Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-resistant Staphylococcus aureus (MRSA) in vitro. To randomly generate A. oryzae mutant strains, spores were treated with ethyl methanesulfonate (EMS). Over 3000 EMS-treated A. oryzae cultures were tested in the screen, and one isolate, CAL220, exhibited altered morphology and antibacterial activity. Culture supernatant from this isolate showed antibacterial activity against Methicillin-sensitive Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa, but not Klebsiella pneumonia or Proteus vulgaris. The results of this study support our hypothesis and suggest that the screen used is sufficient and appropriate to detect secreted antibacterial fungal compounds resulting from mutagenesis of A. oryzae. Because the genome of A. oryzae has been sequenced and systems are available for genetic transformation of this organism, targeted as well as random mutations may be introduced to facilitate the discovery of novel antibacterial compounds using this system.

Purpose: Use of ampicillin in outpatient parenteral antimicrobial therapy (OPAT) has historically been complicated by frequent dosing and short beyond use dates. However historic stability data relied on inaccurate testing methods. The purpose of this study is to evaluate the stability of ampicillin using high-pressure liquid chromatography (HPLC), the gold standard, in a real-world OPAT dosing model using continuous infusion at room temperature over 24 hours immediately following preparation compared to batches stored under refrigeration for 24 hours, 72 hours, and 7 days. Methods: An HPLC method was developed and validated as stability – indicating according to guidance in USP general Chapter < 1225 >. Method development included linearity, precision, accuracy, repeatability and forced degradation. Four batches were prepared using 4 different lots from 2 different manufacturers for each storage condition (immediate, 24 hours, 72 hours, and 7 days). Three 2-gram vials were each reconstituted with 10 mL of sterile water for injection (SWFI) and added to 250 mL of normal saline by a licensed pharmacist and stored in a laboratory refrigerator (2 – 8oC). A pump system was used to continuously circulate the solutions through medical grade tubing at room temperature. One milliliter aliquots were removed from each batch at time 0, 4 hours, 8 hours, 12 hours and 24 hours and analyzed for ampicillin concentration using the aforementioned HPLC method. The samples were filtered prior to analysis using a 0.22-micron syringe filter and analyzed in triplicates along with freshly prepared calibration samples (24 – 12 mg/mL). Peak area was used to determine percent recovery for each sample. Results:Each batch was assayed for initial concentration (20.34 – 21.50 mg/mL) upon preparation, and percent recovery was compared to that initial concentration thereafter. Acceptable recovery was defined as 90 – 110% of initial concentration. On the day of product preparation (immediate use), the average percent recovery over 24 hours was 96.4%. The other average percent recoveries were as follows: 95.8% (24-hour storage), 94.6% (72-hour storage) and 90.3% (7-day storage). These data represent the average percent recovery for all time points during the 24 hours sampling (n = 60 for each experiment). When evaluating individual time points, the percent recovery remained above 90% for all batches and time points except for the 7-day storage experiment. Under 7-day storage conditions, the percent recovery fell below 90% after 4 hours of circulation through the medical grade tubing. Furthermore, 95% confidence interval for percent recovery for ampicillin in the samples stayed within 90 – 110% of the initial concentration for the duration of the experiment for all test groups except 7-day storage. Conclusion:Ampicillin can be prepared and stored in a refrigerator for up to 72-hours prior to continuously infusing at room temperature over 24 hours with less than a 10% loss of potency over the dosing period. This model supports twice weekly OPAT delivery of ampicillin.

