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Polymeric films as vehicle for buccal delivery: swelling, mechanical, and bioadhesive properties

Peh KK, Wong CF

J Pharm Pharm Sci, 1999 May-Aug;2(2):53-61.
PMID: 10952770

To investigate the suitability of an SCMC (sodium carboxymethyl cellulose/polyethylene glycol 400/carbopol 934P) and an HPMC (hydroxypropylmethyl cellulose/polyethylene glycol 400/carbopol 934P) films as drug vehicle for buccal delivery.
Application of similarity factor in development of controlled-release diltiazem tablet

Peh KK, Wong CF

Drug Dev Ind Pharm, 2000 Jul;26(7):723-30.
PMID: 10872090

Controlled-release grade hydroxypropylmethylcellulose (HPMC) or xanthan gum (XG) and microcrystalline cellulose (MCC) were employed to prepare controlled-release diltiazem hydrochloride tablets. The similarity factor f2 was used for dissolution profile comparison using Herbesser 90 SR as a reference product. Drug release could be sustained in a predictable manner by modifying the content of HPMC or XG. Moreover, the drug release profiles of tablets prepared using these matrix materials were not affected by pH and agitation rate. The f2 values showed that only one batch of tablets (of diltiazem HCl, HPMC or XG, and MCC in proportions of 3.0:3.0:4.0) was considered similar to that of the reference product, with values above 50. The unbiased similarity factor f2* values were not much different from the f2 values, ascribing to a small dissolution variance of the test and reference products. The amount of HPMC or XG incorporated to produce tablets with the desired dissolution profile could be determined from the curves of f2 versus polymer content. Hence, the f2 values can be applied as screening and optimization tools during development of controlled-release preparations.
Simple high-performance liquid chromatographic method for the determination of ranitidine in human plasma

Wong CF, Peh KK, Yuen KH

J Chromatogr B Biomed Sci Appl, 1998 Oct 23;718(1):205-10.
PMID: 9832378

A simple high-performance liquid chromatographic method was developed for the determination of ranitidine in human plasma. Prior to analysis, ranitidine and the internal standard (metoprolol) were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M potassium dihydrogenphosphate-acetonitrile (88:12, v/v) adjusted to pH 6.5. Analysis was run at a flow-rate of 1.3 ml/min and at a detection wavelength of 229 nm. The method is sensitive with a detection limit of 1 ng/ml at a signal-to-noise ratio of 3:1, while the quantification limit was set at 15 ng/ml. The calibration curve was linear over a concentration range of 15-2000 ng/ml. Mean recovery value of the extraction procedure was about 90%, while the within-day and between-day coefficients of variation and percent error values of the assay method were all less than 15%.
An in-vitro method for buccal adhesion studies: importance of instrument variables

Wong CF, Yuen KH, Peh KK

Int J Pharm, 1999 Mar 25;180(1):47-57.
PMID: 10089291

A method using a texture analyzer equipment and chicken pouch as the biological tissue was investigated for measuring the bioadhesive properties of polymers under simulated buccal conditions. The method was evaluated using two polymers, namely Carbopol 974P and Methocel K4M while the instrument variables studied included the contact force, contact time and speed of withdrawal of the probe from the tissue. The parameters measured were the work of adhesion and peak detachment force. Longer contact time and faster probe speed not only gave better reproducibility of results, but also better sensitivities for both parameters measured. On the other hand, a certain level of contact force was found essential for achieving good bioadhesion, above which there was no further contribution to the bioadhesion process. When the method was applied to determine the bioadhesiveness of several polymers, the values obtained for the work of adhesion and peak detachment force were quite consistent in the ranking of the polymers. The Carbopols were found to have the highest values, followed by gelatin, sodium carboxymethyl celluloses and hydroxypropylmethyl celluloses. On the other hand, Alginic acid, Eudragit RLPO and RSPO, and Chitosan appeared to have low bioadhesive values.
Formulation and evaluation of controlled release Eudragit buccal patches

