**1. Introduction**

The latest World Health Organization's (WHO) global report estimated that approximately 10 million people developed new tuberculosis (TB) infections that progressed to TB disease with about 1.5–2 million deaths recorded per annum [1]. So far, TB is the deadliest infectious disease globally and millions of people continue to fall sick and die annually. It is amongs<sup>t</sup> the first ten primary causes of death from a single infectious agen<sup>t</sup> worldwide, ranking above HIV/AIDS [1–3] (WHO, 2019; Swaminathan and Rekha, 2010; Kumar et al., 2017;). It remains a global threat with approximately 1.7 billion people having latent TB infection that can turn into active TB disease at any time [1]. Tuberculosis is an airborne, infectious disease that usually affects the lungs (pulmonary TB) leading to

severe coughing, fever and chest pains or in some rare cases, other body parts (extra-pulmonary TB). It is caused by *Mycobacterium tuberculosis* also called tubercle bacilli. It is preventable and curable if diagnosed early and managed with the correct medicines [1,4,5].

Generally, TB infection within the pediatric population is considered to be a major cause of morbidity and mortality [6]. According to the latest WHO's global TB report, at least one million children under the age of 15 (accounting for about 11% of the a ffected population) contract active TB infection with about 230,000 fatalities recorded annually [1] (WHO, 2019). Children may have TB disease at any age, but most often under 5 years old in TB-endemic countries. TB disease is also prevalent amongs<sup>t</sup> children infected with the human immunodeficiency virus (HIV) who are usually at a twenty times greater risk of contracting active TB infection compared to children who are HIV negative [7,8]. Children often contract TB from actively infected adult household members, during birth or when they present with weak immune systems; such as in infants, those infected with HIV or the severely malnourished who are at greater risk of developing TB disease or even dying. Pulmonary TB is the most common in children although extra-pulmonary TB may occur. Pediatric TB is more common in developing countries where there is overcrowding, poverty and malnutrition than in developed states [2]. Treatment and prevention of TB in children is considered neglected regardless of the alarming statistics as there are few scientifically justified studies focusing on: (i) accurate pediatric dosing; (ii) designing desirable drug formulations suitable for use in children of all ages; (iii) developing effective diagnostic tools for this age group as they usually do not manifest any symptoms or signs of disease early; plus (iv) the belief that childhood TB is not important for TB control [9–11].

To date, commonly used pharmaceutical formulations are either liquid dosage forms (e.g., solutions, suspensions), fixed dose dispersible tablets and in most instances, adult tablets are often broken, crushed or mixed with food or water (co-administration) to make pediatric managemen<sup>t</sup> possible [12–15]. Despite the availability of a few commercialized pediatric preparations, considerable global scarcity still exists, meaning that many children are unable to access these medicines [15–19]. Moreover, studies have shown that co-administration (with food, water etc.) is a common global practice for treating children with TB and that it is performed without appropriate instructions. In most case, caregivers just choose any food or drink without any assessment of its impact on safety and e fficacy [13,15,20]. This may potentially lead to inaccurate dosing, resulting in reduced e fficacy or adverse e ffects often caused by under-dosing and over-dosing respectively, disruption of the outer coating leading to physicochemical instabilities, and potential active pharmaceutical ingredient (API) wastage [2,13,21].

The use of alternative dosage forms such as suspensions or solutions can potentially help us overcome some of these challenges but they are also known to be generally less stable even when refrigerated, di fficult to taste mask, expensive for safe transportation and have short shelf lives; all of which limit their applicability [2,22]. Dispersible tablets on the other end are deemed more child-friendly but still limited in that they are di fficult to administer while in transit or when there is reduced/no access to potable water—like in most underdeveloped and developing countries where TB is endemic. They usually contain additives that are either not safe for use in children or hygroscopic in nature, making them prone to atmospheric moisture/water absorption that can lead to active drug instability, eventual inactivity and possible pharmacotherapeutic ine fficacy [17,19,23]. Other potentially applicable delivery systems for children include chewable tablets, which are often more suitable for older children (>3 years) with teeth, and sprinkles, though they are more acceptable for older children that can eat solid food [23,24].

