Gayatri C.Patel, M.Pharm, PhD, is associate professor in the Department of Pharmaceutics & Pharmaceutical Technology, Ramanbhai Patel College of Pharmacy at the Charotar University of Science and Technology, Changa Campus, Changa- 388 421, Anand, Gujarat, India, gayatripatel.ph@charusat.ac.in, +91 2697 265162.

Gayatri C. Patel

Round white pills and plastic pill bottle | Image Credit: © ironstealth - Stock.Adobe.com

Submitted: June 8, 2023.
Accepted: January 5, 2024.

Cefpodoxime proxetil (CFPD PRXL) is a third-generation cephalosporin antibiotic with broad-spectrum activity. However, its bitter taste often leads to poor patient compliance. This study aimed to develop a taste-masked drug resin complex (DRC) using the ion exchange resin Kyron T-114. Various variables, including resin ratio, pH, temperature, soaking time, and stirring time, were investigated for their impact on drug loading and taste reduction. A dispersible tablet was then formulated using the DRC and other pharmaceutical excipients. The formulated tablet underwent evaluation for weight variation, hardness, dimension uniformity, friability, content uniformity, wetting time, 

 drug release. Results demonstrated that the wetting and disintegration times of the tablets, as well as the 

 drug release, were comparable to those of the commercially available CFPD PRXL product. In conclusion, the developed method for taste masking CFPD PRXL and preparing orodispersible tablets proved to be simple, cost-effective, and potentially suitable for industrial application.

Cefpodoxime proxetil (CFPD PRXL) is a third-generation cephalosporin antibiotic with broad-spectrum activity commonly prescribed in both solid and liquid forms to treat various bacterial infections, including upper respiratory infections, ear infections, skin infections, and urinary tract infections (1–3). However, its extremely bitter taste poses a challenge for patient compliance, particularly among pediatric and geriatric populations. Solid dosage forms such as tablets are frequently used for these patients (1–3).

Ion exchange resins are solid, high-molecular weight polyelectrolytes that can reversibly and stoichiometrically exchange their mobile ions with the surrounding medium. These resins are available in various sizes and can be utilized to adsorb bitter cationic drugs onto weak cation exchange resins containing carboxylic acid. This interaction forms a complex that effectively masks the bitterness of active ingredients (4). Resinates can then be formulated into lozenges, chewing gum, suspensions, or dispersible tablets, effectively concealing the taste (3).

The binding of drugs to the resin can be achieved through repeated exposure or prolonged contact with the resin. By forming weak ionic bonds between the drugs and the oppositely charged resin substrates or resinates, the drug-resin complex (DRC) remains stable under salivary pH conditions, successfully masking the unpleasant taste and odor of the drugs.

The objective of this study was to incorporate the DRC into a dispersible tablet to enhance patient compliance (5–6). In a previous study, the preparation and evaluation of the DRC was performed using Kyron T-114, a weak acid derivative of methyl acrylic acid cross-linked polymer having a carboxylic acid functional group, which is responsible for its taste masking property for a pharmaceutical suspension; however, its use in tablet formulation was not investigated. Therefore, the specific objective of this present study was to develop and evaluate an orally disintegrating tablet (ODT) of CFPD PRXL using the optimized DRC that were utilized for suspension formulation (6).

 is a research scholar, both at Charotar University of Science and Technology, Ramanbhai Patel College of Pharmacy, Department of Pharmaceutics & Pharmaceutical Technology, CHARUSAT Campus, Changa-388421, Gujarat, India.


*To whom all correspondence should be addressed.


Vol. 48, No. 3
March 2024
Pages: 22–26

When referring to this article, please cite it as Patel, G.; Patel, D. Development of Taste-Masked Oral Dispersible Tablets of Cefpodoxime Proxetil. 

Articles

This study aimed to develop a taste-masked drug resin complex using the ion exchange resin Kyron T-114.

Development of Taste-Masked Oral Dispersible Tablets of Cefpodoxime Proxetil

In the present investigation, the fixed-dose combination (FDC) tablet of Atorvastatin calcium and Ezetimibe was prepared by a quality-by-design approach using 2

 factorial design. Precompression and post-compression parameters were determined indicating excellent flow properties of granules as well as good tableting characteristics of the final tablets. Dissolution study showed higher drug release nearly 80% within 30 min for all prepared batches attributed to conversion from crystalline to amorphous form of the drug. ANOVA studies revealed that there was a linear correlation between the predicted, and the observed responses gave higher values of r

, indicating excellent fitting of the model (P < 0.001). Thus, the prepared FDC tablet executed a good relationship between selected critical formulation parameters and critical quality attributes. The drug release rate obtained met the selected dissolution criteria.

Submitted: August 5, 2020.
Accepted: October 12, 2020.

Rajendra Kotadiya,* rajendrakotadiya.ph@charusat.ac.in; +91 8160701232, is assistant professor, Krunal Jasani is PG student, and Gayatri Patel is professor, all at Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology (CHARUSAT), Changa, Gujarat, India.

