Pharmaceutical Sciences

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    Genetic Influences on Opioid Use in Low Back Pain: OPRM1 rs1799971
    (2019-03-05) Phillips, Nicole R.; Licciardone, John C.; Romanowski, Benjamin
    Purpose: The OPRM1 rs1799971 single nucleotide polymorphism (SNP) has been studied for its influence on drug abuse and opioid use. Opioids have a questionable long-term risk-benefit profile in chronic low back pain, and a genetic predisposition to taking higher doses could place a patient at higher risk of complications related to opioid use. Studies have demonstrated higher dosages of self-administered opioids in an acute post-surgical setting in subjects with the rs1799971 AG polymorphism. We hypothesize that subjects with the rs1799971 AG polymorphism will use higher doses of opioids than subjects with other rs1799971 polymorphisms in the setting of chronic low back pain. Methods: This study was conducted using data from the PRECISION Pain Research Registry at UNTHSC. A saliva sample from each subject was obtained to determine genotypes, including the OPRM1 rs1799971 SNP. Additionally, numerical ratings of low back pain intensity and opioid use in morphine milligram equivalents (MMEs) were measured. The MMEs were computed in accord with CDC guidelines, which further indicate that opioid doses greater than 50 MMEs per day double the risk of opioid complications as compared with doses under 20 MMEs per day. We analyzed pain intensity, daily MMEs, and the proportion of subjects taking doses in excess of 50 MMEs per day based on allele status at SNP rs1799971. Results: Of 351 subjects with subacute or chronic low back pain, 279 were AA and 72 were AG. There was no significant difference in MMEs between rs1799971 AA subjects (x̅=5.90, SD=17.03) and AG subjects (x̅=9.53, SD=22.85). AG subjects had statistically significant lower pain intensity (x̅=5.2777 SD=1.74) compared to AA subjects (x̅=5.985, SD=1.9489 Mann-Whitney U=7897.5 p=0.005). rs1799971 AG subjects were more likely to be taking opioid doses greater than 50 MMEs per day than AA subjects (OR=3.40 95% CI:1.01-11.46 p=0.04). Five and 6 subjects with AG and AA, respectively, were taking doses greater than 50 MMEs per day (OR, 3.40; 95% CI, 1.01-11.46; Fishers exact p=0.11) Conclusion: There was no significant difference in mean MMEs among rs1799971 AG and AA subjects. However, AG subjects were marginally more likely than AA subjects to be taking doses greater than 50 MMEs per day. This SNP could potentially place rs1799971 AG patients at higher risk of complications relating to higher opioid doses, indicating a less favorable risk-benefit profile for long-term opioid therapy.
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    An Alternative Method To Quantify Surface Properties of Anti-Cancer Drugs
    (2019-03-05) Curry, Stephen; Kastellorizios, Michail; Mishra, Ina
    Purpose Physicochemical properties such as size and size distribution affect liposome formulations’ physical stability and accumulation in the target tissue. The FDA’s “Liposome Drug Products, Guidance for Industry”, 2018 emphasized size and size-distribution as “critical quality attributes”, however, it does not mention the criteria for an acceptable polydispersity index (PDI), currently measured using size-scattering technique. A monodisperse, homogenous size distribution population (PDI≤0.3) is desired. In this work, we measured surface tension of two different size distributions of liposome populations, with lipid composition similar to clinically approved anticancer formulation DOXIL, as a method to quantify liposome surface properties. This work establishes a building block in our long-term goal of obtaining insight into and facilitating the translation of nanoparticles from animal to human studies by offering additional preclinical characterization techniques based on surface properties of nanoparticles. Method Liposome formulations A and B, consisting of hydrogenated (Soy) phosphatidyl choline, cholesterol, and DSPE-PEG (7.64:5.17:1 molar ratio) were formulated in-house using thin film hydration method and probe sonication. Average particle size (PS) and PDI was obtained using dynamic light scattering (Mobius122, Wyatt Technology). The two formulations were fabricated with small differences in probe sonication process, yielding slightly different PDIs but the same average PS. The formulations were tested at two different lipid concentrations: 1mg/ml and 0.1mg/ml. Surface energy measurements were obtained on all four using pendant drop method, reported as mean with standard error (n=3) against the PDI. Results Average PS for A and B was similar (92.9±1.6 nm, 90.3±0.4 nm respectively), with PDIs 0.1±0.02 and 0.04±0.02 respectively. As expected, the surface tension was significantly decreased with concentration. PDI was found to significantly affect the total surface tension at higher concentration tested (1mg/ml) while it did not play a role at the lower concentration tested (0.1mg/ml). Interestingly, this trend was reversed when the surface tension was broken into its polar and dispersive components. Conclusions This work confirmed that small PDI changes, arising from slight variations in fabrication/manufacturing process, can translate into measurable changes in surface properties that can be obtained more rapidly and with higher accuracy than conventional DLS-based sizing techniques.
