The suspensions could be easily re-dispersed with gentle shaking. The visual and microscopic appearance were off-white in colour, with no lumps of the tablet core. The appearance of the two suspensions did not change during the entire 30-day study period at either temperature. As shown in Table 3, the potency of the sugar and sugar-free PMD formulations ranged from 99.7 to 103.6% and 99.6 to 102.8%, respectively, of the theoretical concentration over the 30-day storage, demonstrating that the aliquots withdrawn were re-dispersible and uniform. The pH ranged from 6.91 to 7.22 in the sugar formulation, and from 4.28 to 4.46 in the sugar-free formulation of PMD.

Table 3.

Potency of pretomanid in two liquid formulations

Study day	Theoretical concentration remaining (n = 9)
	Sugar formulation		Sugar-free formulation
	Room temperature % Mean ± SD	30°C % Mean ± SD	Room temperature % Mean ± SD	30°C % Mean ± SD
0	100.94 ± 0.77	100.44 ± 0.50	101.34 ± 0.54	101.24 ± 0.74
15	101.76 ± 0.69	101.12 ± 0.40	100.70 ± 0.78	101.31 ± 0.67
30	102.46 ± 0.60	102.78 ± 0.44	101.37 ± 0.80	101.34 ± 0.57

SD = standard deviation.

Microbial tests for six specific microorganisms and Burkholderia cepacia complex (BCC) showed no growth during the 30-day study period in either of the two formulations at room temperature and at 30^°C. TAMC and TYMC results (Table 4) met the USP<1111> limits for aqueous oral liquids in both formulations during the 30-day storage period. Therefore, both formulations were acceptable for storage up to 30 days.

Table 4.

Results of microbial limits tests of two pretomanid liquid formulations *

Microbial counts	Microbial limits test results
	Sugar formulation (room temperature and 30°C)			Sugar-free formulation (room temperature and 30 °C)
	Day 0 cfu/g	Day 15 cfu/g	Day 30 cfu/g	Day 0 cfu/g	Day 15 cfu/g	Day 30 cfu/g
TAMC	<10	<10	<10	<10	<10	<10
TYMC	<10	<10	<10	<10	<10	<10

* Per USP<1111>, acceptable criteria for microbiological quality of oral aqueous liquids are TAMC <200 cfu/g and TYMC <20 cfu/g, and absence of E. coli.

cfu = colony-forming unit; TAMC = total aerobic microbial count; TYMC = total yeast and mold count.

DISCUSSION

Drug-resistant TB remains a health concern in LMICs. Swallowing difficulties is a key contributor to the burden of being on treatment, which can lead to sub-optimal adherence or inaccurate dosing – both risks for the development of resistance. A population-based survey found that 16.3% of adults experienced dysphagia, and 86% of them used drinking fluids as the most common compensatory manoeuver for oral intake.⁶ We performed this study to develop a feasible PMD dose administration alternative for older patients with dysphagia.

Two extemporaneous oral liquid formulations of PMD (sugar and sugar-free) were developed. Our findings demonstrated that PMD at 20 mg/mL was stable in both simple syrup and sugar-free suspension formulations after preparation and storage over a 30-day period. These formulations can be used effectively for patients with dysphagia. The sugar-free suspension formulation offers a choice for patients such as those with diabetes or on calories restriction.

We intentionally used easily accessible and affordable ingredients (e.g., sugar, Thick & Easy and preservatives used in foods) to prepare the formulations to maximise the application of our findings in high TB prevalence countries. Cane sugar can be substituted with powdered sugar, or the pharmacy can source simple syrup from a commercial supplier. These PMD suspension formulations were pharmaceutically elegant, and no marked changes occurred in appearance or potency of these formulations as documented by stability-indicating accurate and reproducible HPLC analyses. Uniform re-dispersibility of the suspensions was demonstrated by testing samples withdrawn from each of the three bottles at each time-point and over the storage period. We followed the American Society of Health-System Pharmacists (Bethesda, MD, USA)⁷ and USP⁸ guidelines for compounded aqueous non-sterile preparations. The selection of the preservatives and their concentrations were based on the recommended concentrations from literature⁹ and commercially available compounding vehicles.¹⁰ We conducted the stability studies for 30 days to meet the beyond-use date (BUD) requirements for USP<795>.¹¹ The assigned BUD was confirmed by stability testing and microbial count/absence of specific organisms. The use of potassium sorbate and methylparaben as preservatives in the sugar-free suspension formulation ensured no substantial growth of microorganisms during the storage period of 30 days. Citric acid was added to the sugar-free formulation to maintain the pH below 6, which is optimum for antimicrobial activity of potassium sorbate.⁹ The simple syrup formulation did not require any preservatives for microbial stability.

The plastic bottles used for storing the suspensions were representative of the prescription dispensing bottles routinely used in pharmacies, including in LMICs.

CONCLUSIONS

PMD liquid formulation in simple syrup and sugar-free suspensions were easily prepared with the use of widely available and affordable ingredients. The PMD suspensions were chemically and physically stable during the 30-day study period and microbial counts met USP limits. A liquid formulation of PMD is thus a viable alternative to tablets for patients with dysphagia.