Evaluation of potency and metabolic stability of diphyllin-derived Vacuolar-ATPase inhibitors.

Diphyllin is a naturally occurring lignan comprised of an aryl naphthalene lactone scaffold that demonstrates beneficial biological activities in disease models of cancer, obesity, and viral infection. A target of diphyllin and naturally occurring derivatives is the vacuolar ATPase (V-ATPase) complex. Although diphyllin-related natural products are active with in vitro models for viral entry, the potencies and unknown pharmacokinetic properties limit well-designed in vivo evaluations. Previous studies demonstrated that diphyllin derivatives have the utility of blocking the Ebola virus cell entry pathway. However, diphyllin shows limited potency and poor oral bioavailability in mice. An avenue to improve the potency was used in a new library of synthetic derivatives of diphyllin. Diphyllin derivatives exploiting ether linkages at the 4-position with one-to-three carbon spacers to an oxygen or nitrogen atom provided compounds with EC50 values ranging from 7 to 600 nM potency and selectivity up to >500 against Ebola virus in infection assays. These relative potencies are reflected in the Ebola virus infection of primary macrophages, a cell type involved in early pathogenesis. A target engagement study reveals that reducing the ATPV0a2 protein expression enhanced the potency of diphyllin derivatives to block EBOV entry, consistent with effects on the endosomal V-ATPase function. Despite the substantial enhancement of antiviral potencies, limitations were identified, including rapid clearance predicted by in vitro microsome stability assays. However, compounds with similar or improved half-lives relative to diphyllin demonstrated improved pharmacokinetic profiles in vivo. Importantly, these derivatives displayed suitable plasma levels using oral administration, establishing the feasibility of in vivo antiviral testing.

Investigating the Replacement of Carboxylates with Carboxamides to Modulate the Safety and Efficacy of Platinum(II) Thioether Cyanide Scavengers.

Cyanide represents a persistent threat for accidental or malicious misuse due to easy conversion into a toxic gas and access to large quantities through several industries. The high safety index of hydroxocobalamin is a cornerstone quality as a cyanide scavenger. Unfortunately, intravenous infusion of hydroxocobalamin limits the utility in a mass casualty setting. We previously reported platinum(II) [Pt(II)] complexes with trans-directing sulfur ligands as an efficacious alternative to hydroxocobalamin when delivered by a bolus intramuscular injection in mice and rabbits. Thus, to enable Pt(II) as an alternative to hydroxocobalamin, a high safety factor is needed. The objective is to maintain efficacy and mitigate the risk for nephrotoxicity. Platinum amino acid complexes with the ability to form five- or six-membered rings and possessing either carboxylates or carboxamides are evaluated in vitro for cyanide scavenging. In vivo efficacy was evaulated in the zebrafish and mice cyanide exposure models. In addition, Pt(II) complex toxicity and pharmacokinetics were evaluated in a cyanide naive Sprague-Dawley model. Doses for toxicity are escalated to 5x from the efficacious dose in mice using a body surface area adjustment. The results show the carboxamide ligands display a time and pH dependence on cyanide scavenging in vitro and efficacy in vivo. Additionally, exchanging the carboxylate for carboxamide showed reduced indications of renal injury. A pharmacokinetic analysis of the larger bidentate complexes displayed rapid absorption by intramuscular administration and having similar plasma exposure. These findings point to the importance of pH and ligand structures for methionine carboxamide complexes with Pt(II).

Intramuscular administration of glyoxylate rescues swine from lethal cyanide poisoning and ameliorates the biochemical sequalae of cyanide intoxication.

