Evaluation of the bioavailability of a Tamiflu taste-masking pediatric formulation using a juvenile pig model and LC-MS/MS

Abstract

Aim: To improve the palatability and increase compliance in pediatric patients, different taste-masking technologies have been evaluated to support the NIH Pediatric Formulation Initiative.

Methods: This bioavailability approach combined a juvenile porcine model which represented the pediatric population, and an advanced UHPLCMS/MS method. Juvenile pigs were administered with either commercial Tamiflu or its taste-masking formulation and plasma samples were obtained from 0 to 48 h. The mass spectrometer was operated in positive mode with electrospray ionization.

Results: The bioavailability profiles were not significantly different between the two formulations which demonstrated that taste-masking by forming an ionic complex was a promising approach for formulation modification.

Conclusion: The pre-clinical study revealed a promising model platform for developing and screening taste-masking formulations.

Plain language summary

Article Highlights.

• Compared with the more straightforward development of adult formulations, the development of pediatric formulations is far more challenging due to a broad range of physiological and pharmaceutical factors such as the development stage of the child, safety of the excipients, palatability and ease of swallowing.

• Palatability is a critical quality attributes for pediatric formulations and poor palatability caused by the bitter taste of many drugs has been a major obstacle for the acceptability of medicines intended for pediatric use.

• As part of the NIH Pediatric Formulation Initiative and the later NIH Pediatric Formulation Initiative to enhance research in pediatric-friendly formulations through the Best Pharmaceuticals for Children Act's Framework to Enable Pediatric Drug Development to help advance stable and compliant focused pediatric formulations, the FDA has evaluated different taste-masking techniques used in pediatric formulation development.

• In the FDA, an oseltamivir phosphate taste-masking formulation was developed by forming an ionic complex of oseltamivir using Amberlite IRP64 resin by the FDA laboratories and was shown to be a promising pediatric taste-masking formulation based on its in vitro physicochemical and organoleptic evaluation.

• The focus of our present work was to conduct patient centric bioavailability studies to evaluate product quality and product performance of the ionic complex taste masking formulation.

• The bioavailability study was carried out on a preclinical animal model using a bioanalytical method with sufficient sensitivity, selectivity and high-throughput capability for the simultaneous quantitation of oseltamivir and oseltamivir acid.

• Because of pharmacokinetics and pharmacodynamics differences between the pediatric population and adults, a juvenile porcine model was chosen to represent the pediatric population by mimicking the developmental stages of gastrointestinal conditions in the child and by providing valuable in vivo data recognizing the fundamental differences in the ADME process between children and adults.

• The outcome of the in vivo study found that the bioavailability profiles were not significantly different between the two formulations demonstrating that taste-masking by forming an ionic complex offered promise for modification of the adult formulation for pediatric application.

1. Background

The number of pharmaceutical products approved for pediatric use in USA has increased significantly since the passage of the Best Pharmaceuticals for Children Act in 2002 [1]. Compared with the more straightforward development of adult formulations, the development of pediatric formulations is far more challenging due to a broad range of physiological and pharmaceutical factors such as the development stage of the child, safety of the excipients, palatability and ease of swallowing [2–5]. Palatability is a critical quality attributes for pediatric formulations, and poor palatability caused by the bitter taste of many drugs has been a major obstacle for the acceptability of medicines intended for pediatric use. The noncompliance by children caused by unpleasant taste has accounted for one of the most important causes of therapeutic treatment failure [6,7]. Palatable drugs using taste masking formulations have been clinically proven to encourage increased patient compliance by minimizing medication loss due to spitting [7–10]. Numerous taste-masking approaches and technologies have been considered to improve the palatability of various drug products and thus increase pediatric patient compliance such as chemical modification, sensory masking with sweetener or physical barrier coating [6,8,9,11–18].

