DOI: 10.31038/JPPR.2019231

Body Text

Many medicines used for pediatric patients are not available in pharmaceutical forms adapted to their needs. Actually, most oral medications are developed for adults as tablets and capsules. These forms are lack of dosing flexibility and it does not meet dosage requirements for pediatric patients from neonates to adolescents [1]. According to the updated review that including new pediatric formulations marketed in the United States (US), the country of European Union (EU), and Japan spanning the years 2007 to 2018, 16 kinds of pediatric oral formulations of which 7 drugs are ready-to-use and manipulation is required in 9 drugs, and 51 total new pediatric oral formulations of which 21 drugs are ready-to-use and manipulation is required in 30 drugs [2]. Furthermore in Nigeria, that is one of the low-middle income countries, 121 of 143 oral essential medicines (85%) were not available as flexible solid oral dosage forms and manipulation is forced [3].  When this manipulation is forced, the adult dosage forms are manipulated by either a health care provider, such as pharmacist, or by the parents and caregivers (e.g., crashing or grinding a tablet, capsule opening and sprinkling it into some foods or drink). These processes are called compounding and are commonplace for those medicines that lack pediatric formulations. Compounding procedures are regulated by provincial pharmacy standards, based on guidelines published by the National Pharmacy Regulatory Authorities (NPRA) in Canada or the US Pharmacopeia (USP). However, compounded medicines are not approved by rigorous process in each country such as Food and Drug Administration (FDA), European Medical Agency (EMA), Health Canada, Pharmaceuticals and Medical Devices Agency (PMDA) and the other region’s regulatory authorities.

The compounded medication’s characteristics and its physical property and specifications are not always known, and its compounding procedure is not well established or controlled and validated before their use in children. This is particularly true with reference to: stability, potency, content uniformity, purity or bioavailability, and so on. First and most important, the administration of the appropriate dose cannot be guaranteed. Moreover, most of the compounded medicines have an unpleasant and bad taste, which leads to adherence challenges. Even if every compounding process is taken to ensure, errors have a potential to do occur. Some compounding is needed in oncologic drugs with the concerns and obvious health risks to health providers and/or caregivers. It has the potential to expose the entire family to these toxic chemical agents.  Furthermore, the vehicle (e.g., juice, milk or yogurt) used to dissolve drugs and administer these compounded medicines, or to mask the bad taste of compounded drugs, has a potential to influence drug absorption. Especially, physicians and pharmacist need to be aware of the consequences of compounded medicines for drugs with a narrow therapeutic and safety drug index. Compounding at home also increase the variability in the product by inaccurate measurement, issues with stability or errors in instruction for manipulation [4]. Caregivers often mistake the procedure of the extemporaneous preparation [5]. A lack of bioequivalence study of compounded drugs is also concerned [6]. It is important as the formulation can lead the difference between successful treatment and therapeutic failure.

According to our survey results of 328 hospitals that have a pediatric department in Japan, that account for approximately a half of pediatric department, a total of 320 compounded drugs were identified and most of them were administered as a powder formulation. Top five percentile of compounded drugs were briefly indicated in Table 1. In Canada, the Goodman Pediatric Formulation Center which is a not-for-profit organization that is working as a facilitator between industry, regulatory and reimbursement agencies to bring commercialized pediatric formulations into Canada, also conducted a survey with hospital pharmacists from a dozen pediatric Canadian institutions in 2017. A total of 12 drugs were identified as a priority by at least one third of investigated institutions. Noteworthy, 11 of 16 drugs were same with the result of our survey, suggesting that a lack of appropriate formulation for pediatric patient is common issue in the world and global drug development may be an effective solution (Table 1, the 5^th column).

Table 1. The current state of compounding in 328 hospitals that have pediatric departments in Japan and availability in other regulatory authorities.

a. The information about compounding in Canadian hospital was provided by the Goodman Pediatric Formulations Centre. Check marks (✔) indicate the common drugs that are desired from both Canada and Japan.
b. Approved drug information was searched using the websites provided from Food and Drug Administration (FDA) in the United States, Medicines and Healthcare products Regulatory Agency (MHRA) in the United Kingdom, and European Medical Agency (EMA)
Compounded drugs in the top 5 percentile was indicated. No., number; N/A, not available.

If the global product development is proceeding, which formulation is acceptable?

In many cases, commercialized pediatric formulations are available in other jurisdictions, such as in the US and in Europe and these are often developed as liquids or suspensions (Table 1, right column). Oral liquid medicines have some disadvantages over solid medicines. The major barrier in development of oral liquid formulations is taste-masking of drugs as almost all of health provider for pediatric patients in the US reported that a taste and palatability were the greatest barriers to appropriate medication [7]. The excipients used in the development of a product need to be safe and acceptable for use in children. Excipients are typically used to optimize the formulation of the medicine to improve palatability, shelf-life and/or manufacturing processes [8]. Another problem is that liquid medicines are less chemically stable than solid medicines and require refrigeration in hot climates to guarantee their quality and efficacy. When a company manufactures develop a product for different regions, it may be necessary to adapt tastes and flavors in order to different regional preferences. Having knowledge about caregivers’ perceptions would also be needed. These issues become a bottleneck restricting to facilitating the age-appropriate drug development. In 2008, the challenges of ensuring access to appropriate drug formulations for pediatric patients led the World Health Organization (WHO) to propose flexible solid oral dosage forms as the preferred formulations for them [9]. The use of oral solid dosage forms such as dispersible tablets, powders, granules, films or sprinkles for reconstitution have a potential to be an excellent substitute for liquid formulations, because the solid product has typically better stability compared with a liquids. However, the instructions for reconstitution can be complicated for untrained or uneducated individuals, yet it is important that the final product contains the correct dosage for the patients. If these forms are administered in the absence of water they are only applicable to infants who are accepting solid such as a baby food. The risk of any error also remains.

From these compelling issues, the Academy of Pharmaceutical Science and Technology in Japan and its subcommittees named ‘the individualized medicine focus group’ and ‘the clinical formulation focus group’, decided to prepare countermeasures in medically ensured compounding procedures. We aimed to collect accurate information about the present status of compounding and unclose what information is needed to the medically ensured compounding procedure.

To facilitate the drug development globally, no regulatory and financial drivers to develop age-appropriate medicines for pediatric patient become a heavy drag, especially about off-patent drugs. In the European countries, there is a significant number of existing drugs where age-appropriate formulations are needed [10, 11]. Almost all of these drugs are generic drugs developed in the remote past for which there are no incentives or any intellectual property protection, making these drugs less interesting to invest time and cost. Requesting an age-appropriate drug development to industrial companies is one of the best solutions, however, ensuring the quality of compounded formulation by the health professionals is required as an urgent issue. While the future of ideal pediatric oral formulations may increasingly be with taste-masked, preservative-free, and user-friendly formulations including multi-particulate solid dosage forms such as mini-tablets, orally disintegrating tablets, and granules, to assure the quality of compounded drug that is not on the radar of manufacturer is needed.

Acknowledgement

This work was supported by a Research Program from the Japanese Agency for Medical Research and Development under Grant Number JP19mk0101134, awarded to H.N. We are grateful to collaborated 328 hospital pharmacists who supported us to perform our survey.

