Pharmacokinetic Comparison of Novel Tablet-in-Tablet and Conventional Ketorolac Tromethamine Tablets in Beagle Dogs

Abstract

Background and Objective

Ketorolac tromethamine (KT), a nonsteroidal anti-inflammatory drug (NSAID) and cyclooxygenase inhibitor, is commonly used for the management of moderate to severe pain. The objective of this study was to compare the pharmacokinetic characteristics of KT in beagle dogs following oral administration of conventional tablets and a novel tablet-in-tablet (TIT) formulation.

Methods

A comparative dissolution study was conducted to evaluate the release profiles of both formulations. Non-compartmental analysis was used to determine the pharmacokinetic parameters of each formulation.

Results

Approximately 20% of the administered KT from the TIT formulation was released within the first 30 min, with a cumulative release exceeding 90% at 16 h. In contrast, the conventional tablets released about 50% of the drug within 30 min and completed the release at 4 h. In the single-dose study, the time to reach maximum plasma concentration (T_max) for conventional tablets was 1 h, while T_max for the TIT formulation was 5 h. Both maximum concentration (C_max) and area under the concentration–time curve (AUC) for the TIT formulation were lower than those for conventional tablets. In the repeated-dose study, when equivalent doses (35 mg) of conventional tablets were administered in divided daily doses, the TIT formulation showed no significant differences in most steady-state pharmacokinetic parameters, except for T_max,ss.

Conclusion

The results of this study suggest that the development of a novel KT tablet formulation utilizing the push-pull osmotic pump (PPOP) and tablet-in-tablet techniques warrants further investigation in clinical trials.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40268-025-00516-7.

Key Points

A comparative study of conventional and novel tablet-in-tablet (TIT) formulations of ketorolac tromethamine was conducted.

The TIT formulation showed extended-release properties in vitro and in vivo.

Introduction

Effective management of postoperative pain is crucial for promoting faster recovery [1]. Adequate pain control not only alleviates discomfort but also facilitates healing, restores mobility, and enhances overall patient outcomes. By mitigating pain, recovery is expedited, and the risk of complications following surgery is reduced. Postoperative pain management is typically individualized, with an initial focus on multimodal, non-opioid analgesics. Opioid analgesics are commonly used for acute postoperative pain [2, 3]; however, their long-term use is associated with several drawbacks, including the development of tolerance, physical and psychological dependence, withdrawal symptoms, and other adverse effects [4]. Therefore, nonsteroidal anti-inflammatory drugs (NSAIDs), which lack these opioid-related complications, are often preferred for managing postoperative pain.

Ketorolac tromethamine (KT, CAS #74103-07-4) is a NSAID commonly utilized for managing moderate to severe pain, particularly in postoperative scenarios. As a potent analgesic, KT exerts its effects through the inhibition of cyclooxygenase enzymes COX-1 and COX-2, facilitating significant pain relief equivalent to that of opioids and steroidal medications. It has been extensively employed in preoperative, intraoperative, and immediate postoperative settings, notably for pain relief associated with abdominal, gynecologic, oral, orthopedic, and urologic surgeries. Additionally, KT is indicated for alleviating acute renal colic pain resulting from trauma and visceral pain linked to cancer [5]. The advocacy for using nonopioid analgesics like KT in perioperative pain management aims to minimize or entirely replace opioid consumption in postoperative care, thereby addressing the growing concern of opioid dependence.

KT is classified as a Class I drug in the biopharmaceutical classification system (BCS) [6, 7], characterized by good aqueous solubility and in vivo permeability. Despite its favorable absorption properties, KT has a relatively short plasma elimination half-life of 5–6 h [8]. It is commercially available in various formulations, including an injectable solution (15–30 mg), oral tablets (10 mg), and an ophthalmic solution (0.4–0.5%) [5]. Due to its short half-life, KT necessitates repeated daily dosing to maintain therapeutic plasma concentrations and ensure consistent pain relief. However, the need for frequent dosing (oral: 10–20 mg per time, twice per day) and the resulting fluctuations in blood drug concentrations may limit the clinical utility of these current dosage forms. Recent research has demonstrated that continuous intravenous infusion of ketorolac serves as an effective postoperative analgesic, particularly in older patients [1].

To reduce the frequency of oral dosing, a novel extended-release (ER) formulation of KT has been developed to provide sustained release over 16 h following oral administration. This formulation is designed as a tablet-in-tablet (TIT) system (Fig. 1), consisting of an immediate-release (IR) outer layer and an ER core. The core tablet, which functions as a push–pull osmotic pump (PPOP), is capable of releasing the drug in vitro for 16 h after the initial release from the outer layer. The IR outer layer could mitigate the initial lag time associated with the ER PPOP system. However, there is a lack of in vivo data regarding the performance of this novel KT dosage form. Therefore, a preliminary pharmacokinetic study of the KT TIT is necessary to better understand its in vivo behavior and to inform potential adjustments to its formulation.

Fig. 1.