Recent studies show that the most popular age appropriate delivery systems are small sized, solid oral drug delivery systems e.g., minitablets and multi-particulates and orally disintegrating formulations like orodispersible tablets or films [18,25]. Particularly, orodispersible formulations are of choice because of their characteristic advantages such as water free administration, easy to use anywhere and at any time without the need for external help or specialized caregivers, improved stability, easier transportation, cost e ffectiveness and rapid disintegration when placed within the oral cavity releasing incorporated API for absorption. This definitely allows easy administration to

pediatric patients with or without teeth [26]. Orodispersible delivery systems offer advantages such as enhanced pediatric compliance, possibility of local action, dosage accuracy, reduced choking risks, easy handling and portability [27,28]. They also allow rapid onset of action and increase in bioavailability due to rapid dispersion within the mouth and significant pre-gastric absorption, all leading to desirable pharmacotherapeutic efficacy [29]. Furthermore, antitubercular agents are administered at low doses in children so, orodispersible formulations will not be outsized or pose a choking hazard [19,23–25,30,31].

Therefore, this study details the design, optimization and systematic in vitro evaluation of a polymer-based, orodispersible film formulation containing pyrazinamide (PZA) as a potential alternative for flexible pediatric dosing. It is a first line antitubercular agen<sup>t</sup> often used in combination with isoniazid, rifampicin and ethambutol for the treatment of active TB infection [32,33]. PZA is highly bactericidal, and acts by sterilizing slowly metabolizing tubercle bacilli, resulting in low incidence of bacteriological relapse post completion of chemotherapeutic regimen. It facilitates treatment shortening, leading to greater patient compliance [32,34–39]. It is a prodrug which undergoes conversion into active pyrazinoic acid by the bacterial enzyme pyrazinamidase at or below pH 5.6 [33]. Typically, it is administered for the initial 2 months of a 6-month treatment for drug-susceptible infections. PZA is a Class III drug according to the Biopharmaceutics Classification System (BCS) characterized by its high aqueous solubility (15 mg/mL at 25 ◦C), relatively low permeability (logP = −1.88) and linear absorption over a broad spectrum of doses [36,38] (Becker et al., 2008; Adeleke et al., 2016). The PZA loaded orodispersible matrices were prepared using the solvent casting method [27,40,41]. The PZA loaded formulation was prepared using a combination of pharmaceutical excipients which included copolymer polyvinyl alcohol-polyethylene glycol as a matrix and film forming agent, citric acid as a natural preservative, sodium starch glycolate as a superdisintegrant and xylitol as a sweetener acceptable for pediatric use as documented by Dixit and Puthli [42]. Formulation preparation and optimization were facilitated using a response surface method based on a 4-factor, 3-level Box Behnken experimental design (Minitab® 18 Statistical Software (Minitab LLC, State College, PA, USA), a robust, high performance quadratic template widely applied in the development of viable drug carriers [38,43,44]. The optimized orodispersible film formulation was then physicochemically characterized in vitro by determining its mass, dimensions (inner and outer diameter), disintegration time, drug release and kinetics, drug content, dissolution pH, surface morphology changes, thermal behavior, crystallinity and structural chemical backbone transitioning. Furthermore, we studied the stability of the optimized formulation under common environmental storage conditions, its organoleptic qualities and cytobiocompatibility in vitro.

### **2. Results and Discussion**

### *2.1. Orodispersible Formulation Variants*

Employing the initial one-variable-at-a-time screening together with the systematic 4-factor, 3-level Box Behnken experimental design template, 27 pyrazinamide loaded orodispersible formulations were successfully prepared using the solvent casting method. Through these approaches, the independent variables affecting the response parameters, namely disintegration time (Y1), dissolution pH (Y2) and formulation weight (Y3), were identified. In general, the orodispersible formulations appeared as whitish, dense and bendable, hollow cup-shaped matrices that were light weight (<122 mg) and had average inner and outer diameters of 11 ± 1 mm and 10 ± 0.81 mm respectively. Overall, the 27 orodispersible formulations had average weights ranging between 121.4 ± 8.00 mg and 60.87 ± 3.80 mg, disintegrated with a total matrix structure collapse within 0.20 ± 0.09 to 5.67 ± 0.42 minutes and presented dissolution pHs spanning from 6.59 ± 0.23 to 7.43 ± 0.01 which is relatively close to that of the oral cavity (saliva). Differences observed in the measured response parameters showed that the selected independent variables applied at the varying factors levels and combinations, according to the quadratic design template, had noteworthy effects on the nature of each formulation. Numerical values of response parameters measured for all 27 formulations are presented in Table 1.