Cyanide-a fast-acting poison-is easy to obtain given its widespread use in manufacturing industries. It is a high-threat chemical agent that poses a risk of occupational exposure in addition to being a terrorist agent. FDA-approved cyanide antidotes must be given intravenously, which is not practical in a mass casualty setting due to the time and skill required to obtain intravenous access. Glyoxylate is an endogenous metabolite that binds cyanide and reverses cyanide-induced redox imbalances independent of chelation. Efficacy and biochemical mechanistic studies in an FDA-approved preclinical animal model have not been reported. Therefore, in a swine model of cyanide poisoning, we evaluated the efficacy of intramuscular glyoxylate on clinical, metabolic, and biochemical endpoints. Animals were instrumented for continuous hemodynamic monitoring and infused with potassium cyanide. Following cyanide-induced apnea, saline control or glyoxylate was administered intramuscularly. Throughout the study, serial blood samples were collected for pharmacokinetic, metabolite, and biochemical studies, in addition, vital signs, hemodynamic parameters, and laboratory values were measured. Survival in glyoxylate-treated animals was 83% compared with 12% in saline-treated control animals (p < .01). Glyoxylate treatment improved physiological parameters including pulse oximetry, arterial oxygenation, respiration, and pH. In addition, levels of citric acid cycle metabolites returned to baseline levels by the end of the study. Moreover, glyoxylate exerted distinct effects on redox balance as compared with a cyanide-chelating countermeasure. In our preclinical swine model of lethal cyanide poisoning, intramuscular administration of the endogenous metabolite glyoxylate improved survival and clinical outcomes, and ameliorated the biochemical effects of cyanide.

Identification of Platinum(II) Sulfide Complexes Suitable as Intramuscular Cyanide Countermeasures.

The development of rapidly acting cyanide countermeasures using intramuscular injection (IM) represents an unmet medical need to mitigate toxicant exposures in mass casualty settings. Previous work established that cisplatin and other platinum(II) or platinum(IV)-based agents effectively mitigate cyanide toxicity in zebrafish. Cyanide's in vivo reaction with platinum-containing materials was proposed to reduce the risk of acute toxicities. However, cyanide antidote activity depended on a formulation of platinum-chloride salts with dimethyl sulfoxide (DMSO) followed by dilution in phosphate-buffered saline (PBS). A working hypothesis to explain the DMSO requirement is that the formation of platinum-sulfoxide complexes activates the cyanide scavenging properties of platinum. Preparations of isolated NaPtCl5-DMSO and Na (NH3)2PtCl-DMSO complexes in the absence of excess DMSO provided agents with enhanced reactivity toward cyanide in vitro and fully recapitulated in vivo cyanide rescue in zebrafish and mouse models. The enhancement of the cyanide scavenging effects of the DMSO ligand could be attributed to the activation of platinum(IV) and (II) with a sulfur ligand. Unfortunately, the efficacy of DMSO complexes was not robust when administered IM. Alternative Pt(II) materials containing sulfide and amine ligands in bidentate complexes show enhanced reactivity toward cyanide addition. The cyanide addition products yielded tetracyanoplatinate(II), translating to a stoichiometry of 1:4 Pt to each cyanide scavenger. These new agents demonstrate a robust and enhanced potency over the DMSO-containing complexes using IM administration in mouse and rabbit models of cyanide toxicity. Using the zebrafish model with these Pt(II) complexes, no acute cardiotoxicity was detected, and dose levels required to reach lethality exceeded 100 times the effective dose. Data are presented to support a general chemical design approach that can expand a new lead candidate series for developing next-generation cyanide countermeasures.

Multispecific targeting of glioblastoma with tumor microenvironment-responsive multifunctional engineered NK cells.