References

1. Venables R, Marriott J, Stirling H (2012) FIND OUT: key problems with children’s medicines formulations … It’s a taste issue! Int J Pharm Pract 20: 23.
2. Strickley RG (2019) Pediatric oral formulations: an updated review of commercially available pediatric oral formulations since 2007. J Pharm Sci 108: 1335–1365.
3. Orubu ES, Tuleu C (2017) Medicines for children: flexible solid oral formulations. Bull World Health Organ 95: 238–240.
4. Richey RH, Shah UU, Peak M, Craig JV, Ford JL, et al. (2013) Manipulation of drugs to achieve the required dose is intrinisic to paediatric practice but is not supported by guidelines or evidence. BMC Pediatr 13: 1–8.
5. Tomlin S, Cokerill H, Costello I, Griffith R, Hicks R, et al. (2009) Making medicines safer for children – guidance on the use of unlicensed medicines in paediatric patients. Guidelines 1–12.
6. Batchelor HK, Marriott JF (2015) Formulations for children: problems and solutions. Br J Clin Pharmacol 79: 405–418.
7. Milne CP, Bruss JB. (2008) The economics of pediatric formulation development for off-patent drugs. Clin Ther 30: 2133–2145.
8. Fabiano V, Mameli C, Zuccotti GV. (2011) Paediatric pharmacology: remember the excipients. Pharmacol Res 63: 362–365.
9. WHO. Annex 5. (2012) Development of paediatric medicines: points to consider in formulation. World Health Organ Tech Rep Ser 970: 197–225.
10. EMA. (2013) Revised provisional priority list for studies into off-patent paediatric medicinal products. EMA/98717/2012: 1–12.
11. WHO. (2015) WHO Model List of Essential Medicines for Children. 5^th Edition: 1–42.

DOI: 10.31038/JPPR.2019224

Abstract

Currently, novel drug design focused on the searching pharmacological compounds acting via influence on GPCRs heteromers. The strategy allows obtaining highly selective effects since these heteromers appear only on specific cells and tissues. Therefore, human monoclonal scFv antibodies able to recognizing GPCRs heteromers may constitute a valuable tool in modern therapies. Antibody phage display technique together with high throughput screening play a key role in the development of clinically useful immunomolecules. Therefore in the present work we focused on the comparison of various strategies used for biopanning process during phage display procedure, dedicated to isolation scFv antibodies specifically recognizing GPCRs heteromers. Experiments were conducted in two different cell lines (CHO-K1 and HEK 293) and six various selection procedures were described. Elimination of nonspecific bindings constitutes a key point during the process. Results obtained duing selection conducted in the conditions promoting internalization process were the most satisfactory.

Keywords

Phage Display, scFv antibody, GPCRs, Hetromer, Biopanning

1. Introduction

Recently heteromers (receptor heterodimers) formed by human G-Protein Coupled Receptors (GPCRs) constitute extremely important targets in the design of modern treatment strategies [1]. Alteration of pharmacological properties of the receptors included in the heterocomplex have been proven and widely described in the literature [1–3]. Research focused on finding therapeutic compounds able to selective recognition of GPCRs heteromers are currently very popular. Such strategy allows to obtain a tissue-specific acting, since the interaction between receptors engaged in the complex formation can only take place when the receptors are simultaneously expressed on the same cell. Recent data indicate the existence of clinically relevant GPCRs heteromers, important in the treatment of, among others, pain, asthma or Parkinson’s disease [3–7].

Creation of the human monoclonal antibodies with specificity towards membrane GPCRs heteromers still remains sizable challenge. To fulfil its role, the kind of antibody must recognize the structural epitope formed within the GPCRs heteromeric structure and, at the same time, not show specificity for monomeric or homomeric forms of the receptors. The phage display technology provided the best conditions for the isolation of human monoclonal antibody specifically recognizing the spatial epitope formed by GPCRs heteromers.

Currently phage display technology attracts most attention since the methods is a powerful tool, among others, in drug discovery, nanotechnology, immunology, agriculture, diagnostics, neurobiology, molecular imaging etc [8–12]. The technology developed by George P. Smith in 1985 [13] constitutes a very useful tool for the study of protein–protein, protein–peptide, and protein–DNA interactions [14]. The methodology is based on the fact that phage phenotype and genotype are physically linked [14]. A gene encoding a protein of interest inserted into a gene of bacteriophage coat protein is expressed and presented on the phage surface. The concept is simple: a population of phage is engineered to express random-sequence peptides, proteins or antibodies on their surface [8]. From this population, a selection is made of those phage that bind the desired target [8]. Hereby, large proteins libraries can be screened and unique molecules which bind to their targets with high affinity and specificity can be isolated [15]. The advantage of the method is the possibility of the production of monoclonal antibodies recognizing antigens that cannot be used to immunize an animal due to their toxicity, non-immunogenicity or presence in complexes on the surface of cell membranes [16].

ScFvs (Single Chain Variable  Fragment) are small monoclonal antibody fragments composed of immunoglobulin-heavy (VH) and light chain-variable (VL) regions with a flexible peptide linker designed to connect the two chains such that the antigen binding site is retained in a single co-linear molecule [17]. The kind of antibodies can be derived from phage display libraries [18,19]. ScFvs are very useful in pharmacology and diagnostic fields as well as in drug delivery issues since they can function as targeting ligands. Functionalization of the surface of drug carriers by scFvs enable controlled transport of pharmacological compounds directly to the desired place of action [20].  ScFvs, in comparison to the much larger Fab, F(ab)₂, and IgG forms, are characterized by better tissue penetration, lower retention times in non-target tissues, faster blood clearance and, above all, reduced immunogenicity. These features cause that they are very useful for therapeutic applications [21].

The main purpose of presented work was the description of different strategies which may be used during phage display procedure for the isolation of scFvs antibodies specifically recognizing human GPCRs heteromers. To separate the phages that effectively bind defined heteromer it is extremely important to carry out the selection rounds in conditions most similar to those in which desirable receptors occur naturally in the cells, which allowed to preserve the native spatial conformation of the heteromer. Elimination of nonspecific binding without losing rare specific ones seems a serious challenge. Therefore, in the work several various types of selection were presented. The experiments were independently conducted for two GPCRs pair: dopamine D₂ (D₂R) and serotonin 5-HT_1A (5-HT_1AR) receptors as well as  dopamine D₂  and serotonin 5-HT_2A receptors. Similar results were obtained for both cases. For simplicity in the work the outcomes for D₂–5-HT_1A were presented.

2. Materials and Methods

2.1 Cell culture and Transfection

CHO-K1 cells (ATCC) were grown in RPMI (Sigma) medium; HEK 293 cells (ATCC) were grown in minimal essential medium (MEM) (Sigma) with 1% L-glutamine. Both medium were supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma). All cells were cultured at 37 °C inside a humidified incubator in an atmosphere of 5% CO₂. Transient and stable transfections were made by using the TurboFect reagent (Thermo Sci.) according to the manufacturer’s protocol. Early passages of CHO-K1 as well as HEK 293 cells were stably transfected (1.5 µg DNA) with the plasmid pcDNA3.1(+) encoding the human 5-HT_1AR or the human D₂R (UMR cDNA Resource Centre) separately or cotransfected with both vectors. Stable cell lines expressing D₂R and/or 5-HT_1AR were obtained after the addition of the selection antibiotic, G418 (Sigma), at a final concentration of 0.75 mg/ml. Cells resistant to the antibiotic and stably expressing investigated receptors were analysed by RT-PCR (data not shown). Forty-eight hours before the selection experiment, stable cell lines were, additionally, transiently transfected with 0.5 µg DNA (per 10 mm plate area) encoding the desired receptors.