As part of the NIH Pediatric Formulation Initiative and the later NIH Pediatric Formulation Initiative to enhance research in pediatric-friendly formulations through the Best Pharmaceuticals for Children Act's Framework to Enable Pediatric Drug Development to help advance stable and compliant focused pediatric formulations, the US FDA has evaluated different taste-masking techniques used in pediatric formulation development. In this study, oseltamivir phosphate, a front-line antiviral drug for seasonal flu has been selected as one of the model drugs for pediatric formulation development. Oseltamivir phosphate, a prodrug of oseltamivir acid, was approved under trade name Tamiflu for the treatment of flu in children 2 weeks old or older. The bitter taste was previously masked by using pea starch maltodextrin-Kleptose Linecaps^® (Mld) [19]. In the FDA, an oseltamivir phosphate taste-masking formulation was developed by forming an ionic complex of oseltamivir using Amberlite IRP64 resin by the FDA laboratories and was shown to be a promising pediatric taste-masking formulation based on its in vitro physicochemical and organoleptic evaluation [20–22]. The focus of our present work was to conduct patient centric bioavailability studies to evaluate product quality and product performance of the ionic complex taste masking formulation. The bioavailability study was carried out on a preclinical animal model using a bioanalytical method with sufficient sensitivity, selectivity and high-throughput capability for the simultaneous quantitation of oseltamivir and oseltamivir acid. This approach aimed to verify the in vitro product quality assessment and provide additional information on product performance by including the bioavailability of the active metabolites. Because of pharmacokinetics and pharmacodynamics differences between the pediatric population and adults, a juvenile porcine model was chosen to represent the pediatric population by mimicking the developmental stages of gastrointestinal conditions in the child and by providing valuable in vivo data recognizing the fundamental differences in the ADME process between children and adults [23]. As another essential part of the study, a sensitive, reproducible and high throughput method for oseltamivir and oseltamivir acid in porcine plasma was designed to provide accurate bioavailability data. Several methods have been reported for the determination of oseltamivir and/or its active metabolite oseltamivir acid in pharmaceutical formulations and different biological matrices using HPLC-UV [24–29], HPLC-FLD with precolumn derivatization [30,31], CE [32,33] and HPLC/UPLC-MS/MS [34–41]. As of 2000, tandem mass spectrometry using multiple reaction monitoring (MRM) has become the dominant choice of detection technology for the quantitation of oseltamivir and oseltamivir acid in preclinical/clinical studies using protein precipitation, LLE and SPE or a combination of protein precipitation and SPE for sample preparation [34–41]. MRM detection was chosen for the development of the analytical method in our study because of its superior sensitivity and selectivity over other detection methods. A Waters Cortecs 1.6 μm UPLC C18+ Column with sub-2μm core-shell particles and a 3.0-min gradient elution time was used for the chromatographic separation resulting in excellent resolution and column selectivity for both analytes and allowing the use of one simple protein precipitation for plasma sample preparation. The sensitive and efficient UHPLC-MS/MS method developed and validated here was successfully applied to our preclinical bioavailability study with juvenile pigs receiving a single dose of commercial oseltamivir phosphate or its pediatric taste-masking formulation.

In summary, a bioavailability approach consisting of an appropriate animal model and advanced UHPLC-MS/MS assay technology was developed and successfully applied to the product quality assessment of an oseltamivir phosphate taste-masking pediatric formulation.

2. Experimental

2.1. Chemicals & reagents

Oseltamivir phosphate reference standard, USP grade, was purchased from the US Pharmacopeia (MD, USA). Reference standards of oseltamivir acid, oseltamivir-d3 phosphate and oseltamivir-d3 acid were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). All standards were used without further purification or other modification. Optima LC/MS grade acetonitrile, methanol and formic acid (FA) were purchased from Fisher Scientific (PA, USA). Filtered 18 mΩ water was supplied in house by a Millipore Milli-Q System (MA, USA). All other chemicals and reagents were of analytical grade.

