Final Report. Prepared by

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1 Final Report Proof-of-Concept Study for a Fixed Dose Combination Product Containing Sulfamethoxazole, Trimethoprim, Isoniazid and Pyridoxine. Executive Summary Prepared by Daniel T. Smith, Ph.D. Department of Industrial and Physical Pharmacy Purdue University West Lafayette, IN USA The overall purpose of this research was to support the development of a pharmaceutical fixed dose combination (FDC) product containing the active ingredients sulfamethoxazole (800 mg), trimethoprim (160 mg), isoniazid (300 mg) and pyridoxine (25 mg). One of the technical challenges that needs to be overcome in creating this FDC is minimizing the size of the final dosage form since the amount of active ingredients (APIs) is large (1.285 g in total). The specific purpose of this project was to explore manufacturing strategies that might be used to increase the densities of the active ingredients in order to reduce the physical size of each dose. An addition goal of the project was to assess the chemical stability of the API mixture. To that end, the project had three specific aims, which were: 1. Determine if a granulation strategy will be useful in reducing the size of the final dosage form 2. Develop an initial tablet formulation for the mixture of APIs 3. Analyze the granulation mixtures as well as the tablets for chemical stability under ambient and stress conditions The appropriate ratios of APIs were successfully granulated using both wet and dry methodologies. Density measurements taken before and after the granulations indicate that a 9% increase in density was achieved by wet granulating the APIs and a 2% increase was achieved using dry granulations. When formulated into tablets, both the dry granulated product and the tablets made from the as received APIs were of similar thickness. In contrast, tablets made from the wet granulated APIs were quite a bit thicker. These results suggest that even though wet granulation leads to a greater increase in density, is not the best method to minimize the size of the final dosage. Finally, the stability of API mixture was explored at various stages of the manufacturing process. Samples of granulation pre- blends, dried granules and

2 tablets were stored at 40 C and 75% RH for 30 days in order to explore the chemical stability of the APIs in the formulations. It was noted that materials made by wet granulation (both granules and tablets) exhibited significant yellowing on aging. Samples containing the as received APIs or the dry granulated blend showed very slight discoloration on aging. HPLC analysis of the all the aged samples showed no signs of decomposition products, indicating that the APIs were chemically stable despite the observed coloration. Results and Discussion A novel FDC product containing 800 mg of sulfamethoxazole, 160 mg of trimethoprim, 300 mg of isoniazid and 25 mg of pyridoxine has been proposed for the treatment of TB. Due to the large amount of API in the proposed product, it may be a challenge to manufacture a final dosage form that is not too large for patients to safely ingest. One part of this project was to determine if a granulation strategy could be used to increase the density of the active ingredients and, therefore, reduce the size of the final dosage form. To that end, appropriate ratios of the four APIs were subjected to both wet and dry granulations and the densities of the resulting granules were compared with those of the starting powder blends to determine the magnitude of change in density. Additionally, tablets were made from the initial powder blend and the granulation mixtures in order to determine if granulation had an impact on the size of the final dosage. Another focus of this project was to assess the chemical stability of the API mixture. Currently, there is no product containing all four of the proposed APIs, so establishing that these compounds are compatible with each other will be critical for the development of a successful product. To explore this issue, an existing HPLC method was successfully adapted to separate all four APIs. This method was then used to analyze API/excipient mixtures. Chemical stability testing was carried out under accelerated aging conditions (40 C, 75% RH, 30 days). API/excipient blends (pre- granulation and post- granulation) as well as tablets were subjected to stability testing followed by HPLC analysis to determine if degradation occurred. Dry Granulation On a manufacturing scale, dry granulation is typically accomplished through roller compaction. The active ingredient(s) are blended with a binder then the dry blend is fed between the faces of two closely spaced rollers that compact the mixture into a cohesive ribbon. Granules are formed from the ribbon by milling to achieve the desired particle size. The granules can then be blended with additional excipients and manufactured into the desired final dosage form. Since a roller compactor capable of handling laboratory- scale granulations was not available for this project, dry granulation was accomplished through an alternate method known as slugging that closely mimics roller compaction. In this case, the API/binder blend is compressed into large pellets using flat- faced tooling and the

