Permeability of Fresh and Frozen Porcine and Human Gingiva and the Effect of Storage Duration.

The gingiva is the target site for some topical drugs, but the permeability of human gingiva has not been systematically evaluated. Pigs are a common animal model for in vitro membrane transport studies. The objectives of this study were to: (a) determine the permeability coefficients of freshly excised human gingiva using model permeants, (b) compare the permeability coefficients of fresh human gingiva with those of fresh porcine gingiva, (c) evaluate the effect of freezing duration on the permeability of porcine gingiva, and (d) compare the permeability coefficients of fresh and cadaver (frozen) human gingiva. A goal was to examine the feasibility of using porcine gingiva as a surrogate for human gingiva. The potential of using frozen tissues in permeability studies of gingiva was also examined. Fresh and frozen porcine gingiva, fresh human gingiva, and frozen cadaver human gingiva were compared in the transport study with model polar and lipophilic permeants. The fresh porcine and human tissues showed similarities in the "permeability coefficient vs. octanol-water distribution coefficient" relationship. The porcine gingiva had a lower permeability than that of the human, with a moderate correlation between the permeability of the fresh porcine and fresh human tissues. The permeability of the porcine tissues for the model polar permeants increased significantly after the tissues were frozen in storage. Moreover, the frozen human cadaver tissue could not be utilized due to the high and indiscriminating permeability of the tissue for the permeants and large tissue sample-to-sample variabilities.

Iontophoresis on Porcine and Human Gingiva.

PURPOSE: Iontophoresis is a noninvasive method that enhances drug delivery using an electric field. This method can improve drug delivery to the tissues in the oral cavity. The effects of iontophoresis on gingival drug delivery have not been investigated. The objectives of this study were to (a) determine the flux enhancement of model permeants across porcine and human gingiva during iontophoresis, (b) examine the transport mechanisms of gingival iontophoresis, and (c) evaluate the potential of iontophoretically enhanced delivery for three model drugs lidocaine, ketorolac, and chlorhexidine. METHODS: Passive and iontophoretic fluxes were determined with porcine and human gingiva using a modified Franz diffusion cell and model drugs and permeants. To investigate the transport mechanisms of iontophoresis, the enhancement from the direct-field effect was determined by positively and negatively charged model permeants. The electroosmosis enhancement effect was determined with neutral permeants of different molecular weight. The alteration of the gingival barrier due to electropermeabilization was evaluated using electrical resistance measurements. RESULTS: Significant flux enhancement was observed during gingival iontophoresis. The direct-field effect was the major mechanism governing the iontophoretic transport of the charged permeants. Electroosmosis was from anode to cathode. The effective pore radius of the iontophoretic transport pathways in the porcine gingiva was ~0.68 nm. Irreversible electropermeabilization was observed after 2 and 4 h of iontophoresis under the conditions studied. CONCLUSION: Iontophoresis could enhance drug delivery and reduce transport lag time, showing promise for gingival drug delivery.

Characterization of Porcine Gingiva for Drug Absorption.

Gingiva or gum is a part of the periodontium that surrounds the tooth. Its main function is to provide an effective barrier to both mechanical trauma and bacterial invasion. Gingiva is the target site for some topical drugs. The most common disease in gingiva is periodontal diseases (gum infections). Understanding the gingiva barrier properties could provide insights into approaches to effective drug delivery for the gingiva. Porcine gingiva was chosen as the model in the present membrane transport study. The permeability coefficients of gingiva were determined using a modified Franz diffusion cell with small diffusional area (0.03 cm2) and 12 model permeants with different physicochemical properties. The influences of edge effect and aqueous boundary layers were not observed in the modified diffusion cell setup for the small pieces of gingiva tissue samples. Lipophilic permeants exhibit higher permeability coefficients than hydrophilic permeants. A correlation was observed between the Log permeability coefficient (Log P) and Log octanol-water distribution coefficient (Log Dow) in the analysis. The permeant molecular weight (MW) was also a factor in the Log P vs. Log Dow relationship. The coefficient of Log Dow in this three-factor relationship (0.42) suggested that the gingiva barrier was less lipophilic than octanol.