2.2 Screening of phage-displayed scFv libraries

The human antibody scFv phagemid library Tomlinson I+J (Geneservice) was used. The library J was amplified and titrated (used the library size was 1.9×10¹² cfu) according to the manufacturer’s protocols using E. coli TG1 cells [18]. Biopanning was performed on positive (+) and negative (-) cells expressing desired receptors. CHO-K1 as well as HEK 293 cell lines were used. CHO+ and HEK+ cells constituted cells expressing D₂–5–HT_1A heteromers whilst CHO- and HEK- cells expressed separately D₂R or 5–HT_1AR and were mixed before experiment in a 1:1 ratio. Four – six positive rounds of selection followed by prenegative selection and one final negative selection were performed independently on both mentioned above cell lines. Briefly, during preselection amplified phages were blocked for 2 hr at room temperature (RT) in 3% MPBS (with stirring from time to time) and then were added to the negative cells (growing on 150 mm plates (15x) 95% confluence) or to the cells suspension – 10⁸ cells) for 2 hr at RT (with stirring from time to time). Next, the cells were collected and centrifuged (10 min at 1000 rpm). Supernatant containing unbound phages was used to positive selection. The final negative selection were performed similarly to preselection.

2.2.1 Positive selection – type A and B

Phages derived from preselection were added to the culture medium of positive cells growing on 150 mm plates (10x, 95% confluence) and were incubated with shaking for 2 hr at 37 °C (type A) or at RT (type B). After that time, unbound phages were washed away with PBS buffer. The number of washes after each round of selection has been shown in Table 1. In the next step, the cells were collected, centrifuged and resuspended in PBS containing 1 mg/ml trypsin. The suspension was incubated on the rotator for 10 min at RT and then centrifuged (10 min, 1000rpm, 4 °C). The supernatant containing the desired phages after titration and amplification was used for another round of biopanning.

Table 1. Number of washing steps performer after each round of selection during phage display procedure.

2.2.2 Positive selection – type C

Positive cells growing on 150 mm plates (10x, 95% confluence) were used. Phages after preselection were added to the cell medium for 2 hr (at 0 °C). Then, the medium was removed and cells were washed 3 times with cold PBS. Between washings, RPMI medium was added, and the cells were incubated on ice for 10 min. Then, the temperature of incubation was changed to 37 °C for 20 min. In the next step, the cells were washed 4 times using elution buffer (100 mM glycine, 150 mM NaCl, pH 2.8). The number of washes increased (twice each time) with subsequent rounds of selection. Finally, cells were harvested from the plates and resuspended in PBS containing trypsin (1 mg/ml) for approximately 15 min (until cell lysis). Then, the obtained suspension was centrifuged (10 min, 4000 rpm, 4 °C), and the supernatant containing the desired phages after titration and amplification was used for another round of biopanning.

2.2.3 Positive selection – type D

The experiment was performed in the cells suspension expressing both desired receptors (positive cells). The number of used cells (in the first round was 2 × 10⁷) increased twice with subsequent rounds of selection. Phages were incubated with the cells for 2 hr with shaking at RT. Then unbounded phages were eliminated by washing (PBS) and centrifugation (10 min, 1000 rpm, RT) (Table 1). In the next step cell pellet was resuspended in PBS containing trypsin (1 mg/ml) for approximately 5 min. Then the suspension was centrifuged (10 min, 4000 rpm, RT), the supernatant was collected and after titration and amplification was used for another round of biopanning.

2.2.4 Positive selection – type E and F

In case E and F experiments were performed similarly to type D. Differences appeared at the stage of acquiring bounded phages. In type E, after washing steps cell pellet was incubated with H₂O (caused cell lysis) for 10 min with shaking at RT. Then trypsin (1 mg/ml) in PBS was added to the suspension for 10 min incubation at RT. Finally, the desired phages were obtained from the supernatant after centrifugation (10 min, 4000 rpm, RT).

In case F, after washing steps the cell pellet was incubated for 15 min at RT with clozapine (10^–9 M). Then, after centrifugation (10 min, 1000 rpm, RT) the supernatant was collected and the trypsin (1 mg/ml) in PBS was added. Obtained phages after titration and amplification were used for another round of biopanning.

2.3 Polyclonal phage ELISA

The quality of the biopanning process was monitored using polyclonal phage ELISA. Amplificated phages (50 µl) obtained after selection rounds were incubated with 50 µl of 4% MPBS for 2 hr at 37 °C. Then, 1.5 × 10⁵ cells (resuspended in 50 µl of medium with 5% FBS) were mixed with previously blocked phages. Both the positive (CHO+ or HEK+ cells expressing D₂–5-HT_1A heteromers) and the negative (CHO- or HEK- cells  expressing a single type of receptor mixed at the 1:1 ratio) probes were used. After 1 hr ice incubation, the washing step was conducted 3 times at 4 °C using 200 µl cold PBS. Each washing round ended with centrifuging (1000 rpm x g, 10 min, 4 °C), and the supernatant rejection. Detection of bound phages were determined by horseradish peroxidase (HRP)-conjugated anti-M13 monoclonal antibodies (GE Healthcare). Briefly, after washing, the probes were incubated with the antibody resuspended in a 1:5000 ratio in 3% MPBS for 30 min on ice and then washed 4 times as described above. Finally, 100 µl of TMB substrate (GE Healthcare) and 100 µl of 1 M HCl (per well) were used to induce the reaction. The absorbance was measured at 450 nm. Experiments were performed in triplicate.

2.4 Monoclonal phage ELISA

Based on the results of polyclonal phage ELISA, phage clones from rounds characterized by the highest affinity against positive cells (CHO+ or HEK+) cells were randomly selected for monoclonal phage ELISA experiments. Individual bacterial colonies were inoculated into 96-well plates containing 100 µl 2xTYAG (2xTY (Bioshop) with 100 μg/ml ampicillin (Sigma) and 1% glucose (Bioshop)) medium per well and cultured overnight at 37 °C (250 rpm shaking). Then, 5 µl of the culture (from each well) was added to fresh 200 µl 2xTYAG medium and cultured with shaking (250 rpm) at 37 °C for 2 hr. Next, 10⁹ helper phages were added to the each well and incubated for 1 hr and at 37 °C with shaking at 250 rpm. After centrifugation (1800 x g, 10 min), the supernatants were removed, and bacterial pellets were resuspended in 200 µl 2xTYAKG (2xTY containing 100 μg/ml ampicillin, 50 µg/ml kanamycin (Sigma) and 1% glucose) medium and incubated overnight at 30 °C (250 rpm). Finally, after plates centrifugation (1800 x g, 10 min), the 50 µl of supernatants (containing monoclonal phages) were used in the phage ELISA as described above (2.3).

3. Results and Discussion

The phage display technology has provided the ability to create antibody libraries that contain a great number of phage particles, from which each one encodes and displays different molecules (10⁶–10¹¹ different ligands in a population of > 10¹² phage molecules) [14]. Finding the most suitable molecule that reflects desired properties depends largely on proper conduction of biopanning experiments. It is extremely important especially in case of isolation of monoclonal scFv antibodies directed towards GPCRs heteromers.  Because the kind of antibody must recognize spatial epitope that naturally occurs within heteromer structure, the key issue constitute such choice of experimental conditions which would ensure a natural environment in which heteromers may be formed. Generally the biopanning method is based on repeated cycles of incubation, washing, amplification and reselection of bound phage [14]. The target molecule may be immobilized on solid support as microtiter plate wells, PVDF membrane column matrix or immunotubes magnetic beads and even on whole cells [14]. In our case target antigen (defined heteromer) was presented on the surface of living cells. This kind of biopanning process is more complicated than in case when purified antigen is immobilized on the plate surface. A large number of variables can affect the behaviour of cells, which can translate into the quality of expressed heteromers and the key parameter here is the presentation of the ideal heteromer structure for the selection.

