<Emphasis Type="BoldItalic">In Vitro</Emphasis> and <Emphasis Type="BoldItalic">In Vivo</Emphasis> Drug Release from a Novel <Emphasis Type="BoldItalic">In Situ</Emphasis> Forming Drug Delivery System

Purpose The objective of this work was to investigate the influence of various preparation and formulation parameters on the in vitro and in vivo release of bupivacaine hydrochloride from an injectable in situ forming microparticle system (ISM). Methods The in vitro drug release of ISM was investigated as a function of various formulation and process parameters and was compared to the drug release from in situ forming implants and conventional microparticles. In vivo studies were carried out in male Sprague–Dawley rats. Results Upon contact with an aqueous medium, the internal polymer phase of the ISM system solidified and formed microparticles. The initial drug release from ISM systems was reduced with decreasing polymer phase/external oil phase ratio. An advantage of the ISM system compared to in situ implant systems was the significantly reduced burst effect, resulting in drug release profiles comparable to microparticles prepared by conventional methods. The in vivo drug release studies were in good agreement with the in vitro drug release. With the ISM system, the analgesic effect of the bupivacaine hydrochloride was prolonged when compared to the injection of a drug solution or drug-polymer solution. Conclusions ISM are an attractive alternative for parenteral drug delivery systems.     

Exploring the Carbamazepine Interaction with Human Pregnane X Receptor and Effect on ABCC2 Using <Emphasis Type="BoldItalic">in Vitro</Emphasis> and <Emphasis Type="BoldItalic">in Silico</Emphasis> Approach

Purpose

Over expression of ATP-binding cassette transporters is considered one of the major reasons for non-responsiveness to antiepileptic drugs. Carbamazepine (CBZ), one of first line antiepileptic drug is known to influence ABCC2 expression but its exact molecular mechanism is unknown.

Methods

We investigated the effect of CBZ on expression of ABCC2 and pregnane X receptor (PXR) in HepG2 cell line and compared with hyperforin (agonist of PXR) and ketoconazole (antagonist of PXR) through realtime PCR and western blot assay. Involvement of PXR was demonstrated through nuclear translocation and RNA interference and related effect of CBZ on ABCC2 through functional activity assay. Molecular docking and dynamic simulation approach was used to understand the interaction of CBZ with PXR.

Results

CBZ and hyperforin increased the PXR and ABCC2 expression whereas reversed when present it in combination with ketoconazole. Experiments confirmed CBZ induced ABCC2 expression is PXR dependent. Molecular dynamic (MD) simulation and in vitro experiment indicated possibility of CBZ to be PXR agonist and PXR residue Gln285 to be important for CBZ-PXR interaction.

Conclusions

CBZ alters the functional activity of ABCC2 through PXR, which in turn can interfere with therapy. Mutational analysis of residues revealed the importance of Gln285 in ligand interaction.

     

<Emphasis Type="BoldItalic">In Vitro</Emphasis> Transport Activity and Trafficking of MRP2/<Emphasis Type="BoldItalic">ABCC2</Emphasis> Polymorphic Variants

Purpose

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is an efflux pump that removes drugs and chemicals from cells. We sought to characterize the expression, trafficking and in vitro activity of seven single nucleotide polymorphisms (SNPs) in the ABCC2 gene.

Methods

ABCC2 SNPs were generated using site-directed mutagenesis and stably expressed in Flp-In HEK293 cells, which allows targeted insertion of transgenes within the genome. Total and cell surface expression of MRP2 as well as accumulation of substrates (calcein AM and 5(6)-carboxy-2′,7′-dichlorofluorescein diacetate, CDCF) were quantified in cells or inverted membrane vesicles expressing wild-type (WT) or variant forms.