Schematic representation of the novel tablet-in-tablet formulation of ketorolac tromethamine

In this study, we conducted a feasibility assessment for developing a novel ER dosage form (TIT) of KT through comparative pharmacokinetic analyses in beagle dogs. We performed comparative dissolution tests of KT-loaded ER TITs and commercially available conventional tablets in dissolution media. The pharmacokinetic characteristics of KT were evaluated in beagle dogs following the oral administration of both a conventional KT tablet and a novel KT TIT. Non-compartmental modeling and analysis approaches were employed to determine the pharmacokinetic parameters of KT. The findings from this study are crucial for studying the bioequivalence between the conventional tablet and the novel tablet formulation. Given that beagle dogs can tolerate dosages comparable to human scales, they represent an appropriate animal model for pharmacokinetic studies aimed at developing innovative formulations of KT [9].

This study is innovative in both the development of a novel drug delivery system for KT and its comprehensive pharmacokinetic evaluation. The findings offer valuable insights into the potential clinical application of this novel formulation.

Materials and Methods

Materials

Ketorolac tromethamine tablets (10 mg) were obtained from Shandong New Era Pharmaceutical Co. (China). The ketorolac tromethamine powder was supplied by Hainan Zhuoke Pharmaceutical Co. (China). The internal standard, [2H5]-Ketorolac, was purchased from Shanghai Zhenzhun Biotechnology Co. (China) for in vivo sample analysis. Polyethylene glycol (PEG) 4000 was sourced from Nanjing Weier Chemical Industry Co. (China). Polyethylene oxide (PEO) was purchased from Dow Chemical Company. Magnesium stearate, hydroxypropyl methylcellulose (HPMC), and polyvinylpyrrolidone (PVP-K30) were procured from Anhui Shanhe Pharmaceutical Excipients Co. (China). Sodium chloride was provided by Jiangsu Qinfeng Pharmaceutical Co., Ltd. (China). Cellulose acetate was purchased from Eastman Chemical Company (Kingsport, TN, USA).

Methanol (high-performance liquid chromatography [HPLC] grade) and acetonitrile (HPLC grade) were supplied by Fisher Scientific Inc. (Waltham, MA, USA) for the determination of both in vitro and in vivo ketorolac concentrations via HPLC and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, respectively. All other reagents used were of analytical or HPLC grade.

Animals

All animal procedures were approved by the Animal Care Committee of Wuhan Hongren Bio-pharmaceutical Co. (protocol ID: IACUC202400052) and were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Wuhan Hongren Bio-pharmaceutical Co.

Eighteen beagle dogs (9 males and 9 females), with a mean weight of 9.1 ± 0.8 kg and an average age of 10.1 ± 2.3 months, were purchased from Hubei Yizicheng Biotechnology Co. (SCXK2021-0020; China). All animal experiments were approved by the Wuhan Hongren Bio-pharmaceutical Co. and conducted in accordance with the Guide for the Care and Use of Laboratory Animals from Hongren Bio-pharmaceutical Co.

The dogs were housed with ad libitum access to food and water but were fasted for 12 h prior to the experiment and fed 4 h post-dose in the single dosing study (free access to water and food in the repeated dosing study). The animals were maintained on a 12-h light/dark cycle (lights on from 8:00 to 20:00) in an environment with a temperature of 20–25 °C and relative humidity of 40–60%.

Preparation of Tablet-in-Tablet Containing Ketorolac Tromethamine

A KT-loaded TIT was prepared according to our previously reported method [10]. Its formulation is displayed in Table 1. Briefly, the drug layer was prepared by accurately weighing the designated amounts of drugs and excipients (PEO, NaCl, HPMC). These components were sieved through an 80-mesh screen and thoroughly mixed using a Turbula T2F blender (Willy A. Bachofen AG, Switzerland). A solution of (5%, W/V) polyvinylpyrrolidone (PVP-K30) was prepared by dissolving the prescribed amount in an appropriate volume of ethanol (95%, V/V). This solution was then mixed with the drug layer powder mixture to form a cohesive soft mass. The wet mass was passed through a 20-mesh sieve to form granules, which were subsequently dried in a 40 °C oven (Jinghong Instrument Ltd., China) for 4 h. After drying, the granules were passed through an 18-mesh sieve to achieve uniform particle size. The dried granules were then blended with magnesium stearate (1%), resulting in the final drug layer granules. The pull–push layer (polyethylene oxide, NaCl) was prepared using the same procedure as the drug layer. The drug-containing granules were placed in the tablet press mold (8 mm), and a pre-pressing step was applied to flatten the surface. The pull–push layer granules were then added, and compression was performed to form the core tablet. To ensure tablet integrity, the core tablet hardness was maintained within the range of 50–60 N during the compression process. A coating solution was prepared by dissolving the required amount of polyethylene glycol (PEG 4000) in water. Cellulose acetate was then slowly added to the solution (acetone water, 95/5, V/V) with continuous stirring until it was completely dissolved. This coating solution (PEG 4000 1.5 g/L and cellulose acetate 30 g/L) was applied to the compressed tablet core. Coating parameters were as follows: coating pan temperature was maintained at 40 °C, the rotation speed of the coating pan was set at 40 rpm, and the spraying rate was controlled at 2–4 mL/min. After the desired weight gain (14%) was achieved, the tablets were dried in an oven at 40 °C for 24 h. Finally, the coated tablets (PPOP) were perforated with a 0.8-mm hole on the side of the drug layer to facilitate controlled drug release.