When referring to this article, please cite it as Rajendra Kotadiya, Krunal Jasani, and Gayatri Patel, “Using Quality by Design to Develop Fixed-Dose Combination Tablets,” 

In the present investigation, the fixed-dose combination (FDC) tablet of Atorvastatin calcium and Ezetimibe was prepared by a quality-by-design approach using two-level factorial design.

Using Quality by Design to Develop Fixed-Dose Combination Tablets 

	Submitted: January 21 2019    
	Accepted: July 9 2019

The concept of pulsatile release, in which active ingredient is fully and quickly released after a predetermined lag time, is gaining popularity as a drug-delivery approach. Pulsatile release offers a way to counteract the “first-pass” effect, and to enable nocturnal dosing. This article summarizes research into optimizing the formulation of pulsatile release tablets that contain diclofenac sodium (DS), an anti-inflammatory used to treat rheumatoid arthritis. Studies focused on optimizing the formulation of a time-controlled tablet containing the active ingredient in its inner core, surrounded by a coating of hydrophilic polymers. 

	As the fields of chronopharmaceutics and chronopharmacology (1) reveal more about the importance of circadian rhythms with respect to human physiology, disease state, and drug action, time-controlled pulsatile-release tablets are being considered as a desirable drug-delivery mode. Research discussed in this article focused on compression coating, which can involve direct compression of both the tablet’s core and its coat, obviating the need for separate coating processes and the use of coating solutions. Compression-coated formulations can be used to protect hygroscopic, light-sensitive, oxygen- or acid-labile drugs, or to separate incompatible drugs from each other. They can also be used to achieve controlled release, and a number of studies (2) have evaluated compression-coated time-controlled drug delivery systems. Most of these formulations release the drug after a lag phase. 

	Compression is easy to achieve on a laboratory scale. The technique requires relatively large amounts of coating materials, however, and it is difficult to position the cores correctly (3).  For pulsatile release, modifications are made to the outer layer of the tablet during formulation development to control the lag time prior to release of the drug from the tablet’s inner core (4–5). 

	Studies used Penwest’s TIMERx technology (6) (

) to formulate the tablets, which are comprised of an inner core that contains the drug and an outer layer that has been compression-coated with a hydrophilic matrix of the heteropolysaccharides xanthan and locust bean gum (7). The goal of this work was to formulate compression-coated tablets, taking human circadian rhythms into account, using pulsatile delivery of DS to modulate the drug level for optimal treatment of rheumatoid arthritis (8, 9).   

	In this study, a time-dependent compression coated system of DS was developed to target drug release in the colon. If the formulation could be developed so that the required dose of DS could be administered at night, at around 10 PM, it was believed that patients’ morning pain symptoms could be avoided. The drug’s therapeutic effect could also be prolonged by continuously releasing the medication over an extended period of time after the administration of a single dose. 

	Numerous variables are known to affect drug release from time-controlled compression coated tablet formulations. They include the viscosity grade of polymer, the amount of polymer, the drug-polymer ratio, and the nature of the drug used in the tablet system (10, 11). The research summarized in this article set out to investigate the influence of the type and amount of polymer and the viscosity grade of the polymer in the coat on the time-controlled swelling or rupturing of compression coated tablets. 

DS and hydroxypropylmethylcellulose (HPMC K4M and K15M) were donated by Welable Pharmaceutical Pvt. Ltd. Mehsana. Lactose anhydrous, micro crystalline cellulose (Avicel PH-102), cross-carmellose sodium (Ac-Di-Sol) and magnesium stearate all were purchased from SD Fine Chem Ltd., Mumbai. 

Direct compression was used to prepare the inner-core tablets of DS. Preliminary trials were run to optimize the core tablet (

. Optimized formula for core tablet of diclofenac sodium.			

	A powder mixture of the drug, microcrystalline cellulose (Avicel PH-102), lactose anhydrous, cross-carmellose sodium (Ac-Di-Sol), and sunset yellow coloring was dry blended for 30 min in a double-cone blender, after which magnesium stearate and aerosol were added. It is very important to control the mixing rate with magnesium stearate. The blend was further blended for 5 min and compressed into tablets (with an average tablet weight of 200 mg) using a rotary tablet machine equipped with 7-mm diameter flat punches. 

	Sufficient pressure was applied to maintain tablet hardness at 6 ± 1 kg/cm². After ejection, the tablets were stored over silica gel in a desiccator for 24 hrs, to allow for elastic recovery and hardening. Prepared tablets were then evaluated for physical properties such as weight and content uniformity, hardness, friability, disintegration time, diameter, and thickness (12,13).

Selection of polymers for compression coating. 

Studies used two grades of directly compressible polymer hydroxypropylmethylcellulose (HPLC) K4M (3000–5000 mPas for 2% aqueous solution at 20 °C) and HPMC K15M (12,000–21,000 mPas for 2% aqueous solution at 20 °C) for the time-controlled compression-coated formulation of DS. Both grades are known to form gel under aqueous conditions (14–17). 

Preparation of compression coated tablet. 