Results

The cell surface expression of the C-24T-, G1249A-, G3542T-, T3563A-, C3972T- and G4544A-MRP2 variants was similar to WT-MRP2. While expression was similar, transport of calcein AM was enhanced in cells expressing the G3542T-, T3563A-, C3972T-, and G4544A-MRP2 variants. By comparison, cells expressing the C2366T-MRP2 variant had 40–50% lower surface expression, which increased the accumulation of calcein AM up to 3-fold. Accumulation of CDCF in inverted membrane vesicles expressing the C2366T-MRP2 variant was also reduced by 50%. In addition, the G1249A-MRP2 variant had decreased transport of CDCF.

Conclusions

Taken together, these data demonstrate that genetic variability in the ABCC2 gene influences the in vitro expression, trafficking, and transport activity of MRP2.

     

PEGylated PAMAM Dendrimer-Doxorubicin Conjugates: <Emphasis Type="BoldItalic">In Vitro</Emphasis> Evaluation and <Emphasis Type="BoldItalic">In Vivo</Emphasis> Tumor Accumulation

Purpose  

To investigate the effects of PEGylation degree and drug conjugation style on the in vitro and in vivo behavior of PEGylated polyamidoamine (PAMAM) dendrimers-based drug delivery system.     

Targeted Delivery of Epirubicin to Cancer Cells by Polyvalent Aptamer System <Emphasis Type="BoldItalic">in vitro</Emphasis> and <Emphasis Type="BoldItalic">in vivo</Emphasis>

Purpose

The clinical use of Epirubicin (Epi), as an anthracycline drug, is limited because of its cardiotoxicity. Here, an Epirubicin (Epi)-modified polyvalent aptamer system (MPAS) conjugate was developed for the treatment of both murine colon carcinoma cells (C26) and breast cancer cells (MCF-7).

Methods

Epi-MPAS conjugate formation was evaluated by fluorometric analysis. Release profiles of Epi from the developed conjugate were analyzed at pHs 5.4 and 7.4. For MTT assay (cytotoxic study) C26 and MCF-7 (target cells) and CHO cells (Chinese hamster ovary cell, nontarget) were treated with Epi, MPAS and Epi-MPAS conjugate. Internalization was assessed by fluorescence imaging and flow cytometry analysis. The designed conjugate was used for prohibition of tumor growth in vivo.

Results

Release of Epi from the Epi-MPAS conjugated was pH-dependent (more release at pH 5.5). Flow cytometry analysis and MTT assay showed that Epi-MPAS conjugate could significantly enhance the cellular uptake of Epi and increase its cytotoxicity in target cells as compared with non-targeted cell (CHO). Additionally, this complex could efficiently prohibit the tumor growth in vivo.

Conclusion

In conclusion, the developed drug delivery system had the characteristics of efficient Epi loading, pH-dependent drug release and tumor targeting in vitro and in vivo.

     

Novel Inhibitors of Human Organic Cation/Carnitine Transporter (hOCTN2) <Emphasis Type="BoldItalic">via</Emphasis> Computational Modeling and <Emphasis Type="BoldItalic">In Vitro</Emphasis> Testing

Purpose  The objective was to elucidate the inhibition requirements of the human organic cation/carnitine transporter (hOCTN2). Methods  Twenty-seven drugs were screened initially for their potential to inhibit uptake of l-carnitine into a stably transfected hOCTN2-MDCK cell monolayer. A HipHop common features pharmacophore was developed and used to search a drug database. Fifty-three drugs, including some not predicted to be inhibitors, were selected and screened in vitro. Results  A common features pharmacophore was derived from initial screening data and consisted of three hydrophobic features and a positive ionizable feature. Among the 33 tested drugs that were predicted to map to the pharmacophore, 27 inhibited hOCTN2 in vitro (40% or less l-carnitine uptake from 2.5 μM l-carnitine solution in presence of 500 μM drug, compared to l-carnitine uptake without drug present). Hence, the pharmacophore accurately prioritized compounds for testing. K _i measurements showed low micromolar inhibitors belonged to diverse therapeutic classes of drugs, including many not previously known to inhibit hOCTN2. Compounds were more likely to cause rhabdomyolysis if the C _max/K _i ratio was higher than 0.0025. Conclusion  A combined pharmacophore and in vitro approach found new, structurally diverse inhibitors for hOCTN2 that may possibly cause clinical significant toxicity such as rhabdomyolysis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="BoldItalic">In Silico</Emphasis> Absorption Analysis of Valacyclovir in Wildtype and <Emphasis Type="BoldItalic">Pept1</Emphasis> Knockout Mice Following Oral Dose Escalation

