Development and Optimization of a Biphasic-Release Acetazolamide Tablet-in-Tablet Formulation

Abstract

Background and Objective

High-altitude illness (HAI) poses health risks to individuals at high altitudes, and acetazolamide (ACZ) is the only Food and Drug Administration (FDA)-approved prophylactic drug. Conventional immediate-release (IR) tablets and extended-release (ER) capsules do not simultaneously provide early drug availability and prolonged coverage. This study aimed to develop a biphasic-release ACZ tablet-in-tablet (ACZ-TIT) formulation as a proof-of-concept oral dosage form integrating IR and ER.

Methods

ACZ–excipient compatibility was assessed using Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and powder X-ray diffraction (PXRD), and solubility was evaluated in physiologically relevant media. Formulation optimization was performed via single-factor studies and response surface methodology (RSM). Quality control included weight, hardness, friability, content, and related substances. In vitro dissolution studies were conducted, and drug release kinetics were analyzed using multiple models.

Results

No new incompatibility signals were observed between ACZ and the selected excipients, and only small solubility differences were found across the tested media. ACZ-TIT showed a biphasic-release profile, with 25.3% drug release at 0.5 h and 86.5% at 10 h. Physical properties were within pharmacopeial limits (weight variation ≤ 2.65%, hardness 112.0 ± 21.2 N, friability 0.18%, and assay relative standard deviation < 1.2%), and the related substances were found to be within the specified limits. Release kinetics were best described by the Ritger-Peppas model (R² = 0.993), supporting diffusion-dominated ER.

Conclusion

ACZ-TIT may provide rapid initial release followed by sustained exposure, which could help address limitations of currently available ACZ dosage forms. In vivo pharmacokinetic and clinical studies are still required to establish clinical applicability.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40268-026-00546-9.

Key Points

The proposed tablet releases part of the dose quickly and the rest more slowly over time.

The optimized formulation met routine quality tests and showed reproducible in vitro release behavior.

The design may offer earlier symptom coverage than current extended-release capsules, but this still needs in vivo confirmation.

Introduction

High-altitude illness (HAI) is a collective term encompassing a spectrum of hypoxia-related disorders that occur following exposure to high-altitude environments. HAI is broadly classified into acute mountain sickness (AMS) and chronic mountain sickness (CMS) based on the duration and pattern of hypoxic exposure [1].

AMS arises from acute exposure to hypobaric hypoxia caused by a rapid decrease in ambient oxygen pressure, leading to disturbances in cerebral and cardiopulmonary function. Common clinical manifestations include headache, dizziness, nausea, vomiting, palpitations, and dyspnea [2, 3]. If not promptly recognized and managed, AMS may progress to life-threatening complications such as high-altitude pulmonary edema (HAPE) and high-altitude cerebral edema (HACE), both of which are associated with high mortality rates [4]. Globally, more than 100 million individuals travel annually to high-altitude regions for occupational, recreational, religious, or other purposes. Epidemiological studies report that the incidence of AMS ranges from 10 to 85%, with a strong positive correlation between disease prevalence and altitude [5, 6]. In contrast, CMS primarily affects individuals residing at altitudes above 2500 m for prolonged periods, typically months or years [7]. The hallmark pathological feature of CMS is excessive erythrocytosis (EE), which increases blood viscosity and predisposes patients to serious complications, including pulmonary hypertension, cor pulmonale, left ventricular dysfunction, and thromboembolic events [8]. The incidence of CMS increases with both altitude and age [7], affecting approximately 10% of the estimated 140 million long-term high-altitude residents worldwide [6].

Several pharmacological agents have demonstrated efficacy in the prevention or treatment of HAI, including carbonic anhydrase inhibitors (CAIs), glucocorticoids, phosphodiesterase inhibitors, and calcium channel blockers. Additional agents, such as spironolactone, acetaminophen, ibuprofen, Ginkgo biloba extract, procaterol, salmeterol, and salbutamol, have also shown potential benefits [9, 10]. The primary therapeutic goal in AMS is rapid alleviation of hypoxia-related symptoms and prevention of HAPE and HACE, commonly achieved using acetazolamide (ACZ), dexamethasone, or nifedipine [11]. In contrast, CMS management focuses on long-term control of polycythemia and pulmonary hypertension, typically employing low-dose ACZ, endothelin receptor antagonists, phosphodiesterase-5 inhibitors, and phlebotomy [12]. Notably, ACZ remains the only drug approved by the US Food and Drug Administration (FDA) for both prevention and treatment of HAI.

ACZ, a diuretic developed in the 1950s, belongs to the CAI class. Its pharmacological action involves potent inhibition of carbonic anhydrase activity, forming the basis for its applications in controlling atrial aqueous secretion in certain glaucomas, treating specific epilepsies (e.g., absence seizures), alleviating heart failure-related edema, and, crucially, preventing and treating AMS [13–15]. ACZ exerts its effects through multiple mechanisms: inhibiting carbonic anhydrase, improving ventilation and oxygenation, reducing HAI symptoms, attenuating pulmonary hypertension, enhancing exercise performance, and modulating central nervous system functions [16–20], collectively making it effective for HAI management.

ACZ exhibits favorable pharmacokinetic (PK) properties, with an oral bioavailability of 70–90% and an elimination half-life of approximately 3–9 h, making it a suitable candidate for oral formulation development [21]. From a PK/pharmacodynamic (PD) perspective, the rapid attainment and sustained maintenance of effective systemic exposure are closely associated with timely prophylaxis and reliable symptom control in HAI [22]. Studies have shown that the minimum effective plasma concentration of ACZ for glaucoma treatment is approximately 4 µg/mL, and that the incidence of adverse effects increases markedly when plasma concentrations exceed 20 µg/mL [23]. However, the therapeutic concentration window for the prevention and treatment of HAI has not yet been clearly established. Available evidence suggests that a daily dose of 250–500 mg is an appropriate regimen for the prophylaxis and treatment of altitude illness [9]. Currently available oral ACZ products mainly include immediate-release (IR) tablets and extended-release (ER) capsules. ER capsules have been reported to exhibit a relatively delayed onset of action, typically exceeding 2 h, which may limit their ability to provide prompt preventive protection during rapid ascent. In contrast, IR tablets generally achieve a faster onset (within approximately 1 h) but require frequent dosing, thereby reducing convenience and potentially compromising patient adherence [24]. To our knowledge, no ACZ formulation integrating both IR and ER within a single dosage form has been reported. This unmet need highlights a key formulation challenge: achieving both rapid onset and prolonged, stable systemic exposure in a single oral preparation.