2.2. Preparation of taste masked formulations

The taste masked formulations were prepared in-house at FDA's laboratories. The ionic complex of oseltamivir phosphate and Eudragit EPO polymer was prepared with the drug-to-polymer ratio of 1:2 (w/w) by the solvent evaporation method. Briefly, Eudragit EPO polymer-Oseltamivir phosphate granules were prepared by adding polymer in 80 ml ethanol and the dispersion was sonicated for 10 min. When the polymer granules disappear to form either clear or translucent solution, drug was added under stirring to obtain dispersion of the drug in the polymer. The dispersion was allowed to stir for 1 h at room temperature. The dispersion was centrifuged at a speed of 4500 rpm for 40 min to obtain sediments containing insoluble drug mixed with polymer. The sediment was then harvested and dried in hot air oven at 40°C for 48 h. The dried mass was then broken into small pieces before drying in vacuum oven at 37°C for additional 48 h. The dried product was crushed using mortar and pestle and collected the granule sizes between 1000 and 500 μm after passing through a USP sieve. Granules equivalent to 45mg Oseltamivir phosphate were filled in size 4L hard gelatin capsules.

2.3. Preparation of calibration, quality control, system suitability & internal standards

Oseltamivir, oseltamivir acid and their respective internal standards oseltamivir-d3 and oseltamivir acid-d3 stock solutions, 1 mg/ml, were prepared with 18 mΩ water and stored at -80°C. A working standard solution containing both oseltamivir and oseltamivir acid, 10 μg/ml each, was diluted form stock solutions with DI water. A working quality control standard solution with both oseltamivir and oseltamivir acid, 8 μg/ml and the internal standard working solution, containing 10 μg/ml each of oseltamivir-d3 and oseltamivir acid-d3 were prepared in 18 mΩ water. These working solutions were then aliquoted and stored at -80°C. Ten more spiking standard solutions were prepared by serial volume to volume dilutions from the working standard solution aliquot to provide concentrations of 5000, 2000, 1000, 500, 200, 100, 50, 20, 10 and 5 ng/ml in DI water. Quality control spiking standards were prepared in similar fashion from quality control working standard solutions to provide concentrations of 1000, 10 and 5 ng/ml with DI water. A 100 ng/ml spiking internal standard solution was prepared by dilution from the internal standard working solution using acetonitrile. These spiking standard solutions and quality control spiking solutions were prepared freshly daily prior to sample analysis.

2.4. Instrument conditions

The LC-MS/MS system consisted of an Agilent 6410B Triple Quadrupole mass spectrometer (Agilent Technologies, CA, USA) interfaced by an electrospray ionization (ESI) probe with an Agilent 1290 1290 Infinity Binary LC System (Agilent Waldbronn, Germany). The chromatographic separation was achieved on a CORTECS UPLC C18+ Column (2.1 × 50 mm, 1.6 μm) equipped with a CORTECS UPLC C18+ VanGuard Pre-column (2.1 mm × 5 mm, 1.6 μm). The mobile phase consisted of 0.1% formic acid in water and methanol. The total run time was 3.0 min. The column temperature was maintained constant at 55°C during sample analysis. The MS/MS parameters in the positive-ion ESI mode were tuned to maximize generation of protonated analyte molecules and the production of characteristic product ions for each analyte by means of Agilent MassHunter Optimizer software. The two most abundant precursor-to-product ion pairs were chosen for multiple reaction monitoring of oseltamivir and oseltamivir acid, one for quantitation and one for qualification. In a similar fashion, the most abundant precursor-to-product ion pairs were chosen for selected reaction monitoring of oseltamivir-d3 and oseltamivir-d3 acid. The MRM channel and MS parameters are listed in Table 1.

Table 1.

Multiple reaction monitoring channels and MS parameters for analytes and internal standards.

Compound name	Precursor ion	Product ion	Dwell	Fragmentor	Collision energy	Cell accelerator voltage
Oseltamivir	313.21	225.1	100	97	4	7
Oseltamivir	313.21	166.1	100	97	16	7
Oseltamivir acid-d3	288.2	139.1	100	93	16	7
Oseltamivir acid	285.18	197.1	100	93	4	7
Oseltamivir acid	285.18	138.1	100	93	16	7
Oseltamivir-d3	316.23	167.1	100	97	16	7

2.5. Porcine plasma sample preparation

Porcine plasma samples from the bioavailability study were removed from the -80°C freezer and thawed on ice. After vortexing for 30 s, 200 μl of each plasma sample was transferred to a 1.7 ml micro-centrifuge tube containing 100 μl of spiking internal standard solution and 20 μl of DI water. Samples were vortexed for 30 s and then extracted with 1 ml of ice-cold acetonitrile, The mixture was vortexed for 30 s and followed by 10 min centrifugation at 17,850 rpm at 4°C. The supernatant was then transferred to another 1.7 ml microcentrifuge tube and dried using a standard SpeedVac. The completely dried residue was reconstituted with 200 μl of mobile phase. The reconstituted samples were spun down for 10 min and 150 μl of the clear supernatant taken for UHPLC-MS/MS analysis.