Tumor antigen heterogeneity, a severely immunosuppressive tumor microenvironment (TME) and lymphopenia resulting in inadequate immune intratumoral trafficking, have rendered glioblastoma (GBM) highly resistant to therapy. To address these obstacles, here we describe a unique, sophisticated combinatorial platform for GBM: a cooperative multifunctional immunotherapy based on genetically engineered human natural killer (NK) cells bearing multiple antitumor functions including local tumor responsiveness that addresses key drivers of GBM resistance to therapy: antigen escape, immunometabolic reprogramming of immune responses, and poor immune cell homing. We engineered dual-specific chimeric antigen receptor (CAR) NK cells to bear a third functional moiety that is activated in the GBM TME and addresses immunometabolic suppression of NK cell function: a tumor-specific, locally released antibody fragment which can inhibit the activity of CD73 independently of CAR signaling and decrease the local concentration of adenosine. The multifunctional human NK cells targeted patient-derived GBM xenografts, demonstrated local tumor site-specific activity in the tissue, and potently suppressed adenosine production. We also unveil a complex reorganization of the immunological profile of GBM induced by inhibiting autophagy. Pharmacologic impairment of the autophagic process not only sensitized GBM to antigenic targeting by NK cells but promoted a chemotactic profile favorable to NK infiltration. Taken together, our study demonstrates a promising NK cell-based combinatorial strategy that can target multiple clinically recognized mechanisms of GBM progression simultaneously.

Development of a Pediatric Mini-Tablet Formulation for Expedited Preclinical Studies.

Multiple considerations are essential to address the main challenges of dose flexibility and patient adherence in pediatric drug development, particularly for oncology. Mini-tablets, 2 mm in diameter, were manufactured using a rotary tablet press at a set weight and compression force level. The physical characteristics were consistent for mini-tablets throughout multiple batches. Polymeric amorphous solid dispersion (ASD) was used as a solubility enhancing technique to increase solubility and exposure of lapatinib. The effects of the polymeric excipient and disintegrant on drug release properties were investigated. While having a lower apparent solubility and shorter storage stability, hydroxypropyl methylcellulose E3 (HPMCE3) formulation provided a higher percentage of drug release compared to hydroxypropyl methylcellulose phthalate (HPMCP). The intermolecular interaction within the ASD system plays a role in the level of apparent solubility, physical stability, and concentration of free drug available in an aqueous environment. Juvenile porcine models at two different weight groups (10 and 20 kg) were used to obtain the pharmacokinetic parameters of lapatinib. While the dose-normalized exposure of drug was found to be lower in the pig study, the dose flexibility of mini-tablets enabled a constant dose level to be administered to achieve equivalent plasma concentration-time profiles between the two groups. This linear scaling in the amount of drug in pediatric and adult population has also been observed in human clinical studies.

The CUL3/neddylation inhibitor MLN4924 reduces ethanol-induced locomotor sensitization and inflammatory pain allodynia in mice.

Heterologous sensitization of adenylyl cyclase (AC) is defined by an enhanced cAMP response following persistent activation of Gαi/o-coupled receptors. This phenomenon was first observed in cellular models, and later reported in animal models of inflammatory pain or following chronic exposure to drugs of abuse including opioids and cocaine. Recently, we used genome-wide siRNA screening to identify Cullin3 signaling as a mediator of AC sensitization in cellular models. We also showed that pharmacological inhibition of Cullin3 with the neddylation inhibitor, MLN4924, abolished heterologous sensitization of several AC isoforms, including AC1, AC2, AC5, and AC6. Because ACs, especially AC1, have been implicated in alcohol-induced locomotor sensitization and inflammatory pain, we assessed the potential activity of MLN4924 in both murine models. We found that MLN4924 (30 mg/kg, i.p.) accumulated in the brain and reduced both locomotor sensitization induced by repeated alcohol administration and allodynia in an inflammatory pain model. Based on our previous findings that MLN4924 potently blocks AC sensitization in cellular models, we propose that the activity of MLN4924 in both animal models potentially occurs through blocking AC sensitization. Our findings provide the basis for understanding the molecular mechanism and yield a new pathway for drug development for pathological disorders associated with AC sensitization.

Considerations for Determining Direct Versus Indirect Functional Effects of Solubilizing Excipients on Drug Transporters for Enhancing Bioavailability.