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    Microbial Natural Product Drug Discovery Through Systematic Sampling of Diverse Texas Soils
    (2019-03-05) Patel, Rinkal; Cheng, Yi-Qiang; Currens, Grant
    Purpose: Microorganisms have long been a valuable source of new pharmaceutically relevant small molecules. Because of their intrinsic need to compete for scarce resources in their microenvironment, they have often adapted to produce secondary metabolites capable of exerting cytotoxic effects against competing microorganisms. Microbial natural products and their derivatives account for nearly half of the currently approved anti-infective and anti-cancer drugs. Because of the State of Texas’ rich soil diversity, systematically collecting and fermenting bacteria and fungi collected around the state should yield a library of interesting and diverse molecules which can then be screened for desirable activities and identified for use as lead compounds in pharmaceutical research. Methods: To validate this project, the chemical crude extracts from 80 fungal species were separated through reverse-phase flash chromatography and screened for cytotoxicity against MIA PaCa-2 (ATCC® CRL-1420), SH-SY5Y (ATCC® CRL-2266), and COLO 829 (ATCC® CRL-1974) cancer cell lines as measured by Promega’s CellTiter-Glo® Luminescent Cell Viability Assay (ATP-Luciferase assay). Subsequently, a short-list of fractions containing promising cytotoxic compounds was identified and those fractions were subjected to further purification through multi-step activity-guided reverse-phase high-performance liquid chromatography (HPLC). Accurate masses and isotope distributions for compounds were assessed through time-of-flight mass spectrometry (TOF MS) and compared to known compound databases for dereplication. The purified compounds then underwent high-resolution proton and carbon NMR studies to determine their structure and novelty. Results: The first 80 fungal species collected from around the state were separated into 800 flash chromatography fractions of which 17 exhibited consistent cytotoxicity against MIA PaCa-2, a human pancreatic carcinoma cell line. Early study of a further narrowed test-group of 8 fungal species has thus far yielded a few interesting compounds. Notably of these species, one produces a compound with a mass of 401.257 which has been observed in multiple HPLC generated fractions exhibiting [greater than] 99% cell clearance after 48 hours. Based upon the accurate mass and isotope distribution, this compound preliminarily does not appear to have been previously described. Sufficient quantities of purified compound have been produced to perform high-resolution proton and carbon NMR to determine compound structure. Conclusions: We have seen from our preliminary study that we can systematically construct and screen a library of natural products derived from Texas microbes as way to identify lead compounds which exert cytotoxic effects on established cancer cell lines. Further, through collaboration, this library can be screened against a wider variety of targets to aid in identifying valuable lead compounds.