3 appropriate die. These pellets, which are similar to the ribbon produced by roller compaction, are then milled to the appropriate particle size. The binder chosen for this study was microcrystalline cellulose (MCC 200), a widely used excipient in the pharmaceutical industry. The amount of binder that was blended with the APIs (20 weight percent of the active ingredients) was chosen in an effort to minimize the amount of binder used without negatively impacting the binder s ability to form strong granules. No attempt was made to optimize the nature of the binder or the amount of binder used. A physical mixture of API and binder was made then easily formed into pellets using standard methodology (i.e. a tool and die set mounted in a Carver press). The ultimate size of the pellets was dictated by the size of tooling on hand and how much of the dry blend the die could hold. In this case, the pellets were 1.58 cm in diameter and approximately 0.7 cm in height. Once formed, the pellets were milled to produce a powder. Bulk (tapped) density measurements were performed on the pre- blend and on the milled granules. The tapped density of the pre- blend was found to be g/ml while the tapped density of the dry granulated mixture was g/ml. This represents a 2.4% increase in density. Wet Granulation High- shear wet granulations were explored as a potential alternative to roller compaction as a way of increasing API density. A binder is necessary in wet granulations to help with particle adhesion within the granule, and, as the name implies, a solvent is also required for the formation of granules. Methylcellulose was chosen as the binder for the wet granulations and was used at 20 weight percent of the API weight. For convenience, the dry binder was blended with the APIs prior to solvent addition. Water was used as the solvent and amount used was empirically determined based upon the amount needed to produce granules without overwetting. As with the dry granulation binder, no attempt was made to optimize the nature of the binder or the amount used. The tapped density of the wet granulation pre- blend (including binder) was determined then compared to the tapped density of the dried granules. For the pre- blend, the density was found to be g/ml. The density of the milled granules was found to be g/ml, a 9.6% increase in density. HPLC Separation of all Four APIs The HPLC method used to separate all four APIs is the same as described in the Assay section of the USP monograph Sulfamethoxazole and Trimethoprim Oral Suspension. This method readily separated the sulfamethoxazole, trimethoprim and isoniazid components of the mixture. The small pyridoxine peak elutes very closely to the isoniazid peak, however resolution was sufficient for this project.

4 During the course of the project, it was found that a slightly lower ph for the mobile phase (ph 5.5 vs. ph 5.9) improved the separation of these two peaks. Accelerated Aging Samples of the blends, granules and tablets used in this study were subjected to accelerated aging conditions (40 C and 75% RH) for 30 days to determine if chemical degradation of the active ingredients would occur. A humidity chamber was constructed from a desiccator containing a saturated NaCl solution. Open petri dishes containing the samples were stored on a rack above the brine solution in the desiccator. The desiccator was then placed in an oven at 40 C. After 30 days, samples were removed from the desiccator and analyzed by HPLC. Samples subjected to the accelerated aging conditions include a blend of the APIs alone, the API/excipient blends prior to granulation, granules produced from both wet and dry granulations, mixtures of the APIs (or granules) with the excipients used for tableting and the final tablets. After aging, very slight discoloration (yellowing) was noticed in most samples. This yellowing was significantly more noticeable in the granules/formulations that were produced by wet granulation. Despite the discoloration, HPLC analysis of each of the samples indicated that no significant changes had occurred during aging and no new peaks appeared in the chromatograms. It is not clear at this time what the source of color is. FDC Tablet Formulation A basic formulation was used to manufacture tablets in order to compare the size of the final dosage form and to allow for stability testing of the tablets. In addition to the APIs or granules, MCC was added as a binder and croscarmellose sodium was included as a disintegrant. The amount of croscarmellose used per tablet was held constant (~3% of final blend weight) in each of the tablets while the weight percent of MCC used varied (~30% or ~19% of final blend weight) depending on whether a blend of the APIs alone was being used or if granules were being used. The extra MCC used in the API alone blend was necessary to keep the final tablet weights equal as the granules contained additional excipients as a result of the granulation step. It was found that when MCC represented 15% of the final blend weight, the resulting compact was weak and did not hold together well. The MCC and croscarmellose sodium were physically mixed with either the API blend or the granules then compressed into tablets using 2.54 cm round, flat- faced tooling and a Carver press. The size of the tooling was determined by the amount of material that needed to be compressed. Additionally, flat- faced tooling was chosen to simplify measurements used for comparing final tablet size. Tablet thicknesses were measured with calipers across the center of each tablet and are summarized in Table 1. Of the three sets of tablets made, the tablets made from wet granulated materials were significantly thicker than the rest. Tablets made from either the APIs alone or from dry granulation were of similar thickness, with the dry granulated tablets being marginally (3%) thinner.