Wong CF, Yuen KH, Peh KK

Int J Pharm, 1999 Feb 01;178(1):11-22.
PMID: 10205621

Controlled release buccal patches were fabricated using Eudragit NE40D and studied. Various bioadhesive polymers, namely hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose and Carbopol of different grades, were incorporated into the patches, to modify their bioadhesive properties as well as the rate of drug release, using metoprolol tartrate as the model drug. The in-vitro drug release was determined using the USP 23 dissolution test apparatus 5 with slight modification, while the bioadhesive properties were evaluated using texture analyzer equipment with chicken pouch as the model tissue. The incorporation of hydrophilic polymers was found to affect the drug release as well as enhance the bioadhesiveness. Although high viscosity polymers can enhance the bioadhesiveness of the patches, they also tend to cause non-homogeneous distribution of the polymers and drug, resulting in non-predictable drug-release rates. Of the various bioadhesive polymers studied, Cekol 700 appeared to be most satisfactory in terms of modifying the drug release and enhancement of the bioadhesive properties.
Possible mechanism for drug retardation from glyceryl monostearate matrix system

Peh KK, Wong CF, Yuen KH

Drug Dev Ind Pharm, 2000 Apr;26(4):447-50.
PMID: 10769787

Lipophilicity was evaluated as a possible mechanism for drug retardation from a glyceryl monostearate matrix system. Lipophilicity of the glyceryl monostearate matrix system was studied using contact angle measurement of water droplets on the surface of compressed disks, extrudate ascension of water, and movement of water through a powder mixture packed in a high-performance liquid chromatographic (HPLC) column. Increase in glyceryl monostearate content resulted in an increase in water droplet contact angle, decrease in the rate of water ascending the extrudate, and increase in the pressure values as a function of flow rate of water moving through the powder mixture. These could be due to the increase in lipophilicity of the matrix, rendering the matrix less wettable. As a result, the rate of water penetration into the matrix decreased, and the drug release could be sustained.
Fulltext Role of α-helical structure in organic solvent-activated homodimer of elastase strain K

Rahman RN, Salleh AB, Basri M, Wong CF

Int J Mol Sci, 2011;12(9):5797-814.
PMID: 22016627 DOI: 10.3390/ijms12095797

Recombinant elastase strain K overexpressed from E. coli KRX/pCon2(3) was purified to homogeneity by a combination of hydrophobic interaction chromatography and ion exchange chromatography, with a final yield of 48% and a 25-fold increase in specific activity. The purified protein had exhibited a first ever reported homodimer size of 65 kDa by SDS-PAGE and MALDI-TOF, a size which is totally distinct from that of typically reported 33 kDa monomer from P. aeruginosa. The organic solvent stability experiment had demonstrated a stability pattern which completely opposed the rules laid out in previous reports in which activity stability and enhancement were observed in hydrophilic organic solvents such as DMSO, methanol, ethanol and 1-propanol. The high stability and enhancement of the enzyme in hydrophilic solvents were explained from the view of alteration in secondary structures. Elastinolytic activation and stability were observed in 25 and 50% of methanol, respectively, despite slight reduction in α-helical structure caused upon the addition of the solvent. Further characterization experiments had postulated great stability and enhancement of elastase strain K in broad range of temperatures, pHs, metal ions, surfactants, denaturing agents and substrate specificity, indicating its potential application in detergent formulation.
Fulltext Construction of New Genetic Tools as Alternatives for Protein Overexpression in Escherichia coli and Pseudomonas aeruginosa