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    Development and Characterization of In Situ Self-Assembly Nanoparticles for Oral Tissue-Targeting Delivery
    (2019-03-05) Dong, Xiaowei; Le, Sophia
    Purpose: Tissue-targeting delivery system is an advanced method that improves drug concentration in tissues of interest and minimizes systemic toxicity. Drugs are encapsulated in a carrier and will be released at the site of action. Up to now, very few studies have reported on oral formulations of tissue-targeting delivery systems. Oral dosage form is preferred for the ease in administration and increased patient compliance. The objective of this study is to develop an orally administered nanoformulation that can deliver drug to targeted tissues using the in situ self-assembly nanoparticle (ISNP). Methods: Docetaxel (DTX), an anticancer agent, was used as a model drug for this study. DTX ISNP granules were prepared using surfactant, lipid, solid carrier and DTX. Long term stability of the granules was characterized in term of particle size, drug loading, entrapment efficiency and solid structure using particle size analyzer, HPLC and differential scanning calorimetry (DSC). Particle size stability in simulated physiological environment (2 hours in pH 1.2 followed by another 3 hours in pH 6.8) were studied. In-vitro release of DTX from NPs were also measured using HPLC. After oral administration of DTX ISNP granules, DTX concentration in plasma and tissues of rats were measured using LC-MS. Results: Average size of DTX ISNPs were around 187 nm with narrow distribution and polydispersity index in-vitro release study, up to 20% of DTX was released from NPs after 20 minutes. Plasma concentration of DTX fluctuated throughout the study without significant difference between DTX ISNP granules and DTX powder. However, compared to DTX powder, DTX ISNP granules remarkably increased drug concentrations in liver, lung and kidney at 1 hour after oral administration. Conclusions: The results demonstrated that ISNP nanotechnology has the potential applications in developing an oral formulation that selectively delivers the drugs to the targeted tissues.
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    Asymmetric syntheses identify preferred stereochemistry in small molecule allosteric modulators of the neuropeptide Y4 receptor
    (2019-03-05) Schubert, Mario; Vu, Oanh; Du, Yu; Stichel, Jan; Schüß, Corinna; Weaver, C. David; Beck-Sickinger, Annette; Meiler, Jens; Emmitte, Kyle; Mishra, Nigam
    PURPOSE: The neuropeptide Y4 receptor (Y4) is a GPCR belonging to a family of five receptors that bind ligands neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP). These ligands are hormones that play important roles in the regulation of feeding behavior and energy homeostasis. Small molecule ligands that selectively activate the Y4 receptor are potential therapeutics for obesity. Currently, there is a lack of non-peptide Y4-selective ligands available for studying Y4. Having recently identified multiple small molecule Y4 ligands via HTS, optimization of these hits is ongoing. The objective of the work described here is to develop asymmetric syntheses of two confirmed small molecule Y4 ligands, enabling determination of the preferred stereochemistry for Y4 activity and facilitating further optimization efforts. METHODS: Compounds were prepared via solution phase chemistry. Microwave reactions were run in an Anton Paar Monowave 200. Flash chromatography was carried out on either a CombiFlash® EZ Prep or CombiFlash® Rf+ system and utilized RediSep® Rf normal phase disposable columns. Preparative HPLC was carried out on a CombiFlash® EZ Prep system using a RediSep® Prep C18 10 x 250 mm, 100Å, 5 μm HPLC column from Teledyne ISCO. Compounds were characterized via NMR on a Bruker Fourier 300HD NMR spectrometer and via LCMS on an Agilent 6230 Accurate-Mass TOF LC/MS. Compound pharmacology was assessed in a two-addition protocol via Ca2+ flux assays in COS7 cells stably expressing Y4 and a chimeric G-protein. RESULTS: Four diastereomers of Y4 PAM tBPC were synthesized to [greater than] 87% d.e. utilizing a synthetic route employing an asymmetric alkylation, chelation controlled addition, and a Grubbs RCM reaction for the formation of key bonds. (1S,2S)-tBPC was found to be the preferred diastereomer for Y4 activity. Two enantiomers of Y4 NAM VU0637120 were synthesized to [greater than] 99% e.e. by employing commercially available chiral starting materials. (S)-VU0637120 was highly preferred for Y4 activity compared to (R)-VU0637120. A library based on (S)-VU0637120 was subsequently synthesized. CONCLUSIONS: Identification of the preferred stereoisomers for these Y4 ligands were successfully accomplished through asymmetric syntheses. These results enable the further optimization of these compounds in the context of the preferred stereochemistry, enhancing the probability of identifying optimized tools for studying the Y4 receptor.
