• Contributions of dissolution and intestinal permeation to oral drug product quality

      Ginski, Mark Joseph; Polli, James E. (1999)
      Dissolution and intestinal permeation are the primary biopharmaceutical factors controlling oral drug product performance. The objective of this research was to develop in vitro dissolution and Caco-2 cell monolayers, both separately and in combination, as tools to characterize the biopharmaceutical performance of oral drug products. Firstly, in vitro dissolution methods were developed to elucidate the degree of drug solubility-controlled or formulation-controlled drug dissolution from immediate release dosage forms. Results indicate dissolution from formulations may be controlled by disintegration or diffusion through the aqueous boundary layer. Drug solubility and formulation design dictated the degree of disintegration or diffusion control. Secondly, Caco-2 monolayer permeability was quantitatively related to human drug absorption kinetics. Results suggest a rank-order relationship between Caco-2 permeability and in vivo intestinal permeation rate, providing a kinetic interpretation of Caco-2 monolayer permeability. Caco-2 monolayer methods were also used to develop a simple kinetic model to elucidate P-glycoprotein effects on intestinal drug transport. Results suggest P-glycoprotein substantially reduced net fexofenadine transport, which was well characterized by the simple kinetic model. Thirdly, "static" and "dynamic" dissolution/Caco-2 systems were developed. Each system integrated in vitro dissolution and Caco-2 monolayers to predict the relative contributions of dissolution and intestinal permeation to overall drug absorption kinetics. Results indicate the "static" system accurately predicted the relative contributions of dissolution and intestinal permeation to overall drug absorption and the "dynamic" system simultaneously assessed the effect of formulation changes on dissolution and intestinal permeation. Hence, in vitro dissolution and Caco-2 cell monolayer methods were successfully developed to characterize the biopharmaceutical performance of oral drug products. Separately, these methods identified factors controlling drug product dissolution, estimated the absorption potential of oral drug candidates and elucidated P-glycoprotein effects on intestinal drug transport. In combination, these methods elucidated the degree to which dissolution and intestinal permeation limits overall drug product performance and characterized excipient effects on dissolution and intestinal permeation.
    • Investigation of sigma and dextromethorphan-like neuroprotection using glutamate-induced LDH release, cellular morphology and dynamic calcium signaling

      Klette, Kevin Louis; Moreton, J. Edward (1995)
      The role of the putative sigma receptor in mediating neuroprotection against glutamate induced neuronal injury was examined in mature cultured rat cortical neurons. With the exception of the sigma1, selective ligand (+)-3-PPP, all of the sigma receptor ligands tested were neuroprotective, preventing glutamate induced morphological changes and increases in LDH release. When corrected for relative sigma versus PCP binding site affinity, it appears that a positive correlation exists between neuroprotective potency and sigma1, site affinity. None of the sigma ligands were neurotoxic when tested alone at concentrations at least 5-30 times their respective neuroprotective EC{dollar}\sb{lcub}50{rcub}{dollar} values. The effect of neuroprotective sigma ligands on the unique calcium responses evoked by glutamate, NMDA, potassium chloride (KCl) and trans-ACPD were investigated to elucidate the mechanism of sigma-mediated neuroprotection. In general, except for (+)-3-PPP all of the sigma ligands studied interfered with glutamate and NMDA induced (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} signaling, but, highly sigma{dollar}\sb1{dollar} selective ligands also lacking substantial PCP binding site affinity (i.e. carbetapentane, DTG and haloperidol) were much less effective in altering calcium influx induced by 80 {dollar}\mu{dollar}M glutamate. In contrast to glutamate, KCl (50 mM) produced changes in (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} which were not neurotoxic to the neurons as measured by LDH release. Sigma ligands which lack substantial PCP site afflinity were very effective in altering KCl induced calcium signaling while the sigma/PCP site ligand (+)-cyclazocine was ineffective or, in the case of (+)-SKF 10047, much less effective. Similar to the effects of sigma ligands on KCl induced calcium dynamics, the sigma selective ligands DTG, haloperidol, (+)-pentazocine, and carbetapentane were very effective in altering intracellular calcium dynamics evoked by trans-ACPD while the sigma/PCP ligand (+)-SKF 10047 was ineffective or, in the case of (+)-cyclazocine, much less effective. Importantly, (+)-3-PPP, a non-neuroprotective sigma selective ligand, actually potentiated the calcium response elicited by trans-ACPD. The ability of sigma ligands applied at maximal neuroprotective concentrations to attenuate receptor and/or voltage-gated changes in calcium dynamics suggests that modulation of neurotoxic (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} plays a significant role in sigma-mediated neuroprotection. The unique modulatory effects of sigma ligands on the buffering of neuronal (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} will likely have numerous therapeutic applications in the treatment of CNS injury and other neurodegenerative disorders.
    • PC cell derived growth factor (PCDGF/granulin precursor) expression, antiestrogen resistance and tumorigenesis in human breast cancer cells