Modification of small dissolution chamber system for long-acting periodontal drug product evaluation.

Conventional dissolution testing methods may not be suitable for long-acting periodontal drug products due to the small volume, slow fluid flow rate, and environment in the periodontal pocket. The objective of this study was to evaluate a 3D-printed small volume flow-through dissolution chamber system (modified from a previous study) for biorelevant and dose-discriminating testing. Three periodontal drug products with different dosage forms were tested: Atridox, Arestin, and PerioChip. Modifications were made to suit the specific characteristics of these dosage forms. No significant differences were observed between the % drug release profiles in vitro and in vivo except for Atridox. The differences observed with Atridox could be related to the exposing surface area of the drug product. Similar differences were observed from this effect in COMSOL model simulations. Overall, the drugs show reasonable in vitro-in vivo correlations (R2 ≥ 0.91) with linear regression slopes close to unity. For dose discrimination between 75% and full dosing, significant differences were observed in the drug release data at specific time points of the products (p ≤ 0.05). The present results suggest that a small volume dissolution chamber with slow flow rate could potentially provide biologically relevant and dose-discriminating evaluations for periodontal drug products.

Evaluation of Heat Effects on Fentanyl Transdermal Delivery Systems Using In Vitro Permeation and In Vitro Release Methods.

Experimental conditions that could impact the evaluation of heat effects on transdermal delivery systems (TDS) using an in vitro permeation test (IVPT) and in vitro release testing (IVRT) were examined. Fentanyl was the model TDS. IVPT was performed using Franz diffusion cell, heating lamp, and human skin with seven heat application regimens. IVRT setup was similar to IVPT, without using skin. Dissolution study was conducted in a modified dissolution chamber. The activation energy of skin permeation for fentanyl was determined using aqueous solution of fentanyl. In IVPT, the increase of temperature from 32 °C to 42 °C resulted in a 2-fold increase in flux for fentanyl TDS, consistent with the activation energy determined. The magnitude of flux increase was affected by the heat exposure onset time and duration: higher flux was observed when heat was applied earlier or following sustained heat application. Heat induced flux increases could not be observed when inadequate sampling time points were used, suggesting the importance of optimizing sampling time points. Drug release from TDS evaluated using IVRT was fast and the skin was the rate-limiting barrier for TDS fentanyl delivery under elevated temperature.

Evaluation of Heat Effects on Transdermal Nicotine Delivery In Vitro and In Silico Using Heat-Enhanced Transport Model Analysis.

A combined experimental and computational model approach was developed to assess heat effects on drug delivery from transdermal delivery systems (TDSs) in vitro and nicotine was the model drug. A Franz diffusion cell system was modified to allow close control of skin temperature when heat was applied from an infrared lamp in vitro. The effects of different heat application regimens on nicotine fluxes from two commercial TDSs across human cadaver skin were determined. Results were interpreted in terms of transport parameters estimated using a computational heat and mass transport model. Steady-state skin surface temperature was obtained rapidly after heat application. Increasing skin surface temperature from 32 to 42°C resulted in an approximately 2-fold increase in average nicotine flux for both TDSs, with maximum flux observed during early heat application. ANOVA statistical analyses of the in vitro permeation data identified TDS differences, further evidenced by the need for a two-layer model to describe one of the TDSs. Activation energies associated with these data suggest similar temperature effects on nicotine transport across the skin despite TDS design differences. Model simulations based on data obtained from continuous heat application were able to predict system response to intermittent heat application, as shown by the agreement between the simulation results and experimental data of nicotine fluxes under four different heat application regimens. The combination of in vitro permeation testing and a computational model provided a parameter-based heat and mass transport approach to evaluate heat effects on nicotine TDS delivery.