A rotary tablet machine was used for compression coating, and the device was maintained at constant pressure, using a two-step direct compression procedure. In the first step, using the weights listed in 

, the die was filled with half of the weight amount of polymer to make a powder bed, and the core tablet was placed manually in the center of that bed. In the second step, the remaining half of polymer was added to the die above the core tablet. The powder was then compressed under a sufficient compression force, using a 10-mm diameter flat punch to maintain the coated tablet’s hardness at 10 ± 0.50 kg/cm². A standard manual process was used for die filling, core centralization, and machine operation. Data were then analyzed using a 32 full factorial design. In this approach, two factors were evaluated, each at three levels, and experimental trials were performed at all nine possible combinations (Batches F1 to F9). The experimental design with the corresponding formulations is outlined in 

: Composition for compression coated formulation.			

					Weight ratio of HPMC K15M toHPMC K4M(%) 

					Weight of tablet (after compression coating) mg

Research examined the effects of varying excipient and polymer weight in coatings designed to optimize the release of diclofenac sodium. The goal was to release it at night to improve the treatment of rheumatoid arthritis. 


Optimizing Compression Coating for Pulsatile Release Tablets

	Submitted: Dec. 22, 2018.
	Accepted: Jan. 8, 2019.

	Pegylation is a well-known technology used to increase residence time of L-asparaginase in blood circulation and to reduce hypersensitivity reactions; however, it is important to address manufacturing problems associated with the shorter shelf-life of pegylated L-asparaginase (pegaspargase) upon long-term storage in the form of a solution. Exposure to the sudden excursions encountered during storage and shipping may affect stability of the pegaspargase drug product material. In this article, the effect of freeze-thawing and high temperature on the stability of pegaspargase protein was studied. Differences in the degradation pathways for the storage conditions were identified, and manufacturing issues associated with the degradation are discussed. It was observed that pegaspargase follows different degradation pathways when exposed to freeze-thawing and high temperature stress. These differences in the degradation pathways have different implications on the manufacturing process. 

L-asparaginase has been extensively investigated for the treatment of acute lymphoblastic leukemia (ALL) (1). The role of L-asparaginase present in guinea pig serum in the reduction of lymphoma was first studied by Broome, and the inhibitory effect was attributed to depletion of asparagine due to the presence of L-asparaginase in the serum (2). L-asparaginase enzymatically cleaves amino acid L-asparagine into aspartic acid and ammonia. Depletion of L-asparagine in blood serum results in inhibition of protein-synthesis, DNA-synthesis, and RNA-synthesis, especially in leukemic blasts that are not able to synthesize L-asparagine and thus undergo apoptosis (3). Normal cells, in contrast, are capable of synthesizing L-asparagine and are less affected by its rapid withdrawal during treatment with the enzyme L-asparaginase (4).

-asparaginase: Kidrolase, EUSA Pharma; Elspar, Ovation Pharmaceuticals; Crasnitin, Bayer; Leunase, Sanofi-Aventis; Asparaginase medac, Kyowa Hakko) has been used in the treatment of ALL (5). Although L-asparaginase is an effective antineoplastic agent used in chemotherapy of ALL, development of antibodies against L-asparaginase and hypersensitivity reactions may lead to discontinuation of the treatment (6). Such adverse events along with the other toxicities like thrombosis, pancreatitis, hyperglycemia, and hepatotoxicity led to the development of alternative sources of L-asparaginase (7). The development of L-asparaginase from Erwinia chrysanthemi (Erwinase, EUSA Pharma) is an outcome of such efforts (8). The disadvantage of frequent intramuscular injections and the adverse events of hypersensitivity reactions led to development of a pegylated version of native 

-asparaginase (polyethylene glycol [PEG]-asparaginase: Oncaspar, Enzon Pharmaceuticals Inc) (9). Oncaspar is an asparagine-specific enzyme indicated as a component of a multi-agent chemotherapeutic regimen for treatment of patients with first line ALL and hypersensitivity to asparaginase. Oncaspar is available as a solution in a single-use glass vial with the strength of 3750 IU per 5 mL of solution. The recommended dose of Oncaspar is 2500 IU/m

intramuscularly or intravenously. Oncaspar is not recommended to be used when it is frozen or stored at room temperature 15–25 °C (59–77 °F) for more than 48 hours.

	L-asparaginase is a homotetrameric enzyme comprised of four identical subunits with a mass of 34,592 Da coupled by weak, non-covalent, largely hydrophobic interactions (10). The tetrameric structure of the L-asparaginase enzyme is required for enzymatic activity (11). Pegaspargase (pegylated L-asparaginase) is a covalent conjugate of 

-derived L-asparaginase with monomethoxypolyethylene glycol (mPEG) succinimidyl succinate (PEG-SS; molecular weight of 5000 Da). Approximately 69 to 82 molecules of mPEG are linked to L-asparaginase. The succinyl linker between PEG and L-asparaginase contains an ester linkage that can lead to hydrolytic removal of the PEG moiety from the PEG-protein conjugate (12). The release of PEG from the protein molecule (i.e., depegylation) upon storage in the form of solution leads to a shorter shelf-life. Also, stability of the protein molecule can be affected by chemical as well as physical factors (13). Proteins can be denatured readily by stresses encountered in solution, or in a frozen or dried state (14). It is also important to know the effect of sudden excursions encountered during shipping of the drug product material, which may affect stability of the product. Drug product may be exposed to lower or higher temperature from its real-time storage conditions during shipment. It is known that L-asparaginase shows changes in its structural and biological properties upon repeated freeze-thaw. In this article, the effect of freeze-thaw stress and high temperature on the stability of pegaspargase (pegylated 

-derived L-asparaginase) has been investigated; manufacturing issues associated with these degradation pathways are discussed. 