Several parameters affect biopanning efficiency, including antigen concentration, temperature, washing stringency (washing number and composition of wash buffer) as well as blocking and elution buffer composition [22]. Therefore, in the present work six various strategies (types A-F) of selection were described. Experiments were performed depending on the temperature (0 ^oC, RT, 37 ^oC), in the conditions that promote internalization process (type C), in the conditions where heteromer-bounded phages were isolated from interior of the cells after water lysis (type E), in the conditions where heteromer-bounded phages were displaced by clozapine (type F). Moreover during experiments washing stringency was maintained (Table 1). Additionally, two different cell line (CHO-K1 and HEK 293) were adopted to the procedure. Experiments were performed for attached cells as well as in the cells suspension. CHO-K1 cells are well attached to the surface than HEK 293 cells which makes them better for experiments conducted on plates where rigours washing steps are made. Comparison both used cell lines indicate that results obtained for such experiments using HEK 293 cells were definitely worse (Table 2 A,B). Probably most phages were lost during washing steps which was related to the easy detachment of cells from the plate.

Table 2. Titre of phages after each selection round (I-VI positive selection followed by negative preselection, VII – negative selection). Procedure performed on A) CHO-K1 cells, B) on HEK 293 cells.

The selection process was assessed by monitoring the enrichment ratio and polyclonal phage ELISA. The increasing titre of phages as well as polyclonal phages ELISA results indicates the correctness of the biopanning process and corresponds with the enrichment of phages that specifically recognized the defined heteromer. Preselection conducted on negative cells provided initial elimination of phages exhibited binding affinity towards monomeric forms of receptors included into D₂–5-HT_1A heteromers as well as towards other molecules presented on the cell surface. It was very important and critical move because it enrich the amount of phages acquiring potentially, desired binding properties before the actual positive selection. Results obtaining during experiments performed without negative preselection was not as satisfying as expected (data not shown). The biopanning rounds were repeated until the obtained results (phages titre and polyclonal ELISA) related to positive (specific to defined heteromer) phages reached a plateau or started to decline (Figure 1,2, Table 2A,B). In case of experiments performed on CHO-K1 cells the plateau was achieved faster (after 4 round of selection) (Table 2A). For both cell lines. the level of polyclonal phages binding to positive cells increase with the number of selection. Moreover, in the initial rounds the difference between “phages” binding affinity to positive vs. negative cells was much smaller than in case of further rounds. Similarly to preselection, the last, only negative selection plays also an important role in the elimination of nonspecific bounded phages. As we can see the phages titre as well as binding specificity significantly increased after the last negative selection. Conducting further, only negative selection did not caused further increase of phages titre and binding affinity (data nor shown).

Figure 1. Polyclonal phage ELISA. Enrichment of phages that specifically recognized the D₂–5-HT_1A heteromer. Experiments performed in CHO-K1 cell line. A-F various selection types.

Figure 2. Polyclonal phage ELISA. Enrichment of phages that specifically recognized the D₂–5-HT_1A heteromer. Experiments performed in HEK 293 cell line. A-F various selection types.

After the selection process, the specific binding of individual monoclonal phages to cells presenting defined heteromers was determined by monoclonal phage ELISA techniques. Such tests were conducted on various cell lines (CHO-K1, HEK293) expressing desired receptors in pairs or individually. The kind of experiment enable real identification of monoclonal phages displaying desired scFv molecules on the surface. About 1000 phages obtained after each type of selection were tested. Table 3 presents the results obtained for the three best phages for a given type of selection. The most satisfactory results (the highest heteromer specificity) were obtained in case of selection in conditions conducive to internalisation (type C) as well as in case of selection F where phages were displaced by clozapine. Clozapine is a pharmacological compounds which well-known affinity towards both D₂R and 5-HT_1AR [23–25]. Moreover its influence on various GPCRs heteromer formation has been documented [26–27]. As we can see here (Fig 1,2, Table 2), the titre of phages after final selection round (type F) was not as higher as in C case, however, the quality of isolated scFvs were very promising (Table 3).

Table 3. Binding level of various monoclonal phages specific to D₂–5HT_1A heteromer (results for 3 the best phages) presented on positive cells (CHO+ or HEK+ cells) in relation to: CHO-K1, CHO- cells – or HEK 293, HEK- , determined by ELISA technique. [R] –ratio of positive (absorbance 450nm positive cells) vs negative signal (absorbance 450nm negative cells).

Comparison of the results obtained for both used cell lines indicates that in case of experiments performed on HEK 293 cells, effects were not as promising as in case of CHO-K1 cells. The visible differences appeared only at the monoclonal phages analysis stage. Phages, isolated based on selection on HEK 293 cells were less specific to desired heteromer (Table 3). The phenomenon, beyond the quality of the experiment itself, may be correlated with endogenous expression of D₂R on the HEK 293 cell surface.

4. Conclusion

In conclusion presented results indicate the phage display technique as a valuable tool for isolation of human monoclonal scFv antibodies towards GPCRs heteromers. At the same time, they point to the key role of appropriate conditions during biopanning process. Based on our experience the best binding parameters were obtained for phages isolated after selection in the conditions promoting internalization process. A very important is also a proper choice of cell line dedicated to such procedure.  Elimination of nonspecific bindings by negative preselection as well as the last round of the only negative selection constitutes a key point during the biopanning process.

5. Acknowledgment

The Faculty of Biochemistry, Biophysics and Biotechnology is partner with the Leading National Research Centre (KNOW) supported by the Ministry of Science and Higher Education. The work was also co-financed from European Union within Regional Development Fund – Grants for innovation – PARENT/BRIDGE Programme – POMOST/2011–4/5 and N N401 009640 project.

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DOI: 10.31038/JPPR.2019223

Abstract

Guaifenesin is used as an expectorant. One of its available over-the-counter tablets is immediate- and extended-release bilayer in design. The purposes of this project were [1] to compare the in vitro release profiles between one-stage dissolution (using water as medium) and two-stage dissolution (using 0.1 N HCl and phosphate buffer pH 6.8), and [2] to explore the polymeric release mechanism from the tablet. We also proposed a less acidic liquid chromatographic mobile phase, 50% methanol (which stability indication method was validated), to compare with the mobile phase described in the Guaifenesin Tablets monograph (methanol/water/glacial acetic acid, 40:60:1.5 v/v/v). With the dissolution duration, temperature, paddle stir rate, and sampling schedule being kept the same, the release profiles using Monograph mobile phase to quantify the samples collected from both dissolution methods were found similar (n = 4). When the same set of two-stage dissolution samples were subject to two different mobile phases, the profiles in the acid stage were similarly. But 50% methanol quantified the Buffer Stage samples less than Monograph mobile phase since hour 3, when 250 mL of 0.2 N tribasic sodium phosphate was added and the medium adjusted with 2 N NaOH to pH 6.8. The differences were 12.7% ± 1.4% at hour 4, and 20.9% ± 1.6% at hour 12 (n = 4, p < 0.001). As to the polymeric control, the computed exponent (n value) in the Peppas power law approximation was in the range of 0.7, which suggested release mechanism is anomalous transport. The cross-section of the retrieved tablets at end of the dissolution studies supported the inference.

Keywords

Extended-Release Bilayer Tablets, Liquid Chromatography, One-Stage Vs. Two-Stage In Vitro Dissolution, Peppas Power Law Approximation, Polymeric Control.

