This study focuses on the development of mathematical models that explain and predict the combined effects of pH control and complexants (cyclodextrin), pH control and surfactants, as well as pH control and cosolvents on the total solubility of the drug. The total solubility of the drug is expressed as the sum of the concentrations of individual species. In complexant-water and surfactant-water systems free unionized drug [Dᵤ] and free ionized drug [Dᵢ] are present along with either complexed unionized drug [DᵤL] and complexed ionized drug [DᵢL], or micellar unionized drug [DᵤM] and micellar ionized drug [DᵢM], respectively. On the other hand, in cosolvent-water system the only species present are free unionized drug [Dcᵤ] and free ionized drug [Dcᵢ]. The equations developed show that a pH change favoring ionization of the drug not only increases the concentration of the ionized species in water, but also increases the concentration of the ionized species in cyclodextrins, micelles, or cosolvents. In fact, the concentration of the ionized species in the complexant, micelle, or cosolvent can be greater than those of the unionized species. The solubility data of flavopiridol and several other drugs reported in the literature support these conclusions. A mathematical model is also developed to describe the combined effect of cosolvency and complexation on non-polar drug solubilization. The total drug solubility is determined by the summation of three drug species present in the solution: free drug [D], drug-ligand binary complex [DL], and drug-ligand-cosolvent ternary complex [DLC]. The proposed equation describes the dependencies of these three species upon the intrinsic drug solubility, [Dᵤ], the cosolvent solubilizing power, sigma, the binary and ternary intrinsic complexation constants, K(b)ⁱⁿᵗ and K(t)ⁱⁿᵗ, and the cosolvent destabilizing powers for the binary and the ternary complexes, ρ(b) and ρ(t). The equation explains the decline in the solubility of fluasterone (a non-polar drug) produced by low cosolvent concentrations as well as the increase in the solubility produced by high cosolvent concentrations that are observed at all cyclodextrin concentrations.

Tuberculosis has been one of the most commonly encountered infectious diseases of humans throughout history. The discovery and introduction of effective treatments in the 1940s resulted in a decline of the incidence of the disease until the 1980s, which marked a sharp turn in this trend with an increase in the number of reported cases in the world. The prevalence of mycobacterial infections in poorer nations and centers of urban decay, and especially in immunologically compromised patients indicates the need for new and better treatments. In this study, to contribute to the worldwide effort to discover more effective medications against tuberculosis, 26 previously unreported compounds were synthesized and tested against Mycobacterium tuberculosis. The synthesized compounds were designed to act as bioisosteres or prodrugs of pyridine and pyrazinecarboxylic acid derivatives and contained acidic or neutral hetero- or carbocyclic ring systems attached to pyridine or pyrazine rings at different positions. The synthesized ring systems included 1,2,4-oxadiazole-5-ones, 1,2,4-thiadiazole-5-ones, 1,2,4-oxadiazole-5-thiones, 1,3,4-oxathiazoline-2-ones and cyclobutene-1,2-diones, which were documented in the literature to act as carboxylic acid isosteres or could act as prodrugs thereof. Pivaloyloxymethyl derivatives of the compounds were also prepared in order to increase their lipophilicity and therefore improve their bioavailability. The synthesized compounds were expected to be biotransformed by esterases or amidases to the active species after penetration of the mycobacterial cell wan. Biological and pharmacokinetic properties of the compounds were compared with the unmodified polar isosteres of pyrazinoic and nicotinic acids. The majority of the tested compounds exhibited activities ranging from 0.5 to 8 times the activity of pyrazinamide, one of the lead compounds that inspired their design.

