Hemant H. Alur

Transmucosal sustained-delivery of chlorpheniramine maleate in rabbits using a novel, natural mucoadhesive gum as an excipient in buccal tablets

The objective of this study was to evaluate the gum from Hakea gibbosa (Hakea) as a sustained-release and mucoadhesive component in buccal tablets following their application to the buccal mucosa of rabbits. Flat-faced core tablets containing either 22 or 32 mg of Hakea and 40 or 25 mg of chlorpheniramine maleate (CPM) per tablet with either sodium bicarbonate or tartaric acid in a 1:1.5 molar ratio were formulated using a direct compression technique and were coated with Cutina(R) on all but one face. The resulting plasma CPM concentration versus time profiles were determined following buccal application of the tablets in rabbits. The strength of mucoadhesion of the tablets was also quantitated in terms of the force of detachment as a function of time. Following the application of the mucoadhesive buccal tablets, the following values for several pharmacokinetic parameters were obtained. The force of detachment for the mucoadhesive buccal tablets containing 22 mg of Hakea and either 25 and 40 mg CPM, and 32 mg Hakea and 40 mg CPM increased from 1.64+/-0.47 to 7.32+/-0.34 N, 1.67+/-0.30 to 7.21+/-0.36 N, and 2.93+/-0.73 to 7.92+/-0.60 N, respectively from 5 to 90 min following application to excised intestinal mucosa. Addition of either sodium bicarbonate or tartaric acid, as well as higher amounts of CPM, did not affect the mucoadhesive bond strength. These results demonstrate that the novel, natural gum, H. gibbosa, may not only be used to sustain the release of CPM from a unidirectional-release buccal tablet, but also demonstrate that the tablets are sufficiently mucoadhesive for clinical application. The mucoadhesive strength as measured by the force of detachment, can be modulated by altering the amount of Hakea in the tablet. The mucoadhesive buccal tablets evaluated represent an improved transbuccal delivery system for conventional drug substances.

Evaluation of the Gum from Hakea gibbosa as a Sustained-Release and Mucoadhesive Component in Buccal Tablets

The objective of this paper was to evaluate the mucoadhesive and sustained-release properties of the water-soluble gum obtained from Hakea gibbosa (hakea), for the formulation of buccal tablets. Flatfaced tablets containing hakea were formulated using chlorpheniramine maleate (CPM) as a model drug. Two types of tablets were prepared: uncoated tablets (type I) and tablets in which all but one face of the type I tablet was coated with hydrogenated castor oil (Cutina) using a compression coating technique (type II). In an attempt to explain the observed sustained-release effect, the potential for a chemical interaction between hakea and CPM was evaluated by FTIR, differential scanning calorimetry (DSC), UV spectroscopy, and acid-base titrations. Mathematical modeling of the CPM release data was developed to elucidate the mechanism of drug release. The mucoadhesive strength was evaluated by quantitating the force of detachment. Finally, the rates of water uptake and erosion were determined for the buccal tablets. The time required for 90% of the CPM to be released in vitro (t90%) was used as a basis for comparison. For formulations that did not contain hakea, the t90% was 14 min for both directly compressed and wet granulated tablets, whereas the t90% for wet granulated tablets containing 2 or 32 mg hakea/tablet was 36 and 165 min, respectively. Directly compressed tablets containing 2, 12, 22, and 32 mg hakea/tablet displayed t90% values of 48, 120, 330, and 405 min, respectively. DSC, FTIR, UV spectroscopy and acid-base titration experiments suggested the absence of chemical interactions. The force of detachment for directly compressed and wet granulated tablets increased from 0.70 +/- 0.3 to 4.08 +/- 0.52 N and from 0.65 +/- 0.28 to 3.94 +/- 0.31 N as the amount of hakea per tablet was increased from 0 to 32 mg, respectively, at a 5 N attachment compression force. The novel natural gum, hakea, may not only be utilized to sustain the release of CPM from a unidirectional-release buccal tablet, but it also exhibited excellent mucoadhesive properties. The mechanism by which CPM release was sustained was more likely due to slow relaxation of the hydrated hakea. The mucoadhesive strength can be modulated by altering the amount of hakea in the tablet.