Purpose

We developed simulation and modeling methods to predict the in vivo pharmacokinetic profiles of acyclovir, following escalating oral doses of valacyclovir, in wildtype and Pept1 knockout mice. We also quantitated the contribution of specific intestinal segments in the absorption of valacyclovir in these mice.

Methods

Simulations were conducted using a mechanistic advanced compartmental absorption and transit (ACAT) model implemented in GastroPlus?. Simulations were performed for 3 h post-dose in wildtype and Pept1 knockout mice following single oral doses of 10, 25, 50 and 100 nmol/g valacyclovir, and compared to experimentally observed plasma concentration-time profiles of acyclovir.

Results

Good fits were obtained in wildtype and Pept1 knockout mice. Valacyclovir was primarily absorbed from duodenum (42%) and jejunum (24%) of wildtype mice, with reduced uptake from ileum (3%) and caecum/colon (1%), for a total of 70% absorption. In contrast, the absorption of valacyclovir in Pept1 knockout mice was slow and sustained throughout the entire intestinal tract in which duodenum (4%), jejunum (14%), ileum (10%) and caecum/colon (12%) accounted for a total of 40% absorption.

Conclusion

The ACAT model bridged the gap between in situ and in vivo experimental findings, and facilitated our understanding of the complicated intestinal absorption processes of valacyclovir.

     

The <Emphasis Type="BoldItalic">In vitro</Emphasis> Sub-cellular Localization and <Emphasis Type="BoldItalic">In vivo</Emphasis> Efficacy of Novel Chitosan/GMO Nanostructures containing Paclitaxel

Purpose

To determine the in vitro sub-cellular localization and in vivo efficacy of chitosan/GMO nanostructures containing paclitaxel (PTX) compared to a conventional PTX treatment (Taxol^®).

Methods

The sub-cellular localization of coumarin-6 labeled chitosan/GMO nanostructures was determined by confocal microscopy in MDA-MB-231 cells. The antitumor efficacy was evaluated in two separate studies using FOX-Chase (CB17) SCID Female-Mice MDA-MB-231 xenograph model. Treatments consisted of intravenous Taxol^® or chitosan/GMO nanostructures with or without PTX, local intra-tumor bolus of Taxol^® or chitosan/GMO nanostructures with or without PTX. The tumor diameter and animal weight was monitored at various intervals. Histopathological changes were evaluated in end-point tumors.

Results

The tumor diameter increased at a constant rate for all the groups between days 7-14. After a single intratumoral bolus dose of chitosan/GMO containing PTX showed significant reduction in tumor diameter on day 15 when compared to control, placebo and intravenous PTX administration. The tumor diameter reached a maximal decrease (4-fold) by day 18, and the difference was reduced to approximately 2-fold by day 21. Qualitatively similar results were observed in a separate study containing PTX when administered intravenously.

Conclusion

Chitosan/GMO nanostructures containing PTX are safe and effective administered locally or intravenously. Partially supported by DOD Award BC045664

     

A Novel Gastro-Retentive Osmotic Pump Capsule Using Asymmetric Membrane Technology: <Emphasis Type="BoldItalic">In Vitro</Emphasis> and <Emphasis Type="BoldItalic">In Vivo</Emphasis> Evaluation

Purpose  

The purpose of this paper is to develop a novel gastro-retentive osmotic pump capsule using asymmetric membrane technology.     