Revised and Expanded Handbook Provides Comprehensive Introduction and Complete Instruction for Sample Preparation in Vital Category of Bioanalysis Following in the footsteps of the previously published Handbook of LC-MS Bioanalysis, this book is a thorough and timely guide to all important sample preparation techniques used for quantitative Liquid Chromatography–Mass Spectrometry (LC-MS) bioanalysis of small and large molecules. LC-MS bioanalysis is a key element of pharmaceutical research and development, post-approval therapeutic drug monitoring, and many other studies used in human healthcare. While advances are continually being made in key aspects of LC-MS bioanalysis such as sensitivity and throughput, the value of research/study mentioned above is still heavily dependent on the availability of high-quality data, for which sample preparation plays the critical role. Thus, this text provides researchers in industry, academia, and regulatory agencies with detailed sample preparation techniques and step-by-step protocols on proper extraction of various analyte(s) of interest from biological samples for LC-MS quantification, in accordance with current health authority regulations and industry best practices. The three sections of the book with a total of 26 chapters cover topics that include: Current basic sample preparation techniques (e.g., protein precipitation, liquid-liquid extraction, solid-phase extraction, salting-out assisted liquid-liquid extraction, ultracentrifugation and ultrafiltration, microsampling, sample extraction via electromembranes) Sample preparation techniques for uncommon biological matrices (e.g., tissues, hair, skin, nails, bones, mononuclear cells, cerebrospinal fluid, aqueous humor) Crucial aspects of LC-MS bioanalytical method development (e.g., pre-analytical considerations, derivation strategies, stability, non-specific binding) in addition to sample preparation techniques for challenging molecules (e.g., lipids, peptides, proteins, oligonucleotides, antibody-drug conjugates) Sample Preparation in LC-MS Bioanalysis will prove a practical and highly valuable addition to the reference shelves of scientists and related professionals in a variety of fields, including pharmaceutical and biomedical research, mass spectrometry, and analytical chemistry, as well as practitioners in clinical pharmacology, toxicology, and therapeutic drug monitoring.

Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a fatal disease with no available disease-modifying therapies. While destabilizing TTR mutations increase the risk of developing ATTR-CM, the naturally occurring Thr119Met (T119M) variant stabilizes TTR and prevent disease. The two Serine 117 and Serine 117’ (S117/S117’) side chain hydroxyl groups of monomers A and B in T119M variant TTR form direct hydrogen bonds with each other. AG10 is an orally available, small molecule TTR stabilizer in Phase 3 clinical development for ATTR-CM. Structural features that infer high potency of AG10 for stabilizing TTR remains incompletely characterized. In order to investigate the contribution of hydrogen bonds between the pyrazole ring of AG10 and the two S117/S117’ and the salt bridges between carboxyl group and two Lysine 15 and Lysine 15’ (K15/K15’) on the stabilization of TTR, we synthesized three AG10 analogues (compounds 1, 2, and 3). Isothermal titration calorimetry (ITC) was used to determine the binding affinities (Kd) of ligands to TTR. Biochemical studies were used to compare the potency of AG10 relative to other ligands in stabilizing TTR in buffer and human serum. Results highlight the crucial roles played by the carboxyl group and pyrazole ring of AG10 and the importance of the hydrogen bonds it forms with the two TTR dimers, mimicking the interactions in the protective T119M-TTR mutation and enhancing the kinetic stability of the TTR tetramer.

Structure based computational peptide design methods have gained significant interest in recent years with the availability of structural insights of protein-protein interactions obtained from the crystal structures. Most of these approaches design new peptide ligands by connecting the crucial amino acid residues from the protein interface and are generally not based on any predicted receptor-ligand interaction. In this work, a peptide design method based on the Knob-Socket model was used to identify the specific ligand residues packing into the receptor interface. This method enables rational peptide ligand design by predicting amino acid residues that will fit best at the binding site of the receptor protein. Specific peptide ligands for the model receptor CD13 that are overexpressed in several cancer types were designed in this study. From the initial library of designed peptides, three potential candidates were selected based on simulated energies in the CD13 binding site using the programs Molecular Operating Environment (MOE) and AutoDock Vina. In the CD13 enzymatic activity inhibition assay, the three identified peptides exhibited 2.7 to 7.4 times lower IC50 values (GYPAY, 227 µM; GFPAY, 463 µM; GYPAVYLF, 170 µM) when compared to the known peptide ligand CNGRC(C1-C5) (1260 µM). The binding affinities of the peptides (GYPAY, Ki = 54.0 µM; GFPAY, Ki = 74.3 µM; GYPAVYLF, Ki = 38.8 µM) were 10 to 20 times higher than that of CNGRC (C1-C5) (Ki = 773 µM). The double reciprocal plots from the steady state enzyme kinetic assays confirmed the binding of the peptides to the intended active site of CD13. The cell binding and confocal microscopy assays showed that the designed peptides selectively bind to the CD13 on cell surface. The designed peptide-drug conjugates (PDCs) showed lower in vitro cytotoxicity and slightly better in vivo antitumor efficacy as compared to a model drug MMAE. However, the PDCs contributed to much lower weight loss in mice indicating lower side effects in vivo. This study demonstrated the feasibility of a Knob-Socket based rational design of novel peptides ligands in improving the identification of specific binding in comparison to the labor intensive current methods.