Introduction

Guaifenesin, an expectorant, is available over-the-counter in two strengths, 600 mg and 1200 mg [1, 2]. One brand is ER bilayer tablets containing white Immediate Release (IR) and blue ER layers (Figure 1). The Guaifenesin Tablets monograph in USP-NF 2018 (3) describes its dissolution procedure as medium: water; 900 mL; apparatus 2: 50 rpm; time: 45 min and procedure: determine the amount dissolved using UV absorbance at 274 nm, the tolerance was not less than 75% (Q) of the labeled amount dissolved in 45 min [3]. Judging from the dissolution time of 45 min, it is for IR. There are no guidelines specifically written for Guaifenesin ER Tablets in the monograph.

Figure 1. Guaifenesin 1200-mg bilayer ER tablets. (a) Dissecting an intact tablet illustrated no coating was applied to the core tablet, (b) a tablet retrieved from a dissolution vessel at the end of the 12-h study.

Nicholas Peppas introduced a power law approximation to describe drug release from a dosage form. Both the exponent n and the prefactor k of the equation depend on the dosage form geometry, the relative importance of relaxation and diffusion in the pure polymer swelling controlled drug delivery system [4, 5]. Therefore, the first objective of this project was to search within the General Chapters of USP-NF [3] for an in vitro dissolution method, which might be used to study Guaifenesin ER Tablets. Second, a less acidic Liquid Chromatographic (LC) mobile phase (50% methanol, apparent pH 7.0). Both the Monograph mobile phase (methanol/water/glacial acetic acid, 40:60:1.5, apparent pH 3.08) and the proposed mobile phase (50% methanol) were used to establish standard curves of guaifenesin dissolved in the media used for dissolution study, and quantify the dissolution samples to determine the cumulative drug release from the tablet. Third, it aimed at the comparison of the in vitro release profiles between a one-stage dissolution method × 12 h (using water as medium) and two-stage method (composed of Acid Stage × 3 h, and then Buffer Stage × 9 h). Furthermore, the in vitro release data were plotted according to power law approximation to differentiate the drug release mechanism among Fickian diffusion, anomalous transport and polymer chain relaxation (polymer swelling).

Materilas and Methods

Materials

Over-the-counter 1200 mg Guaifenesin ER Bi-layer Tablets containing a white IR layer, and a blue ER layer for 12 h release (Lot BY646, distributed by Reckitt Benckiser, NJ) were purchased from a local pharmacy. Guaifenesin (Spectrum Chemical, Lot 2EC0288), methanol, glacial acetic acid, 10 mL syringes, 0.22 micron 25 mm Nylon syringe filters were obtained from VWR International (Bridgeport, NJ).

Methods

Examination of Tablet Formulation Development

Four tablets randomly taken from the original container were weighed. The tablets were cut vertically to inspect any coat being applied to the tablet core. The inactive ingredients and their pharmaceutical functions were conducted through literature search [3, 6].

Standard Preparations

Guaifenesin powder was dissolved in three different matrices to address the aims of this study. They were deionized water, 0.1 N HCl and phosphate buffer pH 6.8. In deionized water it was made into 2 mg/mL as the stock solution. It was further diluted with a diluent (made of one part of water and four parts of 45% methanol) into different concentrations of standard preparations, 0.0002, 0.002, 0.08, 0.2, 0.4, 1, 1.2, 2 and 10 mg/mL. For constructing the standard curves with 0.1 N HCl and phosphate buffer 6.8, the standard stock solution was prepared into 10 mg/mL. It was further diluted with a diluent: water – 45% methanol (1:4, v/v) to ensure the work ranges were covered.

UV Spectroscopy

The Scan mode of a Cary 50 UV-Vis Spectrophotometer from Agilent Technologies determined the optimal wavelength of guaifenesin, and Sample Read mode recorded the absorbance of standard solutions.

Liquid Chromatographic Conditions

Agilent Series 1100 (Hewlett Packard) contained Vacuum Degasser, Binary Pump, Auto Sampler, Column Thermostated Compartment, and Variable Wavelength Detector. Two different mobile phases quantifying the standard solutions and in vitro dissolution samples were the mobile phases which may be found in Guaifenesin Tablet Monograph (methanol/water/glacial acetic acid, 40:60:1.5, v/v/v [3], apparent pH was 3.08), and our proposed mobile phase (50% methanol, apparent pH was 7.0). The flow rate was set at 1.0 mL/min and run time 7 min/cycle. The selected column was Luna C18 (USP L1, 4.6 × 150 mm, 5 µm) and the injection volume was 20 microliters.

Potency tests determine the drug content in a sample using HPLC, titration or microbial assay. Stability test, shelf-life and beyond-use date are interchangeable.

Methods of determining potency may or may not be stability indicating, but a stability-indication method can determine both potency and stability [7]. Because we proposed using 50% methanol as the mobile phase, which was considered as a new LC method, the stability-indication method must be validated. A know amount of guaifenesin was dissolved in water (one-stage dissolution medium) and in two-stage dissolution media (0.1 N HCl, and phosphate buffer at pH 6.8 respectively). The samples were subject to the following conditions: (a) 50 ^oC Heat for 1 h, then cooled to room temperature quickly, (b) 0.1 N HCl (acid) for 1 h prior to neutralized by 0.1 N NaOH, (c) 0.1 N NaOH (base) for 1 h and neutralized with 0.1 N HCl, and (d) 3% hydrogen peroxide solution for 5 min (8). The samples were then quantified using our proposed mobile phase (50% methanol) to ensure the degradant peaks generated by the experimental conditions were separated from analyte (guaifenesin) peak by the resolution (Rs) ≥ 2 [8].

In Vitro Dissolution Methods

Within the USP-NF 2018 two different dissolution methods are stated. One method is in the section of Extended-release Dosage Forms of General Chapters: <711> Dissolution. It describes as “Procedures and medium are as directed for Immediate-Release Dosage Forms in the monographs. But the test-time points generally are three and are expressed in hours.” The dissolution procedures and medium were searched within Guaifenesin Tablets monographs as directed in General Chapters: <711>. The description was medium: 900 mL water; Apparatus 2: 50 rpm; and time: 45 min. This method will refer as one-stage method in the remaining text. The second method is a two-stage acid-buffer method. It is present in General Chapters: <711> Dissolution, but in the section of Delayed-release Dosage Forms, Method A and Method B. This project followed Method a procedure to avoid contamination, loss of tablets, or breakage of a dissolution vessel. The dissolutions and assays are briefly described in the below.

One-stage Dissolution

The one-stage 12-h dissolution study followed the guidelines in Guaifenesin Tablets monograph (medium: 900 mL water; apparatus 2: 50 rpm), except the time was extended to 12 h to study the drug release mechanism. The sampling schedule was at 0.25, 0.75, 1, 3, 4, 6, 8, 10 and 12 h. One part of each dissolution sample was diluted with four parts of 45% methanol prior to subject to LC assay using mobile phases, 50% methanol as well as methanol/water/glacial acetic acid, 40:60:1.5, respectively. The LC AUC of each time point was converted into the cumulated amount of drug release using the established standard curve which medium in the one-stage dissolution was water (n = 3).

Two-stage Dissolution

For the two-stage 12-h dissolution study, an ER bilayer tablet (Figure 1a) was placed in a vessel of USP Dissolution Apparatus 2 containing 750 mL of 0.1 N HCl at 37.0 ± 0.5 ^oC and stirred at 50 rpm for 3 h. Then 250 mL of 0.2 M tribasic sodium phosphate was added, pH was adjusted to 6.8 with 2 N NaOH. The study continued for another 9 h at 50 rpm while the vessel medium maintained at 37.0 ± 0.5^°C. Sampling schedule was selected the same for both one-stage and two-stage dissolution groups, 0.25, 0.75, 1, 3 h (Acid Stage), and 4, 6, 8, 10, and 12 h (Buffer Stage). One part of a dissolution sample was diluted with four parts of 45% methanol (diluent) prior to LC assay. Both mobile phases stated in Section 2.2.4 were applied in the LC system, respectively. The LC AUC of each time-point dissolution sample was converted into the cumulated amount and cumulative percent of drug release using the established standard curves which media were 0.1 N HCl and phosphate buffer pH 6.8.

