• Application of radiotelemetry in pharmacokinetic-pharmacodynamic modeling of procainamide and N-acetylprocainamide in sedentary and exercise trained rats

      Kharidia, Jahnavi Sanjay; Eddington, Natalie D. (1996)
      Understanding the pharmacokinetics (PK) and pharmacodynamics (PD) of a new drug in an appropriate animal model provides a scientific framework for efficient and rational drug development. Cardiovascular measurements from small animals are difficult or impossible to obtain. The conventional methods, chemical restraint, physical restraint, cuff blood pressure and tethering used to obtain cardiovascular PD have limitations. These methods affect physiological function of animals and introduce stress artifact into data. Recently, radiotelemetry has been proposed as a new method to obtain cardiovascular PD from small animals. The objective of this research was to investigate the application of radiotelemetry in the pharmacokinetic/pharmacodynamic (PK/PD) modeling of cardiovascular agents in small animals. PD of various cardioactive agents such as procainamide (PA) and its active metabolite, N-acetylaprocainamide (NAPA) were evaluated. Additionally, one of the objectives of this dissertation was to evaluate the effects of exercise training on the pharmacokinetics of a model substrate of N-acetylation reactions (phase II enzymes). PA was chosen as a model drug for these studies. Radiotelemetry avoided the stress associated with animal handling and the use of anesthesia during PD studies, which allowed for the attainment of higher quality and more accurate data. PA displayed a significant increase in QT, QR and QS intervals from the baseline. The QT prolongation has been used as a surrogate marker in PA clinical studies. The PK of PA and NAPA were best described by a one compartment model. An Emax model was able to accurately describe the relationship between PA concentration and the QT interval. Effects of exercise training on the PK as well as PD of PA and NAPA were evaluated using radiotelemetry. Rats were exercised for four weeks by treadmill running. These studies noted that four weeks of exercise training did not alter the PK or PD of PA and NAPA suggesting that exercise training may not have significantly affected N-acetyltransferase. In conclusion, radiotelemetry allows the prediction of the PK/PD relationship observed in clinical studies by conducting such studies in small animals.
    • 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.
    • Structural and electrochemical properties of wild-type and mutant cytochromes b-(5)

      Sarma, Siddhartha P.; Guiles, Ronald (1996)
      The main objective of this thesis project was to gain a better understanding of the protein structural factors that modulate heme reduction potentials in b-type cytochromes. Rat liver microsomal cytochrome {dollar}b\sb5{dollar} was chosen as a model system because the availability of a synthetic gene coding for the protein permits mutagenic experiments to be performed. The synthetic gene encoding for cytochrome {dollar}b\sb5{dollar} was subcloned into a high yield bacterial overexpression system that enabled isotopic enrichment of proteins. Site directed mutants of cytochrome {dollar}b\sb5{dollar} were prepared by subcloning the synthetic gene into bacteriophage DNA m 13mp18. Mutants of cytochrome {dollar}b\sb5{dollar} were designed to achieve reorientation of individual axial imidazole ligands. The orientation of the axial ligand planes is thought to modulate the reduction potential of bis(imidazole) axially ligated heme proteins. The A67V (alanine to valine) mutation resulted in a reorientation of the H63 imidazole ring and a shift in reduction potential by {dollar}-20{dollar} mV. Structural characterization of the A67V mutant protein was achieved using homonuclear and heteronuclear NMR methods. Calculation of the orientation of the components of paramagnetic susceptibility tensor showed that the wild type and mutant proteins differed only in the orientation of the z-component. The rotation of the z-component of the susceptibility tensor is in the same direction as the rotation of the H63 imidazole ring. EPR and Near-IR data suggest that the stability of iron d-orbital energy levels in the reduced proteins may be significant in determining the reduction potential of b-type heme proteins. Multidimensional double and triple resonance NMR methods have been applied to assign the backbone and side-chain {dollar}\sp{lcub}13{rcub}{dollar}C resonances for both equilibrium conformers of ferricytochrome {dollar}b\sb5{dollar} On the basis observed NOEs and backbone {dollar}\sp{lcub}13{rcub}{dollar}C chemical shifts, the solution secondary structure of cytochrome {dollar}b\sb5{dollar} has been determined. The {dollar}\sp{lcub}13{rcub}{dollar}C chemical shifts of backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts are observed, is evaluated through calculations of the magnitude of such shifts.