Tablet-in-tablet (TIT) systems, also known as dry-coated tablets or press-coated tablets, provide a promising strategy to overcome this limitation. TITs are produced by compressing a preformed core tablet within an outer layer, enabling spatial separation of formulations with distinct release characteristics [25, 26]. Compared with osmotic pump technologies, TIT systems can be manufactured without a semipermeable membrane coating or laser-drilling step, which makes them attractive for early formulation development and for relatively high-dose active pharmaceutical ingredients (APIs) such as ACZ [27, 28]. Compared with conventional bilayer tablets—where both layers may begin releasing simultaneously—TIT systems allow precise temporal control of drug release through an “outer-to-inner” structural design. In biphasic-release TITs, an IR outer layer provides immediate drug availability, followed by sustained release from the ER core.

Based on these principles, we designed a novel ACZ tablet-in-tablet (ACZ-TIT) formulation comprising an ER skeleton-type core and an IR outer layer containing a disintegrant. After oral administration, the outer layer rapidly disintegrates to achieve prompt therapeutic plasma concentrations, while the exposed ER core gradually hydrates and releases ACZ in a controlled manner via diffusion and/or dissolution mechanisms. This biphasic-release design is intended to maintain effective drug levels over an extended period while ensuring rapid onset of action, thereby addressing the PK shortcomings of existing ACZ formulations.

In this study, pre-formulation investigations were conducted to evaluate drug–excipient compatibility. The ACZ-TIT formulation was optimized using single-factor analysis and response surface methodology (RSM). Comprehensive quality evaluation and in vitro dissolution studies were then performed, and the resulting release behavior was interpreted using multiple kinetic models to characterize the release mechanism. The overall study design is summarized as a block diagram in Fig. 1.

Fig. 1.

Block diagram and technical route for the development and optimization of acetazolamide biphasic-release tablet-in-tablet. ER extended-release, IR immediate-release, RSM response surface methodology

Materials and Methods

Materials

ACZ was obtained from Beijing Double-Crane Pharmaceutical Co., Ltd. (Shangqiu, China). Polyethylene oxide (PEO) and hydroxypropyl methylcellulose (HPMC) were purchased from International Flavors & Fragrances Inc. (IFF, USA). Hydroxypropyl cellulose (HPC) was supplied by Vorm Pharmaceutical Technology Co., Ltd. (Shenzhen, China). Microcrystalline cellulose (MCC, PH 102 grade) and croscarmellose sodium (CCMC-Na) were purchased from Asahi Kasei Chemical Corporation (Japan). All other reagents and solvents were obtained from Shanghai Pharmaceutical Chemical Reagent Co., Ltd. (Shanghai, China) and were of analytical or high-performance liquid chromatography (HPLC) grade.

Pre-formulation Studies

Melting Point Determination

The melting point of ACZ was determined using a capillary melting point apparatus (GM70, Shanghai Zhuoguang Instrument Technology Co., Ltd., China). Dried samples were packed into capillary tubes (inner diameter ≈ 1 mm), sealed at one end, and heated at a controlled rate of 1 ℃/min. The temperature at which complete melting occurred was recorded as the melting point.

Drug–Excipient Compatibility Studies

Potential interactions between ACZ and selected excipients were evaluated using Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), assay determination, and related substances testing [29]. Physical mixtures of ACZ and excipients (PEO, HPMC, HPC, MCC, and CCMC-Na) were prepared at a 1:1 (w/w) ratio. Samples were stored under accelerated conditions (40 ± 2 ℃, 75 ± 5% relative humidity) for 10 days. Analyses were performed on pure ACZ and ACZ–excipient mixtures before and after storage, and results were compared to detect any physicochemical interactions.

FTIR experiment: Infrared spectra were recorded using an FTIR spectrophotometer (WQF-530A, Beijing Beifen-Ruili Analytical Instrument [Group] Co., Ltd., China) over the range of 4000–400 cm⁻¹ with KBr as the background. Characteristic absorption peaks were analyzed to identify functional groups and potential interactions.

DSC experiment. Approximately 10 mg of dried sample was placed in a ceramic crucible, with an empty crucible serving as the reference. Thermal analysis was performed using a DSC instrument (HSC-1, Beijing Hengjiu Experimental Equipment Co., Ltd., China) over a temperature range of 30–300 ℃ at a heating rate of 10 ℃/min under a nitrogen flow of 100 mL/min.

PXRD experiment: Powder PXRD patterns were obtained using an X-ray diffractometer (Empyrean, Malvern Panalytical Ltd., UK). Samples were ground, homogenized, and mounted on a flat sample holder. Diffraction data were collected over a 2θ range of 10°–80° at a scanning rate of 8.67 s per step, with an applied voltage of 45 kV and current of 40 mA. Diffraction peaks were analyzed to assess crystallinity and possible solid-state interactions.

Solubility of ACZ

Because ACZ dissolution testing required selection of a suitable and physiologically relevant medium, solubility was evaluated in media representing different gastrointestinal pH conditions. The aim of this experiment was to determine whether pH had a marked effect on ACZ solubility and to support selection of a candidate medium for subsequent in vitro release studies. ACZ (0.1 g) was added to 50 mL of dissolution media with pH values of 1.2, 4.5, and 6.8, as well as purified water. The suspensions were shaken at 37 ℃ for 24 h using a constant-temperature shaker (SHZ-82, Changzhou Guohua Electric Appliance Co., Ltd., China) to achieve equilibrium. After filtration, the solutions were diluted 100-fold with the corresponding media. Reference solutions (10 μg/mL) were prepared similarly. Absorbance was measured at 265 nm, and solubility was calculated using calibration curves derived from standard solutions.

Formulation Development and Optimization

Preparation of ER Tablet Cores

Direct compression was selected for tablet manufacture due to its simplicity, cost-effectiveness, and suitability for large-scale production. Accurately weighed ingredients were blended for 30 min, dried at 60 ℃ for 2 h, and compressed into tablets using a tablet press fitted with a 10-mm round concave punch (KTP 1X, Romaco Holding GmbH, Germany).

Preparation of ACZ-TITs

For preparation of ACZ-TITs, inner ER cores and outer IR layers were manufactured separately. The IR-layer ingredients were accurately weighed, blended for 30 min, and dried at 60 ℃ for 2 h. Half of the IR powder was pre-compressed in a 14-mm round concave die, the ER core was centrally positioned, and the remaining IR powder was added before final compression to form the TIT structure.

Single-Factor Analysis

Single-factor analysis was conducted by varying one factor at a time while keeping all others constant to evaluate its influence on formulation performance [28]. For the ER core, four factors were investigated: type of ER polymer, amount of ER polymer, amount of filler, and tablet hardness. Formulations tested are summarized in Table 1.

Table 1.