Data Management for Peppas Power Approximation

After the amount of drug release at a sampling time was known (M_t), it was divided by the infinite amount of release (M_∞), which equaled the tablet label strength (1200 mg). This ratio was then plotted against time (in h) to form a power equation (Equation 1) using the power trend line option in the scatter chart of an Excel worksheet. The power exponent (n) and prefactor (k) in the Peppas equation [6] were thus known from the trend line. The obtained values of release exponent from power trend line was further matched the n value in Table 1 [4, 5] to determine the drug release mechanism.

Table 1. The release exponent n of the Peppas power equation and drug release mechanism from polymer-controlled delivery systems in cylinder geometry (4, 5)

Results

Examination of Tablet Rational Development Approach

The ER bilayer tablets taken from its original container were weighed as 1.454 ± 0.011 g (n = 4, relative standard deviation of the tablet weight was 0.76%). Judging from the dissection the tablet cores were not coated (Figure 1a). The inactive ingredients are FD&C blue #1 aluminum lake (coloring agent), hypromellose (controlled-release agent), magnesium stearate (lubricant), microcrystalline cellulose (tablet diluent and disintegrant), and sodium starch glycolate (tablet disintegrant).

Ultraviolet Spectrophotometric and HPLC Linearity

Guaifenesin dissolved in water to prepared into two different concentrations, 0.008 and 0.2 m/mL, then they were scanned from 190 to 790 nm using Cary 50 spectrophotometer (Agilent). In addition to 274 and 276 nm described in the Guaifenesin and Guaifenesin Tablets monographs [3], 270 nm can also be used as the wavelength to quantify guaifenesin. Thererfore, 270 nm was used in the remaining project including assaying dissolution samples. The standard linearity built using water as solvent and quantified using UV spectrophotometer was 0.001 mg/mL to 0.2 mg/mL (100 fold), while that quantified using LC was 0.001 mg/mL to 1.2 mg/mL (1200 fold). The R² (coefficiency of determination) when LC AUC (y-axis) in correlation with UV absorbance (x-axis) ranged from 0.001 to 0.2 mg/mL was 0.9999.

The chromatograms of stability indication method showed the degradant peaks generated by subjecting to 0.1 N HCl (acid), 0.1 N NaOH (base), 50 ^oC (heat) and 3% hydrogen peroxide solution under the exposure time periods described in Section 2.24 either did not produce degradant peak or separated well from analyte (guaifenesin) peak. All the resolution (Rs) were greater than 2 (Please refer to supplemental file).

One-stage In Vitro Dissolution Study Using Water as Medium

The dissolution duration and temperature, paddle stir rate, and sampling schedule were chosen as 12 h, 37.0 ± 0.5 ^oC, 50 rpm and 0.25, 0.75, 1, 3, 4, 6, 8, 01 and 12 h. When the dissolution samples were quantified using Monograph mobile phase, the bilayer ER tablets released 28.0 ± 1.4 % guaifenesin into water at 45 min, 30.4 ± 1.7 % at 1 h, 44.1 ± 6.0 % at 3 h, and 72.1 ± 4.4 % at 12 h (n = 4, Table 2a). When the same samples were assayed using 50% methanol as the mobile phase, the drug releases were: 29.5 ± 1.5 % at 45 min, 32.2 ± 1.6 % at 1 h, 44.7 ± 3.0 % at 3 h, and 75.7 ± 4.3 % at 12 h (n = 4, Table 2a). The dissolution profiles were almost identical and displayed as bi-phasic release when either mobile phase was used to quantify the same set of dissolution samples (n = 4, Figure 2).

Figure 2. Similarity of one-stage 12-h in vitro dissolution profiles between the Monograph mobile phase (methanol/water/glacial acetic acid, 40:60:1.5, v/v/v) and the proposed mobile phase (50% MeOH) when water was the chosen dissolution medium.

Table 2. Cumulative percent of guaifenesin release from bi-layer tablets at key sampling points

(a) One-stage dissolution with different mobile phases

*Two-tailed distribution

(b) Different dissolution methods, but same mobile phase (monograph MP) to assay

*Two-tailed distribution
^ξ Statistically significant

(c) Same two-stage (0.1 N HCl 3 h, then Buffer 9 h) dissolution method with different mobile phases

*Two-tailed distribution
^¥ Statistically significant in both paired and independent t-tests.

One-stage versus Two-stage In Vitro Dissolution Study Using Monograph Mobile Phase

The dissolution duration and temperature, paddle stir rate, and sampling schedule were kept the same as Section 3.4, but only the Monograph mobile phase (methanol/water/glacial acetic acid, 40:60:1.5, v/v/v [3], apparent pH was 3.08) was used. The drug release between one-stage and two-stage methods were 28.0 ± 1.4 % vs. 31.7 ± 4.1 % at 45 min, 30.4 ± 1.7 % vs. 36.3 ± 4.0 % at 1 h, 44.1 ± 6.2 % vs. 56.4 ± 4.4 % at 3 h, and 72.1 ± 4.4 % vs. 79.4 ± 1.9 % at 12 h (n = 4, Table 2b). These releases in these sampling points were different statistically after 45 min dissolution study (Table 2b, Figure 3a).

Figure 3. Dissolution method and mobile phase as factors impacting guaifenesin release profiles: (a) design of dissolution method: one-stage in water versus two-stage (acid and buffer stages) when USP Guaifenesin Tablets monograph mobile phase, and (b) selection of mobile phase: 50% MeOH versus USP Guaifenesin Tablets monograph mobile phase. A depression of 12.7% ± 1.4% (n = 4) present 1 h after the medium pH was adjusted to 6.8 (that is the end of 4 dissolution hours). This depression continued until dissolution ended (see text).

Two-stage In Vitro Dissolution Study Using 50% Methanol versus Monograph Mobile Phase

The dissolution method was a tablet was placed in 750 mL of 0.1 N HCl for 3 h, and then 250 mL of 0.2 M tribasic sodium phosphate was added into the apparatus vessel with the pH being adjusted to 6.8. The dissolution duration and temperature, paddle stir rate, and sampling schedule were kept the same as Section 3.4, but both monograph mobile phase (methanol/water/glacial acetic acid, 40:60:1.5, v/v/v [3], apparent pH was 3.08) and the proposed mobile phase (50% methanol) were used respectively. The resultant AUC were converted into cumulative % of release and compared. The release profiles in Acid stage (time 0 to 3 h) quantified by both mobile phases were almost identical (Figure 3b). The cumulative percent of releases in Acid stage using Monograph mobile phase and 50% methanol as mobile phase were 31.7 ± 4.1 % vs. 32.2 ± 2.8 % at 45 min, 36.3 ± 4.0 % vs. 36.0 ± 3.0 % at 1 h, 56.4.1 ± 4.4 % vs. 55.0 ± 5.6 % at 3 h (Table 2c). Never the less, the profiles were statistically different in the Buffer stage (3 to 12 h, Figure 3b, and Table 2c). The cumulative percent of releases in Buffer stage using Monograph mobile phase and 50% methanol as mobile phase were 55.2 ± 2.1 % vs. 45.8 ± 0.9 % at 4 h (which means one hour after the pH had been adjusted to 6.8), 79.4 ± 1.9 % vs. 62.3 ± 0.7 % at 12 h (Table 2c).

