• Evaluation of In Vitro-In Vivo Correlations in Topical and Transdermal Drug Delivery Systems by In Vitro Permeation Testing and Pharmacokinetic Studies for Bioavailability/Bioequivalence and Heat Effect Assessment

      Zhang, Qingzhao; Stinchcomb, Audra L.; 0000-0003-4061-0690 (2022)
      In vitro-in vivo correlation (IVIVC) has gained great attention in the biopharmaceutical field because of its potential ability to predict drug performance in vivo, eliminating costly and time-consuming clinical trials. However, the regulatory guidance on IVIVC has only been established for extended-release oral dosage forms, and not for topical and transdermal formulations. An in vitro model is necessary to establish IVIVC. The present work showed that the in vitro permeation test (IVPT) using excised human skin can quantify and distinguish bioavailability (BA)/bioequivalence (BE) between a brand-name topical metronidazole gel, a bioequivalent generic gel, and a non-bioequivalent generic cream. A harmonized pharmacokinetic (PK) study in healthy human subjects was conducted to evaluate IVIVC. The scaled average bioequivalence approach was used to establish BE between the gels and distinguish the cream from the gels. The metronidazole PK study didn’t provide reproducible serum levels; therefore, IVIVC’s predictability was weak. The present work also showed IVPT’s ability to predict BA for two rivastigmine TDS under transient heat exposure. A harmonized PK study was conducted. A Level C and a Level A IVIVC were established under baseline temperature (32.0°C). IVPT demonstrated significantly increased maximum flux for both TDS with a comparable fold enhancement. However, the PK study didn’t demonstrate a consistent heat effect on maximum serum concentrations of rivastigmine under elevated temperature (42.0 °C). The IVIVC’s predictability for the heat effect was therefore weak. Encouragingly, findings suggested that IVPT is useful in assessing BE for topical drug products. For hydrophilic drugs, such as metronidazole, it is possible that IVPT may have limited predictability in vivo if significant absorption of the drug occurs via the shunt route, and/or if significant reproducible systemic absorption can’t be quantified from dosing the semisolid over a large surface area. In conclusion, IVPT was able to quantify the magnitude of heat effect on the absorption from rivastigmine TDS, and data also suggested that under baseline temperature, IVPT will likely show good predictability of TDS performance in vivo. However, experimental conditions should be further altered to validate IVPT’s usefulness in the prediction of TDS BA in vivo under the heat exposure.
    • Evaluation of In Vitro/In Vivo Correlations for Transdermal Delivery Systems by In Vitro Permeation Testing and Human Pharmacokinetic Studies, With and Without a Transient Heat Application

      Shin, Soo Hyeon; Stinchcomb, Audra L.; Hassan, Hazem; 0000-0001-5091-8870 (2018)
      An in vitro model that exhibits in vitro/in vivo correlations (IVIVC) is a powerful tool in biopharmaceutical drug development because it can efficiently predict drug product performance in vivo. While the concept of IVIVC has been utilized mostly for oral dosage forms, demonstrations of IVIVC with in vitro permeation testing (IVPT) for transdermal delivery systems (TDS) are emerging. The objective of this work was to evaluate IVIVC for TDS using two model drugs, nicotine and fentanyl, with different physicochemical characteristics (e.g. log P). Additionally, the effect of heat exposure (42 ± 2 °C) on the rate and extent of TDS drug delivery was evaluated. IVPT studies using excised human skin and in vivo pharmacokinetic (PK) studies in human subjects were conducted under harmonized study conditions and designs to evaluate IVIVC. The correlations were evaluated in multiple ways, including a single point comparison of parameters such as steady-state concentration and heat-induced increase in partial AUCs, as well as a point-to-point correlation (Level A IVIVC). Level A IVIVC was examined using multiple approaches. A strong IVIVC was consistently observed for nicotine TDS in presence and absence of heat, suggesting the utility of IVPT as a tool to evaluate and predict in vivo performance of nicotine TDS. The IVIVC results for fentanyl were relatively weaker, especially when IVIVC for heat effects were examined, with greater in vivo heat effects observed compared to the in vitro heat effects. A separate study evaluating IVIVC for fentanyl TDS without a heat exposure component and utilization of some PK parameters obtained directly from study subjects yielded improved IVIVC results. The findings from the present research work suggest that IVPT data generally shows good predictability of in vivo performance of TDS at normal temperature conditions. However, the usefulness of IVPT for assessing and predicting external factors such as heat, especially for lipophilic drug molecules, may have some limitations that could be further improved.
    • In-vitro Efficacy and Intracellular Mechanism of Riboflavin-Conjugated PEGylated Poly- L-Lysine Dendrimer

