Oral candidiasis (OC), caused by the fungal pathogen Candida albicans, is the most common opportunistic infection in HIV؉ individuals and other immunocompromised populations. The dramatic increase in resistance to common antifungals has emphasized the importance of identifying unconventional therapeutic options. Antimicrobial peptides have emerged as promising candidates for therapeutic intervention due to their broad antimicrobial properties and lack of toxicity. Histatin-5 (Hst-5) specifically has exhibited potent anticandidal activity indicating its potential as an antifungal agent. To that end, the goal of this study was to design a biocompatible hydrogel delivery system for Hst-5 application. The bioadhesive hydroxypropyl methylcellulose (HPMC) hydrogel formulation was developed for topical oral application against OC. The new formulation was evaluated in vitro for gel viscosity, Hst-5 release rate from the gel, and killing potency and, more importantly, was tested in vivo in our mouse model of OC. The findings demonstrated a controlled sustained release of Hst-5 from the polymer and rapid killing ability. Based on viable C. albicans counts recovered from tongues of treated and untreated mice, three daily applications of the formulation beginning 1 day postinfection with C. albicans were effective in protection against development of OC. Interestingly, in some cases, Hst-5 was able to clear existing lesions as well as associated tissue inflammation. These findings were confirmed by histopathology analysis of tongue tissue. Coupled with the lack of toxicity as well as anti-inflammatory and wound-healing properties of Hst-5, the findings from this study support the progression and commercial feasibility of using this compound as a novel therapeutic agent.

Microbial species utilize secreted-signaling molecules to coordinate their behavior. Our previous investigations demonstrated a key role for the Candida albicans -secreted quorum-sensing molecule farnesol in modulating Staphylococcus aureus response to antimicrobials in mixed biofilms. In this study, we aimed to provide mechanistic insights into the impact of farnesol on S. aureus within the context of inter-species interactions. To mimic biofilm dynamics, farnesol-sensitized S. aureus cells were generated via sequential farnesol exposure. The sensitized phenotype exhibited dramatic loss of the typical pigment, which we identified as staphyloxanthin, an important virulence factor synthesized by the Crt operon in S. aureus . Additionally, farnesol exposure exerted oxidative-stress as indicated by transcriptional analysis demonstrating alterations in redox-sensors and major virulence regulators. Paradoxically, the activated stress-response conferred S. aureus with enhanced tolerance to H 2 O 2 and phagocytic killing. Since expression of enzymes in the staphyloxanthin biosynthesis pathway was not impacted by farnesol, we generated a theoretical-binding model which indicated that farnesol may block staphyloxanthin biosynthesis via competitive-binding to the CrtM enzyme crucial for staphyloxanthin synthesis, due to high structural similarity to the CrtM substrate. Finally, mixed growth with C. albicans was found to similarly induce S. aureus depigmentation, but not during growth with a farnesol-deficient C. albicans strain. Collectively, the findings demonstrate that a fungal molecule acts as a redox-cycler eliciting a bacterial stress response via activation of the thiol-based redox system under the control of global regulators. Therefore, farnesol-induced transcriptional modulations of key regulatory networks in S. aureus may modulate the pathogenesis of C. albicans-S. aureus co-infections.

Cocaine use disorder (CUD) remains a significant public health challenge. Levo-tetrahydropalmatine (L-THP), a well-tolerated and non-addictive compound, shows promise for the management of CUD. Its pharmacologic profile includes blockade at dopamine and other monoamine receptors and attenuation of cocaine self-administration, reinstatement, and rewarding properties in rats. This study evaluated the safety of L-THP in human cocaine users and its influence on the safety and pharmacokinetics (PK) of cocaine. Twenty-four cocaine-using adult men were randomized to receive L-THP (30 mg BID orally) or placebo double-blind for 4 days, with an intranasal cocaine (40 mg) challenge on the fourth day. Safety and tolerability were evaluated using vital signs, EKG, clinical laboratory tests, and standardized self-report instruments. Peripheral venous blood was collected periodically and later assayed for L-THP and cocaine using highly sensitive and specific ultra-performance liquid chromatography-fluorescence detection (UPLC-FLD) methods. Twenty subjects completed the study, of whom 19 provided complete PK data. The short 3.5-day course of L-THP was safe and well tolerated and did not affect cocaine’s PK or its acute cardiovascular effects. The cocaine AUC0→∞ was 211.5 and 261.4 h*ng/ml, and the Cmax was 83.3 and 104.5 ng/ml for the L-THP and placebo groups respectively. In addition there were no significant difference in the number of side effects reported in each group (L-THP group: 22 [48%], Placebo group: 24 [52%]), or vital signs including, heart rate, blood pressure, complete blood count or EKG. These findings suggest that oral THP has promise for further development as a treatment for CUD.