Wong CF, Rahman RNZRA, Basri M, Salleh AB

Iran J Biotechnol, 2017;15(3):194-200.
PMID: 29845069 DOI: 10.15171/ijb.1524

Background:Pseudomonas protein expression in E. coli is known to be a setback due to significant genetic variation and absence of several genetic elements in E. coli for regulation and activation of Pseudomonas proteins. Modifications in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active Pseudomonas protein possible in both Pseudomonas sp. and E. coli. Objectives: Construction of shuttle expression vectors for regulation and overexpression of Pseudomonas proteins in Pseudomonas sp. and E. coli. Materials and Methods:Pseudomonas-Escherichia shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as E. coli expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZαA-based vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastinolytic activity assay. Results: The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo, each carrying an E. coli lac-operon based promoter, Plac, and a tightly regulated T7(A1/O4/O3) promoter/repressor system was performed to facilitate overexpression study of the organic solvent-tolerant elastase strain K. These constructs have demonstrated an elastinolytic fold of as high as 1464.4 % in comparison to other published constructs. pCon4 and pCon5, on the other hand, are series of pCon2(3)-derived vectors harboring expression cassettes controlled by PT7(A1/O4/O3) promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacIq. Conclusions: The constructs offered remarkable assistance for overexpression of heterogeneous genes in Pseudomonas sp. and E. coli for downstream applications such as in industries and structural biology study.
Organic-solvent stability of elastase strain K overexpressed in an Escherichia-Pseudomonas expression system

Wong CF, Salleh AB, Basri M, Abd Rahman RN

Biotechnol Appl Biochem, 2010 Sep;57(1):1-7.
PMID: 20726840 DOI: 10.1042/BA20100224

The structural gene of elastase strain K (elastase from Pseudomonas aeruginosa strain K), namely HindIII1500PstI, was successfully sequenced to contain 1497 bp. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consists of 301 amino acids, with a molecular mass of 33.1 kDa, and contains a conserved motif HEXXH, zinc ligands and residues involved in the catalysis of elastase strain K. The structural gene was successfully cloned to a shuttle vector, pUCP19, and transformed into Escherichia coli strains TOP10, KRX, JM109 and Tuner™ pLacI as well as P. aeruginosa strains PA01 (A.T.C.C. 47085) and S5, with detection of significant protein expression. Overexpression was detected from transformants KRX/pUCP19/HindIII1500PstI of E. coli and PA01/pUCP19/HindIII1500PstI of P. aeruginosa, with increases in elastolytic activity to 13.83- and 5.04-fold respectively relative to their controls. In addition, recombinant elastase strain K showed considerable stability towards numerous organic solvents such as methanol, ethanol, acetone, toluene, undecan-1-ol and n-dodecane, which typically pose a detrimental effect on enzymes; our finding provides further information to support the potential application of the enzyme in synthetic industries, particularly peptide synthesis.
Molecular investigation of a gene encoding organic solvent-tolerant alkaline protease from Pseudomonas aeruginosa strain K

Rahman RN, Geok LP, Wong CF, Basri M, Salleh AB

J Basic Microbiol, 2010 Apr;50(2):143-9.
PMID: 20082370 DOI: 10.1002/jobm.200900133

A gene encoding an organic solvent-stable protease was amplified from Pseudomonas aeruginosa strain K by polymerase chain reaction using consensus primers based on multiple sequence alignment of alkaline and metalloprotease genes from Pseudomonas species. The gene, which consisted of 1440 bp nucleotides and deduced 479 amino acid residues, was successfully expressed in pGEX-4T-1 expression system in the presence of 1.0 mM IPTG, after an incubation of 6 h at 37 degrees C. Under these conditions, the recombinant strain K protease was, subsequently, released into the periplasm of E. coli BL21 (DE3) with an optimum proteolytic activity detected at 1.0112 U/ml. To date, this is the first reported expression of alkaline protease (aprA) with such remarkable property in Escherichia coli.
Interactions of Flavone and Steroid from A. subintegra as Potential Inhibitors for Porcine Pancreatic Lipase

Ibrahim M, Abdul Azziz SSS, Wong CF, Bakri YM, Abdullah F

Curr Comput Aided Drug Des, 2020;16(6):698-706.
PMID: 31648647 DOI: 10.2174/1573409915666191015112320

