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Many potential therapeutic agents face challenges for their clinical development due to short circulation half-life. As a result, prolonging the half-life of therapeutic drugs in circulation while preserving their hydrophilicity and small size will be a key step toward more effective and safe pharmacological molecules. Our lab developed a new approach for enhancing the safety and efficacy of therapeutic agents. By endowing therapeutic agents with a hydrophilic small molecule (a derivative of the clinical candidate, AG10) which reversibly binds to the serum protein transthyretin (TTR), the half-life of the therapeutic agent should be extended by binding to the TTR in serum. We termed this technology TTR Ligand for half-life extension (TLHEs). The approach involved using TLHE, which binds with TTR by high specificity and affinity. Our group has already shown that this technology extends the half-life of peptides, small molecules, and proteins without seriously affecting their binding activity towards their receptor and efficacy. As we are expanding the applicability of TLHE to extend the half-life of hydrophobic moieties, increasing the polarity of the TLHE linker could be beneficial to maintain overall hydrophilicity. Our main objective here is to see the effect of TTR binding affinity and selectivity of TLHE in serum when we attach a hydrophilic glutamic acid in the TLHE linker.

Cyclin-dependent kinases (CDKs) and cyclins (Cyclins) are the core molecules in the regulation mechanism of the entire cell cycle. Cell cycle dysregulation is a common feature of human cancers, and inhibitors of cyclin-dependent kinases (CDKs) play a crucial role in cell cycle control and are one of the most promising areas of cancer therapy. We aspired to use our cyclin-dependent kinase 8 (CDK8) inhibitor, Q-12, as a probe for biomarker discovery for CDK8 inhibitor sensitive tumor types. Q-12 shows potent inhibition of cell viability and induction of apoptosis process in some triple-negative breast cancer (TNBC) and colorectal cancer cell lines in vitro. Western blot results indicate that Q-12 decrease p-STAT3 (Ser 727) stabilized p-STAT3 (Tyr 705) cause its upregulation. Cytokines are responsible for the increased phosphorylation of STAT3 (Tyr 705). Q-12 inhibit phosphorylation of CDK8 substrates STAT3 (Ser 727), STAT1 (Ser 727), E2F1 (Ser375) and reduce CDK8 protein levels. Q-12 initially increase E2F1 protein levels activated E2F1 and decrease Mcl-1 protein levels. All results suggest that STAT3 may not play a major role in cell death mechanism while E2F1 may play a major role. The main aim of the study is to investigate CDK8 inhibitor Q-12 effects on CDK8 and CDK8 substrates in triple negative breast cancer cell line MDA-MB-468, in order to better understand the mechanism of anti-proliferative effect of Q-12.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates biological responses to planar aromatic hydrocarbon. AHR activates gene transcription by binding to its corresponding enhancer with its partner-aryl hydrocarbon receptor nuclear translocator (ARNT). In addition, this receptor has been shown to regulate xenobiotic-metabolizing enzymes such as cytochrome P450. AHR exists widely in body tissues and affects bioactivation of carcinogenic compounds, T cell differentiation, fatty acid synthesis, cell proliferation, hematopoietic stem cell differentiation, respiratory reactivity, and insulin sensitivity. Although the precise mechanism illustrating the endogenous AHR function remains unclear, there has been intense interest in exploring AHR as a potential target for the treatment of diseases such as cancer and autoimmune diseases. It is known that mouse ahr d-allele possesses low ligand-binding affinity, whereas mouse ahr b-allele has a higher ligand-binding affinity. The d-allele functions more similarly to human AHR than the b-allele, which is most commonly studied. Human AHR can be rather difficult to study since it is relatively unstable and less sensitive to some ligands in vitro. Thus we generated a deletion construct which has the ligand-binding domain of human AHR and hoped that the expression yield could be increased. Here, I present the process and the results of expressing the mouse full-length b-allele of AHR and the human AHR ligand binding domain (LBD, amino acids 108 to 400) in Pichia pastoris. A higher enrichment of the b-allele and LBD was observed in wild-type yeast (yJC100) strain when compared to the protease-deficient yeast (ySMD1163) strain. This observation was consistent with the increased copy number in the wild-type strain. Although the LBD transcript was detected in both the wild-type and protease-deficient strains, the LBD protein was only detected in the wild-type strain.