Poly(L-lysine) as a model drug macromolecule with which to investigate secondary structure and membrane transport, part I: physicochemical and stability studies

Low oral bioavailability of therapeutic peptides and proteins generally results from their poor permeability through biological membranes and enzymatic degradation in the gastrointestinal tract. Since different secondary structures exhibit different physicochemical properties such as hydrophobicity, size and shape, changing the secondary structure of a therapeutic polypeptide may be another approach to increasing its membrane permeation. Poly(L‐lysine) was used as a model polypeptide. The objectives of this study were to induce secondary structural changes in poly(L‐lysine) and to determine the time course over which a given conformer was retained. In addition, the hydrophobicity of each secondary structure of poly(L‐lysine) was assessed. The circular dichroism (CD) studies demonstrated that the conditions employed could successfully induce the desired secondary structural changes in poly(L‐lysine). The α‐helix conformer appeared to be more stable at 25° C whereas the β‐sheet conformer could be preserved at 37° C. On the other hand, the random coil conformer was retained at both temperatures. Significant losses of the α‐helix and the β‐sheet conformers were observed when the pH was reduced. The change in ionic strength did not affect any of the conformers. The octanol/buffer partitioning studies indicated that the α‐helix and the β‐sheet conformers exhibited significantly different (P< 0.05) hydrophobicities. In conclusion, variation of pH and temperature conditions can be used to induce secondary structural changes in poly(L‐lysine). These changes are reversible when the stimuli are removed. The α‐helix and the β‐sheet conformers of poly(L‐lysine) are more lipophilic than the native random coil conformer. Thus, poly(L‐lysine) may represent an ideal model polypeptide with which to further investigate the effects of secondary structure on membrane diffusion or permeation.

Poly(L-lysine) as a model drug macromolecule with which to investigate secondary structure and microporous membrane transport, part 2: diffusion studies.

Peptide drugs are hydrophilic in nature and so their preferred pathway of membrane transport is by the paracellular route, which primarily involves passive diffusion across intercellular pores. The objective of the present study was to investigate the effect of secondary structure on the aqueous diffusion of a model polypeptide, poly(l‐lysine), through a microporous membrane. The primary aim was to systematically evaluate the variables (e.g. viscosity and/or hydrodynamic radius) that may contribute to the difference, if any, in the calculated values of the aqueous diffusion coefficient (Daq) for each conformer of poly(l‐lysine). Variations in pH and temperature of the medium were used to induce secondary structural changes in poly(l‐lysine). Transport studies were conducted for 3 h at 25 or 37°C using side‐by‐side diffusion cells. Hydrophilic microporous polyester membranes with a 1‐μm pore diameter were used to measure the free diffusion of each conformer. The values for the apparent permeability (Papp) and Daq were calculated using standard equations. The viscosity of each conformer solution was determined and the hydrodynamic radius of each conformer was then estimated. At 25°C, both Papp and Daq of the α‐helix conformer were approximately the same as those of the random coil conformer. In contrast, at 37°C, the Papp and the Daq of the β‐sheet conformer were significantly (P < 0.05) less than those of the random coil conformer. At 25°C, the solutions containing primarily either the random coil or the α‐helix conformers had approximately the same viscosity. On the other hand, at 37°C, the solutions containing the β‐sheet conformer had a significantly (P < 0.05) higher viscosity than when this conformer was absent. The random coil and the α‐helix conformers appeared to have comparable sizes, whereas the hydrodynamic radius estimated for the β‐sheet conformer was significantly (P < 0.05) larger than those for the other two conformers. In summary, changing the secondary structure of poly(l‐lysine) from the random coil to the α‐helix did not affect its Papp and intrinsic Daq. On the other hand, appearance of the β‐sheet conformer significantly decreased the values of Papp and Daq. The differences appeared to result from the significantly higher solution viscosity as well as the extended structure associated with the β‐sheet conformer of poly(l‐lysine). This strategy may represent a potential mechanism to sustain the delivery of therapeutic peptide drugs from a controlled drug delivery device.

Permeation of Unfolded Basic Fibroblast Growth Factor (bFGF) Across Rabbit Buccal Mucosa—Does Unfolding of bFGF Enhance Transport?

AbstractPurpose. To investigate whether recombinant human basic fibroblast growth factor (rhbFGF) would permeate freshly-excised rabbit buccal mucosa. In addition, the effect of a permeation enhancer (Na+ glycocholate) and the possibility of reversibly unfolding the globular protein to a more linear conformation to increase the permeability of the test protein was evaluated. Methods. Thein vitro flux of bFGF through freshly-excised rabbit buccal mucosa was determined using side-by-side diffusion systems. Detection of bFGF was performed using gradient elution, reversed-phase high-pressure liquid chromatography (RP-HPLC). Fluorescence spectroscopy and heparin affinity chromatography were used to assess the tertiary structure of bFGF. Results. Preliminary in vitro results have demonstrated that the bFGF flux increased from 1.4 ± 0.13 ng min−1 cm−2 to 3.2 ± 0.38 ng min−1 cm−2 with the addition of 15 mM Na+ glycocholate (NaG) to the donor solution. Subsequent addition of guanidine HC1 (GnHCl) to the donor solution (3 M) was not followed by a further increase in the flux of bFGF (2.9 ± 0.26 ng min−1 cm−2). However, when the order of addition of the additives was reversed (GnHCl first followed by NaG), the flux of bFGF across rabbit buccal mucosa was increased. Upon addition of GnHCl, there was a significant (p < .05) increase in bFGF flux from 1.2 ± 0.15 ng min−1 cm−2 to 5.0 ± 0.58 ng min−1 cm−2. Addition of NaG further increased the flux to 8.5 ± 1.1 ng min−1 cm−2 which was approximately 3- to 3.5-fold greater than that determined with the protein alone in the absence of any donor phase additives. The percent of parent bFGF remaining following a 3-hr exposure of a bFGF solution to either the mucosal, serosal, or both sides of rabbit buccal mucosa were 54.3 ± 5.7%, 71.8 ± 6.3%, and 36.2 ± 5.4%, respectively with the majority of parent bFGF lost during the first 15 minutes. A model endopeptidase (endoproteinase Arg-C from mouse submaxillary gland) was shown in vitro to contribute to the loss in parent bFGF. Conclusions. The permeation of bFGF across rabbit buccal mucosa may be significantly increased by initially unfolding the protein with GnHCl and then treating the tissue with the permeation enhancer, NaG. Refolding and possible reactivation of bFGFs bioactivity may occur following membrane transport and subsequent dilution into an infinite sink.