      Pak, Yewon; Swaan, Peter W. (2017)
      Chemotherapeutic drugs have advanced using different drug delivery methods to treat breast cancer specifically. This development has arisen because many classical drugs exhibit physicochemical limitations including solubility, specificity, stability, biodistribution, and therapeutic efficacy. There were numerous adverse effects associated with these limitations because chemotherapeutic drugs enter normal tissues. In order to eliminate off-target side-effect,nanoparticles were developed to target anticancer drugs to a specific carcinogenic area. As one of developing nanomedicines, dendrimers possess ability to be utilized in different administration routes and has potential to stay in the blood circulation longer while showing increased accumulation in tumor cells. Commercially available poly (amidoamine) (PAMAM) dendrimers have the potential to cause toxicity in vivo due to lack of biodegradation at sites of accumulation. Poly-L-Lysine (PLL) dendrimers are an alternative class of dendrimers that possess a biodegradable structure. PEGylated poly-l-lysine (PLL) dendrimers are known to be more favorable due to lessened cytotoxicity manifested by masking of cationic charges and avoiding uptake by Reticulo Endothelial System (RES). Using this biodegradable dendrimer, we sought to examine the effect of PEGylation as well as delivering anti-cancer drug, Doxorubicin (DOX), to a targeted internalization pathway in human breast cancer cells effectively. PEGylated PLL dendrimers also have their limitation, in which some tumor cells are not dependent upon enhanced permeability and retention (EPR) effect. As a result, riboflavin receptor, which is found to be upregulated in the exterior of breast and ovarian cancer cells, was utilized by attaching a riboflavin ligand to PEGylated PLL dendrimers in order to be actively uptaken by breast cancer cells. To target chemotherapeutic drug selectively and efficaciously, riboflavin conjugated PLL dendrimers were assessed in-vitro by investigating cytotoxicity, uptake accumulation, and intracellular colocalization. Further investigation on the endocytosis mechanism and detailed intracellular trafficking in different compartments of the cells were analyzed in order to fully understand the machinery behind delivering chemotherapeutic drugs successfully.
    • Itraconazole-HPMCAS amorphous spray dried dispersions: composition and process factors impacting performance

      Honick, Moshe Avraham; Polli, James E. (2019)
      Despite their potential for improving the oral bioavailability of poorly water soluble drugs, spray dried dispersions (SDDs) have properties that make them challenging to formulate. The objective of this dissertation was to elucidate composition and process factors for favorable SDD performance and to develop fast-, medium-, and slow-release formulations for an IVIVC study. Itraconazole (ITZ) was used as a model poorly soluble drug and hypromellose acetate succinate (HPMCAS) was used as a carrier polymer for the SDDs. Film casting proved to be a useful screening method for demonstrating the feasibility of producing amorphous SDDs of ITZ and HPMCAS as well as for rank ordering the grades of HPMCAS (i.e. HPMCAS-L > HPMCAS-M > HPMCAS-H) in terms of in-vitro dissolution performance. Producing solid oral dosage forms of ITZ-HPMCAS SDDs proved challenging due to the low particle size, poor flowability, and low bulk density of the SDDs. Initial tableting on a Natoli hand-operated press showed that drug release from tablets containing SDDs of ITZ and HPMCAS-L were very sensitive to small differences in compaction pressure and porosity. Interestingly, the same sensitivity was not observed in SDDs of ITZ and HPMCAS-M. Using a compaction simulator, reproducible fast-, medium- and slow-release tablet formulations of ITZ and HPMCAS SDDs was developed by varying polymer grade (HPMCAS-L, HPMCAS-M), slugging pressure (20, 40 MPa), and compaction pressure (70, 85, 100 MPa). The performance of SDDs was further evaluated by comparing the compaction behavior of ITZ-HPMCAS SDDs and physical mixtures of ITZ and HPMCAS. Although the compressibility of both the SDDs and physical mixtures were similar, the SDDs had a greater tendency to laminate, especially at higher compression speeds. Tablets of SDDs containing ITZ and HPMCAS-L were particularly prone to lamination compared to the SDDs containing HPMCAS-M or HPMCAS-H. Interestingly, when the SDDs were not laminated they had a greater tensile strength than tablets produced with the physical mixtures. In conclusion, there are significant challenges associated with formulating SDDs of ITZ and HPMCAS. In addition to elucidating composition and process factors impacting performance, fast-, medium-, and slow-release formulations for an IVIVC study were developed.
    • Mechanisms of Dendrimer-Mediated Oral Drug Delivery