Power Law Approximation

On order to fit Power Law Approximation equation, the data had to be taken from extended release region. Since the studied tablet was designed as IR/ER bilayer, we subtracted the cumulative amount of release from the dissolution study at a particular sampling point from the cumulative amount of drug release in the first hour (immediate layer) as M_{t iin} Equation 1, and further divided M_t by M_∞. M_∞ was the label amount (1200 mg) minus cumulative amount in 1 hour. The fraction was then plotted against the time of drug released from the ER layer (the total dissolution time minus 1 hour in immediate release layer). Power equations in different dissolution methods and mobile phases were obtained using Excel scatter plot trendline options. The value of the power exponent (n) was 0.767 for both mobile phases, while the prefactor (k) was 0.1007 for Monograph mobile phase and 0.1068 for the proposed mobile phase (50% methanol). The reason of choosing data from hour 2 to hour 12 to fit Peppas power law was based on General Chapter <1088> In Vitro and In Vivo Evaluation of Dosage Forms describes that “For immediate-release dosage forms the in vitro dissolution process typically requires no more than 60 min…” [3]. According to Table 1, the obtained power exponent illustrate that the ER layer of this bi-layer tablet follows Fickian diffusion (Table 3). The Peppas power law was also applied to two-stage dissolution from hour 2 to hour 12 as well as hour 4 to hour 12 using both mobile phases. But the data from hour 2 to hour 12 in the two-stage dissolution method using either mobile phase to quantify are not reported here due to the transition of Acid stage into Buffer stage at the end of hour 3 (Table 3) to avoid misguiding.

Table 3. The release exponent n of the Peppas power equation and drug release mechanism using the ER layer dissolution data from hour 2 to hour 12, and hour 4 to hour 12

*Due to the transition between acid stage and buffer stages at hour 3 (see text).

Discussion

Hydrogels are polymer networks that contain a substantial amount of water. Dry polymer networks can absorb tens, hundreds, or even thousands of times their weight in water without or with dissolving. They have the properties to those of soft biological tissues and of great utility in pharmacy due to a low interfacial tension and less irritation [9]. The polymer network was able to sustain its own structural integrity through cross-linkage [10]. According to General Chapters: <1088> In Vitro and In Vivo Evaluation of Dosage Forms describes that “For immediate-release dosage forms the in vitro dissolution process typically requires no more than 60 min…”. Using this definition, the drug load in an ER bilayer tablet was determined as approximate ≤ 30% in IR white layer, and the remaining in ER blue layer. The residual tablets were retrieved at the end of the 12-h dissolution study showed white layer of the ER bilayer tablet disappeared, but the blue layer swelled but the integrity was still kept (Figure 4). When the bilayer tablet was cut vertically, the polymers in the core were still densely packed reflecting that dissolution medium had penetrated into the tablet core, but the polymer had not yet fully swelled or disintegrated, which resulted in the tolerance for only about 70% to 75% (Q) of the labeled amount. In addition, the leaching of the colorant and erosion of polymer were evidenced by the dissolution medium changed from clear into light blue and the medium became more viscous and slightly sticky in the 12-h release study.

Figure 4. A studied tablet was retrieved from a dissolution vessel: (a) at the end of the 12-h study, and (b) the cross-section shows the core was still densely packed. No significant difference was noticed whether the tablet was retrieved from water or phosphate butter pH 6.8.

The in vitro release data of the ER bilayer tablets from 2 h to 12 h (as the extended release region based on General Chapter < 1088 >) was able to format into a power equation with the power exponent (n) in the range of 0.7 (n = 4, Table 3). Matching the exponent (n value) with those cylindrical geometry in Table 1, its release mechanism was anomalous transport (between Fickian diffusion and polyer swelling), but was closer to polymer swelling mechanism. Peppas and coworkers [4, 5, 6] studied theophylline release from poly(HEMA-co-NVP) [poly(2-hydroxyethylmethacrylate-co-N-vinylpyrrolidone)] disks into distilled water. The tablet geometry of this project was a caplet shape (Figure 1). Siepmann J and Siepmann F also mentioned that the thicker the samples a slightly slowing down of release with time is displayed [6]. This was probably due to drug diffusion becoming increasing more rate limiting. Diffusion is slower at greater distances. When plotting the cumulative amount of drug release versus time, geometry, drug solubility and inhomogeneous initial drug distribution [4, 5, 6] may also impact the value of n (power exponent).

The six inactive ingredients of the ER bilayer tablets and their pharmaceutical functions were stated in Section 3.1. They were composed of coloring agent, controlled-release agent, lubricant, tablet diluent, tablet disintegrant, and gelling agent. Therefore, photos taken after a tablet retrieved from the vessel at the end of a dissolution study were dissected to support the release mechanism of anomalous transport determined from Peppas power law power exponent (n). As seen in Figure 4, there is a clearly defined font between the swollen polymer layer and damped tablet core suggesting that polymer relaxation is required for guaifenesin to be released into either water or phosphate buffer pH 6.8.

Conclusion

In vitro dissolution study may be applied to approximate the drug loaded in IR layer and ER layers of an oral tablet. The current study also supports the use of water as the dissolution medium for extended release dosage forms, because time efficacy and green laboratory practice bring affordable products to our patients. Never the less the selection of a proper mobile phase is of essential. The project suggests that for sake of accuracy and precision, one-stage and two-stage dissolution profiles be compared with the same selected mobile phase. If they are similar, the one-stage study using water as dissolution medium may then be preceded. From Peppa power law as well as the dissection examination of retrieved tablets, the ER layer of the bilayer tablet most likely used anomalous transport mechanism to release guaifenesin.

References

1. https://medical-dictionary.thefreedictionary.com/guaifenesin
2. http://www.mucinex.com/media/854/drug-facts-maximum-strength-mucinex-se.pdf
3. U.S. Pharmacopeial Convention (2018) USP Monographs: Guaifenesin, Guaifenesin Tablets, NF Monographs: Sodium Starch Glycolate, General Chapters: <711> Dissolution. In: USP42-NF37. Rockville MD: U.S. Pharmacopeia; 2018: 2121, 2124, 5962, and 6870.
4. Peppas NA (1985) Analysis of Fickian and non-Fickian drug release from polymers. Pharmaceutica Acta Helvetiae 60: 110–111.
5. Siepmann J, Peppas NA (2001) Modeling of drug release from delivery systems based on hydroxypropyl methycellulose (HPMC). Adv Drug Deliv Rev 48: 139–157.
6. Siepmann J, Siepmann F (2012) Swelling Controlled Drug Delivery Systems. In: Siepmann J, Siegel RA, Rathbone MJ (eds.), Fundamentals and Applications of Controlled Release Drug Delivery. Springer Pg No: 154–162.
7. Rowe RC, Sheskey  PJ, Quinn ME (2009) Handbook of Pharmaceutical Excipients, 6thedn: Pharmaceutical Press: London, UK.
8. Kupiec T, Skinner R, Lanier L (2008) Stability Versus Potency Testing: The Madness is in the Method. Int J Pharmaceutical Compounding, 12: 50–55.
9. L.R, Kirkland JJ, Glajch JL (1997) Completing the Method: Validation and Transfer. In: L.R., Kirkland J.J., Glajch J.L. (Eds.), Practical HPLC Method Development. Snyder John Wiley & Sons Pg No: 709
10. Siegel RA, Alvarez-Lorenzo C (2017) Hydrogels. In: Hillery A, Park K (eds.), Drug Delivery: Fundamentals and Applications CRC Press Pg No: 333.
11. Hydrogel Materials (2014) Drug Delivery: Materials Design and Clinical Perspective. In: Holowka EP, Bhatia SK (eds.), Springer Pg No: 225.