Table 1.

Formulations of tablet-in-tablet containing ketorolac tromethamine (per tablet)

Layer type	Ingredients	Amount (mg)
Drug layer	Ketorolac tromethamine	30
	Polyethylene oxide (100 kDa)	15
	NaCl	6
	Hydroxypropyl methylcellulose	4
Pull-push layer	Polyethylene oxide (7000 kDa)	80
	NaCl	18
	Red iron oxide	0.5
Immediate-release layer (outer tablet)	Ketorolac tromethamine	5
	Microcrystalline cellulose	310
	Magnesium stearate	5

The appropriate amount of KT and microcrystalline cellulose required for the immediate-release layer was weighed. The mixture was sieved through an 80-mesh sieve to ensure uniformity. Next, the sieved powders were blended and the magnesium stearate incorporated, ensuring a homogeneous mixture. Half of the prepared powder blend was transferred into 12-mm punch molds and light pressure applied to compact the powder. The previously prepared PPOP tablet was placed in the center of the mold, then the remaining powder mixture added on top. Finally, further compression was applied to form the KT-loaded tablet with an immediate-release layer.

In Vitro Drug Release Studies

The in vitro dissolution profiles of KT from a TIT and a commercially available conventional tablet were determined using the paddle method of the USP dissolution apparatus (Type II). The dissolution medium (900 mL) was maintained at 37 °C ± 0.5 °C with the aid of a heater circulator, and the paddle rotation speed was set to 50 rpm. The dissolution medium consisted of 900 mL of simulated gastric fluid (SGF; pH 1.2) for the first 2 h, followed by simulated intestinal fluid (SIF; pH 6.8) for the subsequent period [11]. At predetermined intervals (0, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, and 18 h), 5-mL samples were withdrawn from the dissolution vessels. After each sampling, an equal volume of fresh dissolution medium was added to replace the withdrawn volume.

For high-performance liquid chromatography (HPLC) analysis of KT, the samples were filtered using 0.22-μm cellulose syringe filters. The KT concentration was determined using a Shimadzu LC-20AT HPLC system (Shimadzu Corporation, Japan) with a mobile phase composed of methanol, water, and glacial acetic acid in a 65:34:1 (V/V/V) ratio. A 20-μL aliquot of each sample was injected via an auto-injection system. Chromatographic separation was performed on a Diamonsil C₁₈ (5 μm, 4.6 × 250 mm) column (Dikma Technologies Inc., China) with a column temperature of 40 ℃. The absorbance detection wavelength for KT was set at 322 nm, and the mobile phase flow rate was maintained at 1.0 mL/min.

The HPLC method was validated for specificity, accuracy, and intra-day and inter-day precision, in accordance with the standards outlined in the 2020 edition of the Chinese Pharmacopeia. All evaluations were performed in triplicate, and the standard deviations were calculated.

Animal Study Design

Single-dose crossover study: A randomized crossover experimental design was employed to investigate the pharmacokinetic differences between conventional tablets and TITs in beagle dogs. A total of six dogs (3 males and 3 females) were divided into two groups. Dogs in the first group received the conventional tablet (35 mg) orally, while those in the second group were administered the TIT (35 mg) orally. Following a 1-week washout period, the dogs in each group were switched to receive the other drug.

After drug administration, 0.6 mL of anticoagulated (anticoagulant of EDTA-K₂) blood samples were collected from the forelimb veins at the following time points: 0 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 10 h, 14 h, 24 h, 36 h, and 48 h for the TIT dogs, and at 0 min, 5 min, 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 14 h, 24 h, 36 h, and 48 h for the conventional tablet dogs.

Steady-state parallel study: A total of 12 dogs (6 males and 6 females) were divided into two groups. For the conventional tablets (reference group; 35 mg/day), an initial dose of 1.5 tablets (15 mg) was given followed by two additional doses of 10 mg each at 5-h intervals, repeated on a daily basis for 7 consecutive days. In contrast, for the TITs (test group; 35 mg/day), one tablet was administered once daily at a 24-h interval for 7 consecutive days.

After drug administration, 0.6 mL of anticoagulated (anticoagulant of EDTA-K₂) blood samples were collected from the forelimb veins at the following time points:

• Test group (TITs) blood collection time points: Blood samples were collected at the following time points: before and after Day 1 (D1) administration, at 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 10 h, 14 h, and 24 h post-D1 administration; before Day 5 (D5) and Day 6 (D6) administration; before and after Day 7 (D7) administration; at 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 10 h, 14 h, 24 h, 36 h, and 48 h post-D7 administration. In total, 30 blood collection time points were recorded.