Excipients used in drug formulations at clinically safe levels have been considered to be pharmacologically inert; however, numerous studies have suggested that many solubilizing agents may modulate drug transporter activities and intestinal absorption. Here, the reported interactions between various solubilizing excipients and drug transporters are evaluated to consider various potential underlying mechanisms. This forms the basis for debate in the field in regard to whether or not the effects are based on "direct" interactions or "indirect" consequences arising from the role of the excipients. For example, an increase in apparent drug solubility can give rise to saturation of transporters according to Michaelis-Menten kinetics. This is also drawing the attention of regulatory agencies as they seek to understand the role of formulation additives. The continued application of excipients as a tool in solubility enhancement is crucial in the drug development process, creating a need for additional data to verify the proposed mechanism behind these changes. A literature review is provided here with some guidance on other factors that should be considered to delineate the effects that arise from direct physiological interactions or indirect effects. The results of such studies may aid the rational design of bioavailability-enhancing formulations.

Pediatric Formulations: Knowledge Gaps Limiting the Expedited Preclinical to Clinical Translation in Children.

Traditionally, drug discovery and development research have been primarily focused on the mitigation of disease treatment for the general adult population, often overlooking the medical needs of pediatric patients. While remarkable progress toward the discovery of better medicines has been made, the pharmacological differences between children and adults are often neglected as part of the translation process. In fact, until recently, children have been considered therapeutic orphans due to the lack of significant drug discovery, formulation development, and dosage form design specifically tailored for pediatric patients. Perhaps the least understood is the significant physiological changes that occur during the maturation process from birth to adulthood. It requires careful considerations to achieve age-specific-desired therapeutic outcomes with minimal toxicity. This introduces considerable risk into the preclinical and clinical testing of new medicaments, which until recently, was avoided based on the conventional approach where a demonstration of safe and efficacious use in adults over several years potentially would minimize the chance of adverse juvenile responses. However, the lack of appropriate drug products for children has led to off-label use of adult medicines with potential life-threatening adverse reactions and health complications. Recent developments and future considerations regarding pediatric drug discovery and development using a patient-centric approach in the context of ontogenic biopharmaceutical considerations are discussed below.

Correction to: Effects of Dendrimer-Like Biopolymers on Physical Stability of Amorphous Solid Dispersions and Drug Permeability Across Caco-2 Cell Monolayers.

During the production process, an editorial error occurred where the label numbering for the first two tables were inadvertently switched. The original article has been corrected.

Effects of Dendrimer-Like Biopolymers on Physical Stability of Amorphous Solid Dispersions and Drug Permeability Across Caco-2 Cell Monolayers.

The potential applications of dendrimer-like biopolymers (DLB) as stabilizing excipients for amorphous solid dispersion (ASD) of niclosamide, celecoxib, and resveratrol were evaluated based on (1) the formation and physical stability of the ASD and (2) the permeability and flux of the agents across Caco-2 cell monolayers. The evaluation was made by comparing the performance of prototype phytoglycogen derivatives (DLB1, DLB2, and DLB3) with commonly used polymers such as HPMCAS, PVPVA, and Soluplus®. PXRD was used to confirm the formation of the dispersions and detect crystallinity peaks formed during 2- and 4-week storage at 40°C/75% RH. At concentrations below 2 g/mL, the viability of Caco-2 cells remained above 80% for all DLB samples compared to untreated cells in the MTT assay. Permeability studies revealed a repeating pattern in which an increase in the initial concentration (C0) was associated with a concomitant decrease in the apparent permeability (Papp) which we theorize is due to differences in drug-polymer interactions. Niclosamide-DLB1 dispersion had the lowest flux due to a significant reduction in Papp. The high increase in the C0 of celecoxib-DLB2, however, made up for the reduction in the Papp and produced the highest flux values compared to other polymers. Resveratrol-DLB3 had a 5× reduction in Papp, but C0 increased from 25.8 to 176 µg/mL led to a higher flux compared to the crystalline drug without polymer. Collectively, these results provide a "proof-of-concept" basis to demonstrate that DLB excipients have the ability to increase apparent solubility (Solapp), most likely due to drug-binding capacity.