Extended-release (ER) core tablet formulations used in formulation screening and validation

No.	API amounts (mg)	ER material	Amounts (mg)	Filler material	Amounts (mg)	Tablet hardness (N)
F1–F6	200	PEO, HPMC, HPC	80	PH 102	120	55
F7–F10	200	WSR 303, Coagulant, WSR 301, WSR N60K	80	PH 102	120	55
F11–F13	200	WSR 301	50, 60, 70	PH 102	120	55
F14–F16	200	WSR 301	60	PH 102	100, 120, 140	55
F17–F19	200	WSR 301	60	PH 102	120	55, 65, 75
F20–22	200	WSR 301	63	PH 102	117	55

APIs Active Pharmaceutical Ingredients F formulation, HPC hydroxypropyl cellulose, HPMC hydroxypropyl methylcellulose, PEO polyethylene oxide

Based on the dissolution profile of the marketed ER capsule Diamox^® (500 mg), which showed < 15% drug release at 0.5 h and > 80% release at 10 h [30], these values were used as formulation-development benchmarks for screening the ER core rather than as PK-equivalence targets.

For the IR outer layer, two factors were evaluated: disintegrant content and filler content. The corresponding formulations are listed in Table 2. Optimal IR-layer composition was determined by balancing disintegration time and compression performance.

Table 2.

Immediate-release outer layer formulations and levels used in the single-factor experiments

No.	API amounts (mg)	Disintegrants material	Amounts (mg)	Filler material	Amounts (mg)
F23–F25	50	CCMC-Na	5	PH 102	395, 445, 495
F26–F28	50	CCMC-Na	5, 15, 25	PH 102	445

APIs Active Pharmaceutical Ingredients, F formulation, CCMC-Na croscarmellose sodium

RSM Optimization

Factors identified as important in the single-factor studies were further optimized using RSM. A 3² full factorial design with replicated center points was employed to investigate interactions between variables and to identify formulations meeting the predefined release targets (≤ 15% at 0.5 h and ≥ 80% at 10 h). Each formulation was tested in triplicate. Drug release at 0.5 h (Y₁) and 10 h (Y₂) served as response variables. Experimental design, regression analysis, analysis of variance (ANOVA), and response surface visualization were performed using Design-Expert^® software (version 13.0, Stat-Ease Inc., USA).

Quality Control and Testing

All quality evaluations were conducted in accordance with the Chinese pharmacopoeia (2025 edition) [31].

Weight Variance

Twenty tablets from each batch were individually weighed using an electronic balance (BCE223-1CCN, Sartorius Scientific Instruments, China). The mean weight and percentage deviation were calculated.

Thickness

Tablet thickness was measured using a Vernier caliper (DHGDW200F, Delixi Electric Co., Ltd., China) on ten randomly selected tablets.

Hardness

Tablet hardness was measured for ten tablets per batch using a hardness tester (YD-20KZ, Tianjin Tianda Tianfa Technology Co., Ltd., China).

Friability

Ten tablets were tested using a friability tester (FT2000-AF, Tianjin Tianda Tianfa Technology Co., Ltd., China) at 25 rpm for 4 min. Friability was calculated as follows:

$$\text{Friability}\left(\%\right)=\left[\left(\text{initial mass}-\text{final mass}\right)/\text{initial mass}\right]\times 100\%$$ 1

Drug Content

The inner and outer layers of ten tablets were separated and ground into a fine powder, and an amount equivalent to 50 mg of ACZ was accurately weighed into a 250-mL volumetric flask with ~ 200 mL of water. The flask was incubated at 37 ℃ for 24 h to ensure complete dissolution, cooled, and diluted to volume. After filtration, 5 mL was transferred to a 100-mL volumetric flask, 10 mL of 1 mol/L hydrochloric acid was added, and the volume was adjusted with water to yield an approximately 10 μg/mL solution.

The absorbance was measured at 265 nm for the quantification of the ACZ content. The calibration curve revealed a good linear relationship between the ACZ concentration (C, μg/mL) and absorbance (A), with the regression equation expressed as Y = 20.77392X − 0.08565 (where X = absorbance, Y = concentration). The method exhibited excellent linearity, supported by a Pearson’s correlation coefficient (r) of 0.99983 and a coefficient of determination (R²) of 0.99966. These values fully meet the requirements for quantitative analysis specified in the Chinese pharmacopoeia (r ≥ 0.999), verifying that the method is suitable for assay determination, content uniformity assessment, and in vitro release measurements within the validated concentration range of 0–15 μg/mL.

Related Substances

The method and acceptance criteria were based on the Chinese pharmacopoeia (2025 edition) monograph for ACZ tablets [31]. Related substances were determined by the self-control method. The test solution was accurately diluted to prepare the reference solution, and the analysis was performed by HPLC. When the response factors of impurities and the main component were similar, the impurity content was calculated by the peak area ratio. The test solution and reference solution were injected into the HPLC system, respectively, and their chromatograms were recorded. In the chromatogram of the test solution, the area of any individual impurity peak was not to exceed 0.5 times the main peak area of the reference solution (corresponding to an impurity limit of 0.5%), and the sum of all impurity peak areas was not to exceed the main peak area of the reference solution (corresponding to a total impurity limit of 1.0%). The sample met the requirements if the above criteria were satisfied.

Chromatographic conditions: Octadecylsilyl silica gel was used as the stationary phase; the mobile phase consisted of 0.43% anhydrous sodium acetate solution–methanol–acetonitrile (95:2:3, adjusted to pH 4.0 ± 0.05 with glacial acetic acid); the detection wavelength was set at 265 nm; the injection volume was 20 μL.

In Vitro Release Study

Because the solubility study showed only small differences across the tested media, the pH 4.5 acetate buffer/water medium specified in the Chinese pharmacopoeia was selected as the primary medium for formulation screening and routine dissolution testing [31]. Dissolution testing was performed using the paddle method in 900 mL of dissolution medium (150 mL pH 4.5 acetate buffer and 750 mL water) at 37 ± 0.5 ℃ and 100 rpm (SNTR-8600A, Toyama Sangyo Co., Ltd., Japan). Samples (5 mL) were withdrawn at 0.5, 1, 2, 4, 6, 8, 10, 12, 14, and 16 h and replaced with fresh medium. After filtration through 0.45-μm membranes, absorbance was measured at 265 nm. Cumulative release profiles were constructed. In addition to routine dissolution testing in the selected pH 4.5 medium, a multi-medium comparison was performed for both ACZ-TIT and the marketed ER capsule in pH 1.2, pH 4.5, pH 6.8, and water to assess medium-dependent changes in release behavior.

The similarity between the test formulation and the target curve was evaluated via the similarity factor (f₂) method:

$$f_2=50\times \text{log}\left\{{[1+(1/n)\times \sum {(R_t-T_t)}^2]}^{(-0.5)}\times 100\right\}$$ 2

where Rt is the percentage released by the reference formulation at time t, Tt is the percentage released by the test formulation at time t, and n is the number of sampling points. f₂ ≥ 50 indicates similar dissolution profiles.