Dynamic Dissolution Testing To Establish <Emphasis Type="BoldItalic">In Vitro</Emphasis>/<Emphasis Type="BoldItalic">In Vivo</Emphasis> Correlations for Montelukast Sodium,a Poorly Soluble Drug

Purpose  The objectives of the study was to develop a dissolution test method that can be used to predict the oral absorption of montelukast sodium, and to establish an in vitro/in vivo correlation (IVIVC) using computer simulations. Methods  Drug solubility was measured in different media. The dissolution behaviour of montelukast sodium 10 mg film coated tablets was studied using the flow-through cell dissolution method following a dynamic pH change protocol, as well as in the USP Apparatus 2. Computer simulations were performed using GastroPlus™. Biorelevant dissolution media (BDM) prepared using bile salts and lecithin in buffers was used as the dissolution media, as well as the USP simulated intestinal fluid (SIF) pH 6.8 and blank FaSSIF pH 6.5. Dissolution tests in the USP Apparatus 2 were performed under a constant pH condition, while the pH range used in the flow through cells was pH 2.0 to 7.5. The in vitro data were used as input functions into GastroPlus™ to simulate the in vivo profiles of the drug. Results  The solubility of montelukast sodium was low at low pH, but increased as the pH was increased. There was no significant difference in solubility in the pH range of 5.0 to 7.5 in blank buffers, but the drug solubility was higher in biorelevant media compared with the corresponding blank buffers at the same pH. Using the flow through cells, the dissolution rate was fast in simulated gastric fluid containing 0.1% SLS. The dissolution rate slowed down when the medium was changed to FaSSIF pH 6.5 and increased when the medium was changed to FaSSIF medium at pH 7.5. In the USP Apparatus 2, better dissolution was observed in FaSSIF compared with the USP buffers and blank FaSSIF with similar pH values. Dissolution was incomplete with less than 10% of the drug dissolved in the USP-SIF, and was practically non existent in blank FaSSIF pH 6.5. The in vitro results of the dynamic dissolution test were able to predict the clinical data from a bioavailability study best. Conclusions  Dynamic dissolution testing using the flow through cell seems to be a powerful tool to establish in vitro/in vivo correlations for poorly soluble drugs as input function into GastroPlus.     

<Emphasis Type="BoldItalic">In Vitro</Emphasis> and <Emphasis Type="BoldItalic">In Vivo</Emphasis> Release of Vascular Endothelial Growth Factor from Gelatin Microparticles and Biodegradable Composite Scaffolds

PURPOSE: This work evaluated gelatin microparticles and biodegradable composite scaffolds for the controlled release of vascular endothelial growth factor (VEGF) in vitro and in vivo. METHODS: Gelatin crosslinking, VEGF dose, and buffer type were investigated for their effects on VEGF release. Release was also evaluated from microparticles confined within porous polymer scaffolds (composites). In vitro and in vivo studies were conducted using radiolabeled VEGF. RESULTS: The effect of VEGF dose on its fractional release from gelatin microparticles in vitro was minimal, but the addition of collagenase to the buffer resulted in a higher cumulative release of VEGF. Gelatin crosslinking extent was a significant factor on release from both microparticles alone and composite scaffolds in vitro and in vivo. VEGF bioactivity from composite scaffolds in vitro was maintained above 90% of the expected bioactivity over 14 days. CONCLUSIONS: VEGF release kinetics were dependent on the extent of gelatin crosslinking and were characteristic of the specific growth factor due to the effects of growth factor size, charge, and conformation on its complexation with gelatin. These studies demonstrate the utility of gelatin microparticles and their composite scaffolds as delivery vehicles for the controlled release of VEGF for tissue engineering applications.     

Co-Delivery of Ciprofloxacin and Colistin in Liposomal Formulations with Enhanced <Emphasis Type="BoldItalic">In Vitro</Emphasis> Antimicrobial Activities against Multidrug Resistant <Emphasis Type="BoldItalic">Pseudomonas aeruginosa</Emphasis>

Purpose

This study aims to develop liposomal formulations containing synergistic antibiotics of colistin and ciprofloxacin for the treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa.