2.6. Method validation

The bioanalytical method for oseltamivir and oseltamivir acid was validated according to the requirements of the FDA Bioanalytical Method Validation Guidance for Industry [42] for linearity, accuracy, precision, specificity, sensitivity, recovery and guidance specific analytical issues such as carry-over, matrix effect, stability and dilution integrity. The specificity of the assay was determined by comparing extracted blank pooled porcine plasma from six different batches and the plasma calibration standard at the LLOQ level. Carry-over was evaluated by injection of mobile phase blank sample after injecting the upper limit of quantitation (ULOQ) sample. Three independent calibration curves produced from 11 spiked plasma standards (0.5–1000 ng/ml for both analytes) were prepared individually on three separate days. The calibration curve was established over the analytical range 0.5 to 1000 ng/ml for oseltamivir and oseltamivir acid in porcine plasma and the type of calibration and weighting factor for both analytes was determined by Agilent MassHunter Quantitative Analysis Software to determine the best fit between peak area ratio of analyte/internal standard to the analyte calibration standards. The accuracy was determined by comparing the corresponding calculated concentrations with the nominal concentrations. The intra-day and inter-day accuracies and precisions (as RSD%) were assessed by QC samples at 0.5 (LLOQ), 1, 100 and 800 ng/ml for both analytes. The LLOQs were taken as the concentration which gave an accuracy between 80 and 120% with a signal-to-noise ratio greater than 5. Recoveries of the analytes and IS were determined by comparing peak areas of QC samples (0.5, 1, 100 and 800 ng/ml) with those of the of QC solutions of the same concentration but spiked with the extracted blank porcine plasma. Matrix effects were evaluated by comparing the peak areas of extracted blank guinea pig plasma spiked with QC solutions with those of neat QC solutions. Stability tests were evaluated using samples at four QC levels for freeze-thaw stability in plasma (three cycles, -80°C to RT), bench top stability in plasma (4 h at RT) and post-extraction sample stability in the autosampler (4°C for 12 h).

2.7. Animal study

The validated method was applied to a preclinical bioavailability study in juvenile pigs receiving a single dose of commercial oseltamivir phosphate or its taste-masking formulation; an ionic complex of oseltamivir phosphate/Eudragit EPO. The dosage of the taste-masking formulation was calculated based on 6 mg/kg oseltamivir phosphate. This study took place under the guidance of the Purdue Animal Care and Development Use Committee (PACUC) under approved protocol number 1112000407 “Testing and of Pharmaceuticals in the Juvenile Pig” and performed under the care and guidance of a laboratory animal veterinarian. Animals were surgically modified with a jugular vein catheter for blood sampling and housed in the Pigturn-Culex-L^® (Culex) movement responsive caging system. Animal rooms were kept on a 12-h light cycle (6 am–6 pm). The catheter was connected to the Culex system for automated collection of blood at specified intervals. The catheter was connected via a tether arm attached to the animal via a harness such that the cage rotated opposite to the movement of the animal to eliminate tangling and allow for continuous blood collection transfers to sample vials stored in a chilled (4°C) fraction collector outside the cage. A total of 8 male pigs were used with a parallel dosing design. Animals were fasted overnight prior to dosing, with fasting continuing for 1 h after administering the capsules with commercial oseltamivir phosphate or the ionic complex of oseltamivir phosphate/ Eudragit EPO polymer formulation (made in house) followed by a 6-ml flush with water. Blood samples (1 ml) were collected and transferred into tubes containing K₂EDTA as an anticoagulant pre-dose and post-dose at 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 12, 18, 24, 36 and 48 h. Plasma samples were prepared from blood samples within 2 h of collection and stored frozen (-80°C) until analysis.