Disintegration Test

The disintegration time of the IR outer layer was determined using a disintegration tester (ZB-1E, Tianjin Tianda Tianfa Technology Co., Ltd., China). Six tablets were tested at 37 ± 2 ℃, and the time required for complete disintegration of the outer layer was recorded.

Drug Release Kinetics

The release kinetics fitting was performed using Origin software (9.9.0.225, OriginLab Corporation, USA). In vitro release data were fitted to zero-order, first-order, Higuchi, and Ritger-Peppas models to elucidate the drug release mechanism [32]. The best-fitting model was selected based on statistical criteria, including residual sum of squares (RSS), reduced chi-square values, and the coefficient of determination (R²) [33].

Results

Melting Point

Video recordings from the melting point apparatus showed that the ACZ began melting at 254 ℃, transforming from a white powder into a brown liquid. Complete liquefaction occurred at 256 ℃, accompanied by observable thermal decomposition. These observations suggest an approximate melting point of 255 ℃ for the ACZ, with a narrow melting range (254–256 ℃), which is consistent with a relatively pure starting material.

Drug–Excipient Compatibility

The contents of ACZ and its related substances in each batch of samples before and after the accelerated test were determined (Table 3). The results showed that there was no notable change in the content of ACZ before and after the accelerated test, and the related substances were also within the specified range.

Table 3.

HPLC peak areas of ACZ and its physical mixtures in the drug–excipient compatibility study

Sample	Test sample peak area	Max single impurity peak area	Total impurities peak area	Max single impurity (%)	Total impurities (%)
ACZ	27138	9.85	48.85	0.036	0.18
ACZa	27034	11.63	57.32	0.043	0.21
Mixture	26962	13.22	63.78	0.049	0.23
Mixturea	27025	8.65	52.69	0.032	0.19

ACZ acetazolamide, HPLC high-performance liquid chromatography

^aSamples under accelerated conditions after 10 days

The FTIR spectrum of pure ACZ exhibited distinct characteristic peaks, which was consistent with previous studies and confirmed its structural integrity and purity [34]. A broad, intense absorption band at 3350 cm⁻¹ was observed, arising from overlapping N-H stretching vibrations of the sulfonamide (-SO₂NH-) and amide (-NHCOCH₃) functional groups. The symmetric peak shape and stable intensity suggest minimal intermolecular interactions within the sample. The distinct peak at 1660 cm⁻¹ was attributed to the C=O stretching vibration of the amide carbonyl group, which slightly redshifted, likely because of intramolecular hydrogen bonding, which is consistent with typical amide infrared spectral behavior. The twin peaks at 1347 cm⁻¹ (asymmetric) and 1152 cm⁻¹ (symmetric) corresponded to the S=O stretching vibrations of the sulfonamide moiety, with an intensity ratio of approximately 1.2:1. Additional characteristic peaks at 1575 cm⁻¹, 750 cm⁻¹, and 685 cm⁻¹ were assigned to the C=N stretching (thiazole ring), C-S-C out-of-plane bending, and ring deformation modes, respectively. The weak absorption near 2900 cm⁻¹ confirmed the presence of methyl (-CH₃) C-H stretching. Crucially, the baseline above 3500 cm⁻¹ remained flat, with no detectable O-H absorption (indicating negligible residual moisture) or unidentified impurity peaks, further verifying the high purity of the APIs.

The FTIR spectra of the ACZ mixtures with PEO, HPMC, HPC, CCMC-Na, and MCC presented identical peak positions, shapes, and relative intensities relative to those of pure ACZ (Fig. 2). Notably, no new absorption bands or disappearance of characteristic peaks were detected, indicating that there was no chemical interaction between ACZ and the tested excipients.

Fig. 2.

The Fourier transform infrared spectra of ACZ and ACZ–excipient mixtures. ^aSamples under accelerated conditions after 10 days. ACZ acetazolamide

In DSC analysis, pure ACZ exhibited a sharp endothermic peak at 250–260 ℃, corresponding to its melting point, with an enthalpy change (ΔH) of 41.91 J/g (Fig. 3). This is followed by an exothermic peak consistent with the decomposition observed in the melting point determination experiments. These results have also been reported in previous studies [35].

Fig. 3.

The differential scanning calorimetry thermograms of ACZ and ACZ–excipient mixtures. ^aSamples under accelerated conditions after 10 days. ACZ acetazolamide

For physical mixtures of ACZ with MCC, HPC, CCMC‑Na, HPMC, and PEO, the characteristic melting endotherm of ACZ remained observable, with only slight changes in peak shape, intensity, and enthalpy values. No new endothermic or exothermic peaks indicative of drug–excipient interactions or chemical incompatibility were detected. The minor variations in melting enthalpy were attributed to physical dilution effects and weak intermolecular interactions rather than chemical reactions.

The PXRD pattern revealed that the active pharmaceutical ingredient ACZ exhibited multiple sharp, high-intensity diffraction peaks, indicating a highly crystalline nature, with distinct characteristic peaks near 2θ = 20° and 30° [36] (Fig. 4). In the physical mixture, these characteristic peaks remained clearly visible, suggesting weak interactions among the coexisting components. In the accelerated-treated mixture, the characteristic diffraction peaks of ACZ near 2θ = 20° and 30° were still observable, although new overlapping signals appeared. These new signals were considered more likely to reflect storage-related changes in excipient crystallinity than disappearance of the ACZ crystal form. Overall, retention of the major ACZ peaks suggests that the excipients did not induce an obvious phase transition of ACZ during the short, accelerated study.

Fig. 4.

The powder X-ray diffraction spectra of ACZ and ACZ–excipient mixtures. ^aSamples under accelerated conditions after 10 days. ACZ acetazolamide

ACZ Solubility

Drug solubility in physiologically relevant dissolution media is a critical consideration for formulation development, particularly when selecting a candidate medium for in vitro release testing. In this study, the solubility of ACZ was determined in four media simulating different pH environments of the human gastrointestinal tract.

As shown in Table 4, ACZ exhibited slight variations in solubility across the tested media. The highest solubility was observed in phosphate buffer (pH 6.8, 1.59 g/L), followed by 0.1 N HCl (pH 1.2, 1.37 g/L) and acetate buffer (pH 4.5, 1.34 g/L), while the lowest solubility was recorded in purified water (1.27 g/L). However, all values remained within a relatively narrow range (1.2–1.6 g/L), with a maximal difference of only 0.32 g/L. These data suggest only modest pH-related variation in ACZ solubility under the tested conditions.

Table 4.