      Avaritt, Brittany; Swaan, Peter W. (2014)
      Oral administration of chemotherapeutics remains a challenge despite the benefits for both the patient and health care system. To overcome the poor solubility and low oral bioavailability of anti-cancer drugs, polymeric delivery systems have been investigated. Dendrimers, a class of highly branched polymers, have proven useful for drug delivery because of their compact, nanoscopic size. Specifically, poly(amidoamine) (PAMAM) dendrimers have been shown to permeate the intestinal epithelium indicating potential as oral drug delivery carriers. While studies in our laboratory have determined the effects of surface modification on dendrimer transport and uptake, a large gap in knowledge exists in the transport and cytotoxicity mechanisms of PAMAM dendrimers. Additionally, alternatives to PAMAM dendrimers such as biodegradable poly-L-lysine (PLL) dendrimers have yet to be investigated for use in oral delivery. In this work we report the mechanisms of tight junction modulation by PAMAM dendrimers. While anionic dendrimers modulated tight junction proteins, cationic dendrimers opened tight junctions through phospholipase C-mediated calcium signaling allowing for paracellular small molecule transport. In comparison, cationic PLL dendrimers also allowed for small molecule transport with similar decreases in transepithelial electrical resistance. Small generation PAMAM and PLL dendrimers (16 and 32 surface amines) activated Caspase-3 and -7 resulting in apoptosis. In contrast, PLL dendrimers showed less long term toxicity compared to PAMAM dendrimers illustrating the benefits of dendrimer biodegradability. We also investigated the mechanisms of PLL dendrimer internalization and subcellular trafficking and the impact conjugation had on these mechanisms. The pH and enzymes present vary within different intracellular vesicles. Knowledge of the environment a drug delivery system will encounter is crucial for proper drug release. While PLL dendrimers were internalized via cholesterol- and dynamin-mediated endocytosis and macropinocytosis, conjugation site impacted uptake and localization. By conjugating a model compound to either the dendrimer core or surface, the uptake and transport properties of the delivery system were modified. Core-conjugated dendrimers had higher uptake and localized to the lysosomes and nucleus while surface conjugation resulted in higher transport and less accumulation in lysosomes. This research provides important knowledge for designing an effective dendrimer-based oral drug delivery system.
    • Polymer-dual drug conjugates targeted to HER2 overexpressing breast cancer

      Lee, Jun Hyoung; Nan, Anjan (2012)
      Current FDA approved treatments for Human epidermal growth factor receptor type 2 (HER2) overexpressing breast cancers include a humanized monoclonal antibody, Trastuzumab (TRZ) and small molecule tyrosine kinase inhibitors (TKIs). However, TRZ develops rapid drug resistance while TKIs and their metabolites cause hepatotoxicity due to nonselective distribution. To overcome these limitations of each agent it is essential to develop novel strategies to increase efficacy and reduce toxicity. This dissertation evaluates the potential of using conjugates of water soluble, N-(2-hydroxypropylmethacrylamide) (HPMA) copolymers with TRZ and TKI for targeted delivery of dual drugs to HER2 overexpressing tumors. Comb-like random and star-like semitelechelic (ST) HPMA copolymers (PHPMA) and their conjugates to TRZ were synthesized and characterized. Star-like conjugate system showed narrower size distribution, more potent and prolonged anticancer activity in HER2 overexpressing breast cancer cell lines (BT-474 and SK-BR-3) compared to comb-like conjugates. Therefore, the star-like conjugate system was selected to additionally conjugate a second anticancer agent for targeted combination drug delivery. Also fluorescence microscopy studies with Alexa488 labeled star-like conjugate demonstrated cellular internalization. The combination drug delivery system was successfully synthesized by covalently conjugating PKI166 (a model TKI) and TRZ to ST-PHPMA backbone. PKI166 release from the conjugate was observed in the presence of lysosomal enzyme cathepsin B. The polymer dual-drugs conjugate showed synergistic anticancer activity on HER2 overexpressing BT-474 and SK-BR-3 cells. The conjugate also exhibited prolonged anticancer activity up to 72 h by inhibiting the phosphorylation of MAPK and Akt when compared to free TRZ and free PKI166. The in vivo antitumor activity of the conjugates was evaluated in a pilot study in mice bearing SK-BR-3 tumor xenografts. The conjugates showed concentration dependent and synergistic antitumor activity over a 3 weeks treatment. The conjugates also showed no apparent indications of toxicity in non-tumor bearing mice. The results demonstrate the potential of HPMA copolymer based dual mode of action drugs conjugate as a novel combination drug delivery system that could i) prolong anticancer efficacy, ii) synergistically enhance anticancer effects of the drug combination and iii) reduce non-specific toxicity by way of cancer cell specific targeting and synergistic activity.
    • Repurposing Oxaliplatin for the Treatment of Glioblastoma