Human stem cells have revolutionized the study of early neural development, offering a powerful tool to investigate the complex processes involved in the formation of the nervous system. In this study, the human pluripotent stem cell line, TERA2.cl.SP12, was utilized to derive neuroglial cells and to determine their differentiation and functional maturation up to one year in culture; this model was then investigated as a new tool for the study of neurodevelopmental toxicity, for seizure modeling and as a screening method for the investigation of new anticonvulsants. Immunocytochemistry experiments determined that non-differentiated cells expressed the stem cell marker Oct-3/4 and, when exposed to retinoic acid, differentiated into neurons that expressed the neural marker proteins, β-III tubulin, MAP2 and the glial cells expressed GFAP. Immunoblotting analysis of the neural protein βIII-tubulin expression revealed robust expression from 2 months to 12 months of culture, indicating that the neurons remained viable and stable for an extended period. GFAP expression significantly increased at 4 and 8 months of culture, indicating that gliogenesis followed neurogenesis in vitro. These findings support the concept that the neural differentiation of human embryonal carcinoma (EC) stem cells follows a developmental program that closely mimics the intrinsic timescale observed in vivo, as has been observed in human ES and iPS cells.Multi-electrode array (MEA) studies indicated that these neuroglia cultures display complex electrochemical signaling including high frequency trains of action potentials from single neurons, neural network bursts and highly synchronized firing patterns which all increased significantly over one year in culture and indicated complex network formation and neural maturation. Neural activity in this 2D neuron-glia culture was modulated by a variety of voltage-gated and ligand-gated ion channel acting drugs and indicated the presence of inhibitory and excitatory synapses and integrated neural circuits. To test these stem cell derived neurons as a model for epilepsy disease modeling and drug screening, epileptiform activity was induced by the proconvulsant agent 4-aminopyridine (4-AP) in the neural circuits and five antiseizure drugs, representing a diverse group of clinically important agents for epilepsy, were examined. MEA data showed that 4-AP evoked epileptiform-like activity and increased spike frequency, single cell burst firing, and synchronized network bursting in the neuroglial cultures. In addition, spontaneous neural network activity and 4-AP-evoked epileptiform activity were inhibited by first, second and third generation antiseizure agents, consistent with animal and human studies. Pluripotent stem cell-based systems have proven useful in the study of developmental neurotoxicity. Here the impact of 2 widely used anti-epileptic drugs (AEDs), topiramate and gabapentin, on neural development was determined using TERA2.cl.SP-12 stem cell neurons. Transient exposure to gabapentin (3-300µM) or topiramate (3-300µM) did not affect cell viability, proliferation, or differentiation after a 30-day exposure period, except at concentrations of topiramate above the therapeutic range, and which led to a significant reduction in neural differentiation. The impact of chronic exposure to topiramate and on functional activity of neurons was then determined over ultra-long-term cell cultures. Results showed that topiramate decreased firing rate and synchrony index at a concentration of 30µM compared to control. Notably, these effects did not appear until 36 to 52 weeks, implying ‘silent neurotoxicity’ of developmental processes and functional deficits that do not surface until much later. These findings also suggest that human pluripotent stem cells and their neuronal derivatives can advance toxicological research by reducing the time and costs involved, whilst also minimizing the need for animal testing. Brain organoids offer a highly advanced model for the study of early embryonic and fetal brain development as well as human-specific disorders. In the next phase of this study, TERA2.cl.SP-12 stem cells were differentiated into 3D culture (neural organoids) by utilizing Stemcell Technologies STEMdiff cerebral organoid protocol. After 100 days differentiation, immunolabeling of the neural marker protein (βIII-tubulin) and glial cells (GFAP) revealed strong expression and presence of neuroglial cells differentiated from stem cells in the organoid cultures. Interestingly, immunocytochemistry labeling showed GFAP-positive cells surrounding ventricular-like zone (VZ) areas, suggesting VZ formation in the organoids. Multi-electrode array recordings demonstrated spontaneous spiking, single cell bursts and synchronized which all increased significantly over 30 weeks of differentiation. Neural activity in the organoids was modulated by a variety of voltage-gated and ligand-gated ion channel acting drugs and revealed the presence of inhibitory and excitatory synapses and integrated neural circuits. In addition, the convulsant agent 4-AP, evoked epileptiform like activity and increased spike frequency, single cell burst firing, and synchronized network bursting in the organoids, consistent with seizure like activity in these cultures. In further experiments the anticonvulsants, carbamazepine, ethosuximide and diazepam, reduced or blocked 4-AP induced epileptiform-like activity in the organoids at concentrations close to, or within the range of therapeutic dosing for seizure control. Together, the findings reported in this dissertation strongly support the potential value of human pluripotent stem cells in modeling early neural development, neurological disorder and drug investigations in both 2D and 3D cultures.