Imexon is an iminopyrrolidone derivative that has selective antitumor activity in multiple myeloma. The exact mechanism of imexon action is unknown. In human 8226 myeloma cells, the cytotoxicity of imexon was schedule dependent and long exposures ( ≥ 48 hours) to low concentrations of imexon were most effective at inducing cytotoxicity. Our data suggest that imexon does not affect DNA, but it can alkylate thiols by binding to the sulfhydryl group. We have also shown by HPLC studies that in human 8226 myeloma cells, imexon depletes cellular stores of cysteine and glutathione (GSH). Oxidative stress in myeloma cells exposed to imexon was detected by staining with dihydroethidium (HE) and flow cytometry, and by immunohistochemical staining with a monoclonal antibody to 8-hydroxydeoxyguanosine (8-OHdG) followed by confocal microscopy. This antibody can also recognize 8-hydroxyguanine and 8-hydroxyguanosine. The images showed increased levels of oxidized nucleotides in the cytoplasm of cells treated with imexon. Interestingly, 8-OHdG staining was not observed in the nucleus of imexon treated cells, in contrast to the diffuse staining seen with t-butyl hydroperoxide. Myeloma cells exposed to imexon showed classic morphologic features of apoptosis on electron microscopy and phosphatidylserine exposure detected as Annexin-V binding on the cell surface. Mitochondrial enlargement was observed by electron microscopy in the cells treated with 90 μM imexon at as early as 4 hours. Morphometric studies indicated that mitochondrial volume significantly increased from 14.1% in control cells to 18.7% in cells treated with 45 μM imexon for 24 hours. In flow cytometric experiments it was shown that the increase in levels of ROS (measured by staining with dihydroethidium) in myeloma cells exposed to imexon precedes the loss of Δψ(m) (detected by MitoTracker Red labeling). Damage of mitochondrial DNA was detected by semiquantitative PCR assay in myeloma cells treated with imexon; however nuclear DNA remained unaffected by imexon treatment. Moreover, release of cytochrome c from mitochondria to cytosol was observed in imexon treated cells. Partial protection of myeloma cells against imexon cytotoxicity was achieved by treatment with theonyltrifluoroacetone, an inhibitor of superoxide production at mitochondrial complex 11. Myeloma cell line was developed that is stably resistant to imexon. This cell line shows an increase in the expression of mitochondrial proteins (e.g. Bcl-2, thioredoxin 2) with antioxidant properties. These changes are consistent with drug-induced mitochondrial oxidation and apoptotic signaling.

This dissertation represents some of the first work to associate the behavioral stimulant and electrophysiological effects of the putative psychotropic component of many inhalants of abuse, the organic solvent toluene, with changes in mesolimbic dopamine (DA) neurotransmission. To test if the locomotor stimulant properties of toluene are dependent on mesolimbic DA neurotransmission, rats received focal injections of 6-hydroxydopamine (6OHDA) or vehicle into the NaCC, and following recovery received injections of toluene, amphetamine, scopolamine, and vehicle. In 6OHDA-treated animals, the locomotor hyperactivity evoked by toluene and amphetamine, but not scopolamine or vehicle were reduced, and the DA concentration in the NaCC declined ∼90%. To test if toluene directly stimulated ventral tegmental area (VTA) neuronal activity, rat in vitro brain slices containing the VTA were superfused with behaviorally relevant concentrations of toluene. Regions outside the VTA were either mildly stimulated or inhibited by toluene. Toluene was very efficacious at activating DA neurons in the rostral VTA regions, which project to the NaCC. These stimulatory effects appeared unrelated to NMDA receptors, and persisted in ACSF media designed to inhibit synaptic transmission. To examine the effects of repeated exposure to toluene, locomotor hyperactivity in response to daily injections of either toluene or vehicle was assessed. Toluene-induced hyperactivity progressively declined over the 7 days of exposure suggesting the development of tolerance. To determine if these changes involved alterations in the mesolimbic DA system, the same rats were challenged with amphetamine, cocaine and scopolamine. Animals tolerant to toluene were also cross-tolerant to cocaine and amphetamine, but not to scopolamine. Brain slices from similarly treated animals revealed comparatively increased numbers of spontaneously active non-DA neurons with an ∼400 fold increased sensitivity to stimulation by NMDA. In contrast, both the firing rates and the number of spontaneously active VTA DA cells were reduced. VTA DA cells also exhibited hyperpolarized action potential waveforms and a significantly reduced sensitivity to toluene. Taken together it appears that the abuse potential of inhalants containing toluene may stem from the stimulatory actions of this solvent on the mesolimbic dopamine system, a circuit intimately tied to the rewarding effects of most drugs of abuse.