Dissolution Chamber for Small Drug Delivery System in the Periodontal Pocket.

Existing dissolution chambers have relatively large volume compared to the size of the periodontal pocket. A small volume dissolution method that simulates the physiological release environment for periodontal drug delivery is needed. The objectives were to construct a small, more physiologically relevant, dissolution chamber and investigate the properties of the new dissolution chamber for the assessment of sustained drug release systems in periodontal delivery. Flow-through dissolution chambers were constructed using three-dimensional (3D) printing. Drug release experiments were performed using the dissolution chamber and a commercially available long-acting periodontal insert product, PerioChip®. Similar experiments were performed under more traditional larger volume bulk solution conditions for comparison. Computer simulations and experimental results showed that drug clearance from the dissolution chamber was fast compared to drug release from the periodontal product. Drug clearance from the flow-through dissolution chamber and drug release from the sustained release product in the chamber were related to the dissolution medium flow rate and chamber volume. Drug release in the flow-through chamber was slower than that observed in bulk solution, but approached it when the medium flow rate increased. The presence of trypsin in the dissolution medium enhanced drug release from the product. A flow-through dissolution system was constructed that could evaluate drug release from a sustained release product in a small dimension environment by modifying the flow rate and composition of the dissolution medium.

Influencing factors on gelatin matrix for chlorhexidine delivery.

OBJECTIVE: The objective was to evaluate the influencing factors in the fabrication of gelatin matrix (gelatin chips) for drug delivery. The attributes affecting drug release characteristics of the gelatin products were examined. SIGNIFICANCE: Understanding the attributes that affect drug release from gelatin matrix could provide the knowledge base for the development, manufacturing, and performance evaluation of gelatin-based drug products for sustained drug delivery. METHODS: Chlorhexidine (CHX) was the model drug in the gelatin-product testing. The gelatin products were fabricated by two methods: a single-pot mixing of all the components and a two-step gelatin crosslinking followed by drug loading. Different gelatin types (Type A porcine and Type B bovine), glutaraldehyde (GTA) crosslinking conditions, glycerin concentration, and CHX concentration in drug loading and loading time were used to fabricate the products. The cumulative amounts of CHX release from the gelatin products were determined using in vitro release testing (IVRT). RESULTS: The attributes affecting CHX release from the gelatin products were gelatin type, GTA crosslinking, and CHX loading concentration. The fabrication methods (two-step method of gelatin crosslinking and drug loading by equilibration vs. direct mixing of the components) also affected CHX release. Other attributes such as glycerin and CHX loading time did not show significant effects on drug release under the conditions studied. In addition, the results in the two IVRT methods employed in this study were comparable. CONCLUSION: Gelatin products of qualitative (Q1) and quantitative (Q2) differences could lead to different drug release behaviors. Drug release was also affected by the ingredient mixing steps during gelatin chip fabrication.

Transepidermal water loss and skin conductance as barrier integrity tests.

In vitro skin permeation studies are commonly used in the risk assessment of toxic compound skin exposure. The present study examined the utility of transepidermal water loss (TEWL) and electrical conductance as barrier integrity tests before skin permeation studies in vitro using a large number of skin samples and fentanyl. TEWL and conductance of the skin samples were measured before the permeation experiments in Franz diffusion cells in vitro with a vapometer and low voltage application, respectively. The data were analyzed based on the in vitro permeation results and in vivo skin absorption information from the transdermal fentanyl product labels. The results showed poor correlations between TEWL and electrical conductance for the skin samples. Weak correlations between fentanyl delivery rate (flux x area) and TEWL and skin conductance were observed. For comparison, TEWL and conductance were also examined after skin perturbation with a syringe needle, and both TEWL and conductance values of the skin samples increased after the perturbation. The data suggest that either TEWL of 10 g/m2/h or skin conductance of 0.07 mS/cm2 can be used as exclusion criteria in skin integrity testing to remove skin samples with high permeabilities under the in vitro conditions studied.