• Reference group (conventional tablets) blood collection time points: Blood samples were collected at the following time points: before Day 1 (D1) administration; at 5 min, 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, and 4 h 55 min post-D1 administration; before the second and third administrations on Day 6; before and after the first administration on Day 7; at 5 min, 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, and 4 h 55 min post-first D7 administration; before and after the second D7 administration at 5 h and 5 h 5 min, respectively; at 5 h 10 min, 5 h 20 min, 5 h 30 min, 6 h, 7 h, 9 h, 9 h 55 min (before the third D7 administration), and 10 h 5 min (after the third D7 administration); at 10 h 10 min, 10 h 20 min, 10 h 30 min, 11 h, 12 h, 14 h, 16 h, 18 h, 20 h, 24 h, 34 h, 46 h, 58 h, and 82 h post-D7 administration. In total, 43 blood collection time points were recorded.

Blood samples were immediately centrifuged at 3000×g for 10 min at 4 °C to separate the plasma, which was then stored at − 20 °C until analysis.

Samples and Standards

KT and the internal standard (IS) were extracted from the plasma matrix using an acetonitrile precipitation method. In each well of a 96-well deep-well plate, 25 μL of plasma sample and 25 μL of IS solution ([2H5]-Ketorolac, 300 ng/mL) were mixed with 400 μL of acetonitrile. The mixture was vortexed for 5 min and then centrifuged at 4 °C for 15 min at 1700×g. After centrifugation, 200 μL of the supernatant was transferred to a new 96-well plate and combined with 100 μL of a methanol–water solution (50:50, v/v). The resulting mixture was vortexed for 5 min. The plate was then placed in an ExionLC AD Multiplate Autosampler (AB Sciex Corporation, MA, USA) at 4 °C. Finally, 5 μL of the sample solution was injected into the HPLC system for analysis.

A 5-mg/mL stock solution of KT was prepared by dissolving 25 mg of the compound in 5 mL of methanol. Working solutions with concentrations of 0.25, 0.50, 2.5, 10, 50, 125, 225, and 250 µg/mL were then prepared through serial dilution of the stock solution with a methanol–water mixture (5:5, v/v). These working solutions were subsequently diluted 1:24 with untreated canine plasma to generate eight standard solutions (0.25 mL each) containing KT at concentrations of 10, 20, 100, 400, 2000, 5000, 9000, and 10,000 ng/mL. Additionally, quality control (QC) samples of KT were prepared at concentrations of 30.0, 3000, and 7500 ng/mL following the same procedure.

Determination of Drug Concentration in Plasma

The HPLC system was an ExionLC AC system (AB Sciex Corporation, MA, USA) and the MS/MS detector was a TRIPLE QUAD 4500 MS/MS system (AB Sciex Corporation, MA, USA). Chromatographic separation was conducted using an Ultimate XB-C₁₈ column (50 × 2.1 mm, 5.0 μm; Welch Technology Corporation, China) and a mobile phase consisting of a mixture of acetonitrile: distilled water with 0.1% formic acid (v/v) at a speed of 0.8 mL/min [12]. The HPLC elution gradient was structured as follows: the initial concentration started at 30% acetonitrile, increased to 90% between 2.0 and 2.5 min, and returned to 30% acetonitrile at 2.51 min. Each sample analysis had a total cycle time of 4 min.

The column oven was maintained at a temperature of 30 °C. The mass spectrometer utilized an electrospray ionization source operating in positive ion mode, specifically detecting the [M+H]⁺ ions. Multiple reaction monitoring was employed to identify ions at the following transitions: m/z 256.3 → 105.0 for KT and m/z 261.3 → 110.1 for the IS. The parameters for collision energy, declustering potential, collision exit potential, ion spray voltage, and entrance potential were set to 50 eV, 25 eV (for KT and IS), 70 V, 10 V, 5500 V, and 10 V, respectively. The dwell time was 200 ms, and the data were analyzed using Analyst 1.6.3 software (AB Sciex Corporation, MA, USA). The sample retention time was approximately 1 min.

Pharmacokinetic Analysis

Chromatographic peak areas for the quantitative analytes and internal standard were obtained using Analyst 1.6.3 software (AB Sciex Corporation, MA, USA). The concentration of the analyte in the corrected sample was determined by performing a linear regression of the analyte concentration in plasma (x) versus the peak area ratio (y) using the weighted least squares method (W = 1/x²). The resulting regression equation (y = a*x + b) was used to construct the standard curve. Analyte concentrations in the plasma of test animals were then calculated based on the standard curve generated from the analysis batch.

Pharmacokinetic parameters were calculated using the noncompartmental analysis of statistical moments with WinNonlin software (version 8.3, Pharsight Corp., Mountain View, CA, USA). The key parameters included half-life (t_½), terminal elimination rate constant (λ_z), clearance (CL), apparent volume of distribution (V_d), mean residence time (MRT), area under the concentration–time curve from 0 to the last measurable time point (AUC_0–t), area under the concentration–time curve from 0 to infinity (AUC_0–∞), degree of fluctuation (DF), accumulation index (R_ac), peak concentration (C_max), and time to peak concentration (T_max).

Statistical Analysis

Similarity factor (f₂) was utilized to assess the similarity of the release profiles [13]. The f₂ value is computed using the following formula:

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

where (n) is the number of time points, (R_t) is the cumulative drug release from the reference formulation at time (t), and (T_t) is the cumulative drug release from the test formulation at the same time point. A similarity factor (f₂) value of 50 or higher indicates that the release profiles of the reference and test formulations are considered similar.