This dissertation will examine the likelihood that tonic activation of descending facilitatory pathways may underlie chronic pain states arising from injuries to peripheral nerves, and that this activation is what maintains neuropathic pain. Activation of these pathways might be the result of plasticity in the RVM that is driven, in part, by release of CCK in response to pain signals from the injured nerve. Hyperactivity of facilitatory ON cells in the RVM may contribute to neuropathic pain and lesions of these cells via the targeted toxin, DERM-SAP, may prevent or reverse neuropathic pain states in the injured animal. Rats treated with DERM or SAP and undergoing SNL exhibited tactile and thermal hypersensitivity. Rats receiving DERM-SAP showed similar responses to sham-operated controls. Administration of RVM DERM-SAP to SNL rats fully reversed established allodynia/hyperalgesia by day 14. Thus, the targeted loss of cells expressing the MOR both prevents and reverses SNL-induced neuropathic pain. Acutely, RVM lidocaine blocked tactile and thermal hypersensitivity on day 6 out to day 12 after SNL, but not on day 3. DERM-SAP pretreatment and DLF lesions did not prevent the onset of tactile and thermal hypersensitivity, but these neuropathic pain signs reversed toward baseline levels beginning on day 4 after SNL. These findings differentiate the mechanisms that initiate neuropathic pain as being independent of supraspinal influences needed to maintain such pain. These descending influences may underlie some of the SNL-induced changes at the spinal level, such as the upregulation of dynorphin and may be key to the maintenance of neuropathic pain. Administration of RVM CCK produces hypersensitivity in naive rats. Pretreatment with RVM DERM-SAP completely blocks the ability of RVM CCK to produce hypersensitivity. These results suggest that the action of CCK in the RVM may be mediated by activation of MOR expressing cells. Enhanced release of CCK in the RVM or upregulation of CCK receptors in RVM neurons after SNL may represent a mechanism of descending pain facilitation to maintain chronic pain states. An observed loss of both MOR and CCK2 receptors after DERM-SAP treatment gives support to the notion that both receptor types may be co-expressed in RVM neurons, and these neurons may be critical for descending facilitatory input from the RVM to maintain neuropathic pain. Understanding the mechanisms of descending facilitation and the spinal effects of, CCK-activated discharge could provide new insight into the modulation of chronic pain, and, furthermore, provide new targets for the development of novel drug therapies.

Selenomethionine, an organic derivative of selenium, has been found to be the predominate selenium component of the dietary cancer chemopreventive agent selenized yeast that has been shown to decrease the incidence and mortality rate of lung, colon, and prostate cancers. Another organic selenium derivative found in the selenized yeast is Se-methylselenocysteine. However, the mechanism of action of the anti-cancer effects of these selenium compounds has yet to be identified. To evaluate the effects of these compounds on growth of the cancer cell types mentioned earlier, various cancer cell lines were treated with either compound. Both selenium compounds were able to induce growth inhibition and alterations in the cell cycle, with selenomethionine being the most potent. Previously, our laboratory has shown that treating cancer cells with selenomethionine depleted polyamines. Polyamines are cations that are needed for various roles in growth and proliferation. To extend these findings to an in vivo model, we gave selenomethionine in the diet (1ppm and 2ppm) for 16 weeks to male F344 rats in the azoxymethane (AOM) rat colon carcinogenesis model. The results showed no significant changes in colonic polyamine levels, however, there were significant changes in the development of microadenomas between control and treated groups. Selenomethionine was able to decrease the promotional effects of colon carcinogenesis through a polyamine-independent mechanism. Next, we tested the hypothesis that selenomethionine might affect cell growth by mechanisms involving cyclooxygenases, specifically the inducible isoform COX-2. Cyclooxygenases (COX-1 and COX-2) are enzymes that metabolize arachidonic acid to various prostaglandins. The human adenocarcinoma cell lines HT-29 and HCA-7, that express variable levels of COX-2, were treated for up to 6 days with selenomethionine. Selenomethionine induced growth inhibition in both cell lines and decreased COX-2 protein expression in the treated groups. Also, prostaglandin (PG) E2 levels were decreased in both treated groups at the latter timepoints. The HCA-7 cell line had a dose and time-dependent decrease in RNA levels treated with selenomethionine, whereas, no effects were observed on RNA expression of the HT-29 cell line. In summary, selenomethionine has chemopreventive effects in colon carcinogenesis by potentially modulating COX-2 proteins in cancer cells.

The disposition kinetics of Amphotericin-B (Am-B) were investigated in rats in three different dose groups. Each group contained four rats. The serum and urine Am-B concentrations were analyzed by reversed-phase HPLC. There were no significant differences for total body clearance and half-lives as a function of dose. Those observations suggested systemic dose-independent behavior of Am-B. However, renal clearance of Am-B decreased significantly as Am-B dose increased; whereas, no renal damage was observed during the experiment. The present studies suggested that renal clearance was dose-dependent and that there may be a saturable tubular secretion process for Am-B renal excretion.