Breast cancer is the second most common type of cancer in the world. Hormone receptor (HR) positive breast cancer (BC) is a prevalent disease accounting for approximately 2 million new cases globally. Almost 70-80% of breast cancer patients are women with a positive score for the estrogen receptor (ER). Triple-negative breast cancer (TNBC) which have a negative score for estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor type 2 (HER2) is considered an aggressive histological breast cancer subtype with limited treatment options.Frequently, doxorubicin (DOXO)-based chemotherapy is utilized in this patient population due to the lack of available molecular targets. While DOXO is an effective chemotherapeutic agent, its efficacy is limited due to acquired drug resistance and cardiotoxicity. Therefore, the identification of other treatment options for TNBC is needed. TNBC is a heterogeneous malignancy, with 70% of cases classified as a basal subtype as they look similar to the epithelial cells of the outermost basal layer of the breast’s milk ducts. This further complicates the search for an effective molecular target. Doxorubicin and other anthracycline derivatives are frequently used as part of the adjuvant chemotherapy regimen for triple- negative breast cancer (TNBC). Although effective, doxorubicin is known for its off-target and toxic side effect profile, particularly with respect to the myocardium, often resulting in left ventricular (LV) dysfunction and congestive 2 heart failure when used at cumulative doses exceeding 400 mg/m2. Ribonucleotide reductase (RR) is a rate limiting enzyme in DNA synthesis consisting of two subunits RRM1 and RRM2. Both RRM1 and RRM2 are encoded by different genes in their chromosomes. Their mRNAs are also differentially expressed throughout the cell cycle. Didox inhibits ribonucleotide reductase subunit 2 (RRM2) which ultimately blocks DNA synthesis. We have observed that the ribonucleotide reductase subunit 2 (RRM2) is significantly over-expressed in estrogen receptor (ER)–negative cells as compared with ER-positive breast cancer cells. Here, we inhibited RRM2 in ER-negative breast cancer cells as a target for therapy in this difficult-to-treat population. We observed that through the use of didox (3,4-dihydroxybenzohydroxamic acid), a ribonucleotide reductase inhibitor, the reduction in RRM2 was accompanied by reduced NFkB activity in vitro. When the ribonucleotide reductase inhibitor didox was used in combination with the chemotherapeutic drug doxorubicin, we observed significant downregulation of NFkB proteins in TNBC. As well, we observed that protein levels of mutant p53 were significantly reduced by didox or combination therapy in vitro. Xenograft studies showed that combination therapy was found to be effective in vivo, resulting in a significantly reduced tumor volume as compared with doxorubicin monotherapy. In addition, the use of didox was also found to ameliorate the toxic myocardial effects of doxorubicin in vivo as measured by heart mass, LV diameter, and serum troponin T protein levels which are released by heart during muscle damage. The data present a novel and promising approach for the treatment of TNBC that merits further clinical evaluation in humans. Hormone receptor positive breast cancers of all stages are selectively treated with endocrine therapy targeting estrogen receptor (ER) activity. But success is limited by the development of acquired resistance owing to long-term therapy. The cyclin D1 and cyclin dependent kinase 4/6 (CDK4/6) complex causes phosphorylation and subsequent inactivation of retinoblastoma (Rb) tumor suppressor protein which promotes progression of the cell cycle from G1 to S phase. This observation led to the development of the first CDK4/6 inhibitor Palbociclib (Ibrance; Pfizer) which induces cell cycle arrest at G1 phase in cancer cells. Intrinsic and acquired drug resistance development, have impacted the therapeutic success rate despite promising clinical outcomes. This situation necessitates the development of potential combination strategies to overcome drug resistance. The combination of didox with palbociclib is a potential strategy to target ER positive and ER negative/triple-negative breast cancer. In our recent study, we confirmed that didox in combination with palbociclib significantly lowers the growth of ER positive and ER negative breast cancer cells along with their palbociclib resistant counterparts compared to no treatment or palbociclib treatment alone. We confirmed that ER positive MCF7 and ER negative MDA-MB-468 parental breast cancer cells exhibit lower IC50 values of palbociclib drug as compared to their palbociclib resistant counterparts. Here, we are reporting that didox alone or in combination with palbociclib decreases cell cycle proteins in ER positive MCF7 and ER negative MDA-MB-468 parental and palbociclib resistant breast cancer cells. This finding opens a novel approach for targeting both ER positive as well as ER negative breast cancer treatment. We are also reporting that didox treatment alters cyclin D1 (CCND1) and RRM2 expression in MCF7 and MDA-MB-468 breast cancer cells along with their palbociclib resistant counterparts. Additionally, we observed that didox alone or in combination with palbociclib alters the cell cycle of MCF7 and MDA-MB-468 parental and palbociclib resistant breast cancer cells. Our data present a novel and promising approach for the treatment of ER positive and ER negative breast cancer that involves inhibition of RRM2, NFkB, and the CDK4/6-cyclin D1/pRb axis that merits further clinical investigation in human models.