Solubility of acetazolamide in different dissolution media

No.	Dissolution media	Solubility (g/L)
1	0.1 N HCl (pH 1.2)	1.37 ± 0.04
2	Acetate buffer (pH 4.5)	1.34 ± 0.03
3	Phosphate buffer (pH 6.8)	1.59 ± 0.05
4	Water (pH 6.3)	1.27 ± 0.03

Formulation Development and Optimization

Drug Dosage

The US FDA recommends a minimum prophylactic daily dose of 500 mg ACZ for the prevention of HAI. Preliminary formulation trials using a single tablet (500 mg ACZ) combined with conventional excipients resulted in tablets with excessive size, which could negatively affect patient acceptability and compliance. To address this limitation, a split-dose strategy was adopted, and the target strength of each tablet was set at 250 mg. Currently marketed IR ACZ tablets are available at a strength of 125 mg. Based on conventional dosing regimens, an initial IR dose of approximately half this amount (62.5 mg) was considered for the outer IR layer. However, given the biphasic-release design, rapid drug release from the IR outer layer and then the initial burst from the ER core could lead to an undesirably high peak plasma concentration (C_max), potentially increasing the risk of adverse effects. To mitigate this risk, the IR dose should be reduced.

Based on these considerations, the optimized formulation consisted of 50 mg ACZ in the IR outer layer and 200 mg in the ER core, yielding a total tablet strength of 250 mg and satisfying the split-dose requirement. Future in vivo PK studies are planned to further refine the dose distribution and to evaluate whether the formulation can provide an acceptable plasma concentration–time profile.

Selection of ER Matrix Materials

The in vitro drug release profiles of ACZ core tablets formulated with different ER materials are presented in Fig. 5. PEO, HPMC, and HPC were selected as candidate ER materials for evaluation. Among the tested formulations, PEO-based batches exhibited the release profiles closest to the targets (≤ 15% at 0.5 h and ≥ 80% at 10 h). Specifically, the cumulative release rate reached 71.57% within 10 hours for the WSR 301 formulation, and 96.65% for the WSR N60K formulation. Compared with the other polymer systems evaluated, PEO provided the most favorable balance between release retardation and overall drug availability, and was thus selected as the ER material for subsequent formulation optimization.

Fig. 5.

In vitro release profiles of different extended-release materials (F1–6). F formulation, HPC hydroxypropyl cellulose, HPMC hydroxypropyl methylcellulose, PEO polyethylene oxide

The in vitro drug release profiles of ACZ core tablets prepared with different grades of PEO are illustrated in Fig. 6. Four grades, namely WSR 303, Coagulant, WSR 301, and WSR N60K, were included in the assessment. Among the tested formulations, the batch containing WSR 301 showed the release profile that best matched the targets (≤ 15% at 0.5 h and ≥ 80% at 10 h), leading to the selection of WSR 301 as the optimal ER material.

Fig. 6.

In vitro release profiles of different PEO (F7–10). F formulation, PEO polyethylene oxide

Effects of ER Material Amount

The in vitro drug release profiles of ACZ ER core tablets containing different amounts of WSR 301 are presented in Fig. 7. A pronounced reduction in the ACZ release rate was observed as the WSR 301 content increased from 50 to 70 mg. Compared with the 50 mg formulation, the similarity factor (f₂) values for the 60-mg and 70-mg formulations were 44.97 and 32.83, respectively, both below the acceptance criterion of 50, indicating notable differences in dissolution behavior. These results show that the amount of WSR 301 had a notable influence on the drug release profile. Among the three formulations evaluated, the tablet containing 60 mg of WSR 301 most closely matched the predefined target release profile. Accordingly, this concentration was selected for subsequent formulation optimization studies.

Fig. 7.

In vitro release profiles of different amounts of extended-release materials (F11–13). F formulation

Effects of Filler Amount

The in vitro drug release profiles of ACZ ER core tablets containing different amounts of MCC are shown in Fig. 8. A notable trend was observed in which the drug release rate increased with increasing MCC content. Using the formulation containing 100 mg MCC as the reference, the f₂ values for the 120-mg and 140-mg MCC formulations were 55.48 and 37.18, respectively. These results indicate that a moderate increase in MCC content (to 120 mg) did not result in a notable change in the dissolution profile (f₂ ≥ 50), whereas further increasing the MCC content to 140 mg produced a distinct alteration in drug release behavior (f₂ < 50).

Fig. 8.

In vitro release profiles for different amounts of filler (F14–16). F formulation

Among the tested levels, the formulation containing 120 mg MCC most closely matched the target release profile and was therefore selected as the optimal filler level for subsequent studies.

Effects of Tablet Hardness

The in vitro release profiles of ACZ ER core tablets with different hardness levels are presented in Fig. 9. A slight delay in drug release was observed with increasing tablet hardness; however, the overall variation among formulations was minimal. Using the formulation with a hardness of 55 N as the reference, the f₂ values for tablets with hardness levels of 65 N and 75 N were 93.17 and 77.31, respectively. Both values exceed the acceptance threshold of 50, indicating that increasing tablet hardness within this range did not materially change in the drug release profile. Accordingly, tablet hardness was maintained within the range of 55–75 N in subsequent experiments.

Fig. 9.

In vitro release profiles at different hardness levels (F17–19). F formulation

Formulation Optimization

Results from the single-factor experiments showed that both the PEO content and the MCC content had notable effects on the in vitro drug release behavior of the ER tablet core. Consequently, these two variables were selected as the critical formulation factors for further optimization using RSM. A three-level factorial design was employed, with the independent variables PEO content (X₁) and MCC content (X₂) evaluated at low (−1), medium (0), and high (+1) levels. This design generated nine experimental formulations, including a center-point formulation (F14) that was replicated three times to estimate experimental variability. The design layout, factor levels, and corresponding response values are summarized in Table 5.

Table 5.

Three-level factorial response surface design layout and observed responses for the core tablets

Run	Actual values		Responses
	X1	X2	Y1	Y2
4	50	100	18.1	85.2
9	50	120	21.3	97.7
7	50	140	25.0	101.4
5	60	100	10.2	73.2
6	60	120	14.9	86.3
10	60	120	14.2	85.1
11	60	120	14.7	86.1
1	60	140	19.5	97.5
2	70	100	7.7	60.9
3	70	120	9.7	72.8
8	70	140	13.2	84.3

X₁ polyethylene oxide content, X₂ microcrystalline cellulose content, Y₁ cumulative drug release at 0.5 h, Y₂ cumulative drug release at 10 h

The cumulative drug release at 0.5 h (Y₁) and 10 h (Y₂) was selected as the response variables. Model fitting indicated that a quadratic model best described Y₁, whereas a linear model was most appropriate for Y₂. For both responses, the model significance probabilities were below 0.05, indicating statistically significant fits. ANOVA and regression analysis further confirmed the adequacy and significance of the proposed models, as summarized in Table 6.

Table 6.