Inhibition of a model protease--pyroglutamate aminopeptidase by a natural oligosaccharide gum from Hakea gibbosa.

The purpose of this study was to investigate the effect of the oligosaccharide gum from Hakea gibbosa on the activity of a model protease enzyme pyroglutamate aminopeptidase (5-oxoprolyl peptidase; EC 3.4.19.3) and to elucidate the mechanism responsible for the decreased activity. Enzyme kinetic studies were conducted at 37 degrees C in 100 mM potassium phosphate buffer with 10 mM EDTA, 5% (v/v) glycerol, and 5 mM DTT (pH 8) for 15 min and were performed both in the presence and absence of the gum. Enzymatic activity was determined by a colorimetric assay using the specific substrate L-pyroglutamic acid beta-napthylamide. The enzyme kinetics was studied at various substrate and gum concentrations. The velocity of the reaction was determined by the amount of the product (beta-napthylamine) liberated at each substrate and gum concentration. The Ks and Vmax of the enzyme in the absence of the gum were 24.40+/-2.14 microM and 502.95+/-28.90 nmoles x min(-1) x mg protein(-1), respectively. As the concentration of the gum was gradually increased from 0.1 to 2%, the value of the Vmax decreased from 318.94+/-21.46 to 158.83+/-24.51 nmoles x min(-1) x mg protein(-1) while Ks increased from 17.42+/-4.6 to 63.03+/-1.89 microM. The mechanism for the inhibition of the enzyme by Hakea appeared to be a mixed-linear type (a type of non-competitive inhibition) as suggested from Hanes-Woolf, Dixon and Cornish-Bowden plots. The turnover number, kcat, calculated for the enzyme also decreased from 14.09+/-0.81 to 4.45+/-0.69 min(-1) as the concentration of the inhibitor was incrementally increased from 0 to 2% (w/v). The K(i) and alphaK(i) calculated from Dixon and Cornish-Bowden plots were found to be 0.31+/-0.11% (w/v) and 1.33+/-0.42% (w/v), respectively. The natural gum from Hakea gibbosa was effective in non-competitively inhibiting the enzyme pyroglutamate aminopeptidase. Thus, the natural gum may be a promising additive not only for its sustained-release and mucoadhesive properties as shown previously, but also for its ability to slow the enzymatic degradation of therapeutic polypeptides incorporated in dosage forms.

Inducing a change in the pharmacokinetics and biodistribution of poly-L-lysine in rats by complexation with heparin.

The aim of our study was to induce changes in the plasma elimination half‐life (t1/2 elim), rate and extent of urinary excretion, and biodistribution of a model macromolecule, poly‐l‐lysine, in rats following complexation with heparin. Male Sprague‐Dawley rats were dosed intravenously with either unfractionated [3H]heparin, FITC‐labelled poly‐l‐lysine, or an [3H]heparin:FITC‐labelled poly‐l‐lysine complex. Serum and blood concentration vs time and urinary excretion profiles were determined as well as the resulting patterns of biodistribution to liver, spleen, kidney, and muscle tissue. While the mean values for the total body clearance of poly‐l‐lysine and the complex were not significantly different, the volume of distribution and the half‐life associated with elimination from the serum were increased greater than 2‐fold for the complex compared with free poly‐l‐lysine. The rate and extent of elimination in the urine followed the relative rank order; heparin >poly‐l‐lysine> heparin:poly‐l‐lysine complex. Thirty minutes following intravenous administration, there was significantly more tissue deposition/uptake of the complex in the liver, kidney, and muscle, but not the spleen, when compared with poly‐l‐lysine administered alone. Complexation of heparin to poly‐l‐lysine effectively increased the fraction of an administered dose of poly‐l‐lysine that was deposited in liver, kidney, and muscle tissue. Due to the macromolecular complex being nontoxic and uncharged, potentially it might serve as a suitable carrier for both conventional and peptidic drugs to increase drug distribution to liver, kidney, or muscle tissue.

