The studies in this thesis research were conducted to investigate if sensitivity to induced cortical spread depression (CSD) or the consequence of a CSD event is affected by sumatriptan induced latent sensitization. Previous studies in our lab showed persistent exposure of sumatripan to rats produced a latent state of sensitization. Using persistent sumatripan exposed rats as a model for medication overuse headache, behavior, electrical stimulation threshold to provoke a CSD event and the immunoreactivity of c-Fos in the trigeminal nucleus caudalis (TNC) were characterized. Current results showed no statistical difference of electrically induced CSD thresholds in anesthetized rats measured at day 20 in sumatripan exposed rats compared with saline treated rats. Topiramate (80 mg/kg, i.p.) used clinically for prophylaxis of migraine headache significantly increased CSD threshold in both saline and sumatriptan infused rats. CSD events appear to be associated with trigeminal vascular system activation in TNC because c-Fos expression significantly enhanced in rats with electrically stimulated CSD events. As compared to saline treated rats, sumatriptan-exposed rats demonstrated a significantly higher number of c-Fos positive cells following the electrically stimulated CSD event. Under environmental stress (bright light), sumatripan exposed rats demonstrated decreased response thresholds to periorbital and hindpaw tactile stimuli (i.e., allodynia) and enhanced c-Fos expression in TNC. A single dose of topiramate (80 mg/kg, i.p.) reversed environmental stress induced allodynia and c-Fos over-activity. Taken together, these results suggest that latent sensitization induced by persistent sumatripan exposure seems not correlated to the threshold of electrically stimulated CSD in current model. However, CSD enhanced the responses of trigeminal system in rats with sumatriptan-induced latent sensitization. The protective effects of topiramate shown in this model may be related to blocking the initiation of CSD events resulting from environmental stimulation as well as inhibiting the consequences of CSD events in primary afferents. These findings correlate with clinical observations of protective effects of topiramate for migraine prophylaxis.

Chronic pain affects approximately 100 million Americans. Opioids are the mainstay therapy for the treatment of chronic pain. While physicians and patients alike are apprehensive about using opioids due to their side effects including respiratory depression and addiction, 259 million opioid prescriptions were written in 2012. Although opioids are the most efficacious available analgesics, they increase both positive and negative reinforcement, ultimately leading to addiction. The pro-nociceptive neurotransmitter, Substance P (SP) and its corresponding receptor (NK₁R), are not only found on pain pathways to promote pain but also found in the ventral tegmental area associated with dopamine neurons. Studies have shown that Substance P can potentiate positive reinforcement of opiates and may play a role in opioid reward. Here using in vivo microdialysis, we show that systemic morphine significantly increases SP release in the VTA, an effect mediated by ventral midbrain GABAergic neurons. Substance P administered to the VTA results in a significant increase in dopamine release in the nucleus accumbens (NAc). Using CRISPR-Cas9 knockdown of NK₁R in the VTA we prevent the induction of opiate reward as tested using a conditioned place preference paradigm (CPP). Finally, we developed a novel opioid agonist/NK₁R antagonist bifunctional compound, TY032, which inhibits acute and chronic pain in male rats. Importantly, TY032 microinjection into the VTA did not increase extracellular dopamine release in the NAc and did not produce a positive CPP score. These data indicate dual targeting of the dopamine reward circuitry and pain pathways with multifunctional opioid-NK₁R compounds may be an effective strategy in developing future analgesics that lack the potential for abuse.

The weak selectivity of chemotherapeutic drugs against tumors has sustained efforts to develop better chemotherapeutic agents that are more potent and selective at destroying tumor cell populations versus normal tissues. This project focuses on evaluating the cell killing effects of the microtubule inhibitor, stilbene 5c, against melanoma cancer. We utilized an in vitro murine melanoma model to study the effects of stilbene 5c on tumor proliferation and survival, as well as growth arrest and cell death. Our findings indicate that stilbene 5c promotes dose dependent cell death in melanomas with the induction of apoptosis and autophagy. The role of autophagy was further assessed using the pharmacological autophagy inhibitor, Bafilomycin A1. It was concluded that autophagy was partially cytoprotective as inhibition of autophagy was shown to induce extensive cell death through an increase in apoptosis. Residual surviving cells were shown to be in a state of growth arrest characterized to be senescence. These findings indicate that stilbene 5c could potentially be developed for the treatment of melanoma.