Summary of regression analysis and analysis of variance for responses

Response	Model	R2	SS	DF	MS	F value	P value	Model significance
Y1 (%)	Quadratic	0.9870	273.25	5	54.65	76.20	0.0001	Significant
Y2 (%)	Linear	0.9980	1413.15	2	706.58	128.94	< 0.0001	Significant

DF degree of freedom, MS mean square, R² coefficient of determination, SS sum of squares, Y₁ cumulative drug release at 0.5 h, Y₂ cumulative drug release at 10 h

The resulting polynomial equations describing the relationships between the independent variables and the responses were as follows:

$$Y_1=14.55-5.63\ast X_1+3.62\ast X_2-0.35\ast X_1\ast X_2+1.03\ast X_1^2+0.3816\ast X_2^2$$ 3

$$Y_2=84.59-11.05\ast X_1+10.65\ast X_2$$ 4

These equations indicate that increasing the PEO content exerts a negative effect on drug release, whereas increasing the MCC content has a positive effect, consistent with the trends observed in the single-factor studies. The combined influence of PEO and MCC on ACZ release from the ER tablet cores was further visualized using three-dimensional response surface plots (Fig. 10).

Fig. 10.

Effects of WSR 301 (PEO) and PH 102 (MCC) on acetazolamide release at 0.5 h (a) and 10 h (b). MCC microcrystalline cellulose, PEO polyethylene oxide, X₁ PEO content, X₂ MCC content, Y₁ cumulative drug release at 0.5 h, Y₂ cumulative drug release at 10 h

Upon inputting the predefined release targets (≤ 15% at 0.5 h and ≈ 80% at 10 h) into the optimization module, the software generated an optimal formulation comprising 63.009 mg PEO and 117.622 mg MCC, with predicted in vitro release profiles of 12.52% at 0.5 h and 79.99% at 10 h. Three batches of tablets were fabricated in accordance with this predicted formulation, and their in vitro release behaviors were determined: 11.69%, 13.26%, and 14.22% at 0.5 h and 81.67%, 81.22%, and 80.65% at 10 h; these experimental results were consistent with the model-predicted values (Fig. 11).

Fig. 11.

In vitro release profiles of three batches of the optimal extended-release tablet core obtained by response surface methodology (F20–22). F formulation

Furthermore, this predicted formulation was in close agreement with the experimentally optimized composition (60 mg PEO and 120 mg MCC) obtained from single-factor experiments. Consequently, the core formulation of the ER tablets employed in subsequent studies was finalized as 60 mg PEO and 120 mg MCC.

Outer Filler Dosage

For coated tablet formulations, a key consideration is whether the IR outer layer can completely and stably encapsulate the ER core, with the filler playing a critical role in achieving this. An excessive filler amount can produce oversized tablets, whereas insufficient filler may fail to fully cover the core, increasing the risk of tablet breakage. In this study, the quantities of ACZ (50 mg) and CCMC-Na (5 mg) in the IR layer were fixed, while the amount of PH 102 was varied across 395 mg, 445 mg, and 495 mg to evaluate its effect on final tablet quality. The results showed an 18% tablet breakage rate at 395 mg PH 102, whereas no breakage occurred at 445 mg or 495 mg. Therefore, to balance process stability, tablet integrity, and cost-effectiveness, 445 mg PH 102 was selected as the optimal filler dosage for the IR outer layer in subsequent optimization studies.

The Impact of Disintegrants

Disintegration is a prerequisite for effective drug release from IR formulations. An inappropriate disintegrant level may result in excessively rapid or delayed drug release, potentially altering absorption kinetics and systemic exposure, and thereby affecting therapeutic efficacy. In this study, the quantities of ACZ (50 mg) and PH 102 filler (445 mg) in the IR outer layer were fixed, while the disintegrant (CCMC-Na) concentration was varied at 1%, 5%, and 10% (w/w). The corresponding disintegration times were 8 s, 5 s, and 5 s, respectively. All formulations exhibited disintegration times well below the pharmacopeial limit of 15 min for conventional tablets, indicating rapid and effective disintegration.

Given that increasing the disintegrant content beyond 1% did not yield a meaningful reduction in disintegration time, and considering formulation cost-effectiveness and process efficiency, a disintegrant concentration of 1% was selected for the IR outer layer in subsequent formulation development.

In Vitro Release Behavior of ACZ-TIT

To achieve an “IR followed by ER” release profile, ACZ-TITs were prepared using the optimized ER core formulation. The IR outer layer contained 20% of the total ACZ dose (50 mg per tablet), while the ER core accounted for the remaining 80% (200 mg per tablet). The detailed composition of the final formulation is presented in Table 7, and the corresponding in vitro dissolution profile is shown in Fig. 12.

Table 7.

Formulation components of ACZ tablet-in-tablet

Type	Component	Amount (mg)
Core tablet	ACZ	200
	PEO WSR 301	60
	MCC PH 102	120
Outer layer	ACZ	50
	CCMC-Na	5
	MCC PH 102	445

ACZ acetazolamide, CCMC-Na croscarmellose sodium, MCC microcrystalline cellulose, PEO polyethylene oxide

Fig. 12.

In vitro release profiles of acetazolamide tablet-in-tablet (ACZ-TIT) and acetazolamide extended-release capsule

The in vitro dissolution profiles of ACZ-TITs randomly selected from the same batch were highly consistent. The mean cumulative release was 25.3% (approximately 63.2 mg) at 0.5 h and 86.5% at 10 h, with a maximum intrabatch relative standard deviation (RSD) of 6.1%, indicating good batch uniformity and reproducible release behavior. A comparative analysis of the in vitro release profiles of ACZ-TIT and the marketed ER capsule revealed distinct differences in release kinetics. ACZ-TIT exhibited a substantially higher release rate during the initial 1 h, followed by a more gradual later release phase than the reference ER capsule. These in vitro differences were consistent with the intended biphasic design of faster initial release followed by sustained release, rather than with strict point-by-point matching to the reference product.

Drug Release Kinetics

To elucidate the drug release mechanism of the ACZ ER formulation, the in vitro dissolution data of the ER core tablets were fitted to several commonly applied kinetic models, including zero-order, first-order, Higuchi, and Ritger-Peppas models. The fitting results and corresponding statistical parameters are summarized in Table 8.

Table 8.

Kinetic model fitting results for acetazolamide tablet-in-tablet in vitro release data

Model	Equation	RSS	Reduced Chi2	R2
Zero order	Y = 0.06 * X + 0.23	0.1	0.0110	0.901
Higuchi	Y = 0.25 * X1/2 + 0.04	0.009	0.0009	0.992
First order	Y = 1 * (1 − e−0.21 * X)	0.048	0.0053	0.953
Ritger-Peppas	Y = 0.3 * X0.45	0.007	0.0007	0.993

R² coefficient of determination, RSS residual sum of squares, Y cumulative drug release percentage, X time

Among the evaluated models, the Ritger-Peppas model provided the best overall fit to the release data, as evidenced by the highest coefficient of determination (R² = 0.993) and the lowest RSS and reduced chi-square values. These results indicate that the Ritger-Peppas model most accurately describes the release behavior of ACZ-TIT during the 0.5–16 h time interval.