**Table 1.** Response parameters generated based on the quadratic experimental design template.

 represents the centrepoint experimental runs.

### *2.2. Selection and Validation of the Optimized Orodispersible Formulation*

\*

The ANOVA analysis revealed that the percentage of polyvinyl alcohol polyethylene glycol, sodium starch glycolate, citric acid and xylitol contained in each formulation variant significantly (*p* < 0.05) impacted the response parameters. Based on the statistical method and constraints applied on the formulation weight, disintegration time and dissolution pH, a formula was developed for the preparation of the optimized orodispersible formulation using the Minitab® 18 Statistical Software. An overall desirability of 0.991 indicating the robustness of the optimization platform and design template was obtained. The optimized formula containing the levels of each independent variable is shown in Table 2. To further confirm the validity and suitability of the optimization process, the optimized formulation was prepared in triplicate following earlier described method and measurements of the formulation weight, disintegration time and dissolution pH were performed experimentally. The magnitude of the observed response parameters measured against that of the predicted values displayed a high degree of correlation, further supporting the precision and robustness of the statistical design employed for generating the desired optimized formulation.


**Table 2.** Optimized formula and model summary of fitted and experimental outputs as it relates to the experimental design template.

**Note**: **X1** = Polyvinyl alcohol polyethylene glycol (Kollicoat® IR); **X2** = Sodium starch glycolate (Primojel®); **X3** = Citric acid; **X4** = Xylitol, **Y1** = Disintegration time; **Y2** = Disintegration pH; **Y3** = Formulation weight.

### *2.3. Physical Properties of Optimized Formulation*

The optimized orodispersible formulation was thin, flexible making handling possible, whitish in color with a hollow/concave shape as well as inner and outer diameters of 10.00 ± 0.52 mm and 11.00 ± 0.43 mm. The formulation was made up of sodium starch glycolate as a super disintegrate, co-polymer polyvinyl alcohol polyethylene glycol as a matrix and film forming agent, xylitol as a sweetener suitable for pediatrics, citric acid as a natural preservative and pyrazinamide (500 mg) as a model antitubercular agent. The optimized formulation was light weight and small enough for orodispersible applications. It rapidly disintegrated in less than 60 seconds (i.e., 0.58 min ≡ 34.98 s) when placed in simulated saliva at 37 ± 0.1 ◦C. The dissolution was close to neutral (7.0) and saliva pH (6.8), meaning that the formulation has no potential to irritate the buccal mucosa (Table 2). Digital photographs of the PZA loaded and drug free (placebo) optimized formulations are shown in Figure 1.

**Figure 1.** Digital photograph of ( **A**) drug loaded and (**B**) placebo orodispersible film formulation.

### *2.4. Drug Content and Release Behavior*

The PZA content of the optimized orodispersible formulation was 25.02 ± 0.71 mg equaling 101.13 ± 2.03%*<sup>w</sup>*/*<sup>w</sup>*, indicating that uniform drug distribution occurred among replicate test samples and drug remained stable during and after preparation. In vitro drug release was carried out under biorelevant conditions to determine the rate at which PZA molecules were released in simulated saliva (pH 6.8) at 37 ± 0.1 ◦C, mimicking the buccal environment. The generated release profile is illustrated with Figure 2. Figure 2B, which is an expanded form of segments of Figure 2A, shows that drug liberation was initiated under 10 s (0.17 min) with 1.94 ± 0.28% released almost immediately after the formulation came in contact with the dissolution media (onset of matrix disintegration). Subsequently, the amount of drug released continued to increase rapidly, reaching its first peak at 5 min (72.01 ± 11.93%) and then maintained a relatively plateaued profile until 60 min when complete matrix dissolution and drug release (102.50 ± 5.19%) occurred.