The use of valproic acid (VPA) (also known as Depakote, Depakene, and others) frequently results in elevated plasma ammonia. In some people, hyperammonemia may be clinically significant, resulting in hyperammonemic encephalopathy, which may be severe. Valproic acid-induced hyperammonemic encephalopathy may occur in people with normal liver function, despite normal doses and serum levels of VPA. We describe 2 cases of valproic acid-induced hyperammonemic encephalopathy in patients with supratherapeutic VPA levels, although the condition has been described in people with normal VPA levels. With the increasing indications and off-label uses of VPA, family physicians should be aware of this potential complication of VPA and check ammonia levels in patients taking VPA who present with alterations in mental status. Treatment with L-carnitine may be beneficial in reducing ammonia levels. Valproic acid (VPA) is effective in the treatment of seizure disorders, bipolar disorder, migraine headache prophylaxis, neuropathic pain, restless legs syndrome, dementia-related agitation, and social anxiety disorder, among other conditions. VPA has numerous drug interactions and toxicities; severe toxicities include hepatic damage, pancreatitis, teratogenicity, thrombocytopenia, and hyperammonemia. Here we depict 2 case reports of VPA-induced hyperammonemic encephalopathy (VHE), both occurring in patietns with no history of underlying liver disease. In one instance, the patient was able to function, but with significant cognitive limitations. In the second case, the patient was comatose. Both of the patients we describe also had supratherapeutic VPA levels, but VHE is a well-documented potential complication of the use of VPA in the medical literature, and it may occur in people with normal VPA levels.1 Because of the wide spectrum of symptoms associated with VHE, physicians should consider hyperammonemia in the differential diagnosis of any patient taking VPA who shows changes in behavior, cognition, or orientation

Abstract available thorugh the American Journal of Pharmaceutical Education.

The 2018-2019 Research and Graduate Affairs Committee (RGAC) was charged with critically evaluating the leadership development support necessary for pharmacy researchers, including postdoctoral trainees, to develop the skills needed to build and sustain successful research programs and analyzing how well those needs are being met by existing programs both within AACP and at other organizations. The RGAC identified a set of skills that could reasonably be expected to provide the necessary foundation to successfully lead a research team and mapped these skills to the six domains of graduate education in the pharmaceutical sciences established by the 2016-2017 RGAC (Table 1). In addition, the RGAC identified competency in team science and the bench-to-bedside-to-beyond translational spectrum as being critical elements of research leadership. The universality of these skills and their value prompted the RGAC to make two related recommendations to AACP