Solubility is the amount of solute in the solvent system at phase equilibrium with certain temperature and pressure. Many of the new chemical entities are lipophilic molecules that require techniques to enhance solubility. Solubility enhancement can be achieved by either physical and/or chemical modification of the drug. Various techniques are available for solubility enhancement of poorly soluble drugs include particle size reduction, salt formation, solid dispersions, use of surfactants, prodrug, crystal modification, etc. In this study, the three model drugs belong to BCS class II and IV having low solubility with a certain range of physicochemical properties were studies in solubility enhancement using fusion method, co-precipitation, nano-milling and spray drying techniques. The two different polymers employed for solubility enhancement are PEG 8000 and PVP 40,000. Solubility was determined by Shake Flask method at the temperature of 37±0.1 °C. The objective is to investigate the enhancement of solubility of the three model drugs namely Glipizide, Carvedilol and Furosemide in 1:1, 1:5 and 1:10 drug-polymer ratios and are characterized by Differential Scanning Calorimetry (DSC). The Solubility of Glipizide was enhanced from 11.18 ± 1.78 µg/ml to 35.73 ± 0.04 µg/ml by 219 % increase with nano-milling technique at 1:5 ratio with PEG 8000 as carrier whereas with PVP 40000 as carrier, 286 % increase in solubility to 43.26 ± 7.87 µg/ml was observed at 1:1 ratio by fusion method. The solubility of Carvedilol was enhanced from 5.04 ± 0.55 µg/ml to 17.51 ± 0.94 µg/ml by 246 % at 1:5 ratio by fusion method with PEG8000 as carrier and 2924 % enhancement in solubility to 152.70 ± 9.09 µg/ml at 1:10 ratio by nano-milling with PVP40000 as the carrier. Furosemide showed an increase in solubility from 55.94 ± 2.48 µg/ml to 164.11 ± 9.18 µg/ml by 193 % at 1:10 ratio by nano-milling technique with PEG8000 as carrier whereas with PVP40000 as carrier, 444 % increase was observed at 1:1 ratio by nano-milling technique with solubility of 304.52 ± 23.11 µg/ml. The data showed that the decrease in percentage crystallinity and enthalpy of fusion of the model drugs upon implementing solubility enhancement techniques with the effect of particle size and the carrier used resulted in the increase of aqueous solubility of the model drugs.

Proteins are large biomolecules that have great therapeutic potential in treating many human diseases. However, chemical/enzymatic degradation, denaturation, and poor penetration into cells are some of the challenges for clinical use of intracellular proteins. Previously, our group has developed cationic lipid-coated magnesium phosphate nanoparticle (LP MgP NP-CAT) formulations to enhance the intracellular delivery of the negatively charged protein catalase. The goal of the current research is to develop a formulation to deliver cytochrome c (CytC), a positively charged protein into lung cancer cells A549. Specifically, this thesis research prepares and tests liposome-coated magnesium phosphate nanoparticle for delivery of cytochrome c (CytC LP/MgP). CytC LP/MgP was designed, prepared and characterized, showing that it had an average diameter around 150 nm and ζ-potential around +30 mV. The morphology of CytC LP/MgP was validated by transmission electron microscopy. CytC LP/MgP successfully led to the attachment of CytC to A549 cells, as supported by fluorescence imaging. Intracellular delivery of CytC alleviated the cytotoxicity of cationic lipids in A549 cells, as suggested by the MTS assay on cell viability, which could facilitate the clinical use of cationic lipids in drug delivery systems.