**Figure 2.** (**A**) In vitro drug release behavior in simulated saliva and (**B**) illustrates an expanded segmen<sup>t</sup> of the drug release profile.

The mathematical models applied to represent the release mechanisms of the active drug from the formulation were zero-, first-, second-order as well as Higuchi, Korsmeyer–Peppas and Hixon–Crowell models yielded R values of 0.59, 0.47, 0.33, 0.40, 0.90 and 0.51, respectively. The optimized formulation displayed a good fit to the Korsmeyer–Peppas (*R* = *0.90*) and the computed *n-value* was 0.83, meaning that drug release after formulation hydration was controlled by an anomalous drug diffusion process followed by matrix relaxation (disentangling polymer chains or disintegration) and erosion (matrix dissolution) [45].

### *2.5. Optimized Formulation Characterization*

### 2.5.1. Thermal Behavior Using Differential Scanning Calorimetry and Thermogravimetry

Generated differential scanning calorimetry (DSC) thermograms were employed in the assessment of typifying thermal quantity changes for pure PZA, pharmaceutical excipients (citric acid, xylitol, poly vinyl alcohol polyethylene and sodium starch glycolate), optimized drug loaded and placebo formulations. Measured key thermal quantities include melting point (Tm) and glass transition temperature (Tg). Typical DSC thermograms are shown in Figure 3. The DSC scans of citric acid and xylitol represented in (Figure 3A,B) show sharp endothermic peaks corresponding to their Tm at 153.38 ◦C and 92.64 ◦C, respectively, indicating their purity and stable states. Polyvinyl alcohol polyethylene glycol (Kollicoat® IR) thermogram (Figure 3C) depicts multiple broad endothermic and exothermic peaks with a small endothermic peak at 92.3 ◦C and a more prominent endothermic peak at 212.65 ◦C as Tm. The presence of two endothermic peaks shows that the Kollicoat® IR is made up of two different natured polymers. The appearance of Tg noted at 44.24 ◦C can be associated with its amorphous co-polymeric transitioning into a crystalline material. Sodium starch glycolate thermogram (Figure 3D) displayed characteristic exothermic peak at 298.75 ◦C and glass transition was observed at 46.45 ◦C. Pure pyrazinamide shows two endothermic peaks at 154.00 ◦C, corresponding to solid–solid transition and a sharp endothermic peak at 190.64 ◦C (Figure 3E), which corresponds to the Tm, indicating its α- polymorphic form and purity of PZA [38,46,47]. Thermal peaks identified for each excipient and pure drug are indicative of their purity and stability as individual compound before their inclusion in the orodispersible formulation mix. The placebo thermogram (Figure 3F) presented broad endothermic and exothermic peaks, revealing its semi-crystalline nature which can be related to its crystalline, semi-crystalline and amorphous constituents already mentioned above. Likewise, the optimized PZA loaded orodispersible formulation thermogram (Figure 3G) also displayed a semi-crystalline trend with broad endothermic and exothermic peaks occurring within the melting point region of excipients. This finding further supports its blended pure drug and excipient content. The slight shift of the PZA peak in the mixture may have been influenced by polymeric crystallization occurring during the formation of the orodispersible formulation resulting in the endothermic peak slightly shifting to 193 ◦C, accounting as the Tm of the newly prepared drug loaded formulation further confirming drug stability within the drug delivery matrix. The disappearance of melting peaks of individual excipients in the drug loaded formulation shows complete solubilization of the drug and excipients within the matrix. It also supports the formation of a new compound, hence the physical transitioning of PZA from crystalline to the amorphous form potentially accounting for the rapidly disintegrating quality of the developed formulation [48]. The obtained DSC thermograms demonstrate the compatibility of the drug (PZA) and pharmaceutical excipients used in the formulation development with no evidence of possible adverse interactions.