L-asparaginase was procured in the form of lyophilized powder. PEG of 5000 Da size was procured in the activated form. Pegaspargase was prepared by conjugating (pegylation) multiple units of 5 kDa activated PEG (m-PEG-N-hydroxysuccinimidyl ester) at different sites of the α-NH

group of Lysine residues of the L-asparaginase enzyme using the pegylation techniques known in the art (15, 16). Pegylation occurs in all four subunits at the specified sites of the enzyme. The pegylated L-asparaginase (crude solution) was purified to remove excess free PEG using column chromatography (AKTA system, GE Healthcare). Buffer exchange of the purified pegylated L-asparaginase protein solution was performed in the desired formulation media (with final concentration of 5.58 mg of dibasic sodium phosphate, 1.20 mg of monobasic sodium phosphate, and 8.50 mg of sodium chloride in 1 mL of water for injection) by membrane ultrafiltration and diafiltration steps. Ultrafiltration and diafiltration were performed in a controlled manner at room temperature with 50 kDa molecular weight cut off membrane by using a tangential flow filtration system (Sartorius Stedim Biotech). At the end of buffer exchange, the protein solution was concentrated through an ultrafiltration step, and the final concentration was adjusted to approximately 8.8 mg/mL. The protein solution was finally filtered through a 0.2-µm sterile filter (Sartopore 2, Sartorius Stedim Biotech). The final formulation composition was maintained to be the same as that of the commercial product Oncaspar. All the excipients for the final formulation (i.e., dibasic sodium phosphate, monobasic sodium phosphate, and sodium chloride) were obtained from Merck, Germany. 

Freeze-thawing and exposure to high temperature. 

In order to check the effect of freezing and thawing, pegaspargase solution was aliquoted with 500 µL solution in cryo-vials of 1-mL capacity (Nunc CryoTube vials, Thermo Scientific). Samples were frozen at or below –20 °C in a deep freezer (Thermo Electron Corporation, Model No.: ULT1740-3-V40). Thawing was performed at room-temperature rapidly until the frozen mass was converted into the liquid solution. In order to check the effect of high temperature (forced degradation due to heat), formulated pegaspargase protein solution was filled in 2-mL glass vials (borosilicate USP Type I clear flint glass, Schott) with coated stoppers (fluorinated polymer-coated butyl rubber stopper, West Pharmaceuticals) and sealed with flip-off seals (aluminium seals with flip-off plastic cap, West Pharmaceuticals). Samples were exposed to a controlled high temperature of 40 °C ± 5 °C with 75% ± 5% relative humidity (RH) and 25 °C ± 5 °C with 60% ± 5% RH in the stability chamber (Thermo). Samples were withdrawn at different time intervals and stored between +2 °C and +8 °C before analysis.

Samples were analyzed by high-performance size exclusion chromatography (HP-SEC) using two different detectors: an ultraviolet (UV) detector and a refractive index (RI) detector. The RI detector was used to detect an increase in free PEG due to exposure of pegaspargase to freeze-thawing and high temperature, if any. Polypeptide profile of pegaspargase protein was also evaluated by analyzing samples using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).

	HP-SEC of pegaspargase was performed on a high-performance liquid chromatography system (LC 2010-CHT series, Shimadzu) equipped with a YMC Pack Diol-300 column (8.0 × 300 mm; 5 µm). Before injecting the sample, the column was pre-equilibrated with buffer containing 90% 50 mM sodium phosphate (pH 6.8) with 300 mM sodium chloride and 10% 2-propanol at a flow rate of 0.5 mL/min under an oven temperature of 25 °C. After equilibrating the column, 10 µg of sample was injected and analyzed in isocratic mode at a flow rate of 0.5 mL/min, and chromatographic separation was monitored at 214 nm with UV detection or using the RI detector to monitor free PEG in the samples. 

	The polypeptide profile of pegaspargase was obtained under non-reducing conditions. SDS-PAGE was carried out essentially in accordance with the Laemmli method (1970) (17). For non-reducing SDS-PAGE, 4–20% gradient polyacrylamide gel (Bio-Rad) was used. A total of 10 µg of pegaspargase protein was loaded for different test samples on the gel. After electrophoresis, protein bands were developed by either Coomassie R250 (Merck) staining or barium iodide staining using barium chloride (Merck) and iodine (Merck). Coomassie R250 is known to be more sensitive compared to Coomassie G250 and is commonly used to stain the protein molecules during SDS-PAGE. Barium iodide forms a complex with PEG and is a well-known technique to stain PEG after gel electrophoresis. In the present work, degradation of pegylated L-asparaginase molecule (aggregation or fragmentation) upon exposure to freezing and thawing or high temperature was detected using Coomassie R250 staining, while the presence of free PEG was detected by barium iodide staining. 

	The effects of freeze-thaw and high temperature on pegaspargase protein were investigated, and differences in the degradation pathways were evaluated. 