3. Results & discussion

3.1. Bioavailability for product quality assessment

A key mission of the FDA is to regulate pharmaceutical drug products to ensure an uninterrupted supply of high-quality, safe and effective drugs, free of contamination and defects. To fulfill this mission, scientists at FDA have applied various approaches to evaluate pharmaceutical product quality. Depending on the nature of the drug API and the complexity of the drug formulation, different in vitro and in vivo approaches including dissolution, drug binding and equilibrium, in vitro release testing (IVRT) and in vitro permeation testing (IVPT), the characterization of physicochemical properties, IVIVC, bioavailability (BA) and bioequivalence (BE) can be selected as a final determinate to evaluate product quality. In this study, we chose the bioavailability approach because it could provide a more comprehensive product quality assessment over in vitro methods by including the bioavailability of the active metabolite. In addition, because of pharmacokinetics and pharmacodynamics differences between the pediatric population and adults, the use of the juvenile porcine model has been proven to be a good animal model to represent the pediatric population since it mimics the developmental stage of gastrointestinal conditions in children [23].

3.2. Analytical method development

Successful BA studies rely on the accurate measurement of drug concentrations in biological samples such as plasma, serum and tissue. To achieve this goal, a precise and reproducible bioanalytical method is critical. As part of our continuous efforts for the development of a bioanalytical platform using a systematic approach, key aspects of the method development such as identifying and minimizing sources of method variability and improving method reproducibility have been emphasized to best ensure the intended acceptable method performance. For the preclinical bioavailability of oseltamivir taste-masking pediatric formulation the critical method attributes have been determined to be method sensitivity, linearity range and throughput. A high sensitivity was required for the method due to the much lower pediatric dosage strength compared with the adult formulation and the limited plasma samples available from the required multiple sampling of the test animals. Based on previously reported LC-MS/MS methods [34–41], a tandem mass spectrometer with MRM detection was first considered for the sample analysis. Both oseltamivir and oseltamivir acid exhibited excellent MS response using positive ESI as expected. The MS response was further enhanced by adding formic acid to the mobile phase as proton donor which also helped to reduce the peak tailing during chromatographic separation. After testing several commercial UPLC and HPLC columns, the Waters Cortecs 1.6 μm UPLC C18+ Column with sub-2μm core-shell particles was selected because it provided optimal chromatographic peaks and efficient chromatographic resolution. To improve the retention of oseltamivir acid, a polar analyte, methanol was used in place of acetonitrile and the column temperature was raised to 55°C to reduce the elevated column backpressure. To improve the method throughput, a 3.0-min gradient program was used to achieve fast elution of oseltamivir while maintaining the appropriate retention of the polar metabolite, and a cleanup section with strong solvent composition was added to reduce the column memory effect and improve the reproducibility of the bio-sample analyses. The use of sub-2 μm core-shell silica particles also afforded improved chromatographic separation by reducing potential matrix effects and allowing for a simple one step protein precipitation for plasma sample preparation. The optimized UHPLC-MS/MS method also provided a wide dynamic range for both analytes from 0.5 to 1000 ng/ml which covered the sample concentration range from the bioavailability study. To help ensure specificity and achieve a zero or near-zero carryover, an acidified needle wash solvent was used on the Agilent system to successfully remove residue in the autosampler. In practice, no such carryover peak was observed at the retention times of analytes and internal standards in the mobile phase blank injected after the upper limit of quantitation sample. Overall, the UHPLC-MS/MS method provided fast chromatographic separation, acceptable assay selectivity and high throughput capability.

3.3. Analytical method validation

Specificity was verified by the absence of endogenous peaks in the plasma blank and absence of co-eluting peaks in the sample or calibration standards by mass detection. Typical chromatograms of extracted blank plasma and calibration standards of oseltamivir and oseltamivir acid in porcine plasma at LLOQ (0.5 ng/ml) are shown in Figures 2 & 3. Carryover was evaluated by injecting a mobile phase blank immediately after the upper limit of quantitation. The results indicated no significant carryover. The plasma calibration curves were validated over the range 0.5 to 1000 ng/ml for oseltamivir and oseltamivir acid in porcine plasma with a weighted (1/x²) power equation. The correlation coefficients were greater than 0.99 (Figures 1 & 2) and the lower limit of quantification was 0.5 ng/ml for both analytes. with correlation coefficients (r²) greater than 0.99. Values for intra- and inter-day accuracy and precision were satisfied at all QC concentrations for both analytes (Table 2) and no significant carryover for analytes or internal standard was observed in the mobile phase blank injected after plasma calibration standard at ULOQ.