Delta9-tetrahydrocannbinol (THC), the primary active constituent of Cannabis sativa, has long been known to reduce opioid withdrawal symptoms. Although THC produces most of its pharmacological actions through the activation of CB1 and CB2 cannabinoid receptors, the role these receptors play in reducing opioid withdrawal symptoms remains unknown. The endogenous cannabinoids, N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonylglycerol (2-AG), activate both cannabinoid receptors, but are rapidly metabolized by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The objective of this dissertation was to test whether increasing AEA or 2-AG, via inhibition of their respective hydrolytic enzymes, reduces morphine withdrawal symptoms in in vivo and in vitro models of opiate dependence. Morphine-dependent ICR mice subjected to acute naloxone challenge or abrupt withdrawal (via pellet removal) reliably displayed a profound withdrawal syndrome, consisting of jumping, paw tremors, head shakes, diarrhea, and weight loss. THC and the MAGL inhibitor, JZL184 dose-dependently reduced the intensity of precipitated withdrawal measures through the activation of CB1 receptors. The FAAH inhibitor, PF-3845, reduced the intensity of a subset of precipitated signs through the activation of CB1 receptors, but did not ameliorate the incidence of diarrhea or weight loss. In the next set of experiments, MAGL inhibition dose-dependently reduced the intensity of all spontaneous withdrawal signs (i.e jumps, paw flutters, head shakes, weight loss and diarrhea) in a CB1 receptor dependent manner. However, FAAH inhibition reduced the intensity of head shakes and paw flutters, but did not affect other signs. Strikingly, a combination of low-dose JZL184 and high-dose PF-3845 reduced abrupt withdrawal signs in a manner similar to complete MAGL inhibition, which suggests potential therapeutic advantages of dual enzyme inhibition. This combination elevated appropriate eCB levels and caused moderate CB1 receptor desensitization, but did not affect receptor number in whole brain. Since MAGL, but not FAAH inhibition, blocked diarrhea during opioid withdrawal in vivo, we investigated whether inhibitors of each enzyme would differentially attenuate naloxone-precipitated contractions and secretion in morphine-dependent ilea in vitro. Both enzyme inhibitors attenuated the intensity of naloxone-induced contractions, and blocked naloxone-precipitated hypersecretion. Thus, these models offer useful tools for investigating in vitro end-ponts of withdrawal, but not for elucidating anti-diarrheal mechanism of these inhibitors.If targeting endocannabinoid catabolic enzymes is indeed a viable approach to treat other abuse disorders, it is important to know whether these inhibitors would themselves have abuse or dependence liability. In the final series of experiments we tested whether prolonged elevation of endocannabinoid leads to the development of cannabinoid dependence, based on the occurrence of somatic withdrawal signs upon challenge with rimonabant, a CB1 receptor antagonist. Repeated treatment with high doses, but not low doses, of JZL184 led to cannabinoid dependnece. These results indicate that the strategy of increasing endogenous cannabinoids through the inhibition of their catabolic enzymes represents a promising approach to ameliorate opioid withdrawal symptoms.