Evaluation of 25% Poloxamer As a Slow Release Carrier for Morphine in a Rat Model.

The objectives of this study were to evaluate poloxamer as a slow release carrier for morphine (M) and potential tissue irritation after subcutaneous poloxamer-morphine (PM) injection in a rat model. Based on the result of a previous in vitro work, 25% poloxamer, with and without morphine, and saline were administered in 14 rats' flanks. Blood for morphine concentrations was automatically sampled at multiple preprogrammed time points using the Culex™ unit for 48 h. Skin tissues from the injection sites were harvested and evaluated for histopathological changes. Following M or PM administration, it was determined that the half-life (t1/2) was significantly longer in the PM (5.5 ± 7.2 h) than M (0.7 ± 0.8 h) indicated a slow dissolution of poloxamer with morphine. The tmax was within 15 min and Cmax was approximately three times higher with M than with PM, reaching 716.8 (±153.7 ng/ml) of plasma morphine concentrations. There was no significant difference in total area under the curve and clearance of M versus PM. Histology inflammatory scores were similar between M, PM, and poloxamer but were significantly higher than saline control. We concluded that 25% poloxamer was capable of increasing the t1/2 of morphine, without a significant tissue irritation.

Development of a direct contact astrocyte-human cerebral microvessel endothelial cells blood-brain barrier coculture model.

OBJECTIVES: In conventional in-vitro blood-brain barrier (BBB) models, primary and immortalized brain microvessel endothelial cell (BMEC) lines are often cultured in a monolayer or indirect coculture or triculture configurations with astrocytes or pericytes, for screening permeation of therapeutic or potentially neurotoxic compounds. In each of these cases, the physiological relevancy associated with the direct contact between the BMECs, pericytes and astrocytes that form the BBB and resulting synergistic interactions are lost. We look to overcome this limitation with a direct contact coculture model. METHODS: We established and optimized a direct interaction coculture system where primary human astrocytes are cultured on the apical surface of a Transwell® filter support and then human cerebral microvessel endothelial cells (hCMEC/D3) seeded directly on the astrocyte lawn. KEY FINDINGS: The studies suggest the direct coculture model may provide a more restrictive and physiologically relevant model through a significant reduction in paracellular transport of model compounds in comparison with monoculture and indirect coculture. In comparison with existing methods, the indirect coculture and monoculture models utilized may limit cell-cell signaling between human astrocytes and BMECs that are possible with direct configurations. CONCLUSIONS: Paracellular permeability reductions with the direct coculture system may enhance therapeutic agent and potential neurotoxicant screening for BBB permeability better than the currently available monoculture and indirect coculture in-vitro models.

Absorption of Transdermal Fluoxetine Compounded in a Lipoderm Base Compared to Oral Fluoxetine in Client-owned Cats.

The objective of this study was to compare serum concentrations of transdermal fluoxetine compounded in Lipoderm base versus commercially available oral fluoxetine tablets. Sixteen clinically healthy, client-owned cats that were at least one year of age were enrolled. Cats weighed between three and seven kilograms, had no comorbidities, and were behavior medication naïve. Cats were recruited from January 2016 through April 2016. Eight cats were assigned to each medication group based on owner preference. The cats received either oral (1 mg/kg) or transdermal (5 mg/kg; maximum 25 mg daily) fluoxetine compounded in a transdermal base (PCCA Lipoderm), administered daily for 60 days. Serum levels of fluoxetine and norfluoxetine were assessed as a surrogate for relative efficacy. Serum was collected and analyzed by high-performance liquid chromatography-mass spectrometry/mass spectrometry at baseline and days 5, 10, 30, 45, and 60 post-drug start. Adverse effects were monitored during physical exams, speaking with owners, and laboratory analysis of liver function tests at baseline and days 5, 30, and 60 post-drug start. Serum fluoxetine concentrations significantly differed between the treatment groups at days 45 and 60 post-drug start. Norfluoxetine concentrations significantly differed at days 30, 45, and 60 post-drug start. Blood concentrations of fluoxetine and norfluoxetine significantly differed between oral and transdermal routes after 30 days of treatment. Oral fluoxetine concentrations were consistently higher. Transdermal fluoxetine appeared to be well-tolerated, but a lack of knowledge regarding effective blood levels makes it unclear if a clinical effective response would be obtained at the blood concentrations achieved.