Characterization of Temperature Profiles in Skin and Transdermal Delivery System When Exposed to Temperature Gradients In Vivo and In Vitro.

PURPOSE: Performance of a transdermal delivery system (TDS) can be affected by exposure to elevated temperature, which can lead to unintended safety issues. This study investigated TDS and skin temperatures and their relationship in vivo, characterized the effective thermal resistance of skin, and identified the in vitro diffusion cell conditions that would correlate with in vivo observations. METHODS: Experiments were performed in humans and in Franz diffusion cells with human cadaver skin to record skin and TDS temperatures at room temperature and with exposure to a heat flux. Skin temperatures were regulated with two methods: a heating lamp in vivo and in vitro, or thermostatic control of the receiver chamber in vitro. RESULTS: In vivo basal skin temperatures beneath TDS at different anatomical sites were not statistically different. The maximum tolerable skin surface temperature was approximately 42-43°C in vivo. The temperature difference between skin surface and TDS surface increased with increasing temperature, or with increasing TDS thermal resistance in vivo and in vitro. CONCLUSIONS: Based on the effective thermal resistance of skin in vivo and in vitro, the heating lamp method is an adequate in vitro method. However, the in vitro-in vivo correlation of temperature could be affected by the thermal boundary layer in the receiver chamber.

Completely humanizing prolactin rescues infertility in prolactin knockout mice and leads to human prolactin expression in extrapituitary mouse tissues.

A variety of fundamental differences have evolved in the physiology of the human and rodent prolactin (PRL) systems. The PRL gene in humans and other primates contains an alternative promoter, 5.8 kbp upstream of the pituitary transcription start site, which drives expression of PRL in "extrapituitary" tissues, where PRL is believed to exert local, or paracrine, actions. Several of these extrapituitary PRL tissues serve a reproductive function (eg, mammary gland, decidua, prostate, etc), consistent with the hypothesis that local PRL production may be involved in, and required for, normal reproductive physiology in primates. Rodent research models have generated significant findings regarding the role of PRL in reproduction. Specifically, disruption (knockout) of either the PRL gene or its receptor causes profound female reproductive defects at several levels (ovaries, preimplantation endometrium, mammary glands). However, the rodent PRL gene differs significantly from the human, most notably lacking the alternative promoter. Understanding of the physiological regulation and function of extrapituitary PRL has been limited by the absence of a readily accessible experimental model, because the rodent PRL gene does not contain the alternative promoter. To overcome these limitations, we have generated mice that have been "humanized" with regard to the structural gene and tissue expression of PRL. Here, we present the characterization of these animals, demonstrating that the human PRL transgene is responsive to known physiological regulators both in vitro and in vivo. More importantly, the expression of the human PRL transgene is able to rescue the reproductive defects observed in mouse PRL knockout (mPRL(-)) females, validating their usefulness in studying the function or regulation of this hormone in a manner that is relevant to human physiology.

Estrogen regulation of the dopamine-activated GIRK channel in pituitary lactotrophs: implications for regulation of prolactin release during the estrous cycle.

Prolactin (PRL), synthesized and secreted from lactotrophs of the anterior pituitary gland, is tonically inhibited by hypothalamic dopamine (DA) throughout the female reproductive (estrous) cycle. Our laboratory has shown that DA hyperpolarizes these cells by activating G protein-coupled inwardly rectifying K(+) (GIRK) channels; however, this response is only observed on proestrus. While the cellular mechanisms that allow for functional expression of this unique DA-signaling pathway are unclear, we hypothesized that activation of the DA-GIRK effector pathway is due to the rise in circulating estrogen (E2) during the preceding day of diestrus. Thus, we examined the effects of E2 on primary lactotrophs isolated from female rats. Treatment with a physiological concentration of E2 (40-80 pg/ml, in vivo or in vitro) induced a proestrous phenotype in diestrous lactotrophs. These cells exhibited a DA-induced membrane hyperpolarization, as well as a secretory rebound of PRL following DA withdrawal (characteristic of proestrous cells). Internal dialysis of GTPγS demonstrated that E2 exposure enabled functional expression of GIRK channels, and this regulation by E2 did not involve the D2R. The effect of E2 was blocked by the receptor antagonist, ICI 182,780, and by the protein synthesis inhibitor, cycloheximide. Single-cell analysis revealed increased mRNA expression of GIRK channel subunits in E2-treated lactotrophs. While E2 is known to have multiple actions on the lactotroph, the present findings illuminate a novel action of E2 in lactotrophs-regulation of the expression of a DA effector, the GIRK channel.