Data are presented as means ± standard deviations (SD). C_max and T_max were extracted directly from the pharmacokinetic data. The pharmacokinetic parameters (continuous variables) for both the reference formulation (conventional tablet) and the test formulation (TIT) were logarithmically transformed (Ln) and compared using Student’s t-test. Specifically, an independent samples t-test was applied for the single-dose study, and a paired samples t-test was used for the repeated-dose study. For T_max, nonparametric tests were employed: the Wilcoxon signed-rank test for the single-dose study and the Mann–Whitney U test for the repeated-dose study [14]. A p value of < 0.05 was considered statistically significant. All statistical analyses were conducted using SPSS software (version 23.0; SPSS Inc., Chicago, IL, USA).

Results

In Vitro Drug Release Studies

The comparative in vitro drug release profiles of KT from TITs and conventional tablets in dissolution media at different time intervals are illustrated in Fig. 2. The dissolution media included pH 1.2 simulated gastric fluid (SGF) for the initial 2 h, followed by pH 6.8 simulated intestinal fluid (SIF) for the remaining duration. For the TITs, approximately 20% of the loaded KT was released within the first 30 min, followed by a sustained release of the remaining drug over 16 h, ultimately achieving a cumulative release exceeding 90% at 16 h. In contrast, conventional tablets dissolved about 50% of the drug within 30 min in the dissolution medium and completed the drug release at 4 h. The similarity factor (f₂) between the release profiles of the TITs and conventional tablets was 16.4, which is below the threshold of 50. This indicates that the release profiles of the reference and test formulations were not similar.

Fig. 2.

Comparative dissolution profiles of ketorolac tromethamine (mean ± SD) from conventional tablets and the tablet-in-tablet formulation (n = 6)

Pharmacokinetic Analysis

The bioanalytical method was validated in accordance with the United States Food and Drug Administration (FDA) Bioanalytical Method Validation Guidance [15]. Linearity equation (y = 0.00125*x + 0.000179) was established across the range of 10–10,000 ng/mL, with a correlation coefficient (R²) greater than 0.996. Each analytical run included six QC samples at three concentration levels (30.0, 3000, and 7500 ng/mL), representing low, medium, and high concentrations within the assay range, to evaluate assay performance. Inter-assay precision and accuracy were assessed using QC samples from all study sample runs. The intra- and inter-batch precision (n = 6) was found to be between 4.1 and 6.0%, while the intra- and inter-batch accuracy (n = 6) ranged from 98.6 to 109.7%. Stability assessments indicated that short-term stability (room temperature for 22 h), post-extraction stability (at 4 °C for 96 h), freeze-thaw stability (at − 20 °C and − 80 °C for four cycles), and long-term stability (at − 80 °C for 1 month) were all satisfactory.

The mean plasma concentration–time profiles of KT following both single and multiple doses of reference tablets and test formulations (tablet-in-tablet, TIT) are shown in Figures 3, 4 and 5.

Fig. 3.

Mean plasma (mean ± SD) concentration–time profiles of ketorolac tromethamine following a single oral dose of either conventional tablets (35 mg) or the tablet-in-tablet formulation (35 mg) (n = 6)

Fig. 4.

Day 1 mean plasma (mean ± SD) concentration–time profiles of ketorolac tromethamine in the repeated-dose study, following a single oral dose of either conventional tablets (15 mg) or the tablet-in-tablet formulation (35 mg) (n = 6)

Fig. 5.

Mean plasma (mean ± SD) concentration–time profiles of ketorolac tromethamine (35 mg) after 7 days of repeated dosing (n = 6)

The main pharmacokinetic parameters following a single oral administration of 35-mg KT in either TITs (T) or conventional tablets (R) to beagle dogs are summarized in Table 2. The time to reach peak concentration (T_max) for the reference formulation (R) was 1.00 h, while T_max for the test formulation (T) was delayed to 5.00 h. Additionally, the maximum concentration (C_max) for the test formulation was significantly lower than that of the reference formulation, with values of 2940 ± 554 ng/mL and 8317 ± 1454 ng/mL, respectively. Following the single oral administration of 35 mg KT, drug exposure in beagle dogs was reduced for the test formulation (AUC_0–∞ of 39.2 ± 9.7 h*μg/mL) compared with the reference formulation (AUC_0–∞ of 59.60 ± 29.3 h*μg/mL). The relative bioavailability (F) of the test formulation was 59.2%.

Table 2.