Effect of freeze-thaw on stability of pegaspargase. 

Pegaspargase protein formulated at about 8.8 mg/mL concentration in 49-mM sodium phosphate buffer in presence of 146-mM sodium chloride was exposed to freeze-thaw stress as described in the previous section (Materials and methods). After thawing, samples were analyzed by HP-SEC and SDS-PAGE. 

	Chromatographic profiles obtained with the samples of pegaspargase before and after exposure to the freezing and thawing stress are shown in 

. The chromatographic profile shows that the peak corresponding to pegaspargase appears as a single sharp peak when not exposed to freezing and thawing stress; however, upon exposure to freeze-thawing, the peak appears as a split peak with an increase in a shoulder peak and a decrease in the principal peak corresponding to the pegaspargase. 

	Results obtained with SDS-PAGE analysis of the pegaspargase samples before and after exposure to the freezing and thawing stress are shown in 

. Qualitative analysis of pegaspargase samples by SDS-PAGE under non-reducing conditions shows no significant change in the polypeptide profile of the pegaspargase upon exposure to freezing and thawing stress.

	Chromatographic profiles obtained with samples of pegaspargase analyzed by HP-SEC followed by RI detection are shown in 

. The chromatogram shows no increase in the level of free PEG in the pegaspargase samples exposed to freeze-thaw stress.

Effect of high temperature on stability of pegaspargase. 

Pegaspargase formulated at about 8.8 mg/mL concentration in 49-mM sodium phosphate buffer in presence of 146-mM sodium chloride was exposed to high temperature conditions (40 °C ± 5 °C; 75% ± 5% RH) for a maximum up to one month as described in the previous section (Materials and methods). Samples were withdrawn at different time intervals and analyzed by HP-SEC and SDS-PAGE. 

	Chromatographic profiles obtained with samples of pegaspargase withdrawn at different time intervals are shown in 

. It is observed that the retention time and shape of the peak corresponding to pegaspargase changes with time of exposure at high temperature. The peak corresponding to pegaspargase shifts toward higher retention time with increase in exposure time at high temperature conditions. The deformation of the peak corresponding to pegaspargase also increases with time when exposed to high temperature. Results obtained with SDS-PAGE analysis of the pegaspargase samples withdrawn at different time intervals after exposure to high temperature over the period of one month are shown in 

. Qualitative analysis of pegaspargase samples by SDS-PAGE under non-reducing conditions show an increase in low molecular-weight species and the presence of free PEG as analyzed by two different staining methods using Coomassie R250 dye and barium iodide, respectively.

	In separate set of experiments, pegaspargase samples were exposed to high temperature conditions (i.e., 40 °C ± 5 °C; 75% ± 5% RH and 25 °C ± 5 °C; 60 % ± 5 % RH) for up to 15 days and 30 days, respectively. Samples were withdrawn at different time intervals and analyzed by HP-SEC followed by detection by an RI detector to check the effect of high temperature on release of PEG from the PEG-protein conjugate. Chromatographic profiles obtained with samples of pegaspargase exposed to high temperature conditions and withdrawn at different time intervals are shown in 

. It is observed that the level of free PEG increases with increase in exposure time under high-temperature conditions. 

	When exposed to higher temperature, pegaspargase shows changes in the retention time and shape of the peak corresponding to the pegaspargase protein as analyzed by HP-SEC. The deformation of the principal peak corresponding to the pegaspargase protein indicates a loss of structural integrity, and the shift toward the higher retention time suggests fragmentation of the protein molecule over the period of time upon exposure to the high-temperature conditions. The results obtained with SDS-PAGE analysis also show generation of low molecular weight species or fragmentation upon exposure to high-temperature conditions and corroborate the observations made through analysis of samples by HP-SEC. 

	Similarly, the significant increase in the shoulder peak and decrease in the peak corresponding to the pegaspargase probably indicates conformational changes in the pegaspargase protein when exposed to freeze-thaw stress. The SDS-PAGE analysis of pegaspargase protein exposed to freeze-thaw stress does not indicate any fragmentation or generation of low molecular weight species upon exposure to freeze-thaw stress. 

	The increase in the peak corresponding to the free PEG in the chromatograms obtained through analysis of the pegaspargase samples using HP-SEC followed by RI detection indicates removal of PEG from the protein backbone as a function of time when stored at high temperature conditions. When exposed to freeze-thaw stress, pegaspargase shows no increase in the peak corresponding to free PEG indicating no dissociation of the PEG from the protein backbone due to freezing and thawing. These results indicate that pegaspargase degrades through depegylation along with loss of structural integrity when exposed to high temperature; however, under freezing and thawing stress it does not show any depegylation. Under freezing and thawing conditions, the changes in the peak profile for the peak corresponding to pegaspargase were observed to be different than the changes observed when pegaspargase was exposed to high temperature stress, which indicates different effects on the structural properties of pegaspargase under each stress condition. 