Figure 2.

Chromatograms of extracted blank porcine plasma (top row) and oseltamivir acid calibration standard at 0.5 ng/ml (bottom row): I, oseltamivir acid; II, oseltamivir acid-d3.

Figure 3.

Mean oseltamivir plasma concentration profiles in juvenile pigs after a single oral dose of commercial oseltamivir phosphate or its taste-masking formulation.

Figure 1.

Chromatograms of extracted blank porcine plasma (top row) and oseltamivir calibration standard at 0.5 ng/ml (bottom row): I, oseltamivir; II, oseltamivir-d3.

Table 2.

Intra-day and inter-day accuracy and precision of oseltamivir and oseltamivir acid.

Nominal Conc. (ng/ml)	Intra-day (n=6)			Inter-day (n=18)
	Mean Conc. (ng/ml)	Accuracy%	Precision (RSD%)	Mean Conc. (ng/ml)	Accuracy%	Precision (RSD%)
Oseltamivir
0.50	0.54	109	4.43	0.54	109	7.58
1.00	0.91	91.0	4.51	0.98	98.0	5.09
100	107	107	1.88	107	107	5.38
800	818	102	1.60	824	103	6.50
Oseltamivir acid
0.50	0.54	109	9.41	0.57	113	4.93
1.00	1.01	101	4.39	1.05	105	4.13
100	107	107	2.60	107	107	5.01
800	839	105	2.21	840	105	6.76

Recovery and matrix effect were evaluated with six replicates at selected QC concentrations. The mean recoveries were 95.2–102.9% for oseltamivir at the four QC levels with about 17% ion suppression observed. (Table 3). The mean recoveries were 84.1–96.0% for oseltamivir acid at four QC levels and almost no significant matrix effect was observed. The recovery and matrix effect did not affect the accuracy and precision of the method since stable isotope labeled internal standards were used for both analytes for the compensation of sample extract and MS analysis. Oseltamivir and oseltamivir acid in porcine plasma were found to be stable when subjected to three freeze–thaw cycles and then allowed to remain at room temperature for 4 h. The post-extracted samples were stable for up to 12 h in the autosampler at 4°C (Table 3). The concentrations for the oseltamivir and oseltamivir acid working solutions were found to be unchanged (95.4% remaining for oseltamivir and 97.9% remaining for oseltamivir acid comparing to their freshly prepared working solutions) for up to 14 days at 4°C.

Table 3.

Recovery, matrix effect and stability of oseltamivir and oseltamivir acid.

Characteristic	Quality control level (ng/ml), n = 6				Average	RSD%
	0.50	1.00	100	800
Oseltamivir
Recovery	98.3%	95.9%	102%	103%	99.9%	3.30%
Matrix effect	83.3%	85.2%	83.3%	82.8%	83.7%	1.00%
Benchtop stability	107%	100%	113%	108%	107%	5.37%
Autosampler stability	113%	100%	107%	102%	106%	5.80%
Freeze–thaw (3 cycles) stability	115%	103%	111%	105%	109%	5.31%
Oseltamivir acid
Recovery	89.3%	84.1%	93.6%	96.0%	90.7%	5.20%
Matrix effect	96.1%	96.9%	95.1%	94.5%	95.6%	1.10%
Benchtop stability	117%	108%	111%	108%	111%	4.10%
Autosampler stability	114%	104%	108%	104%	107%	4.68%
Freeze–thaw (3 cycles) stability	117%	106%	111%	109%	111%	4.57%

3.4. Assessment of taste-masked formulation

The formulations were weighted and placed in size 4L capsules for the in vivo animal study. The potency of the complex was 94.49 ± 3.39% and the content uniformity (%) was determined to be 95.95 ± 2.63%. The drug release study found that only 11% of oseltamivir phosphate was released from the complex in 30 s (drug dwell time in oral cavity) at buccal pH 6.8, which was indicative of less bitter sensation. The organoleptic evaluation using α-Astree liquid and taste analyzer (e-Tongue) has further confirmed that the complex effectively suppressed the bitter taste of the drug. The dissolution studies showed that >85% of drug release occurred in 20 min and 93% of drug release in 120 min at BCS gastric conditions which demonstrated the taste-masking formulation could provide desired therapeutic effects [22].