Effect of Different Dissolution Media on In Vitro Drug Release

To explore whether dissolution behavior was sensitive to medium pH, multi-medium dissolution curves were determined for both ACZ-TIT (Fig. 13a) and the marketed ER capsule (Fig. 13b) in pH 1.2, pH 4.5, pH 6.8, and water.

Fig. 13.

In vitro release profiles of acetazolamide tablet-in-tablet (a) and the marketed Diamox^® ER capsule (b) in different dissolution media (pH 1.2, pH 4.5, pH 6.8, and water). ER extended-release

For ACZ-TIT, the release profiles across the tested media remained broadly similar, although pH 6.8 tended to give slightly higher release and water slightly lower release over much of the test interval. Using the dissolution curve in pH 4.5 as the reference, the similarity factor (f₂) values for ACZ-TIT in pH 1.2, pH 6.8, and water were 87.82, 72.60, and 74.21, respectively, all greater than 50, suggesting similar dissolution behavior across the tested media. Using the pH 4.5 profile of the marketed ER capsule as the reference, the corresponding f₂ values in pH 1.2, pH 6.8, and water were 62.09, 69.03, and 88.01, respectively, indicating similar dissolution behavior across the tested media. For the marketed ER capsule, medium-dependent differences were more evident during the early release phase, with pH 6.8 tending to show the fastest release and pH 1.2 the slowest release. However, these differences progressively decreased at later time points, and the dissolution profiles converged toward near-complete release by the end of the test period. Overall, both formulations showed only modest medium-dependent variation under the tested conditions.

Quality Control and Testing

Characterization of ER Tablet Cores

A batch of ER tablet cores was manufactured using a formulation with an ACZ dose of 200 mg, a WSR 301 dose of 60 mg, and a PH 102 dose of 120 mg. The quality attributes of the resulting tablets, including weight, thickness, hardness, friability, content, and related substances, were comprehensively evaluated, as summarized in Table 9.

Table 9.

Characterization of extended-release tablet cores (mean ± SD)

Parameter	Result
Weight (mg)	379.0 ± 11.4
Thickness (mm)	4.45 ± 0.02
Hardness (N)	59.00 ± 3.29
Friability (%)	0.04 ± 0.01
Content (%)	101.1 ± 0.9
Maximum single impurity (%)	0.039
Total impurities (%)	0.19

All measured quality attributes of this batch complied with the standards of the Chinese pharmacopoeia. Notably, content exhibited an exceptionally low RSD of 0.9%, and tablet weight variability was 3%, ensuring accurate and consistent dosing across units. Mechanical integrity was also robust, with friability well below the pharmacopeial limit of 1.0% and hardness values within an acceptable range with minimal variation, indicating strong resistance to abrasion and fracture during handling, packaging, and transportation. Minor deviations in parameters such as thickness further reflect precise manufacturing process control. The related substances were found to be within the specified limits, and the sample complies with the requirements. Collectively, these results confirm that the tablets exhibit high-quality, consistent performance with no apparent risks to product integrity or therapeutic efficacy.

Characterization of ACZ-TITs

The appearance of ACZ-TITs is shown in Fig. 14, and their physical properties are summarized in Table 10. All measured parameters complied with the requirements of the Chinese pharmacopoeia. The tablets exhibited a smooth surface without visible cracks, and the ER cores were centrally positioned within the IR outer layer, which is a critical structural requirement for ACZ-TITs.

Fig. 14.

Appearance, cross-section, and longitudinal section of acetazolamide tablet-in-tablet

Table 10.

Characterization of ACZ-TITs (mean ± SD)

Parameter	Result
Weight (mg)	879.1 ± 23.3
Thickness (mm)	6.16 ± 0.06
Hardness (N)	112.0 ± 21.2
Friability (%)	0.18 ± 0.01
Content (%)	98.3 ± 1.2
Maximum single impurity (%)	0.047
Total impurities (%)	0.25

ACZ-TIT acetazolamide tablet-in-tablet

The weight variation was approximately 2.65%, and was well below the pharmacopoeial limit of < 5%. The tablet hardness (112.0 ± 21.2 N) was sufficient to ensure mechanical integrity during handling, storage, and transportation, while still allowing acceptable patient swallowability. Friability was low (0.18 ± 0.01%), remaining well within the acceptable limit of < 0.5%. In addition, the content result of 98.3 ± 1.2% corresponded to an RSD of approximately 1.2%. The related substances were found to be within the specified limits, and the sample complies with the requirements.

Collectively, these results suggest that the optimized formulation and manufacturing process produced ACZ-TITs with consistent quality, robust mechanical properties, acceptable dosage uniformity, and the related substances were found to be within the specified limits.

Discussion

In the melting point analysis, structural and thermal characterization indicated that thermally labile functional groups in ACZ, including the sulfonamide, thiazole, and acetamide moieties, possess relatively low binding energies. This results in an overlapping melting and decomposition range, consistent with the observed simultaneous melting and thermal degradation. The brown discoloration of the melt is likely due to the formation of small-molecule fragments containing unsaturated bonds, which generate conjugated systems capable of absorbing visible light. These phenomena reflect the inherent thermal behavior of ACZ and align with the “decomposition accompanied by melting” pattern commonly observed in heterocyclic compounds bearing polar functional groups [35].

DSC analysis revealed minor changes in the ACZ melting peak (250–260 ℃), attributed to the low proportion of ACZ in the mixtures and weak physical interactions rather than chemical incompatibility. The slight decrease in melting enthalpy was attributed to the diluent effect of the excipients, without altering the intrinsic thermal properties of ACZ. In several ACZ–excipient mixtures, a broad low-temperature endotherm around 70–80 °C was observed. This event is likely attributable to polymer softening or the loss of residual moisture from the excipients, rather than indicating a new drug–excipient incompatibility [37]. PEO stability also warrants attention because oxidative chain scission can reduce molecular weight and alter gel strength during storage [38, 39]. No antioxidant was included in the present prototype because the current work focused on proof-of-concept development plus short-term compatibility screening. To mitigate these changes in future development, appropriate antioxidants and high-barrier packaging materials will be employed to preserve PEO integrity and maintain consistent release performance.

The PXRD patterns of the physical mixture of the drug and excipients exhibited numerous additional diffraction peaks after exposure to accelerated storage conditions. These phenomena were attributed to recrystallization, partial melting, or crystal perfection of PEO (WSR 301) induced by the accelerated storage conditions (40°C, 75% relative humidity). PEO is a semicrystalline polymer sensitive to heat and humidity; during the aging process, rearrangement of its crystalline regions occurs, resulting in enhanced diffraction intensity, overlapping new peaks, or peak shifts in the PXRD profiles [40]. However, the characteristic diffraction peaks of ACZ remained identifiable and unchanged throughout the storage period, confirming that no crystal form transformation of the drug took place, and there was no incompatibility or chemical reaction between ACZ and the excipients. Therefore, the observed differences reflect physical changes in the PEO polymer matrix rather than instability of the active pharmaceutical ingredient.