Physicochemical and Preclinical Evaluation of a Novel Buccal Measles Vaccine.

The aim of this study is to develop an orally disintegrating film (ODF) containing a microparticulate measles vaccine formulation for buccal delivery. The measles vaccine microparticles were made with biocompatible and biodegradable bovine serum albumin (BSA) and processed by spray drying. These vaccine microparticles were incorporated in the ODF, consisting of Lycoat RS720®, Neosorb P60W® and Tween 80. The yield of the microparticles was approximately 85-95%, w/w. The mean size of the vaccine microparticles was 3.65 ± 1.89 µm and had a slightly negative surface charge of 32.65 ± 2.4 mV. The vaccine particles were nontoxic to normal cells at high concentrations (500 µg/2.5 × 105 cells) of vaccine particles. There was a significant induction of innate immune response by vaccine microparticles which was observed in vitro when compared to blank microparticles (P < 0.05). The vaccine microparticles also significantly increased the antigen presentation and co-stimulatory molecules expression on antigen presenting cells, which is a prerequisite for Th1 and Th2 immune responses. When the ODF vaccine formulation was dosed in juvenile pigs, significantly higher antibody titers were observed after week 2, with a significant increase at week 4 and plateauing through week 6 comparative to naïve predose titers. The results suggest that the ODF measles vaccine formulation is a viable dosage form alternative to noninvasive immunization that may increase patient compliance and commercial distribution.

Cys34-PEGylated Human Serum Albumin for Drug Binding and Delivery.

Polyethylene glycol (PEG) derivatives were conjugated onto the Cys-34 residue of human serum albumin (HSA) to determine their effects on the solubilization, permeation, and cytotoxic activity of hydrophobic drugs such as paclitaxel (PTX). PEG(C34)HSA conjugates were prepared on a multigram scale by treating native HSA (n-HSA) with 5- or 20-kDa mPEG-maleimide, resulting in up to 77% conversion of the mono-PEGylated adduct. Nanoparticle tracking analysis of PEG(C34)HSA formulations in phosphate buffer revealed an increase in the number of nanosized aggregates relative to n-HSA, both in the absence and presence of PTX. Cell viability studies conducted with MCF-7 breast cancer cells indicated that PTX cytotoxicity was enhanced by PEG(C34)HSA when mixed at 10:1 mol ratios, up to a 2-fold increase in potency relative to n-HSA. The PEG(C34)HSA conjugates were also evaluated as PTX carriers across monolayers of HUVEC and hCMEC/D3 cells, and found to have permeation profiles nearly identical to those of n-HSA.

Pharmacogenomics of acetaminophen in pediatric populations: a moving target.

Acetaminophen (APAP) is widely used as an over-the-counter fever reducer and pain reliever. However, the current therapeutic use of APAP is not optimal. The inter-patient variability in both efficacy and toxicity limits the use of this drug. This is particularly an issue in pediatric populations, where tools for predicting drug efficacy and developmental toxicity are not well established. Variability in toxicity between age groups may be accounted for by differences in metabolism, transport, and the genetics behind those differences. While pharmacogenomics has been revolutionizing the paradigm of pharmacotherapy for many drugs, its application in pediatric populations faces significant challenges given the dynamic ontogenic changes in cellular and systems physiology. In this review we focused on the ontogenesis of the regulatory pathways involved in the disposition of APAP and on the variability between pediatric, adolescent, and adult patients. We also summarize important polymorphisms of the pharmacogenes associated with APAP metabolism. Pharmacogenetic studies in pediatric APAP treatment are also reviewed. We conclude that while a consensus in pharmacogenetic management of APAP in pediatric populations has not been achieved, a systems biology based strategy for comprehensively understanding the ontogenic regulatory pathway as well as the interaction between age and genetic variations are particularly necessary in order to address this question.