BACKGROUND: Obesity is one serious health condition that contributes to various chronic diseases. The inhibition of pancreatic lipase is a promising treatment for obesity.
OBJECTIVE: The present study was designed to investigate anti-porcine pancreatic lipase effect of isolated compounds from Aquilaria subintegra and its mechanism.
METHODS: Compounds were isolated with serial column chromatography and their structure were identified using spectroscopic methods. Isolated compounds were tested for anti-lipase potential activity using colorimetric assay. The prediction of energy binding between isolated compounds and enzyme was described using YASARA software.
RESULTS: Four compounds were successfully isolated from the bark of A. subintegra, namely, 5- hydroxy-7,4'-dimethoxyflavone, luteolin-7,3',4'-trimethyl ether, 5,3'-dihydroxy-7,4'-dimethoxyflavone and β-sitosterol. The results indicated that all compounds displayed promising pancreatic lipase inhibitory activity ranging between of 6% to 53% inhibition. Compound 5-hydroxy-7,4'- dimethoxyflavone was a competitive inhibitor and decreases the enzyme catalysis. Meanwhile, β- sitosterol was a non- competitive inhibitor since the latter was bind allosterically toward enzyme.
CONCLUSION: This finding is significant for further investigation of bioactive compounds from A. subintegra on animal study.
C-terminal splice variants of P/Q-type Ca2+ channel CaV2.1 α1 subunits are differentially regulated by Rab3-interacting molecule proteins

Hirano M, Takada Y, Wong CF, Yamaguchi K, Kotani H, Kurokawa T, et al.

J Biol Chem, 2017 06 02;292(22):9365-9381.
PMID: 28377503 DOI: 10.1074/jbc.M117.778829

Voltage-dependent Ca2+ channels (VDCCs) mediate neurotransmitter release controlled by presynaptic proteins such as the scaffolding proteins Rab3-interacting molecules (RIMs). RIMs confer sustained activity and anchoring of synaptic vesicles to the VDCCs. Multiple sites on the VDCC α1 and β subunits have been reported to mediate the RIMs-VDCC interaction, but their significance is unclear. Because alternative splicing of exons 44 and 47 in the P/Q-type VDCC α1 subunit CaV2.1 gene generates major variants of the CaV2.1 C-terminal region, known for associating with presynaptic proteins, we focused here on the protein regions encoded by these two exons. Co-immunoprecipitation experiments indicated that the C-terminal domain (CTD) encoded by CaV2.1 exons 40-47 interacts with the α-RIMs, RIM1α and RIM2α, and this interaction was abolished by alternative splicing that deletes the protein regions encoded by exons 44 and 47. Electrophysiological characterization of VDCC currents revealed that the suppressive effect of RIM2α on voltage-dependent inactivation (VDI) was stronger than that of RIM1α for the CaV2.1 variant containing the region encoded by exons 44 and 47. Importantly, in the CaV2.1 variant in which exons 44 and 47 were deleted, strong RIM2α-mediated VDI suppression was attenuated to a level comparable with that of RIM1α-mediated VDI suppression, which was unaffected by the exclusion of exons 44 and 47. Studies of deletion mutants of the exon 47 region identified 17 amino acid residues on the C-terminal side of a polyglutamine stretch as being essential for the potentiated VDI suppression characteristic of RIM2α. These results suggest that the interactions of the CaV2.1 CTD with RIMs enable CaV2.1 proteins to distinguish α-RIM isoforms in VDI suppression of P/Q-type VDCC currents.
Fulltext Contribution of Coiled-Coil Assembly to Ca2+/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes

Polat OK, Uno M, Maruyama T, Tran HN, Imamura K, Wong CF, et al.

J Am Soc Nephrol, 2019 09;30(9):1587-1603.
PMID: 31266820 DOI: 10.1681/ASN.2018070756

BACKGROUND: TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved.
METHODS: We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca2+-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes.
RESULTS: Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca2+-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca2+ elevations and a disorganized cytoskeleton.
CONCLUSIONS: The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC's coiled-coil assembly which is essential for CaM binding. The resulting excess Ca2+ may contribute to structural damage in the podocytes.

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