Evaluation of TRI-726 as a drug delivery matrix

Background: The TRI-726 polymeric drug delivery matrix is a newly-developed biocompatible hydrogel exhibiting in situ reverse-thermal gelling, mucoadhesivity, and sustained-erosion properties. Methods: Using two model drugs, clindamycin hydrochloride and acetaminophen, we determined the gelling temperatures, in vitro release profiles, kinetics of matrix erosion, rheological properties, mucoadhesive strength, microbiological activity of released clindamycin, and biocompatibility when in contact with cells. Results: It was demonstrated that none of the excipients contained in the TRI-726 polymer matrix caused any loss in clindamycin’s antimicrobial activity following incorporation into the polymer matrix. Thus, the new patent pending TRI-726 drug delivery matrix was both inert and non-reactive toward the incorporated clindamycin in terms of chemical degradation (<10% degradation under accelerated conditions over 6 months) and antimicrobial activity. Conclusions: This new drug delivery matrix is capable of releasing a wide variety of water-soluble drug compounds over an approximate 10-day period, due primarily to protracted dissolution/erosion of the three-dimensional polymer matrix in an aqueous-based biophase. Additionally, TRI-726 exhibits excellent mucoadhesive properties that would allow a candidate drug/TRI-726 formulation to adhere and remain at a potential application site for an extended period of time. Lastly, the biocompatibility tests affirmed the non-toxic and biocompatible nature of TRI-726 when in contact with cells, which suggests its suitability and versatility as a drug delivery matrix for the targeted administration of a wide range of pharmaceutical compounds where in situ gelation, protracted release of the active, and mucoadhesion of the formulation are desired.

An Attempt to Modulate the Microporous Diffusion of a Model Polypeptide by Altering Its Secondary Structure

The purpose of the present study was to determine whether intentional alteration of the secondary structure of a model polypeptide, conantokin-G, influenced the rate and extent of aqueous pore diffusion across a synthetic microporous membrane. Use of a microporous synthetic membrane allowed for analysis of polypeptide transport without the confounding variables of protein binding, acid- and/or enzyme-mediated degradation, endocytotic uptake, and enzymatic inactivation associated with a biological membrane. Conantokin-G was intentionally changed from its native random coil structure to the α-helix structure using calcium, and both structures were verified using circular dichroism. The α-helix structure of conantokin-G was retained even after additional free calcium was removed by equilibrium dialysis. Over the concentration range of 1.25 to 20 mM, there was a linear relationship between the solution calcium concentration and the percent of the α-helix conformer present. The apparent permeability, the apparent aqueous diffusion coefficient with and without inclusion of the Renkin function, and the hydrodynamic radii estimated by diffusion and a computer-software program were calculated for the random coil and α-helix structures of conantokin-G. Calcium-mediated conversion of conantokin-G to its α-helix structure did not significantly (p > .05) change its apparent permeability across a microporous membrane. It is suggested that perhaps complete conversion to the α-helix structure of only a fraction of the conantokin-G molecules (only 0.45 or 45% of the molecules can be converted to the α-helix structure at Ca 2+ concentrations ≥20 mM) may have limited the extent of transport of the α-helix conformer.

Canine Periodontal Disease Control Using a Clindamycin Hydrochloride Gel

Stabilizing or reducing periodontal pocket depth can have a positive influence on the retention of teeth in dogs. A topical 2 % clindamycin hydrochloride gel (CHgel) was evaluated for the treatment of periodontal disease in dogs. The CHgel formulation provides for the sustained erosion of the matrix, but also flows into the periodontal pocket as a viscous liquid, and then rapidly forms a gel that has mucoadhesive properties and also may function as a physical barrier to the introduction of bacteria. A professional teeth cleaning procedure including scaling and root planing was done in dogs with one group receiving CHgel following treatment. Periodontal health was determined before and after the procedure including measurement of periodontal pocket depth, gingival index, gingival bleeding sites, and number of suppurating sites. There was a statistically significant decrease in periodontal pocket depth (19 %), gingival index (16 %), and the number of bleeding sites (64 %) at 90-days in dogs receiving CHgel. Additionally, the number of suppurating sites was lower (93 %) at 90-days for the group receiving CHgel. The addition of CHgel effectively controlled the bacterial burden (e.g., Fusobacterium nucleatum) at both day 14 and 90. Gingival cells in culture were shown to rapidly incorporate clindamycin and attain saturation in approximately 20-minutes. In summary, a professional teeth cleaning procedure including root planning and the addition of CHgel improves the gingival index and reduces periodontal pocket depth.