<p> In recent years, aldehyde oxidase (AO) has continued to grow as an important enzyme in drug metabolism. Herein, we have focused primarily on the inhibition kinetics of AO and also developing tools to better understand AO and its role in human biochemistry and drug metabolism. In the first study, we used a set of small molecule inhibitors to explore the impact of inhibitor structure on mode of inhibition. The results indicated that the mode of inhibition varied greatly with the choice of inhibitor, ranging from mostly uncompetitive to completely competitive Implications for mechanism of inhibition and potential drug-drug interactions in humans is discussed. In the second study, we looked at the impact that the substrate had on measuring inhibition profiles. When comparing one substrate, phthalazine, with another, N-[(2-dimethylamino)ethyl]acridine-4-carboxamide (DACA), we saw a dramatic difference in inhibition mode. In every case, for a given inhibitor the measured mode of inhibition was much more competitive with DACA as the probe substrate. This translated to DDI prediction that varied based on the substrate that was used. Finally, we also developed and employed a trypsin digest and subsequent liquid chromatography tandem mass spectrometry analysis to determine absolute abundance of AO in human liver cytosol. We discussed the future impact of this method for the development of drug metabolism models and understanding the biochemical role of this enzyme.</p>

Potential benefits of developing an avenue for the therapeutic control of angiogenesis are enormous. A search for angiogenesis inhibitors has been the focus of many investigations. One such inhibitor, which caught the attention of many researchers, is 2-methoxyestradiol (2-ME). 2-ME is an endogenous metabolite of estradiol-17beta. The current study was undertaken to characterize the angiostatic effects of 2-ME in human microvascular endothelial cells. Transforming Growth Factor beta (TGF-beta) has been shown to play a vital role in regulating endothelial cell growth and differentiation, as well as apoptosis. We, therefore, investigated whether TGF-beta is modulated by 2-ME using the human microvascular endothelial cell line, HMEC-1. Findings suggest that 2-ME mediated apoptosis in human microvascular endothelial cells involve SAPK/JNK pathway and caspase-3 while the activation of p38 kinase pathway may represent a survival signal to counteract the 2-ME induced apoptotic events. (Abstract shortened by UMI.)

The opioid family of G-protein coupled receptors comprises four known receptor subtype genes (delta, mu, kappa, ORL1) and further receptor heterogeneity within each opioid receptor subfamily has been proposed. All four genes are expressed throughout the central nervous system and are believed to modulate a variety of behavioural responses including analgesia. Opiate drugs such as morphine that are selective for the mu receptor subtype are effective analgesics, but their chronic use is limited by the appearance of side effects such as respiratory depression, constipation and dependence. Consequently, the analgesic potential of agonists selective for other opioid receptors is under investigation. In this regard, previous studies suggest that delta agonists mediate antinociception, yet produce fewer adverse effects than mu agonists. To further investigate the cloned delta opioid receptor (DOR) as a target for novel analgesics, the pharmacological role of DOR in brain was evaluated in rats. / First, we characterized delta agonist binding sites and receptor activation in rat brain membranes. We also introduced a novel antagonist radioligand, [125I]AR-M100613, to label tissues with low delta opioid receptor expression in order to support follow-up studies where radioligand binding was performed on rat brain membranes following antisense treatment. Second, we examined the behavioural response to delta agonists in rats. Deltorphin II and SNC80 (i.c.v.) were shown to induce antinociception in acute pain assays, and to reverse hyperalgesia following tissue inflammation induced by Freund's adjuvant with even greater potency. These findings indicate that delta receptors play an enhanced role in the modulation of descending pain pathways following tissue injury. Deltorphin II and SNC80 (i.c.v. ) were also shown to induce hyperlocomotor activity. Third we used antisense studies to demonstrate that the antinociceptive and locomotor stimulant effects of delta agonists are modulated by the cloned delta opioid receptor (DOR). In contrast to other delta agonists, the antinociceptive effects of DPDPE were not modulated by DOR antisense treatment but rather were blocked by a selective mu antagonist (CTOP) suggesting that DPDPE may activate mu sites in the brain rather than an alternate delta receptor subtype. Finally, we demonstrated that peptide nucleic acids (PNA, i.c.v.) can act as target-specific and sequence-selective antisense agents. In total, these findings demonstrate that DOR is an appropriate target for the development of novel analgesics and that PNA can serve as effective antisense agents for the determination of gene function for CNS targets.