3.5. Preclinical bioavailability study

The mean plasma concentration–time profiles of oseltamivir and oseltamivir acid from juvenile pigs receiving commercial oseltamivir phosphate and its taste-masking pediatric formulation are presented in Figures 3 & 4. The pharmacokinetic parameters were calculated by Phoenix WinNonLin 6.4 using non-compartmental analysis. The PK parameters are summarized in Table 4. No significant statistical difference in the log-transformed C_max, AUC0-t and AUCinf was found between these two formulations (p < 0.05) which demonstrates that the taste-masking Tamiflu formulation can provide similar systemic exposure as a Tamiflu commercial formulation.

Figure 4.

Mean oseltamivir acid plasma concentration profiles in juvenile pigs after a single oral dose of commercial oseltamivir phosphate or its taste-masking formulation.

Table 4.

Pharmacokinetic parameters of taste-masking pediatric Tamiflu and commercial Tamiflu formulations.

Analyte	Formulation	Commercial Tamiflu formulation		Taste-masking pediatric Tamiflu formulation
	Parameters	AVE	SD	AVE	SD
Oseltamivir	AUC0-t (ng*h/ml)	164.04	137.37	119.72	102.83
	AUCinf (ng*h/ml)	166.72	138.55	123.61	104.52
	Cmax (ng/ml)	108.03	102.96	57.87	47.27
	Tmax (h)	0.63	0.68	0.88	0.63
	T1/2 (h)	3.08	2.12	3.83	2.36
Oseltamivir acid	AUC0-t (ng*h/ml)	1256.02	320.06	1289.21	461.31
	AUCinf (ng*h/ml)	1261.06	321.07	1295.13	461.72
	Cmax (ng/ml)	265.68	76.11	280.71	105.07
	Tmax (h)	2.00	0.63	2.17	0.41
	T1/2 (h)	3.13	1.01	3.19	1.86

4. Conclusion

The Best Pharmaceuticals for Children Act (BPCA) became law in 2002 with goals to encourage and support the pharmaceutical industry to perform pediatric studies and for the NIH to identify therapeutic areas and help to sponsor clinical trials. Based on the clinical needs of pediatric patients, the development of a pediatric formulation requires the safety of the excipients, palatability and ease of swallowing to accommodate different developmental stages of the child on resolving the issue of poor palatability caused by the bitter taste of many drugs and the subsequent noncompliance issue. As a result of this collaboration an efficient platform of formulation development, pre-clinical models and high throughput MS procedures was developed as a valuable bioavailability screening approach, to evaluate the in vivo performance, of the taste-masking pediatric formulations. This approach combined a specific juvenile animal model for a targeted patient population and a highly sensitive and selective UHPLC-MS/MS method with high throughput capacity for bio-sample analysis. The outcome of the in vivo study found that the bioavailability profiles were not significantly different between the two formulations demonstrating that taste-masking by forming an ionic complex offered promise for modification of the adult formulation for pediatric application. In conclusion a UHPLC-MS/MS method for the simultaneous quantification of oseltamivir and its active metabolite oseltamivir acid in porcine plasma samples was developed, validated and applied for the bioavailability evaluation of an orally administered taste-masking pediatric oseltamivir phosphate product and the respective commercial adult drug product.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

J Wang, J Gu, PJ Faustino, A Siddiqui, Y Zhao, D Shakleya are employees of the Food and Drug Administration. G Giacoia is an employee of the National Institutes of Health. The authors have no other competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript apart from those disclosed.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Disclaimer

This publication reflects the views of the authors and should not be construed to represent FDA's views or policies.