A phase II trial of docetaxel (Taxotere) as second-line chemotherapy in patients with metastatic breast cancer.

The efficacy and tolerability of docetaxel 100 mg/m(2) every 3 weeks as second-line chemotherapy in patients with metastatic breast cancer was investigated. In addition, the efficacy of a 3-day prophylaxis against cumulative dose-related fluid retention was examined with methylprednisolone 32 mg twice daily for 3 days starting 12 and 3 h before the docetaxel infusion together with oral cetirizine 10 mg 12 and 3 h before start of docetaxel for prevention of acute hypersensitivity reactions. According to the intent to treat-analysis 35% (95%CI: 25; 46) of the 94 patients entered responded to therapy. Their median survival was 12 months (range 0-20 months). The respective response rate for the 87 patients eligible for response evaluation was 37% (95%CI: 27; 48). Their median duration of response was 8 months (range 3-12 months), their median time to progression was 4 months (range 1-12 months). The corresponding response rate in the eligible patient cohort with anthracycline-resistant disease was 28% (95%CI: 15; 45) and increased to 44% (95%CI: 30; 59) in the cohort with non-anthracycline-resistant disease. Patients with visceral metastases responded in 36% and patients with > or = 3 organs involved in 33%. In a retrospective analysis, the 3-day premedication of corticosteroids and antihistamines proved to be as effective as the established but more toxic 5-day regimen in delaying and preventing the occurrence of docetaxel derived toxicities especially the cumulative fluid retention. In conclusion, docetaxel represents one of the most active agents for second-line treatment of metastatic breast cancer, especially for anthracycline-resistant patients. Due to comparable effectiveness of the 5-day regimen which is widely used by others and the 3-day premedication tested in this trial the latter proved to be more favourable and was therefore recommended for future therapies.

Evolution of thiazolidine-based blockers of human Kv1.5 for the treatment of atrial arrhythmias.

Blockade of the Kv1.5 ion channel is a potentially atrial-selective avenue for the treatment of atrial fibrillation and atrial flutter. The development and biological evaluation of a series of thiazolidine-based blockers of Kv1.5 is described.

Discovery and in vitro/in vivo studies of tetrazole derivatives as Kv1.5 blockers.

A novel class of tetrazole-derived Kv1.5 blockers is disclosed. In in vitro studies, several compounds had IC(50)s ranging from 180 to 550 nM. In vivo studies indicated that compounds 2f and 2j increased right atrial ERP about 40% without affecting ventricular ERP.

Phase III trial comparing three doses of docetaxel for second-line treatment of advanced breast cancer.