Solubility studies indicated only modest pH-related variation in ACZ solubility within the tested range. As a sulfonamide derivative, ACZ possesses two weakly acidic ionizable groups: a sulfonamide (pKa ≈ 7.2) and an acetamide (pKa ≈ 10.3) [41]. Because the pH values of the dissolution media (1.2–6.8) are below the primary pKa, ACZ remains largely nonionized, which plausibly explains the relatively small solubility differences observed. Published biowaiver assessments also note that the available data on ACZ absorption and permeability are insufficient to classify the drug with certainty, and no well-established site-specific absorption window has been confirmed [42]. Accordingly, the present formulation was not designed to target a specific intestinal region; instead, it was intended to provide prompt initial release followed by sustained delivery over a broader gastrointestinal transit period. The multi-medium comparison further suggested that ACZ-TIT showed only modest dissolution changes across pH 1.2, 4.5, 6.8, and water, supporting a degree of formulation robustness to luminal pH variation under the tested in vitro conditions. The marketed ER capsule also showed only limited medium-dependent variation overall, although somewhat greater differences were observed during the early phase before the profiles converged at later time points. These observations suggest that the dissolution differences between ACZ-TIT and the marketed ER capsule are more likely to reflect formulation design than marked pH sensitivity alone. However, these in vitro findings should not be interpreted as direct evidence of equivalent in vivo performance, which still requires dedicated PK confirmation.

Selection of the ER matrix polymer suggested that higher-molecular-weight polymers produced stronger release-retarding effects, consistent across PEO, HPMC, and HPC systems. Upon hydration, these polymers form a gel layer around the tablet core. Higher-molecular-weight polymers generate denser, more cohesive gels, increasing diffusion resistance and slowing drug release, whereas lower-molecular-weight polymers form looser gels, allowing faster drug diffusion [43].

Increasing the PEO (WSR 301) content was inversely correlated with drug release rate, attributable to PEO’s gel-forming properties [44]. Higher polymer concentrations create thicker, denser gel layers, enhancing diffusional resistance and slowing drug release. Additionally, higher PEO levels reduce gel erosion rates, and overall release is governed by the combined effects of diffusion and matrix erosion [45].

MCC, a non-gelling hydrophilic filler, influences drug release indirectly. Increasing MCC content decreases the relative proportion of PEO in the matrix, reducing gel layer density and compactness, which lowers diffusion resistance and accelerates ACZ release. While its impact is less pronounced than that of PEO, MCC remains an important factor in tuning release kinetics.

Tablet hardness primarily affects porosity, pore size distribution, and tortuosity, modulating medium penetration and drug diffusion pathways [46]. In hydrophilic matrix systems, rapid polymer hydration and gel formation dominate release behavior. Once a cohesive gel barrier forms, initial porosity differences become negligible, explaining the minimal impact of tablet hardness on drug release within the tested range.

From a translational perspective, the goal of ACZ-TIT was not to reproduce the Diamox^® profile point by point, but to explore a biphasic-release design with faster initial in vitro release than the marketed ER capsule while maintaining prolonged release thereafter. However, a validated minimum effective concentration, C_max target, or steady-state PK target for HAI prophylaxis has not been established. Accordingly, any PK implications of the present biphasic design, such as a potentially earlier T_max than ER capsules, moderated peak exposure relative to a full IR dose, or broader exposure over the dosing interval, should be regarded as hypotheses rather than established advantages, and they require dedicated in vivo PK/PD confirmation. In the present study, the reference ER capsule was therefore used mainly as a formulation-development benchmark rather than as an equivalence target.

The release kinetic analysis is also consistent with the structural design of ACZ-TIT. The rapid initial phase arises from prompt disintegration of the outer IR layer, whereas the later phase is governed mainly by diffusion through the hydrated PEO matrix of the ER core. The Ritger-Peppas exponent (n = 0.45) and the good Higuchi fit therefore align with a diffusion-dominated gel-matrix mechanism rather than with osmotic pumping or erosion-dominated release. This interpretation is consistent with the observed effects of PEO content, MCC content, and tablet hardness during formulation screening. When compared conceptually with osmotic-controlled release oral delivery system (OROS)-based systems, TIT does not offer the same degree of pH- and motility-independent osmotic control. However, TIT has practical advantages at this stage of development: a simpler manufacturing route, no need for semipermeable membrane coating or laser drilling, easier incorporation of an IR outer layer, and lower formulation complexity for a relatively high drug load [27, 28]. These considerations supported selection of TIT as the present development platform.

Conclusions

In this study, a novel ACZ-TIT with an “IR followed by ER” profile was developed and optimized. Pre-formulation investigations suggested adequate purity, acceptable thermal behavior, and compatibility of ACZ with the selected excipients, while solubility assessments showed only modest pH-related variation across the tested media. Systematic formulation optimization, including selection of PEO WSR 301 as the ER matrix, adjustment of filler (MCC) content, and control of IR outer layer composition, enabled the preparation of tablets with acceptable mechanical integrity, assay, and in vitro release behavior.

In vitro dissolution studies showed that ACZ-TIT provided rapid initial drug release from the IR layer followed by sustained, diffusion-controlled release from the ER core, which was consistent with the Ritger-Peppas and Higuchi kinetic models. The optimized formulation showed good batch-to-batch reproducibility and met the applied Chinese pharmacopoeial quality requirements for weight uniformity, hardness, friability, content, and related substances.

Although these results support the feasibility of ACZ-TIT as a biphasic-release formulation, further in vivo PK studies, impurity profiling, and long-term stability evaluations are required before clinical translation. Overall, ACZ-TIT may represent a promising platform for improving convenience and sustaining drug exposure in the prophylaxis and management of HAI.

Supplementary Information

Below is the link to the electronic supplementary material.

Declarations

Authors’ Contribution Statements

Conception and design of the work were performed by Hui Liu, Xiang-yang Xie, and Yuan Zeng. Material preparation, data collection, and analysis were performed by Si-kai Wang, Wei Li, Miao-miao Guo, Man Han, Ze-chun Long, and Jin-hui He. The first draft of the manuscript was written by Hui Liu and Xiang-yang Xie, and all authors edited or commented on versions of the manuscript. All authors read and approved the final version.

Funding

This research was supported by Chutian Elite Program of Hubei Province for Health Professionals (Grant No. 2024-09054).

Competing Interests

The authors have no relevant financial or nonfinancial interests to disclose.

Data Availability

All data generated or analyzed during this study are included in this published article and its supplementary version.

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