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    Using Autodock Tools for Automated Docking of Ligands to D2/D3 Receptors
    (2019-03-05) Hayatshahi, Hamed; Liu, Jin; Kramer, Andrew
    Parkinson’s Disease (PD) is a neurodegenerative disorder of the substantia nigra, affecting roughly ten million individuals worldwide. Considering that the substantia nigra is the main player in dopamine production, progressive erosion of this structure has been shown to dramatically decrease the amount of dopamine in the brain. The direct pathway of movement responds to dopamine through the dopamine receptors, D2 and D3. Therefore, without dopamine, the brain struggles to produce movement, yielding many of the symptoms associated with PD: rigidity, tremors, shuffling gait. Current medications for PD increase dopamine concentrations or artificially activate the D2/D3 receptors; yet they are not without their side effects, most often causing loss of impulse control, confusion, and postural hypotension. Our goal is to better understand the selectivity mechanism of D2/D3 receptors in hopes of generating a more efficient medication in the future. By constructing a series of ligands with a similar molecular backbone but differing in various functional groups, we can utilize computer programming to find the activating energy of each ligand and compare the data in order to determine which functional group specifically activates either the D2 or D3 receptor. In this, roughly ninety distinctive ligands were drawn and converted to a three-dimensional product using Maestro. Utilizing Autodock Tools, these ligands were then computationally bound to both the D2 and D3 receptors, producing binding affinity for each ligand. In order to activate both the D2 and D3 receptors, a salt bridge must form between the protonated nitrogen of the nonaromatic ring of the ligand and aspartate residue 114 on the extracellular aspect of the receptor. Our findings revealed only thirty-six ligands successfully activating the D2 receptor in the correct orientation, and thirty-seven activating the D3 receptor. In total, the ligands drawn were more selective for the D3 receptor. From this data, we found a correlation between the presence of an ortho-methoxy group on the benzene ring of the ligand and its relationship to a serine residue on the receptor on D3R, producing a higher binding affinity. Therefore, by identifying the significance of this serine residue, we can better understand the selectivity of the D2/D3 receptors.
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    Investigating Effects of Pegylation of the Surface Tension of Liposomes used in the Treatment of Breast Cancer
    (2019-03-05) Jameson, Jeffrey; Mishra, Ina; Kastellorizios, Michail; Garrett, Meredith
    Purpose: In recent years there have been many advancements in nanomedicine as potential delivery systems for the treatment of various types of cancer. Two formulations on the market, Doxil® and Myocet®, are made of doxorubicin-loaded liposomes. One them, Doxil® has the hydrophilic polymer polyethylene glycol (PEG) immobilized on its surface, while the other (Myocet®) doesn’t. PEG has been shown to improve liposome circulation in the blood by disguising them from the immune system, creating a ‘stealth’ delivery system for drugs. However, it is unknown whether the PEG changes the way liposomes may interact within the tumor. Here, we fabricated liposomes that mimic Doxil® and Myocet® and measured their surface tension, as surface tension is a driving force behind the interfacial interactions between liposomes and tumor extracellular matrix. Materials and Methods: Two formulations of liposomes (Doxil® and Myocet®) were fabricated using thin film hydration for liposome formation and membrane extrusion for liposome downsizing to nanometer levels. A thin film of each formulation was made and then hydrated with phosphate buffered saline. Extrusion was performed using a 50nm membrane bringing final liposome size of each formulation to approximately 100nm. The Doxil® formulation contained hydrogenated soybean phosphatidylcholine (HSPC), cholesterol and PEG, while the Myocet® contained only HSPC and cholesterol. The two formulations underwent a series of dilutions, and the surface tension of each concentration was calculated using the twin capillary rise method. A custom 3D printed stand coupled with a well plate was used to secure the capillaries while allowing small volumes of the formulations to be used. Results: Data collected showed a significant decrease in surface tension when PEG is removed from the surface of the liposomes. This effect was consistent across all concentrations when compared to the original formulation. Conclusions: Our results show that PEG causes a drastic change in how liposomes interact with surfaces. Further, we demonstrated that this effect is greatly affected by small changes in liposome concentration. The reduction in surface tension observed in Myocet® versus Doxil® may be the reason behind Doxil’s sustained success in the clinic compared to Myocet®. Future studies will focus on quantifying interfacial tension between these liposome formulations and breast cancer biopsies in order to potentially explain their different clinical performance.