Supplemental Material

Validation of HPLC Method to Assay Guaifenesin in Acid Stage, Buffer Stage Media and Water Using Proposed Mobile Phase (50% Methanol)

Stability Indication Method of Guaifenesin in Acid Stage, Buffer Stage Media and Water using HPLC with the proposed mobile phase (50% Methanol): in acid-stage medium and subjected to 0.1 N HCl for 1 h; (b) in acid-stage medium and subjected to 0.1 N NaOH for 1 h; (c) in acid stage medium and subjected to 50^oC for 1 h; (d) acid-stage medium and subjected to 3% hydrogen peroxide for 5 min; (e) in buffer stage medium and subjected to 0.1 N HCl for 1 h; (f) in buffer stage medium and subjected to 0.1 N NaOH for 1 h; (g) in buffer stage medium and subjected to 50 ^oC for 1 h; (h) in buffer stage medium and subjected to 3% hydrogen peroxide for 5 min; (i) in purified water and subjected to 50 ^oC for 1 h; (j) in purified water and subjected to 0.1 N HCl for 1 h; (k) in purified water and subjected to 0.1 N NaOH for 1 h; (l) in purified water and subjected to 3% hydrogen peroxide for 5 min; and (m) in purified water without guaifenesin and subjected to 3% hydrogen peroxide for 5 min as control.

1. Guaifenesin in acid stage medium (0.1 N HCl) – subject to 0.1 N HCl for 1 hour prior to being neutralized with 0.1 N NaOH to neutral pH (n = 3, please refer to pdf version of chromatograms.)

   

2. Guaifenesin in acid stage medium (0.1 N HCl) – subject to 0.1 N NaOH for 1 hour prior to being neutralized with 0.1 N HCl to neutral pH (n = 3, please refer to pdf version of chromatograms.)

   

3. Guaifenesin in acid stage medium (0.1 N HCl) – subject to 50 ^oC for 1 hour prior to cooling to room temperature (n = 3, please refer to pdf version of chromatograms)

   

4. Guaifenesin in acid stage medium (0.1 N HCl) – subject to 3% hydrogen peroxide for 5 min prior to decomposing hydrogen peroxide into water and oxygen and allowing the excess oxygen to escape (n = 3, please refer to pdf version of chromatograms.)

   

5. Guaifenesin in buffer stage medium (phosphate buffer, pH 6.8) – subject to 0.1 N HCl for 1 hour prior to being neutralized with 0.1 N NaOH to neutral pH (n = 3, please refer to pdf version of chromatograms.)

   

6. Guaifenesin in buffer stage medium (phosphate buffer, pH 6.8) – subject to 0.1 N NaOH for 1 hour prior to being neutralized with 0.1 N HCl to neutral pH (n = 3, please refer to pdf version of chromatograms.)

   

7. Guaifenesin in buffer stage medium (phosphate buffer, pH 6.8)– Subject to 50 ^oC for 1 hour prior to cooling to room temperature (n = 3, please refer to pdf version of chromatograms)

   

8. Guaifenesin in buffer stage medium (phosphate buffer, pH 6.8) – subject to 3% hydrogen peroxide for 5 min prior to decomposing hydrogen peroxide into water and oxygen and allowing the excess oxygen to escape (n = 3, please refer to pdf version of chromatograms.)

   

9. Guaifenesin in purified water – subject to 50 ^oC for 1 hour prior to cooling to room temperature (n = 3, please refer to pdf version of chromatograms)

   

10. Guaifenesin in purified water – subject to 0.1 N HCl for 1 hour prior to being neutralized with 0.1 N NaOH to neutral pH (n = 3, please refer to pdf version of chromatograms.)

    

11. Guaifenesin in purified water – subject to 0.1 N NaOH for 1 hour prior to being neutralized with 0.1 N HCl to neutral pH (n = 3, please refer to pdf version of chromatograms.)

    

12. Guaifenesin in purified water – subject to 3% hydrogen peroxide for 5 min prior to decomposing hydrogen peroxide into water and oxygen and allowing the excess oxygen to escape (n = 3, please refer to pdf version of chromatograms.)

    

13. Purified water without guaifenesin – subject to 3% hydrogen peroxide for 5 min prior to decomposing hydrogen peroxide into water and oxygen and allowing the excess oxygen to escape (as a control group)

    

DOI: 10.31038/JPPR.2019222

Abstract

The action of a new pharmaceutical substance of indolinone series, an sGC inducer with antiplatelet activity, on rat blood plasma hemostasis was studied. It was shown that the antiplatelet substance after single oral administration to rats considerably increases thrombin time after 3 hours (24.5 versus 17.3 in control, р < 0.05). Other plasma hemostasis parameters were unchanged.

Key words

antiplatelet, indolinone derivative, plasma hemostasis.

Introduction

Thrombosis plays a key role in the development of acute coronary syndrome, making antiplatelet therapy an important part of prevention and treatment of cardiovascular diseases [1]. One of the main risk factory of cardiovascular disease relapse is insufficiency of existing therapy in people resistant to aspirin and clopidogrel [2,3]. This problem can be solved by using antiplatelet drugs with a novel mechanism of action. Also, increased blood clotting plays a role in cardiovascular complications [4,5]. There were no earlier studies of the action of the indolinone derivative on blood plasma hemostasis.

Study goal – Assess the action of a new antiplatelet compound on rat blood plasma hemostasis after oral administration.

Materials and Methods

Test article – pharmaceutical substance 2-[2-[(5RS)-5-(hydroxymethyl)-3-methyl-1,3-oxazolidine-2-yliden]-2-cyanoethylidene]-1H-indol-3(2H)-one.

Outbred Wistar rats (n=10) were used as test system. Rat handling was performed in accordance with the European Convention and other regulating documents [6].

Intact blood was sampled from common carotid artery of anesthetized rats. The blood was stabilized with 3.8% solution of sodium citrate in 9:1 (v:v) ratio, blood plasma was produced by centrifuging at 2000 g for 20 min.

Plasma hemostasis was assessed by fibrinogen content, activated Partial Thromboplastin Time (aPTT), Prothrombin Time (PT) and Thrombin Time (TT). These parameters were assessed by KG-4 coagulometer (Cormay, Poland). Fibrinogen content was assessed using Claus method.

The antiplatelet drugs were administered to rats once orally in 10 mg/kg dose. At the end of experiment the animals were sacrificed by СО₂.

Statistical analysis was performed by «R» software. The data is presented as mean values and mean standard deviation (M ± m). Significance of difference (р<0.05) between the tests was assessed using Mann–Whitney U test.

Result and Discussion

No significant changes of main parameters of plasma hemostasis: fibrinogen, aPTT and PT, were found 3 hours after single oral administration of the antiplatelet drug in 10 mg/kg dose. TT was increased by 42% compared to the control group (Table 1).

Table 1. Effects of the new antiplatelet drug (10 mg/kg) on rat blood plasma hemostasis.

Note: * –р < 0.05 compared to control.

The new antiplatelet drug doesn’t affect blood plasma fibrinogen content, activated partial thromboplastin time and prothrombin time, but considerably increases the thrombin time, 3 hours after single oral administration in 10 mg/kg dose [7]. The reduction of platelet aggregation by the new drug leads to reduced exit of active components of the coagulation system from the platelets, which may account for the lengthening of thrombin time. There may also be other explanations for this. Additional studies are required for confirmation or discovery of another mechanism, including, possibly, direct inhibition of thrombin.

References

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