Methods

Colistin (Col) and ciprofloxacin (Cip) were co-encapsulated in anionic liposomes by ammonium sulfate gradient. Particle size, encapsulation efficiency, in vitro drug release and in vitro antibiotic activities were evaluated.

Results

The optimized liposomal formulation has uniform sizes of approximately 100 nm, with encapsulation efficiency of 67.0% (for colistin) and 85.2% (for ciprofloxacin). Incorporation of anionic lipid (DMPG) markedly increased encapsulation efficiency of colistin (from 5.4 to 67.0%); however, the encapsulation efficiency of ciprofloxacin was independent of DMPG ratio. Incorporation of colistin significantly accelerated the release of ciprofloxacin from the DMPG anionic liposomes. In vitro release of ciprofloxacin and colistin in the bovine serum for 2 h were above 70 and 50%. The cytotoxicity study using A549 cells showed the liposomal formulation is as non-toxic as the drug solutions. Liposomal formulations of combinations had enhanced in vitro antimicrobial activities against multidrug resistant P. aeruginosa than the monotherapies.

Conclusions

Liposomal formulations of two synergistic antibiotics was promising against multidrug resistant P. aeruginosa infections.

     

Identification of the Promoter of Human Carbonyl Reductase 3 (<Emphasis Type="BoldItalic">CBR3</Emphasis>) and Impact of Common Promoter Polymorphisms on Hepatic <Emphasis Type="BoldItalic">CBR3</Emphasis> mRNA Expression

Purpose  Recent studies suggest that polymorphisms in human carbonyl reductase 3 (CBR3) influence the pharmacodynamics of doxorubicin. First, we sought to identify the promoter of CBR3. Next, we examined whether two CBR3 promoter polymorphisms (CBR3 -725T>C and CBR3 -326T>A) dictate promoter activity and hepatic CBR3 mRNA levels. Methods  The promoter activities of CBR3 reporter constructs were investigated in HepG2 and MCF-7 cells. CBR3 mRNA levels were documented in 95 liver samples from white (n = 62) and black (n = 33) donors. Genotype-phenotype correlation analyses were used to determine the impact of the CBR3 -725T>C and CBR3 -326T>A polymorphisms on hepatic CBR3 mRNA levels. Results  We identified the promoter of human CBR3. Liver samples from black donors showed higher relative CBR3 mRNA levels than samples from whites (CBR3 mRNA_blacks = 3.0 ± 3.1 relative fold vs. CBR3 mRNA_whites = 1.6 ± 1.5 relative fold, p = 0.021). The variant -725C and -326A alleles did not modify the gene reporter activities of engineered CBR3 promoter constructs. In line, hepatic CBR3 mRNA levels were not associated with CBR3 -725T>C and CBR3 -326T>A genotype status. Conclusions  These studies provide the first insights into the regulation and variable hepatic expression of polymorphic CBR3.     

Identification of medicinal <Emphasis Type="Italic">Dendrobium</Emphasis> species by phylogenetic analyses using <Emphasis Type="Italic">matK</Emphasis> and <Emphasis Type="Italic">rbcL</Emphasis> sequences