**Figure 3.** DSC Thermograms of (**A**) citric acid, (**B**) xylitol, (**C**) polyvinyl alcohol polyethylene glycol, (**D**) sodium starch glycolate, (**E**) pyrazinamide, (**F**) placebo and (**G**) drug loaded formulation.

Thermogravimetric curves of pure PZA, pharmaceutical excipients, optimized drug loaded and placebo formulations recorded under a nitrogen saturated atmosphere, purge and heating rates of 25 mL/min and 10 ◦C/min, respectively, are presented in Figure 4. TGA analysis was conducted for additional investigation of thermal degradation events quantified as percentage weight loss as it relates to temperature and its impact of formulation stability. First, important thermal events were identified for individual excipients and pure PZA. The PZA thermal decomposition Tonset was observed at 164 ◦C and complete weight loss occurred at 199.15 ◦C (Figure 4A). The onset of thermal

decomposition for xylitol (Figure 4B) was noted at 240.59 ◦C and final decomposition temperature was observed at about 306.73 ◦C, showing it thermal robustness as Tonset was above 200 ◦C. Citric acid and poly vinyl alcohol polyethylene thermal plots in (Figure 4C,D) commenced decomposition at a Tonset of about 185.99 and 236.39 ◦C, respectively, and complete breakdown only occurred at temperatures greater than 400 ◦C, confirming thermal stability of these excipients. Sodium starch glycolate (Figure 4E) showed two decomposition events, water loss from the polymer was observed at about 125.01 ◦C, corresponding to 8% weight loss, followed by final decay at 257.99 ◦C. Generally, the excipients presented with relatively high, single decomposition temperatures, 199 ◦C and above, indicating their distinct stability and purity. Thermal decomposition of optimized drug loaded and placebo (Figure 4F,G) began at 156.24 and 200.38 ◦C, respectively. Complete weight loss followed at 202.72 ◦C for the PZA loaded formulation and beyond 400 ◦C for the placebo. Onset temperatures considerably below 200 ◦C suggests lower thermal stability which, in this case, can be linked to the loss of residual water molecules from the both matrices due to the presence of hydrophilic components in both drug loaded and placebo formulations. Usually, weak drug and hydrophilic polymer interactions break easily around 100 ◦C [49]. The presence of an additional non-polymeric hydrophilic molecule, PZA, within the drug loaded formulation matrix probably accounts for the reduction in the onset temperature (156.24 ◦C) compared to that recorded for the placebo (200.38 ◦C) which contains all other hydrophilic constituent excluding PZA. This can further influence the temperature at which complete weight loss (100%) occurred for both drug loaded (202.72 ◦C) and placebo formulations (>400 ◦C). From these observations, it appears that the placebo contains more crystalline domains within its molecular structure and its components are more of a physical blend with less amorphous transitioning happening compared to the drug loaded formulation, where a degree of amorphization seems to occur between the PZA and excipient physical blend as also revealed through the DSC (Figure 3G) and XRD (Figure 5G) analytical outputs. Overall, the TGA thermograms exhibited the thermal stability of both drug and pharmaceutical excipients, either as separate entities or blends in the di fferent formulations, as well as presented no visible trace of any destructive chemical interaction. Additionally, the similarity in weight loss patterns plus relative overlap in final decomposition temperatures for both drug loaded formulation (202.72 ◦C) and pure PZA (199.15 ◦C) (Figure 4A,F), can further signify formulation matrix stability and drug intactness which, was also the case with the DSC analyses as the PZA peak remained identifiable in the formulation blend with a slight shift associated with the presence of other excipients (Figure 3G).

**Figure 4.** *Cont.*

**Figure 4.** Thermogravimetric curves of (**A**) PZA, (**B**) xylitol, (**C**) citric acid, (**D**) polyvinyl alcohol polyethylene glycol, (**E**) sodium starch glycolate, (**F**) drug loaded formulation and (**G**) placebo.

**Figure 5.** X-ray diffractograms of (**A**) PZA, (**B**) citric acid, (**C**) xylitol, (**D**) polyvinyl alcohol polyethylene glycol, (**E**) sodium starch glycolate, (**F**) placebo and (**G**) drug loaded formulation.