5 Table 1: Tablet thickness results Tablet Thickness (mm) Tablet 1 Tablet 2 Tablet 3 Average S.D. APIs Alone Wet Granulated Dry Granulated These results suggest that a granulation approach will not lead to a meaningful reduction in dosage form size for the proposed formulation. They also suggest that a wet granulation strategy should be avoided due to increased tablet size and discoloration issues. We will note that since this was a proof- of- concept study, little to no attempt was made to optimize the formulations used, therefore the tablet sizes reported here may not be truly representative of the final commercial product. It is possible that changes in the excipients used and/or the amount of excipients used during the course of manufacturing might lead significant reductions in the size of the final tablet. Procedures Materials Sulfamethoxazole, isoniazid, trimethoprim and pyridoxine HCl were purchased from Sigma- Aldrich (St. Louis, MO) and used as received. HPLC grade water, acetonitrile and triethylamine were purchased for the HPLC analyses and used as received. All excipients were of USP grade. Dry Granulation Procedure Sulfamethoxazole (12.0 g), isoniazid (4.5 g), trimethoprim (2.4 g) and pyridoxine HCl (0.375 g) were combined in a beaker then blended with a spatula until roughly homogeneous. To the blend of APIs was added microcrystalline cellulose (MCC 200, 3.9 g, 20 wt % of the API). This mixture was blended for 1 min using a Mr. Coffee model IDS77 coffee/spice grinder. Tapped density measurements of this blend were taken prior to slugging Slugs were made from the API/MCC blend using a 1.58 cm flat- faced tool and die set mounted in a Carver press. A portion of the blend was transferred to the die and was compressed at 9000 lbs for 30 seconds. This process was repeated until all of the blend had been compressed. The resulting pellets were approximately 0.7 cm tall with a diameter of 1.58 cm. The pellets were then milled in a Spex CertiPrep 8000M ball mill then post- granulation density measurements were then taken. Wet Granulation Procedure Sulfamethoxazole (8.0 g), isoniazid (3.0 g), trimethoprim (1.6 g), pyridoxine HCl (0.25 g) and methylcellulose (2.6 g, Methocel A15 Prem LV, 20 wt %) were weighed then transferred to the bowl of a Cuisinart MiniPrep Plus food processor/chopper.

6 The mixture was blended for 1 min then the tapped density of the mixture was determined. The blend was returned to the food processor then water was added in portions (5 x 0.4 ml, 2.0 ml total) with mixing. The granules were blended for an additional minute prior to being transferred to a drying tray and drying overnight at 40ºC. The dried granules were milled in a Spex CertiPrep 8000M ball mill prior to post- granulation density measurements. As Received Tablets Sulfamethoxazole (12.0 g), isoniazid (4.5 g), trimethoprim (2.4 g) and pyridoxine HCl (0.375 g) were added to the bowl of a Cuisinart MiniPrep Plus food processor/chopper. The mixture was blended for approximately 1 min. To make a single dose, a portion of the blend (1.285 g) was combined with MCC (0.642 g) and croscarmellose sodium (0.064 g) then mixed with a spatula. One dose of the blend was used for stability testing while additional doses were compressed into tablets using a 2.54 cm round, flat- faced tool and die set mounted in a Carver press. As received tablet formulation (per tablet) Sulfamethoxazole 0.8 g Isoniazid 0.3 g Trimethoprim 0.16 g Pyridoxine g MCC g Croscarmellose Na g Total Blend g (64.5 wt % API) Dry Granulated Tablets For each dose of the dry granulated tablets, g of the granules was added to g of MCC and g of croscarmellose sodium. The resulting mixture was blended with a spatula. One dose of the blend was set aside for stability testing and additional doses were compressed into tablets using a 2.54 cm round, flat- faced tool and die set mounted in a Carver press. Dry granulated tablet formulation (per tablet Dry granulated API g MCC g Croscarmellose Na g Total Blend g (64.5 wt % API) Wet Granulated Tablets For each dose of the wet granulated tablets, g of the granules was added to g of MCC and g of croscarmellose sodium. The resulting mixture was blended with a spatula. One dose of the blend was set aside for stability testing and additional doses were compressed into tablets using a 2.54 cm round, flat- faced tool and die set mounted in a Carver press.

7 Wet granulated tablet formulation (per tablet) Dry granulated API g MCC g Croscarmellose Na g Total Blend g (64.5 wt % API) HPLC Method HPLC analyses were performed on an Agilent 1100 HPLC system controlled by Agilent ChemStation software. Mobile phase: Acetonitrile (700 ml), water (200 ml) and triethylamine (1 ml) were mixed then the ph was adjusted to 5.9 with acetic acid. The mixture was then diluted to 1000 ml with water. Flow rate: 1.0 ml/min Detector: 254 nm Column temperature: 25 C Column: Phenomenex Luna C18, 5 µm, 250 x 4.6 mm Run time: 25 min Injection volume: 10 µl

8 Representative HPLC Results API Blend (Not Aged) API Blend (Aged)

9 Tablets from the As Received API Blend (Aged) Wet Granulation Pre- Blend (Not Aged)

10 Wet Granules (Aged) Tablets from Wet Granules (Aged)

11 Dry Granulation Pre- Blend (Not Aged) Dry Granulation (Aged)

12 Tablets from Dry Granulations (Aged)