Currently, there are no FDA approved medications for treatment of cocaine addiction underscoring the dire need to develop such a product. There is an accumulating body of evidence that L-tetrahydropalmatine (L-THP), a non-selective dopamine antagonist, can be used for the treatment of cocaine addiction. Indeed, the FDA recently approved its usage in a Phase I study in cocaine abusers and it was indispensable to develop a simple and sensitive method for the simultaneous determination of L-THP and cocaine in human plasma. We developed a UPLC-FLD method for quantitation of these molecules using an ACQUITY BEH C18 column (2.1 × 50mm, 1.7um) and a mobile phase that consisted of 5 mM ammonium phosphate (PH=4.75), methanol, and acetonitrile (v:v:v, 78:16:6). Venlafaxine was used as the internal standard while hexane was used for the liquid-liquid extraction. The flow rate was 0.4ml/min with fluorescence detection using an excitation wavelength of 230nm and emission detection wavelength of 315nm. This method was selective, linear and sensitive with a lower limit of quantification of 2.5 ng/mL for both cocaine and L-THP. The intra-day precision of cocaine and L-THP was <9.50% while the accuracy was <4.29%. The inter-day precision of cocaine and L-THP was <9.14%, and the accuracy was <12.49%. The recovery for cocaine and L-THP ranged from (43.95 - 50.02%) and (54.65 - 58.31%), respectively. In comparison to forty reported cocaine quantitation methods this method is simple, sensitive and cost-effective and can be used for simultaneous quantitation of L-THP and cocaine. This method meets the FDA guidelines and can be used in current and future clinical studies.

The taro plant, Colocasia esculenta, contains bioactive proteins with potential as cancer therapeutics. Several groups have reported anti-cancer activity in vitro and in vivo of taro-derived extracts (TEs). We reported that TE inhibits metastasis in a syngeneic murine model of Triple-Negative Breast Cancer (TNBC). Purpose: We sought to confirm our earlier studies in additional models and to identify novel mechanisms by which efficacy is achieved. Methods: We employed a panel of murine and human breast and ovarian cancer cell lines to determine the effect of TE on tumor cell viability, migration, and the ability to support cancer stem cells. Two syngeneic models of TNBC were employed to confirm our earlier report that TE potently inhibits metastasis. Cancer stem cell assays were employed to determine the ability of TE to inhibit tumorsphere-forming ability and to inhibit aldehyde dehydrogenase activity. To determine if host immunity contributes to the mechanism of metastasis inhibition, efficacy was assessed in immune-compromised mice. Results: We demonstrate that viability of some, but not all cell lines is inhibited by TE. Likewise, tumor cell migration is inhibited by TE. Using 2 immune competent, syngeneic models of TNBC, we confirm our earlier findings that tumor metastasis is potently inhibited by TE. We also demonstrate, for the first time, that TE directly inhibits breast cancer stem cells. Administration of TE to mice elicits expansion of several spleen cell populations but it was not known if host immune cells contribute to the mechanism by which TE inhibits tumor cell dissemination. In novel findings, we now show that the ability of TE to inhibit metastasis relies on immune T-cell-dependent, but not B cell or Natural Killer (NK)-cell-dependent mechanisms. Thus, both tumor cell-autonomous and host immune factors contribute to the mechanisms underlying TE efficacy. Our long-term goal is to evaluate TE efficacy in clinical trials. Most of our past studies as well as many of the results reported in this report were carried out using an isolation protocol described earlier (TE). In preparation for a near future clinical trial, we have now developed a strategy to isolate an enriched taro fraction, TE-method 2, (TE-M2) as well as a more purified subfraction (TE-M2F1) which can be scaled up under Good Manufacturing Practice (GMP) conditions for evaluation in human subjects. We demonstrate that TE-M2 and TE-M2F1 retain the anti-metastatic properties of TE. Conclusions: These studies provide further support for the continued examination of biologically active components of Colocasia esculenta as potential new therapeutic entities and identify a method to isolate sufficient quantities under GMP conditions to conduct early phase clinical studies.