Species identification of five Dendrobium plants was conducted using phylogenetic analysis and the validity of the method was verified. Some Dendrobium plants (Orchidaceae) have been used as herbal medicines but the difficulty in identifying their botanical origin by traditional methods prevented their full modern utilization. Based on the emerging field of molecular systematics as a powerful classification tool, a phylogenetic analysis was conducted using sequences of two plastid genes, the maturase-coding gene (matK) and the large subunit of ribulose 1,5-bisphosphate carboxylase-coding gene (rbcL), as DNA barcodes for species identification of Dendrobium plants. We investigated five medicinal Dendrobium species, Dendrobium fimbriatum, D. moniliforme, D. nobile, D. pulchellum, and D. tosaense. The phylogenetic trees constructed from matK data successfully distinguished each species from each other. On the other hand, rbcL, as a single-locus barcode, offered less species discriminating power than matK, possibly due to its being present with little variation. When results using matK sequences of D. officinale that was deposited in the DNA database were combined, D. officinale and D. tosaense showed a close genetic relationship, which brought us closer to resolving the question of their taxonomic identity. Identification of the plant source as well as the uniformity of the chemical components is critical for the quality control of herbal medicines and it is important that the processed materials be validated. The methods presented here could be applied to the analysis of processed Dendrobium plants and be a promising tool for the identification of botanical origins of crude drugs.     

<Emphasis Type="Italic">ent</Emphasis>-Abietane diterpenoids from <Emphasis Type="Italic">Isodon xerophilus</Emphasis>

Three new ent-abietanoids, named xerophilusins XIV–XVI, and four known analogues, as well as four known chemical constituents were isolated from the leaves of Isodon xerophilus. Their structures were elucidated by extensive spectroscopic studies, and comparison with literature data. In addition, the cytotoxic activity of the ent-abietanoids against chronic myelogenous leukemia (K562), stomach adenocarcinoma (MKN45), and hepatocellular carcinoma (HepG2) human cell lines was investigated and no activities were observed.     

Formulation and <Emphasis Type="BoldItalic">In-Vitro</Emphasis> and <Emphasis Type="BoldItalic">In-Vivo</Emphasis> Evaluation of a Mucoadhesive Gel Containing Freeze Dried Black Raspberries: Implications for Oral Cancer Chemoprevention

Purpose The purpose of these studies was to formulate mucoadhesive gels containing freeze dried black raspberries (FBR) and to determine optimum parameters for a subset of FBR bioactive compounds including anthocyanin stability, absorption and penetration in-vitro and in-vivo. Materials and Methods Berry gels were prepared having FBR at 5% and 10% w/w and final pHs ranging from 3.5 to 7.5. A HPLC assay was developed to quantify and determine the stability of the anthocyanins in the gels. A single time-point study was performed to determine anthocyanin uptake when the gels were applied to oral mucosa. Penetration of anthocyanins into human oral tissue explants was determined as a function of gel pH and FBR content. A HPLC-mass spectroscopy assay was utilized to quantify the anthocyanin levels in human oral tissue explants, saliva, and blood. Results The stability of anthocyanins in the gel was directly related to gel pH and storage temperature. Maximum stability of anthocyanins was found at lower pH (pH 3.5) and storage temperature (4°C). Anthocyanins contained in mucoadhesive berry gel formulations were readily absorbed into human oral mucosa tissue as evidenced by detectable blood levels within 5 min after gel application. There was a trend for greater penetration of anthocyanins into tissue explants for berry gels with a final pH of 6.5 versus pH 3.5. Conclusions Formulation and characterization of a novel gel formulation for local delivery of chemopreventive compounds to human oral mucosal tissues has been described. The results show anthocyanin stability was dependent upon gel pH and storage temperature and also demonstrate that the gel composition is well-suited for absorption and penetration into the target oral mucosal tissue site.     

<Emphasis Type="BoldItalic">In Vitro</Emphasis> and <Emphasis Type="BoldItalic">In Vivo</Emphasis> Modeling of Hydroxypropyl Methylcellulose (HPMC) Matrix Tablet Erosion Under Fasting and Postprandial Status

Purpose

To develop a model linking in vitro and in vivo erosion of extended release tablets under fasting and postprandial status.

Methods

A nonlinear mixed-effects model was developed from the in vitro erosion profiles of four hydroxypropyl methylcellulose (HPMC) matrix tablets studied under a range of experimental conditions. The model was used to predict in vivo erosion of the HPMC matrix tablets in different locations of the gastrointestinal tract, determined by magnetic marker monitoring. In each gastrointestinal segment the pH was set to physiological values and mechanical stress was estimated in USP2 apparatus rotation speed equivalent.