Pain is a debilitating condition that presents a problem of clinical relevance. Common analgesics include opioids and non-steroidal anti-inflammatory drugs (NSIADs). Despite different degrees of effectiveness, a major drawback of these analgesic classes is their side effects. For example, side effects associated with opioids include pruritus, respiratory depression, hyperalgesia, constipation, dependence. In addition, chronic use of NSAIDs can cause gastric ulcers. Delta-9 tetrahydrocannabinol (THC), the primary psychoactive constituent of marijuana, produces antinociception in various preclinical models of pain. Similarly, many synthetic cannabinoid receptor agonists produce antinociception in preclinical models of pain. However, their psychomimetic effects dampen interest for their therapeutic development. THC and these cannabinoids act upon the endocannabinoid system, which is comprised of the cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptors, endogenous ligands arachidonoylethanolamide (anandamide; AEA), 2-arachidonoyolglycerol (2-AG), and endocannabinoid biosynthetic and catabolic enzymes. In the present study, we evaluated the impact of inhibiting one of the major biosynthetic enzymes of 2-AG, diacylglycerol lipase-b (DAGL-b), and two primary endocannabinoid catabolic enzymes, monoacylglycerol lipase (MAGL), and fatty acid amide hydrolase (FAAH). MAGL is responsible for degrading 2-AG and FAAH is the principal degradative enzyme for anandamide. We hypothesized that inhibiting these enzymes will produce anti-edematous and anti-allodynic effects in preclinical models of inflammatory pain. In Chapter 2, we tested whether the selective MAGL inhibitor JZL184 and FAAH inhibitor PF-3845 would reduce nociceptive behavior in the carrageenan test. JZL184 and PF-3845 significantly attenuated carrageenan-induced paw edema and mechanical allodynia (a nociceptive response to normally non-noxious stimuli), whether administered before or after carrageenan. Complementary genetic and pharmacological approaches revealed that JZL184’s anti-allodynic effects required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-edematous actions. Anti-edematous effects of PF-3845 were mediated through CB2 receptors. Importantly, the anti-edematous and anti-allodynic effects of JZL184 underwent tolerance following repeated injections of high dose JZL184 (16 or 40 mg/kg), but repeated administration of low dose JZL184 (4 mg/kg) retained efficacy. Although the data in the first set of studies demonstrate that inhibition of MAGL or FAAH reduces inflammatory pain, JZL184 and PF-3845 only produced partial effects. In an attempt to increase efficacy, Chapter 3 tested whether combined blockade of FAAH and MAGL would produce enhanced anti-edematous and anti-allodynic effects in the carrageenan model of inflammatory pain. Partial blockade of MAGL, with a low dose of JZL184 (4 mg/kg), and full blockade of FAAH, with a high dose of PF-3845 (10 mg/kg), enhanced the anti-allodynic effects, but no further increases in the anti-edematous effects were found. Importantly, repeated administration of this combination did not result in tolerance. A novel FAAH-MAGL dual inhibitor SA-57, which is far more potent in inhibiting FAAH than MAGL, reversed carrageenan-induced allodynia. Taken together, these findings suggest that dual MAGL and FAAH inhibition represents a promising avenue for the treatment of inflammatory pain. Chapter 4 of this dissertation tested whether inhibition of DAGL-b, a major biosynthetic enzyme of 2-AG, would reverse inflammatory pain. Two DAGL-b inhibitors, KT109, and KT172, which have been previously shown to reduce arachidonic acid, prostaglandins, and TNF-a levels in lipopolysaccharide (LPS)-stimulated murine macrophages, were used to test whether these compounds would elicit antinociceptive effects in the LPS model of inflammatory pain model. Because these drugs also inhibit ABHD6, we assessed KT195, a selective ABHD6 inhibitor that is inactive against DAGL-b. KT109, but not KT172 or KT195, significantly reversed LPS-induced allodynia. Importantly, we found that DAGL-b knockout mice possess an anti-allodynic phenotype, but KT109 did not elicit any further decrease in allodynia in these animals. The anti-allodynic effects of KT-109 did not require cannabinoid receptors. Additionally, the anti-allodynic effects of KT-109 did not undergo tolerance following repeated administration. KT-109 did not produce any gastric hemorrhagic effects when compared to the NSAID diclofenac, which significantly produced gastric hemorrhages. These results suggest that blockade of DAGL-b leads to antinociceptive effects through a cannabinoid receptor independent mechanism of action, with absence of notable side effects. Collectively, the research presented in this dissertation suggests that the endocannabinoid catabolic enzymes MAGL and FAAH, and the endocannabinoid biosynthetic enzyme DAGL-b, represent promising targets to treat inflammatory pain.

The first study examined the interaction between low doses of the multi-kinase inhibitor sorafenib and the histone deacetylase inhibitor vorinostat in colon cancer cells. Sorafenib and vorinostat synergized to kill HCT116 and SW480 cells. In SW480 cells, sorafenib+vorinostat toxicity correlated with CD95 activation and CD95-stimulated autophagy. Drug lethality in SW480 cells was blocked by knock down of CD95. In SW620 cells that are patient matched to SW480 cells, sorafenib+vorinostat toxicity was significantly reduced that correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Overexpression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and tumor cell killing, whereas knock down of LASS6 in SW480 cells suppressed CD95 activation. In HCT116 cells, sorafenib+vorinostat did not increase CD95 plasma membrane levels, weakly induced caspase 8 association with CD95, and knock down of CD95 enhanced drug lethality. In HCT116 cells sorafenib+vorinostat treatment caused CD95-dependent autophagy that was a protective signal. Thus, treatment of tumor cells with sorafenib+vorinostat activates CD95 that promotes viability via autophagy or degrades survival via extrinsic or intrinsic pathways. Drug-induced activation of the de novo ceramide synthesis pathway plays a key role in CD95 activation. The second project explores the mechanism by which the combination of 17AAG, an hsp90 inhibitor, and PD184352, a MEK1/2 inhibitor alters survival in colon cancer cells. 17AAG and PD184352 synergized to kill HCT116 and SW480 cells. In HCT116 cells drug-exposure increased CD95 plasma membrane levels In SW620 cells, 17AAG and PD184352 toxicity was significantly reduced that correlated with a lack of CD95 activation and lower expression LASS6. Overexpression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and tumor cell killing. In Mia Paca2 cells, a pancreatic cell line, inhibition of caspase 8 or overexpression of c-FLIP-s suppressed cell killing by PD184352 and 17AAG exposure. Drug lethality in Mia Paca2 cells was blocked by knock down of CD95. Additionally, overexpression of Bcl-xL or knockdown of caspase 9 decreased cell killing in 17AAG and PD184352 combination treatment. Thus, 17AAG+PD184352 exposure activates the extrinsic and intrinsic apoptotic pathways to kill Mia Paca2 cells. This document was created in Microsoft Word 2000.