Pharmacokinetic parameters of ketorolac tromethamine following a single dose (35 mg)

Parameter (units)	Test (n = 6)	Reference (n = 6)	p value
λz (1/h)	0.0786 ± 0.0226	0.0922 ± 0.0386	0.489
t½ (h)	9.44 ± 2.63	9.30 ± 5.54	0.488
Tmaxa (h)	5.00 (4.00, 6.00)	1.00 (0.500, 2.00)	0.027
Cmax (ng/mL)	2940 ± 554	8317 ± 1454	< 0.001
AUC0–t (h*μg/mL)	33.0 ± 8.4	55.70 ± 21.6	0.006
AUC0–∞ (h*μg/mL)	34.6 ± 9.6	59.60 ± 29.3	0.009
Vd/F (L)	14.40 ± 4.48	7.77 ± 2.10	0.002
CL/F (L/h)	1.09 ± 0.34	0.67 ± 0.20	0.009
MRT0–t (h)	11.60 ± 2.67	8.01 ± 2.92	0.040
MRT0–∞ (h)	13.80 ± 4.20	10.40 ± 6.95	0.097

Data are expressed as mean ± SD

λ_z terminal elimination rate constant, AUC_0–t area under the plasma concentration versus time curve (AUC) from time zero to t, AUC_0–∞ AUC extrapolated to infinity, CL/F apparent total body clearance, C_max peak plasma concentration, MRT_0–t mean residence time from time zero to t, MRT_0–∞ mean residence time from time zero to infinity, T_max the time to reach C_max, t_½ elimination half-life, V_d/F apparent volume of distribution

^aT_max is presented as the median with the corresponding minimum and maximum values (min, max)

After multiple oral administrations of TITs (35 mg/day, once daily at 24-h intervals for 7 consecutive days) or conventional tablets (35 mg/day, with an initial dose of 15 mg followed by two additional doses of 10 mg each at 5-h intervals, repeated on a daily basis for 7 consecutive days), the pharmacokinetic parameters on Day 1 (D1) and Day 7 (D7) are summarized in Tables 3 and 4. On D1, the C_max values of TITs and conventional tablets were 3842 ± 943 ng/mL and 2968 ± 1434 ng/mL, respectively, with no statistically significant difference. The results suggest that the prepared TITs (35 mg) exhibit a similar C_max to that of conventional tablets (15 mg). Compared with the first administration on D1, the accumulation indices (R_ac) of KT in beagles following multiple administrations of either the TITs or the conventional tablets were 1.21 ± 0.840 and 1.33 ± 0.224, respectively. These values indicate that there was no significant drug accumulation in the beagles. For the reference formulation, the steady-state peak time (T_max,ss) after repeated dosing was 0.50 h, while the test formulation exhibited a delayed T_max,ss of 3.00 h. The C_max,ss values for the test and reference formulations were 3030 ± 1030 ng/mL and 3668 ± 1433 ng/mL, respectively, with no statistically significant difference observed. After multiple oral administrations of the test or reference formulations (35 mg/day) in beagle dogs, the in vivo drug exposure for the test formulation was slightly lower than that for the reference formulation, although this difference was not statistically significant.

Table 3.

Pharmacokinetic parameters (Day 1) of ketorolac tromethamine following repeated dosing for 7 days

Parameter (units)a	Test (n = 6)	Reference (n = 6)	p value
λz (1/h)	0.125 ± 0.0273	0.248 ± 0.0712	0.001
t½ (h)	5.79 ± 1.32	2.98 ± 0.84	0.001
Tmaxb (h)	4.00 (2.00, 4.00)	0.42 (0.33, 2.00)	0.003
Cmax (ng/mL)	3842 ± 943	2968 ± 1434	0.156
AUC0-t (h*μg/mL)	36.4 ± 8.5	8.83 ± 3.5	< 0.001
AUC0–∞(h*μg/mL)	39.2 ± 9.7	13.3 ± 7.7	< 0.001
Vd/F (L)	7.81 ± 2.63	5.46 ± 1.55	0.069
CL/F (L/h)	0.94 ± 0.25	1.40 ± 0.64	0.149
MRT0–t (h)	7.90 ± 0.76	2.12 ± 0.217	< 0.001
MRT0–∞ (h)	9.65 ± 1.41	4.35 ± 1.16	< 0.001

Data are expressed as mean ± SD

λ_z terminal elimination rate constant, AUC_0–t area under the plasma concentration versus time curve (AUC) from time zero to t, AUC_0–∞ AUC extrapolated to infinity, CL/F apparent total body clearance, C_max peak plasma concentration, MRT_0–t mean residence time from time zero to t, MRT_0–∞ mean residence time from time zero to infinity, T_max the time to reach C_max, t_½ elimination half-life, V_d/F apparent volume of distribution

^aThe parameters for the test group were calculated over the 0–24 h (35 mg) period on day 1, while those for the reference group were calculated over the 0–5 h (15 mg) period on day 1

^bT_max is presented as the median with the corresponding minimum and maximum values (min, max)

Table 4.

Pharmacokinetic parameters (Day 7) of ketorolac tromethamine following repeated dosing (35 mg) for 7 days