      Tangkeangsirisin, Wisit; Serrero, Ginette (2004)
      Breast cancer is one of the most common malignant diseases in women. Several mechanisms have been proposed for the development and progression of breast cancer. Overexpression of growth factors or their receptors has been widely investigated as a potential pathway of breast malignancy. PCDGF is a novel growth factor characterized in our laboratory. There is significant evidence to suggest a role of PCDGF in human cancers. The purpose of this study was to investigate the role of PCDGF on tumorigenicity, estrogen dependence, endocrine therapy resistance and metastatic potential in human breast cancer. A model system to study the role of PCDGF and estrogen independence was developed by overexpressing PCDGF in human breast cancer MCF-7 cells and cultivating them in estrogen depleted condition. Results presented here show that PCDGF overexpression confers resistance to tamoxifen and fulvestrant in both in vitro and in vivo. PCDGF overexpression and estrogen depletion downregulate estrogen receptor alpha isoform, resulting in estrogen unresponsive cell growth. In addition, doxorubicin resistance was observed in PCDGF overexpressing cells. We found that PCDGF prevents apoptosis induced by tamoxifen, fulvestrant and doxorubicin. The key event in this process is that PCDGF maintains the upregulation of bcl-2 when treated with these agents. In addition, tamoxifen resistant cells express higher level of PCDGF. PCDGF transcriptionally activates estrogen inducible genes such as progesterone receptor and vascular endothelial growth factor (VEGF) via an ERa-dependent pathway. Tumors originated from PCDGF overexpressing cells express higher level of VEGF and angiopoietin-1. In addition, treatment with tamoxifen, in cooperation with PCDGF, stimulates VEGF expression in vitro and in vivo. This may explain why tamoxifen stimulates tumor growth from the PCDGF overexpressing cells but inhibits it in the wild-type cells. PCDGF induces cell migration through matrigel and stimulates matrix metalloprotease-9 secretion, suggesting an important role in metastasis. In summary, these studies provide a possible mechanism of antiestrogens and doxorubicin resistance in human breast cancer by overexpression of PCDGF. The results also show that PCDGF expression correlates with higher tumorigenicity and promotes angiogenesis and metastasis in human breast cancer.
    • Role of endothelial cells in host defense

      Zhang, Bin; Rosen, Gerald M., Ph.D., J.D. (1999)
      Phagocytes, neutrophils and macrophages, are part of the non-specific host defense system in control bacterial infection by the phagocytosis and killing of these microbes through free radical-dependent and free radical-independent mechanisms. In contrast, endothelial cells, in addition to their role in maintenance of homeostasis, have classically been perceived to play a supportive role in host immune response by releasing chemoattractants that recruit phagocytes to the site of infection. Recent studies have, however, demonstrated that the endothelium is capable of responding to stimulation by cytokines as part of their activation responses in host immunity. This dissertation explores the role of endothelia as effector cells in host response. The findings from this dissertation demonstrated that endothelial cells cultured on three dimensional GelfoamRTM are activated by penicillin G to phagocytosis and kill S. aureus. Even though O2-· and NO· are known to exhibit microbicidal activity, it appears that these free radicals do not play an integral part in the observed killing of S. aureus. To further investigate the host defense role of NO·, primary cultures of endothelial cells were transduced with retroviral vector encoding NOS II gene. Upon transduction, these cells released NO· at a constant flux over a long period of time. This provided an excellent model to study the antimicrobial activity of NO· at cellular fluxes without the complications of controlling the rate of NO· from a NO·-releasing compound or those associated with cytokine treatment. When infected with either S. aureus or E. coli, NOS II transduced endothelial cells, producing NO·, phagocytosed both bacteria. However, only E. coli was sensitive to NO· dependent bacterial killing. Taken together, these studies reveal the antimicrobial roles played by endothelial cells upon activation. The mechanisms utilized by these cells include both free radical-dependent and free radical-independent pathways.