Programmed death-ligand 1 (PD-L1) is a type 1 transmembrane protein that has been reported to play a vital role in mediating suppressed immunity. The interaction between PD-L1 and PD-1 delivers a negative signal that reduces the proliferation of these T cells and induces apoptosis at the same time. Antibodies that can block the Programmed death-ligand 1 (PD-L1) on tumor cells have been shown to alleviate cancer-induced immunosuppression. While antibodies have a great potential in various therapeutic uses, many drawbacks such as the high cost of production, huge molecular size, and poor permeability impose restrictions on the extensive use of full-length antibodies. These limitations have necessitated research for finding alternatives to antibodies, such as peptides, that have lower molecular weight and similar properties as antibodies but do not have the lengthy and complicated approach of producing antibodies. In this study, a novel approach based on molecular interactions of the PD1-PD-L1 complex was developed to design peptides against PD-L1 using Knob-Socket model as basis. Three generations of peptides, α-helix, over-packed and salt bridge function peptides, were designed. All designed peptides were docked in the Molecular Operating Environment (MOE) and the AutoDock Vina software for the docking energy and the detail interaction information. Synthesis and characterization of selected peptides were performed after simulation studies. Surface Plasmon Resonance (SPR) studies showed that α-helix and over-packed peptides can’t bind to the PD-L1 protein with no response on sensorgrams, while peptides with salt bridge function had a higher binding response than those two generations of peptides. In confocal microscopic studies, PD-L1 positive breast cancer cell line MDA-MB-231 was used to determine the binding specificity of the salt bridge function peptides to PD-L1 in vitro, while another breast cancer cell line (MCF-7, without PD-L1) was used as a control. After incubation with peptides, significant fluorescence intensities were detected on the MDA-MB-231 cells, while only background fluorescence was observed on MCF-7 cells. In conclusion, this study demonstrated that peptides against PD-L1 designed using the Knob-Socket model and molecular interaction between PD-L1-PD1 complex showed feasibility to bind specifically with PD-L1 receptors.

Proton- and sodium-dependent monocarboxylate transporters (MCTs (SLC16A) and SMCTs (SLC5A)) transport monocarboxylates such as ketone bodies, lactate and pyruvate, as well as drugs such as gamma-hydroxybutyric acid. CD147 acts as an ancillary protein for MCT1 and MCT4, and is involved in membrane trafficking. Previously, it has been shown that MCT expression changes under different sex hormone conditions in skeletal muscle and Sertoli cells. However, it is unknown if MCTs, SMCTs or CD147 demonstrate sex differences in tissues where they play an important role in drug disposition. Monocarboxylate transporter substrates GHB and valproic acid have demonstrated sex differences in pharmacokinetic profiles. We hypothesize that sex hormones regulate monocarboxylate transporters and CD147 expression in drug disposition tissues, including the liver, intestine and kidney. The purpose of the current study is to evaluate sex and sex hormone dependent regulation of MCT1, MCT4, SMCT1 and CD147 mRNA and protein expression in drug disposition tissues. Liver, kidney and intestinal segments (duodenum, jejunum and ileum) were harvested from estrus cycle staged female rats, ovariectomized (OVX) females, males and castrated (CST) male rats. Hormone replacement experiments were performed to investigate testosterone and 17β-estradiol dependent regulation of renal MCTs, SMCT1 and CD147 in OVX females and CST males. mRNA of MCT1, MCT4, SMCT1 and CD147 was evaluated by real time quantitative PCR. Whole cell protein and membrane protein was extracted, target protein expression was evaluated by western blot. We have demonstrated sex and sex hormone dependent regulation of MCT1, MCT4, SMCT1 and CD147 in the liver, intestine regions and kidney occurs in a tissue specific manner. mRNA, protein expression and membrane localization of monocarboxylate transporters and CD147 were regulated differently by sex hormones. Sex differences in MCTs and SMCTs expression are important determinants of drug disposition in the body and sex differences in their regulation may contribute to differences in drug pharmacokinetics.

Niacin is a highly effective, lipid regulating drug associated with a number of metabolically induced side effects such as prostaglandin (PG) mediated flushing and hepatic toxicity. In an attempt to reduce the development of these adverse effects, scientists have investigated differing methods of niacin delivery designed to control drug release and alter metabolism. However, despite successful formulation of various orally based capsule and tablet delivery systems, patient adherence to niacin therapy is still compromised by adverse events such as PG-induced flushing. While the primary advantage of orally dosed formulations is ease of use, alternative delivery options such as transdermal delivery or polymeric micro/nanoparticle encapsulation for oral administration have shown promise in niacin reformulation. However, the effectiveness of these alternative delivery options in reducing inimical effects of niacin and maintaining drug efficacy is still largely unknown and requires more in-depth investigation. In this paper, we present an overview of niacin applications, its metabolic pathways, and current drug delivery formulations. Focus is placed on oral immediate, sustained, and extended release niacin delivery as well as combined statin and/or prostaglandin antagonist niacin formulation. We also examine and discuss current findings involving transdermal niacin formulations and polymeric micro/nanoparticle encapsulated niacin delivery.