Parameter (units)	Test (n = 6)	Reference (n = 6)	p value
λz (1/h)	0.0791 ± 0.046	0.077 ± 0.0388	0.792
t½ (h)	13.7 ± 11.0	10.3 ± 3.3	0.794
Tmax,ssa (h)	3.00 (2.00, 5.00)	0.500 (0.167, 1.00)	0.003
Cmax ,ss (ng/mL)	3030 ± 1030	3668 ± 1433	0.381
Cmin,ss (ng/mL)	477 ± 669	400 ± 522	0.932
Cavg,ss (ng/mL)	1633 ± 932	2023 ± 1301	0.519
AUC0–t (h*μg/mL)	50.0 ± 36.1	60.0 ± 53.1	0.687
AUC0–τ (h*μg/mL)	39.2 ± 22.4	48.5 ± 31.2	0.518
AUC0–∞(h*μg/mL)	60.1 ± 51.3	60.6 ± 53.7	0.881
Vd/F (L)	17.50 ± 9.27	13.90 ± 8.32	0.386
CLss/F (L/h)	1.17 ± 0.66	0.91 ± 0.40	0.521
MRT0–∞ (h)	17.40 ± 10.50	13.80 ± 5.69	0.629
DF (%)	185.0 ± 64.3	184.0 ± 45.0	0.919
Rac	1.21 ± 0.84	1.33 ± 0.22	0.360

Data are expressed as mean ± SD

λ_z terminal elimination rate constant, AUC_0–t area under the plasma concentration versus time curve (AUC) from time zero to t, AUC_0–τ AUC from time zero to τ (dosing interval), AUC_0–∞ AUC extrapolated to infinity, C_avg,ss average plasma concentration at steady state, CLss/F apparent total body clearance at steady state, C_max peak plasma concentration, C_max,ss peak plasma concentration at steady state, C_min,ss minimum plasma concentration at steady state, DF degree of fluctuation, MRT_0–∞ mean residence time from time zero to infinity, R_ac accumulation ratio, T_max,ss the time to reach C_max at steady state, t_½ elimination half-life, V_d/F apparent volume of distribution

^aT_max is presented as the median with the corresponding minimum and maximum values (min, max)

Discussion

In Vitro Drug Release Studies

The comparative in vitro drug release profiles of KT from TITs and conventional tablets were evaluated in dissolution media with distinct pH conditions, simulating the gastric and intestinal environments [11]. The results revealed significant differences (f₂ = 16.4) in the release behavior between the TITs and conventional tablets. As PPOP dosage forms are typically unaffected by release media [16], the KT TITs exhibited similar release profiles in two release media [10] and under the current varying pH conditions.

For the TITs, a controlled and sustained release profile was observed. Approximately 20% of the loaded KT was released within the first 30 min (IR phase of drug release), followed by a slow and steady release of the remaining drug over a 16-h period (ER phase of drug release). By the end of the 16-h dissolution study, the TITs achieved a cumulative release of over 90%, demonstrating an ER profile that could potentially provide therapeutic advantages in terms of maintaining drug levels over a prolonged period. In contrast, the conventional tablets exhibited a much faster release rate, with approximately 50% of the drug dissolved within the first 30 min. This rapid release pattern is characteristic of IR formulations, which, while effective in achieving quick drug absorption, may not offer the same sustained therapeutic effects as the TITs. Increasing the stirring speed may enhance the drug release rate in the pH 1.2 medium of SGF for conventional tablets [6].

These findings highlight the potential of TITs as a promising drug delivery system capable of providing prolonged release, which may improve patient compliance and therapeutic outcomes.

Pharmacokinetic Analysis

In the case of conventional KT tablets, the standard human dosage ranges from 20 to 40 mg per day. Given that ER formulations have the potential to decrease the total daily dose required, we developed the KT TIT formulation with a dose of 35 mg per tablet to control the acute pain. This design aimed to optimize the therapeutic benefits while minimizing the frequency and dose of administration.

The pharmacokinetic evaluation of the test formulation (TITs) and conventional tablets following single or multiple oral administrations provides valuable insights into their respective profiles in beagle dogs. The data presented in Tables 2, 3 and 4 reveal crucial information regarding the absorption and bioavailability of the test formulation.

After a single-dose administration of ER KT-loaded TITs (35 mg), a significantly longer T_max and lower C_max were observed compared with those of conventional tablets (35 mg), as anticipated. This can be attributed to the lower IR dose in the TIT formulation compared with the conventional tablet, as well as the sustained-release properties of the TITs. The relative bioavailability of the ER formulation was 59.2%, which is consistent with similar low bioavailability findings reported in previous studies [16, 17]. This phenomenon may be attributed to variations in drug absorption times in canines, which can be influenced by food intake. A study demonstrated that the gastrointestinal (GI) transit times of sensor-capsules administered to beagle dogs were 13.8 h during the pre-feeding period and 27.8 h post-feeding [18]. In the present single-dose study, the dogs were fasted prior to drug administration, resulting in faster GI transit and insufficient time for the TITs to release their loaded drug in the GI tract (stomach and small intestine), leading to lower bioavailability. This low bioavailability result also indicates that the drug KT may be poorly absorbed in the colon. In contrast, during the repeated dosing study, where the dogs were not fasted before or after drug administration, the AUC_0–∞ values (D7) for both formulations were similar, with no statistically significant difference. This suggests that food intake may influence the pharmacokinetics of the drug when administered in the ER formulation.

Following repeated dosing, on D1 the observed C_max values for the TITs and conventional tablets were 3842 ± 943 ng/mL and 2968 ± 1434 ng/mL, respectively. Notably, although the C_max for the TITs was higher, there was no statistically significant difference between the two formulations. The T_max values on D1 for both formulations were not significantly different from those observed in the single-dose study. These suggested that the initial loading dose of TITs is comparable to the 15-mg dose of the conventional tablets. The statistically significant difference in AUC on D1 between the two formulations can be attributed to the differences in dosing regimens and the time intervals (0–24 h and 35 mg for the test group; 0–5 h and 15 mg for the reference group).