	Oncaspar (PEG-asparaginase, Enzon Pharmaceuticals) in the form of liquid solution is known to have a short shelf-life (eight months) due to removal of PEG from the protein backbone. Also, the storage condition for pegaspargase drug substance is between +2 °C and +8 °C, unlike the majority of biological products that are generally stored in frozen form and are known to have longer shelf-lives of approximately two years. These storage limitations necessitate continuous or immediate conversion of drug substance material to the drug product material. Storage of drug substance material longer then the stipulated time frame between +2 °C and +8 °C reduces the shelf-life available to the final drug product as it is susceptible to depegylation. Unlike other biological products, it is not possible to store the pegaspargase drug substance material under frozen conditions without presence of any cryoprotectant, as freezing and thawing may lead to structural changes in the protein of interest and impact the biological activity or storage stability before it is converted to drug product. Operations such as shipment to distribution centers and pharmacies after manufacturing the drug product also take considerable time before the product gets delivered to the doctors for the treatment. Therefore, it becomes important to have maximum shelf-life available to the drug product material. 

	To bring flexibility in the production of the drug product, it is important to manufacture a drug substance that can be stored for a longer period of time so that the drug product can be manufactured based on the market demand and supply chain efficiency. Also, sudden excursions during shipping of the drug product material may affect the stability of the pegylated L-asparaginase protein. Product may be exposed to lower or higher temperature from the real-time storage conditions during shipment. These can lead to degradation of the pegylated L-asparaginase before it gets delivered to the patient and can show sub-optimal effect during treatment. 

	It is known that pegylation of L-asparaginase increases retention time of L-asparaginase in blood circulation and thereby increases the half-life. However, exposure of pegaspargase protein to both freeze-thaw and high-temperature stress indicated loss of protein integrity, probably due to significant alteration in the structural properties of the protein molecule. The freeze-thaw stress does not lead to any significant removal of PEG from the protein backbone; however, exposure to high temperature leads to depegylation. These observations indicate that the pegaspargase (pegylated L-asparaginase) follows different degradation pathways under different stressed conditions. Further, stabilization of pegaspargase in solution is required to avoid degradation of the molecule during manufacturing process, upon storage, and shipping. Stabilization against freeze-thaw can also help to decouple the manufacturing process of the drug product from that of the drug substance and can bring flexibility for manufacturing of drug product in a multi-product manufacturing facility.

	The authors are thankful to Dr. Sanjeev Kumar Mendiratta, president, and Mr. Chandresh Bhatt, research associate, both in the department of Biotechnology at the Zydus Research Centre, Cadila Healthcare, Ahemdabad, for their scientific guidance and technical support.

	15. Shmuel Zalipsky, Enzon Pharmaceuticals Inc, “Active carbonates of polyalkylene oxides for modification of polypeptides,” US patent 5612460A, Apr. 1989.


	Vol. 43, No. 4
	April 2019
	Pages: 40–48

	When referring to this article, please cite it as C. Patel, S. Bandyopadhyay, and G. Patel, "Degradation Pathways: A Case Study with Pegylated L-Asparaginase," 

, is vice-president, both in the Department of Biotechnology, Zydus Research Centre, Cadila Healthcare Ltd., Ahmedabad, Gujarat, India; 

, is associate professor in the Department of Pharmaceutics & Pharmaceutical Technology, Ramanbhai Patel College of Pharmacy at the Charotar University of Science and Technology, Changa Campus, Changa- 388 421, Anand, Gujarat, India, gayatripatel.ph@charusat.ac.in, +91 2697 265162.

To whom all correspondence should be addressed.

It is important to address manufacturing problems associated with the shorter shelf-life of pegylated L-asparaginase (pegaspargase) upon long-term storage in the form of a solution. 

Degradation Pathways: A Case Study with Pegylated L-Asparaginase

Chronotherapeutic drug delivery systems (CRDDS) have been recognized as potentially beneficial to the chronotherapy (timeoptimized therapy) of widespread chronic diseases that display time-dependent symptoms, such as ulcers, asthma, cardiovascular diseases and arthritis. CRDDS control drug release according to circadian rhythms and the timing of symptoms.

  A number of CRDDS have been developed to synchronize medication with the intrinsic biorhythm of the disease; with conventional and modifiedrelease formulations being administered at different times of the day in accordance with the circadian onset of the disease.

People with rheumatoid arthritis (RA), usually experience peak pain in the morning, which decreases throughout the day. In this scenario, an evening once-a-day nonsteroidal anti-inflammatory drug (NSAID) schedule is recommended. If pain is worse during early afternoon or at night, however, a morning or an evening onceaday NSAID schedule may be recommended. The exact dose depends on the severity of the patient's pain and their individual physiology.

  Most newly developed antiRA agents are now available as oncedaily dosage forms; the absorption kinetics of conventional dosage forms are inadequate in the morning, which is when the need of patient is greatest, and drug levels of once-a-day formulations that have nighttime dosing indication tend to fall off towards the end of the 24h dosing interval, resulting in reduced protection against the pain when the need is more. 

Diclofenac sodium (DS) is a potent NSAID frequently used to treat RA; however, oral administration of DS as a conventional formulation generally fails to achieve the desired clinical effect because it elicits patient incompliance — many fail to take the dosage early enough in the morning to coordinate with the rhythm of RA. Therefore, the development of oral controlledrelease formulations of this drug is highly desirable.  