Nicotine is one of the most commonly used drugs among adolescent populations. Given the fact that adolescence is a unique developmental stage, during which nicotine has long-term effects on future drug-taking behavior, it is essential to understand how early exposure to nicotine during adolescence may affect the abuse liability of other drugs. We hypothesize that repeated exposure to low doses of nicotine in adolescence induce age-specific enhancement of the rewarding effects of several drugs of abuse in the conditioned place preference (CPP) test. Furthermore, we predict that these changes in behavioral responses are mediated by nicotine-induced brain region-specific increases in the expression of ΔFosB, a member of the Fos family of transcription factors, through activation of neuronal nicotinic receptors. We used mice as a model system to investigate the effects of adolescent nicotine exposure on responses to cocaine, amphetamine, and morphine in adulthood. We found that exposure to nicotine during the early phase of adolescence (postnatal day 28) enhanced cocaine CPP, acute locomotor activity, and locomotor sensitization in adulthood. Our data demonstrate that nicotine priming effects on cocaine are affected by the dose, duration, method of administration, age of exposure, and mouse strain. These data strongly suggest that nicotine intake during adolescence may cross-sensitize the brain to the rewarding effects of cocaine. A follow-up study was undertaken to determine if this enhancement applies to other drugs of abuse. The repeated exposure to 0.5 mg/kg nicotine (subcutaneous) during early adolescence resulted in significant enhancement of amphetamine and morphine preference in a CPP test, but had no effect on the somatic signs of morphine withdrawal. In addition, we investigated the possible neuronal mechanisms underlining enhancements to behavioral responses using both in vivo and in vitro techniques. Our results showed that nicotinic antagonists, with varying subtype selectivity, administered during adolescence prior to nicotine exposure diminished cocaine enhancement in CPP. This suggests that the enhancement of cocaine behavioral responses is mediated by neuronal nicotine receptors (mainly β2* and α7). Finally, studies of ∆FosB revealed significant effects of age and nicotine pre-treatment in nucleus accumbens (NAc), but not in the prefrontal cortex (PFC). Indeed, nicotine pre-treatment was able to significantly increase ∆FosB levels in NAc of early adolescent mice compared to adult mice. This accumulation of ∆FosB persisted for several weeks. Further studies are needed to fully examine the mechanisms of action underlying the observed changes in cocaine rewards.

Nicotine is one of the first and most commonly abused drugs in adolescence. According to The Center for Disease Control, every day more than 6000 adolescents try their first cigarette and over 3000 of them become daily smokers. Smoking among adolescents is a strong predictor of future drug abuse and dependence in adulthood. A number of studies has suggests that adolescents pre-exposed to nicotine may suffer permanent disruption of the brain’s reward systems through changes in dopamine receptor function. We hypothesize that nicotine exposure during adolescence causes long lasting neurobiological alterations that increase the likelihood of cocaine use in adulthood. Furthermore, it activates a neurobiological mechanism that is shared by many drugs of abuse, which will increase susceptibility to their rewarding effects. The work in this thesis contributes to the further understanding of this critical developmental period. Conditioned-place-preference, acute locomotor and locomotor sensitization pardigms were used to examine changes in cocaine sensitivity in adulthood. Testing was performed on adult ICR mice that were exposed to nicotine (0.1 or 0.5 mg/kg, S.C., b.i.d.) or saline during adolescence (postnatal days 28 or 46) or adult (postnatal day 70). Data showed that a 7-day exposure to the higher dose of nicotine (0.5 mg/kg) altered cocaine-induced responses. In contrast, neither 1 day exposure nor a low dose of nicotine (0.1 mg/kg) elicited this effect. A follow-up study was undertaken to determine if this enhancement generally applies to other drugs of abuse. Pre-exposure to 0.5mg/kg nicotine during early adolescence demonstrated significant enhancement to morphine reward, but it failed to increase d-amphetamine preference in a CPP model. Further research will be required in order to more fully examine the mechanisms of action for the observed changes in cocaine rewards. In summary, these findings suggest that early adolescent nicotine exposure leads to changes in cocaine reward and sensitivity during adulthood in both dose and duration matters. Indeed, the adolescent brain is uniquely vulnerable to the effects of nicotine on subsequent drug reward.