      Roberts, Nathan; Woodworth, Graeme (2018)
      Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults, accounting for approximately 40% of primary brain tumors. Even with the most aggressive therapy, the mean survival for patients with GBM is still less than 18 months, highlighting the critical need for new therapeutic strategies for this deadly cancer. Among the strategies under consideration is a repurposing of platinum-based chemotherapeutics. Traditionally considered DNA damaging cytotoxic agents, recent findings suggest that platinum-based chemotherapeutics, especially oxaliplatin (OXA), can induce multi-faceted anti-tumor effects, including modulation of cytokines, transcription factors, and tumor immunosuppressive mechanisms, even at lower concentrations that are not directly cytotoxic. Data suggests that a major alternative effect of OXA is the inhibition of signal transducer and activator of transcription 3 (STAT3), a transcription factor at the core of GBM pathobiology. STAT3 signaling is constitutively active in many gliomas and dictates diverse aspects of glioma biology including angiogenesis, invasion, chemotherapeutic resistance, and immunosuppression. STAT3 also controls and co-opts the primary gliomainfiltrating immune cell, the macrophage, which composes up to 40% of the tumor mass. OXA treatment may overcome the pleiotropic glioma-supporting functions of STAT3. It is likely that OXA therapeutic formulations designed to maximize the multi-faceted effects of OXA, including STAT3 inhibition, will have potent anti-GBM effects, including reprogramming of the tumor microenvironment. Although high-dose platinum-based chemotherapeutics have been investigated for CNS tumors, systemic and direct neuronal toxicity at high doses has thus far limited their use. However, new therapeutic delivery strategies including polymeric nanoparticle formulations capable of improving drug delivery to tumor cells, providing a sustained release of chemotherapeutic at the target site, and significantly reducing toxicity are facilitating the adaptation of these compounds in the CNS. We sought to investigate the multi-faceted anti-tumor effects of low-dose OXA in glioma cells and macrophages, with a particular focus on STAT3 modulation. We hypothesized that OXA will reduce STAT3 activity in glioma cells as well as macrophages and that OXA nanoparticle formulations will sustain STAT3 inhibition in vivo, thereby enabling the use of OXA as a biomaterial inhibitor of STAT3 for the treatment of glioma.
    • Water Soluble Polymer Drug Therapies for Targeted Delivery to Pancreatic Cancer

      Stanton, Joseph D.; Swaan, Peter W. (2014)
      Current therapies of advanced staged pancreatic cancer are limited by poor response and high toxicity of chemotherapeutics. As the molecular basis of pancreatic cancer has become better understood the need for a targeted therapy could help provide an increase in therapeutic response while also limiting side effects. N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer drug conjugates have demonstrated their potential use as carriers of small molecule drugs to improve cancer therapies. The overall goal of this research was to develop a polymer peptide drug conjugate based on HPMA copolymers, which can increase the therapeutic index of pancreatic cancer chemotherapy. Previous investigation of the uMUC1 receptor, which is a glycoprotein overexpressed on the surface of pancreatic tumors, has lead to the development of EPPT1, a small peptide has been found to have a strong binding affinity to uMUC1, (Kd=20uM). Our hypothesis HPMA copolymer with an active target EPPT1 to the uMUC1 receptor will enhance therapeutic action of a cancer chemotherapeutic drug such as gemcitabine. In this study, we were successful in the synthesis and characterization of a series of HPMA copolymer-EPPT1-Gemcitabine conjugates. Using model pancreatic cancer cell lines, the binding efficiency, internalization and mechanisms of cellular uptake were evaluated with polymer EPPT1 conjugates. Polymer gemcitabine conjugates were evaluated for efficacy against free gemcitabine. The optimized polymer peptide drug conjugates were evaluated for efficacy and drug release. Results during synthesis and characterization indicated that copolymer yield, solubility and performance were influenced by each incorporation of peptide and drug. Flow cytomentry determined that polymer peptide conjugates were able to bind with Capan-2 and Panc-1 cell lines. Confocal microscopy verified that polymer peptide conjugates were not only getting internalized into the cytoplasm but also routing to the lysosome. Using endocytosis inhibitors, confirmed that polymer peptide conjugates use clathrin mediated endocytosis pathways when getting internalized into the cell. Drug release studies revealed that gemcitabine will detached from the polymer in lysosomal conditions. Polymer drug conjugates compared to free gemcitabine alone against pancreatic cancer cells in MTT assay had equal efficacy. Attachment of the active targeting moiety EPPT1, exhibited that polymer peptide drug conjugates were superior in killing cells to free gemcitabine alone.