The Pulsincap system, developed and registered by R.P. Scherer International Corp. (MI, USA), is a special dosage form comprising a waterinsoluble capsule body enclosing a drug reservoir. The body is closed at the open end with a swellable hydrogel plug, which consists of insoluble, but permeable and swellable polymers (e.g., polymethacrylates), erodible compressed polymers (e.g., hydroxypropylmethyl cellulose, polyvinyl alcohol, polyvinyl acetate, polyethylene oxide), congealed melted polymers (e.g., saturated polyglycolated glycerides, glyceryl monooleate) and/or enzymatically controlled erodible polymers (e.g., pectin). When this capsule comes in to contact with the dissolution fluid, it swells and, after a lag time, the plug pushes itself outside the capsule and rapidly releases the drug. The length of the plug and its position of insertion into the capsule controls the lag time.

In our study, a modified Pulsincap device containing DS was developed to target drug release in the colon. This is a sitespecific and timedependent formulation; by administering the formulation at 10.00 pm, symptoms that are experienced early in the morning are avoided. This therapeutic effect is prolonged by continuously releasing the medication over an extended period of time after administering a single dose. The study objective was to explore the time-and pHdependent controlled drug delivery of DS using the pulsincap system. 

DS was obtained as gift sample from Welable Parma Ltd (India). pHsensitive methacrylic acid copolymers (Acrycoat L 100 and S 100) were supplied as gifts by Corel Pharma Ltd (India). Hydroxypropylmethyl celluloseK4M (HPMC) and hydroxypropyl cellulose (HPCH) were obtained as a gift from Colorcon (India). Sodium carboxymethyl cellulose (NaCMC), cellulose acetate phthalate (CAP), guar gum (GG) and sodium alginate (NaAlg) were purchased from S.D. Fine Chem. Ltd (India). All other chemicals used were of analytical grade. 

Step 1: preformulation study for polymer selection

According to the literature reviewed, the pH in the proximal colon ranges from 6.6 to 7.0 (~6.8).

  A combination of acrycoat L 100 and S 100 was therefore designed to maintain the optimum pH. Accordingly, acrycoat L 100 (soluble in pH 6 and above) and acrycoat S 100 (soluble in pH 7 and above) were mixed in different ratios (1:1, 1:1.5, 1:2 and 1:3) and the final mixtures underwent solubility studies at pH 6.8. Based on the solubility parameters, we ascertained that acrycoat L 100 and acrycoat S 100 at a ratio of 1:2 was highly soluble in pH 6.8. This combination was therefore selected for the preparation of microcapsules.

The acrycoat microcapsules containing DS were prepared using emulsification-solvent evaporation in an oily phase. Accurately weighed acrycoat L 100 and acrycoat S 100 in the ratio of 1:2 were dissolved in 10 mL of acetone to form a homogenous polymer solution. Drug was separately suspended in the polymer solution, which was maintained in a continuous phase consisting of 70 mL of liquid paraffin, 1% (w/w) of span 80 and 0.01% (v/v) simethicon (as antifoaming agent) and was stirred constantly at 1000 rpm using a variable speed propeller stirrer to form a uniform emulsion. Approximately 5–10 mL of acetone was added to the external continuous phase to produce a stable oil/oil emulsion and then stirring was continued (3–4 h) until residual acetone had evaporated. The microcapsules formed were separated by filtration, washed three to four times with nhexane to remove adhering liquid paraffin, and dried for 48 h in a vacuum desiccator.  

Four batches were prepared by varying the drug to polymer ratio (D:P=1:0.5, 1:1, 1:1.5 and 1:2 (w/w) named DM 1 to 4, respectively) and evaluated for their flowability, particle size, drug content, surface morphology, compatibility and 

The body and the cap of the hard gelatin capsules were separated. The body portion of the capsules was placed on a wire mesh into a desiccator, which was then exposed to formaldehyde vapours for 6 h at room temperature and dried at 50 °C for 48 h in a hot air oven. The treated capsule bodies were capped with untreated caps and stored at ambient conditions in a sealed glass container.

The microcapsule formulation that had shown good drug content and release characteristics was placed into the capsule manually. Microcapsules (D:P=1:1.5) equivalent to 100 mg of DS were incorporated into treated bodies of empty capsule shells and plugged with different amounts (10, 15 and 20%) of hydrogel polymers; that is, NaCMC, GG, HPMC, HPCH and NaAlg.  

The joint of the treated body and cap of the capsule was sealed with a small amount of the 5% w/v ethyl cellulose ethanolic solution, and the filled and sealed capsules were then coated with CAP to ensure that drug release occurred in the colon rather than in the stomach. The various parameters of coating conditions were standardized, such as dipping time (5 s), coating solution concentration, temperature (25±10 °C) and drying time (5 min) at 500 °C. Coating was repeated until a 6–8% increase in weight was obtained. The percentage weight gain of the capsules before and after coating was determined using 

is the weight of the capsules after coating, Wo is the initial weight of capsules. The capsules were dried in an oven at 50 °C for 12 h. 