The accumulation indices calculated for each formulation (1.21 ± 0.840 for TITs and 1.33 ± 0.224 for conventional tablets) indicate minimal drug accumulation over the course of the 7-day administration period, suggesting that neither formulation leads to significant drug buildup in the system. This observation is particularly important for chronic use scenarios, as it implies a lower risk of potential toxicity associated with drug accumulation [19].

Moreover, the steady-state peak time (T_max,ss) manifests as a crucial differentiator between the formulations, with the reference formulation achieving T_max,ss at 0.50 h and the test formulation exhibiting a delayed T_max,ss of 3.00 h. This delay in T_max,ss for the TITs may be indicative of a modified-release profile that could result in prolonged therapeutic effects, making it a favorable option in clinical settings where sustained efficacy is desired. The ER formulation exhibited sustained plasma drug concentrations for at least 24 h. Specifically, plasma concentrations of KT from the ER formulation remained consistently above 477 ± 669 ng/mL (C_min,ss) for the entire 24-h period, which was comparable to the conventional tablets administered three times per day (400 ± 522 ng/mL, C_min,ss). Assuming that a 35-mg dosing regimen of conventional KT tablets, administered three times per day, is sufficient to achieve therapeutic effects in beagle dogs, it means that the minimum therapeutic concentration in beagle dogs is 400 ng/mL. This suggests that the TIT formulation may provide sustained analgesic effects throughout this duration. According to the KT label, the C_min,ss for humans receiving a daily dose of 40 mg (10 mg per dose, four times daily) is 290 ng/mL [20], which is similar to the dog C_min,ss observed in this study.

Despite the observed differences in T_max,ss, the C_max,ss of 3030 ± 1030 ng/mL for the test formulation compared with 3668 ± 1433 ng/mL for the reference formulation further underscores that both formulations demonstrate comparable overall exposure levels. While the in vivo drug exposure for the TITs was slightly lower than that for the conventional tablets, the absence of statistical significance reinforces the conclusion that the pharmacokinetic profiles are largely similar, thereby supporting bioequivalence between the two KT formulations.

Regarding the in vitro–in vivo correlation (IVIVC), our study did not specifically aim to establish a direct IVIVC for KT. However, based on the existing literature and the known pharmacokinetics of KT, we anticipate a strong IVIVC due to its BCS Class I classification, which indicates good absorption and predictable systemic availability. Future studies could further investigate this correlation to better validate our findings.

In conclusion, this new sustained release form of KT has suitable pharmacokinetic characteristics to be administered once a day as an effective and safe treatment for pain. The findings indicate that the test formulation maintains therapeutic effectiveness without significant accumulation, highlighting its potential utility in clinical applications. Further studies focusing on different dosing regimens and longer-term safety assessments will be essential to comprehensively evaluate the implications of these pharmacokinetic parameters.

Conclusions

In the comparative dissolution study of KT in SGF and SIF, pharmacokinetic analysis was conducted in beagle dogs to assess the potential for developing a novel tablet dosage form of KT. Approximately 20% of the administered KT was released within the first 30 min, with a cumulative release exceeding 90% at 16 h. This suggests that the novel TIT formulation may be capable of maintaining extended plasma drug concentrations compared with conventional tablets. In the single-dose study, significant differences were observed in the pharmacokinetic parameters between the two formulations. In the repeated-dose study, when equivalent doses of conventional tablets were administered in divided daily doses, the TIT formulation showed no significant differences in most steady-state pharmacokinetic parameters, except for T_max,ss.

Based on these findings, the development of a novel KT tablet dosage form utilizing the PPOP and tablet-in-tablet techniques warrants further investigation. This new sustained-release formulation of KT demonstrates suitable pharmacokinetic characteristics for once-daily administration, potentially offering an effective and safe treatment for pain.

Supplementary Information

Below is the link to the electronic supplementary material.

Declarations

Author Contributions

Conception and design of the work were performed by Hui Liu, Yi-hui Ma, and Guo-wei Zhang. Material preparation, data collection, and analysis were performed by Xiang-yang Xie, Yuan Zeng, Zhi-long Chen, Yu-liang Li, and Wen Lin. The first draft of the manuscript was written by Hui Liu, Yi-hui Ma, and Guo-wei Zhang, and all authors edited or commented on versions of the manuscript. All authors read and approved the final manuscript.

Funding

This research was supported by Chutian Elite Program of Hubei Province for Health Professionals (Grant No. 2024-09054) and Wuhan Natural Science Foundation Exploration Program (Grant No. 2024020801020397).

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethics Approval

All animal procedures were approved by Animal Care Committee of Wuhan Hongren Bio-pharmaceutical Co. (protocol ID: IACUC202400052), a non-clinical contract research organization. The experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals of Wuhan Hongren Bio-pharmaceutical Co.

Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Code Availability

Not applicable.

Data Availability

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