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    Effect of Cholesterol Content on Surface Properties of Doxil-Mimicking Liposomes
    (2019-03-05) Garrett, Meredith; Mishra, Ina; Kastellorizios, Michail; Jameson, Jeffrey
    Purpose:The use of nanoparticles (NPs) for drug delivery has gained a lot of attention from biomedical researchers in the last few decades. This is primarily because NPs are of an extremely small size that enables them to accumulate preferentially inside of the tumors instead of healthy tissue. The reason is unknown as to why research being done on NPs has shown promising results in animal models but does not translate successfully to the clinic. Here, we will study the effect of NPs cholesterol content on their surface tension. Cholesterol is sometimes added to NPs to increase their rigidity and stability, but we do not know if it interferes with other properties such as surface tension. We hypothesize that surface tension can be useful in enriching characterization of novel NPs and help determine which NPs to move on to clinical testing. Methods:We used Doxil®, one of the few successful NPs for cancer therapy, as reference. Doxil® consists of a drug (doxorubicin) encapsulated in nano-sized vehicles (liposomes) made of HSPC, cholesterol (CHOL), and PEG. Two formulations of NPs, HSPC:CHOL:PEG (Doxil®) and HSPC:PEG, were prepared by thin film hydration followed by membrane extrusion. We tested their surface tension at six concentrations using the twin-capillary method. A capillary stand was 3D-printed that was able to hold the capillaries stable. Results: The average change in surface tension for the six different concentrations between the two formulations did not have a noticeable change. The surface tension decreased proportionately at the same concentrations for each of the HSPC:CHOL:PEG and HSPC:PEG formulations. In addition, the same experiment was used to compare the difference in surface tension between HSPC:CHOL and phosphatidylcholine (PC), which is equivalent to HSPC. The difference between the values were also negligible. Conclusion:Based on our results, we conclude that the inclusion of cholesterol in liposomes does not alter their surface energy properties as measured by the twin capillary rise method. This may be due to the fact that cholesterol is a small molecule while PEG is a giant molecule that covers the surface of the entire liposome. Even though cholesterol does not affect surface tension, it is still pertinent for the formulation to be effective. In future studies, an additional technique will be used (pendant drop shape analysis) that gives more insight into surface tension and breaks it into its polar and non-polar components.
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    3D-Printed Insert for Visualization of Liposomal Interactions with Collagen Fibers
    (2019-03-05) Mishra, Ina; Kastellorizios, Michail; Curry, Stephen
    Purpose: Nanoparticles, such as liposome, are commonly used as vehicles for drug delivery to target tissues such as tumors. However, relatively little research has focused on liposomal interactions with the tumor extracellular matrix (ECM), a huge hurdle in the process of perfecting dosage. To our knowledge, there is no method available that allows the observation liposomes against a whole collagen fiber. In order to better study these interactions, we have designed a 3D-printed frame that helps in visualizing liposome transport around a collagen fiber. Observation of our replicated microenvironment will allow us to shed more light on the movements of liposomes within the ECM of tumor cells. Methods: Collagen fiber was removed from adult rat tail and allowed to soak in phosphate-buffered saline (PBS) overnight. Primary design of the frame was done using SketchUp software. Printing of 12mm frame design was done using a Form 2 3D printer (Formlabs). Fiber-in-frame was placed in a glass bottom microwell dish (MatTek Corp.). Research-grade liposomes that mimic the clinical product Doxil (DoxomTM, Liposomics) were used in a background of 0.07mg/ml BSA solution, and added to the channel. Visualization of the interaction was done using confocal microscope (Nikon A1R-MP+ Multiphoton System). Results: As a proof-of-concept, we mounted a collagen fiber into our frame and filled channel with BSA solution. Observation indicates that liposomes prefer to accumulate on the collagen fiber surface. Multiple trial frames were needed to account for height requirements necessary for confocal microscopy. Our built-in sample well allowed for a maximum 20 uL of formulation to be added into the channel environment from one end of the fiber. Our next steps will focus on reducing height and volume of background and sample needed as well as removing the need for a dish or slide altogether. Future studies will focus on kinetic phenomena in this microenvironment. Conclusions: Using the methods described above, we observed that Doxom liposomes accumulated near the fiber surface. This suggests that liposomes can use collagen as a route of travel in the extracellular environment. Future experiments with this frame design and others will be used to study the liposomal interactions with collagen and other ECM components. In addition to new frame designs, instruments with higher intensity and resolution will provide more accurate data.