Results

Erosion was best described by a Michaelis–Menten type model. The maximal HPMC release rate (V_MAX) was affected by pH, mechanical stress, HPMC and calcium hydrogen phosphate content. The amount of HPMC left at which the release rate is half of V_MAX depended on pH and calcium hydrogen phosphate. Mechanical stress was estimated for stomach (39.5 rpm), proximal (93.3 rpm) and distal (31.1 rpm) small intestine and colon (9.99 rpm).

Conclusions

The in silico model accurately predicted the erosion profiles of HPMC matrix tablets under fasting and postprandial status and can be used to facilitate future development of extended release tablets.

     

Characterization of Temperature Profiles in Skin and Transdermal Delivery System When Exposed to Temperature Gradients <Emphasis Type="BoldItalic">In Vivo</Emphasis> and <Emphasis Type="BoldItalic">In Vitro</Emphasis>

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.

     

Bioequivalence Methodologies for Topical Drug Products: <Emphasis Type="BoldItalic">In Vitro</Emphasis> and <Emphasis Type="BoldItalic">Ex Vivo</Emphasis> Studies with a Corticosteroid and an Anti-Fungal Drug

Objective

To examine whether in vitro and ex vivo measurements of topical drug product performance correlate with in vivo outcomes, such that more efficient experimental approaches can be reliably and reproducibly used to establish (in)equivalence between formulations for skin application.

Materials and Methods

In vitro drug release through artificial membranes, and drug penetration into porcine skin ex vivo, were compared with published human in vivo studies. Two betamethasone valerate (BMV) formulations, and three marketed econazole nitrate (EN) creams were assessed.

Results

For BMV, the stratum corneum (SC) uptake of drug in 6 h closely matched data observed in vivo in humans, and distinguished between inequivalent formulations. SC uptake of EN from the 3 creams mirrored the in vivo equivalence in man (both clinically and via similar tape-stripping experiments). However, EN clearance from SC ex vivo did not parallel that in vivo, presumably due to the absence of a functioning microcirculation. In vitro release of BMV from the different formulations did not overlap with either ex vivo or in vivo tape-stripping data whereas, for EN, a good correlation was observed. No measurable permeation of either BMV or EN was detected in a 6-h in vitro skin penetration experiment.

Conclusions

In vitro and ex vivo methods for topical bioequivalence determination can show correlation with in vivo outcomes. However, these surrogates have understandable limitations. A “one-size-fits-all” approach for topical bioequivalence evaluation may not always be successful, therefore, and the judicious use of complementary methods may prove a more effective and reliable strategy.

     

Equivalence-by-Design: Targeting <Emphasis Type="BoldItalic">In Vivo</Emphasis> Drug Delivery Profile

In the United States (U.S.), drug products are considered therapeutically equivalent if they meet regulatory criteria of pharmaceutical equivalence and bioequivalence. These requirements can be traced back to 1977 when the U.S. Food and Drug Administration (FDA) published the regulations on bioavailability and bioequivalence. Over the years, to keep up with the advancement in science and technology, the FDA has been constantly updating the regulatory approaches to assessing and ensuring equivalence. In view of the recent growth in novel pharmaceutical dosage forms and delivery systems, this paper examines the current framework for documentation of therapeutic equivalence and explores the opportunities of further advancing equivalence methods for complex drug products. It is proposed that equivalence may be established by matching the in vivo drug delivery profile (iDDP) between drug products in comparison. This can be achieved by characterizing the iDDP of the reference formulation with application of an equivalence-by-design approach for pharmaceutical development. Critical variables can be identified to serve as in vitro markers or biomarkers for mapping the desired drug delivery profile in vivo. A multidisciplinary approach may be necessary to develop these markers for characterization of iDDPs. The opinions expressed in this article do not necessarily represent the views or policies of the U.S. Food and Drug Administration