PURPOSE: To evaluate whether a relationship exists between docetaxel dose and clinical response in the treatment of patients with advanced breast cancer. PATIENTS AND METHODS: Patients whose cancer had progressed after one prior chemotherapy regimen for advanced breast cancer or had recurred during or within 6 months of adjuvant chemotherapy were randomly assigned to docetaxel 60, 75, or 100 mg/m2 intravenously every 3 weeks. RESULTS: Five hundred twenty-seven patients were randomly assigned (intent to treat [ITT]), and 524 were assessable for toxicity. In the population assessable for efficacy (n = 407), logistic regression analysis showed that increasing docetaxel dose was significantly associated with higher response rate (P = .007) and improved time to progression (TTP; P = .014). In the ITT analysis, a significant dose-response relationship was observed for tumor response (P = .026) but not for TTP (P = .067). The incidences of most hematologic and nonhematologic toxicities were related to increasing dose, with grade 3 to 4 neutropenia occurring in 76.4%, 83.7%, and 93.4% and febrile neutropenia occurring in 4.7%, 7.4%, and 14.1% of patients administered the 60, 75, and 100 mg/m2 doses, respectively. One death was considered treatment related. CONCLUSION: A relationship between increasing dose of docetaxel and increased tumor response was observed across the dose range of 60 to 100 mg/m2 every 3 weeks. Toxicities were related to increasing dose. Depending on the therapy goal, any of the doses studied may be appropriate for second-line treatment of advanced breast cancer.

Discovery and synthesis of tetrahydroindolone derived semicarbazones as selective Kv1.5 blockers.

A novel class of tetrahydroindolone-derived semicarbazones has been discovered as potent Kv1.5 blockers. In in vitro studies, several compounds exhibited very good potency for blockade of Kv1.5. Compound 8i showed good selectivity for blockade of Kv1.5 vs hERG and L-type calcium channels. In an anesthetized pig model, compounds 8i and 10c increased atrial ERP about 28%, 18%, respectively, in the right atrium without affecting ventricular ERP.

Discovery and synthesis of tetrahydroindolone-derived carbamates as Kv1.5 blockers.

A novel class of tetrahydroindolone-derived carbamates has been discovered whose members are potent Kv1.5 blockers. The in vitro data show that compounds 6 and 29 are quite potent. They are also very selective over hERG (>450-fold) and L-type calcium channels (>450-fold).

Synthesis and evaluation of (2-phenethyl-2H-1,2,3-triazol-4-yl)(phenyl)methanones as Kv1.5 channel blockers for the treatment of atrial fibrillation.

A series of novel (2-phenethyl-2H-1,2,3-triazol-4-yl)(phenyl)methanones were prepared and examined for utility as Kv1.5 channel blockers for the treatment of atrial fibrillation.

Adjuvant docetaxel for node-positive breast cancer.

BACKGROUND: We compared docetaxel plus doxorubicin and cyclophosphamide (TAC) with fluorouracil plus doxorubicin and cyclophosphamide (FAC) as adjuvant chemotherapy for operable node-positive breast cancer. METHODS: We randomly assigned 1491 women with axillary node-positive breast cancer to six cycles of treatment with either TAC or FAC as adjuvant chemotherapy after surgery. The primary end point was disease-free survival. RESULTS: At a median follow-up of 55 months, the estimated rates of disease-free survival at five years were 75 percent among the 745 patients randomly assigned to receive TAC and 68 percent among the 746 randomly assigned to receive FAC, representing a 28 percent reduction in the risk of relapse (P=0.001) in the TAC group. The estimated rates of overall survival at five years were 87 percent and 81 percent, respectively. Treatment with TAC resulted in a 30 percent reduction in the risk of death (P=0.008). The incidence of grade 3 or 4 neutropenia was 65.5 percent in the TAC group and 49.3 percent in the FAC group (P<0.001); rates of febrile neutropenia were 24.7 percent and 2.5 percent, respectively (P<0.001). Grade 3 or 4 infections occurred in 3.9 percent of the patients who received TAC and 2.2 percent of those who received FAC (P=0.05); no deaths occurred as a result of infection. Two patients in each group died during treatment. Congestive heart failure and acute myeloid leukemia occurred in less than 2 percent of the patients in each group. Quality-of-life scores decreased during chemotherapy but returned to baseline levels after treatment. CONCLUSIONS: Adjuvant chemotherapy with TAC, as compared with FAC, significantly improves the rates of disease-free and overall survival among women with operable node-positive breast cancer.