Acute behavioral responses to ethanol have predictive value for determining an individual's risk of long-term drinking behavior. Although the neurobiology of alcohol abuse is complex, prior studies from our laboratory demonstrated differential myelin-associated gene expression (MAGE) in medial prefrontal cortex (PFC) as one potential mechanism influencing acute ethanol behaviors between C57BL/6J (B6) and DBA/2J (D2) mice. Our laboratory and others have also shown MAGE is reduced in PFC of alcoholics. Herein, I have extended these findings through expression profiling of PFC into chronic models of ethanol self-administration from non-human primates and mice. Together, these results suggest that regulation of MAGE may be relevant to behavioral phenotypes witnessed in alcoholism. The pathogenesis of alcoholism progresses through multiple stages of drug exposure and withdrawal, however, genetic predisposition is also a major contributing factor for this disease. Therefore, I tested the hypothesis that not only does ethanol have direct effects on MAGE, but also variation in basal MAGE within the PFC is a molecular endophenotype underlying ethanol behavioral sensitivity. Bioinformatics of basal MAGE across the BXD recombinant inbred panel (n=29), derived from B6 and D2 mice, revealed a densely correlated myelin gene network associated with several ethanol behavioral phenotypes. Literature association tools identified Fyn kinase as potential regulator of MAGE. Fyn knockout mice are known to be more sensitive to the sedative-hypnotic properties of ethanol in the loss of righting reflex (LORR) paradigm. Microarray analysis of Fyn knockout mice revealed a significant decrease in MAGE, suggesting MAGE may be an underlying factor for LORR. In support of this premise, microarray analysis of genetic variance in LORR across Inbred Long Sleep and Inbred Short Sleep mice, as well as congenics for the Lore5 quantitative trait locus, also demonstrated an inverse relationship between MAGE and duration of LORR. The hypothesis was further investigated using cuprizone to model demyelination in B6 mice and test them in a battery of acute ethanol behaviors. Cuprizone-treated mice had a significantly greater duration in LORR (p < 0.01), demonstrating that myelin is an important contributor to the genetic variance in LORR. Thus, through genetic, genomic, and pharmacological tools I have ‘molecularly triangulated’ a myelin gene network as a contributing factor influencing acute ethanol behavioral sensitivity. The ability of myelin to alter acute ethanol sensitivity may warrant a prospective study of myelin in humans as a predictive molecular phenotype for an individual’s risk of developing alcohol dependence. Additionally, further genomic dissection of MAGE architecture and associated networks may aid in developing novel pharmacotherapies for an alcohol use disorder. Supported by NIAAA grants F31 AA018615 to SPF

Abstract: Folylpoly-γ-glutamate synthetase (FPGS) catalyzes the formation of polyglutamate forms of the reduced folates and antifolates such as methotrexate (MTX) and pemetrexed; this allows the retention of folates and antifolate cancer drugs inside the cell. The enzyme activity of FPGS is essential for cell proliferation and survival. The mouse fpgs gene contains two promoters spaced 10 kb apart which are activated in a tissue-specific manner. The upstream promoter (P1) and exons A1a and A1b are used in some differentiated tissues, mainly liver and kidney, whereas the downstream promoter (P2) and exon 1 are used in rapidly dividing cells. In contrast, the human fpgs gene expresses virtually all transcripts from the downstream promoter. In order to more faithfully mimic human folate metabolism in the mouse, we have deleted the upstream promoter and the associated two small exons of fpgs in the mouse genome by homologous recombination. Homozygous deletion mice survive embryonic development, grow to adulthood, and reproduce through several generations, they appear to be normal. The results of Q-RT-PCR analysis on RNA from adult mouse liver of three different genotypes (A1aA1b +/+, +/-, -/-) indicated that deletion of P1 results in the release of promoter interference of P2, and activation of the downstream P2 promoter is increased by 3-5 fold. Interestingly, the total FPGS mRNA expression in KO mouse liver is 20-100 fold lower than in liver from wild-type mice. However when the FPGS activity was measured using an FPGS enzyme assay, the liver of knockout mice appeared to have only 2 fold lower enzyme activity than liver from wild-type mice. In conclusion, we have successfully generated a mouse which reflects human folate metabolism much closer than seen in wild type mice. The FPGS- humanized mouse liver model would be an appropriate in vivo tool for the study of the antifolate drug toxicity and inhibition.