Determination of drug entrapment efficiency

  for each batch was calculated in terms of percentage drug entrapment (PDE) using the formula in 

Theoretical drug loading was calculated on the assumption that all of the drug in the polymer solution used was entrapped in the microcapsules and that none was lost during the preparation of the microcapsules. 

Practical drug loading was determined by taking accurately weighed microcapsules equivalent to 100 mg of drug and crushed using a glass pestle and mortar. Methanol was added in small quantities to the mortar and transferred to a 50mL volumetric flask. The mortar was washed with methanol to remove all traces of drug and transferred to the flask. The volume was then increased to 50 mL with the same solvent. The flasks were kept on a sonicator for 5 min. The solution was then filtered using a Whatman filter paper and a suitable dilution was made. Absorbance was read at 281 nm against methanol as a blank. The PDEs of DM1 to DM4 are reported in 

Micromeritic properties of prepared microcapsules

The micromeritic properties of the microcapsules

  were determined by the angle of repose (using the fixedbase funnel method), bulk density, tapped density and Carr's index. Bulk and tapped densities were measured in 10 mL of a graduated glass cylinder. The sample contained in the cylinder was tapped mechanically by a constantvelocity rotating cam. The results are shown in 

Table 1: Physical characteristics of DS microcapsules.			

Particle size and morphology study of microcapsules

The average particle size and morphology of the drugloaded microcapsules

 (n=100) of batches DM1 to DM4 were assessed by using a digital optical microscope (Labomed CX RIII, Ambala, India) and SEM microscopy, respectively. The microcapsules' morphology was characterized by their sphericity and clumping. The results are reported in 

In vitro release profile of microcapsules

  was determined using the USP Dissolution Apparatus 1 Basket method (900 mL of pH 6.8phosphate buffer, 100 rpm, 37±0.5 °C). Microcapsules equivalent to 100 mg of drug were loaded into the basket (covered with Teflon cloth [porous polytetrafluoroethylene (PTFE) cloth]) of the dissolution apparatus. Ten millilitres of the sample were withdrawn from the dissolution media at suitable time intervals and the same amount was replaced with fresh buffer. Samples were filtered though a Whatman filter paper and suitable dilutions were made when required. The absorbance of the samples was determined at the wavelength of 276 nm using UV–Vis spectrophotometer, against pH 6.8 phosphate buffer as a blank. The amount of drug present in the filtrate was then determined from the calibration curve, and cumulative percent of drug release was calculated.  

A compatibility study was employed to assess the physicochemical compatibility between drug and the various excipients used in the formulation.

  FTIR spectrums of drug, acrycoat S 100 and L 100polymer mixture, drug–polymer physical mixture and formulation were taken and are reported in 

The polymer used in the Pulsincap dosage form should be able to swell to its maximum and eject from the capsule body in intestinal fluid. The body portion of the capsules was subjected to disintegration studies at room temperature in a buffer solution of pH 1.2, 7.4 and 6.8.  

Ten capsules were selected randomly from each batch and weighed individually for weight variation.  

In vitro release profile of the pulsatile capsule

Dissolution studies were performed using the USP Dissolution Apparatus 2 Paddle Method (900 mL of dissolution medium, 100 rpm, 37±0.5 °C). Capsules were tied to the paddle with a cotton thread in each dissolution vessel to prevent floating.

To simulate the pH changes along the gastrointestinal tract, three dissolution media with pH 1.2 (simulated gastric fluid (SGF) prepared by dissolving 2 g of NaCl and 3.2 g pepsin in 7 mL of HCl, and then adding deionized water (DW) to 1000 mL, used for 2 h), 7.4 (simulated intestinal fluid (SIF) prepared by dissolving 6.8 g of monobasic potassium phosphate in 250 mL of DW, then adding 190 mL of 0.2 N NaOH, 400 mL of DW and 10 g of pancreatin, and finally adding 0.2 N NaOH to adjust the pH to 7.4, and then diluting with DW to 1000 mL, used for 3 h) and 6.8 (simulated colonic fluid (SCF) prepared by adding 170 mL 1M acetic acid and 160 mL 1M NaOH in DW to 1000 mL, used for subsequent h) were used sequentially.

  Nine hundred millilitres of the dissolution medium was used at each time. Ten millilitres of the sample was withdrawn from the dissolution media at suitable time intervals and the same amount was replaced with fresh buffer. Samples were filtered though a Whatman filter paper and suitable dilutions were made when required. The absorbance of the samples was determined at a wavelength of 276 nm using a UV–Vis spectrophotometer, against a respective buffer as a blank. The amount of drug present in the filtrate was then determined from the calibration curve, and the cumulative percent of drug release was calculated.  

Physicochemical properties of DS microcapsules

The microcapsules of DM1 had a rough surface and the visible drug crystals on the surface indicated that the concentration of the polymeric solution was insufficient for complete encapsulation. DM2 also had rough-surfaced microcrystals, but were less crystalline than DM1. Some clumping of the microcapsules was observed in DM1 and DM2. DM3 and DM4 microcapsules were uniform, spherical, smooth and discrete.  

Chronotherapeutic drug delivery systems (CRDDS) have been recognized as potentially beneficial to the chronotherapy (timeoptimized therapy) of widespread chronic diseases that display time-dependent symptoms.