Persistent pharmacokinetic challenges to pediatric drug development.

The development of new therapeutic agents for the mitigation of pediatric disorders is largely hindered by the inability for investigators to assess pediatric pharmacokinetics (PK) in healthy patients due to substantial safety concerns. Pediatric patients are a clinical moving target for drug delivery due to changes in absorption, distribution, metabolism and excretion (ADME) and the potential for PK related toxicological (T) events to occur throughout development. These changes in ADMET can have profound effects on drug delivery, and may lead to toxic or sub-therapeutic outcomes. Ethical, economical, logistical, and technical barriers have resulted in insufficient investigation of these changes by industrial, regulatory, and academic bodies, leading to the classification of pediatric patients as therapeutic orphans. In response to these concerns, regulatory agencies have incentivized investigation into these ontogenic changes and their effects on drug delivery in pediatric populations. The intent of this review is to briefly present a synopsis of the development changes that occur in pediatric patients, discuss the effects of these changes on ADME and drug delivery strategies, highlight the hurdles that are still being faced, and present some opportunities to overcome these challenges.

Association between hTERT rs2736100 polymorphism and sensitivity to anti-cancer agents.

BACKGROUND: The rs2736100 single nucleotide polymorphism (SNP) is located in the intron 2 of human telomerase reverse transcriptase (hTERT) gene. Recent genome-wide association studies (GWAS) have consistently supported the strong association between this SNP and risk for multiple cancers. Given the important role of the hTERT gene and this SNP in cancer biology, we hypothesize that rs2736100 may also confer susceptibility to anti-cancer drug sensitivity. In this study we aim to investigate the correlation between the rs2736100 genotype and the responsiveness to anti-cancer agents in the NCI-60 cancer cell panel. METHODS AND MATERIALS: The hTERT rs2736100 was genotyped in the NCI-60 cancer cell lines. The relative telomere length (RTL) of each cell line was quantified using real-time PCR. The genotype was then correlated with publically available drug sensitivity data of two agents with telomerase-inhibition activity: Geldanamycin (HSP90 inhibitor) and RHPS4/BRACO19 (G-quadruplex stabilizer) as well as additional 110 commonly used agents with established mechanism of action. The association between rs2736100 and mutation status of TP53 gene was also tested. RESULTS: The C allele of the SNP was significantly correlated with increased sensitivity to RHPS4/BRACO19 with an additive effect (r = -0.35, p = 0.009) but not with Geldanamycin. The same allele was also significantly associated with sensitivity to antimitotic agents compared to other agents (p = 0.003). The highest correlation was observed between the SNP and paclitaxel (r = -0.36, p = 0.005). The telomere length was neither associated with rs2736100 nor with sensitivity to anti-cancer agents. The C allele of rs2736100 was significantly associated with increased mutation rate in TP53 gene (p = 0.004). CONCLUSION: Our data suggested that the cancer risk allele of hTERT rs2736100 polymorphism may also affect the cancer cell response to both TERT inhibitor and anti-mitotic agents, which might be attributed to the elevated telomerase-independent activity of hTERT, as well as the increased risk for TP53 gene mutagenesis conferred by the polymorphism. Detailed mechanisms need to be further investigated.

Assessment of juvenile pigs to serve as human pediatric surrogates for preclinical formulation pharmacokinetic testing.

Pediatric drug development is hampered by biological, clinical, and formulation challenges associated with age-based populations. A primary cause for this lack of development is the inability to accurately predict ontogenic changes that affect pharmacokinetics (PK) in children using traditional preclinical animal models. In response to this issue, our laboratory has conducted a proof-of-concept study to investigate the potential utility of juvenile pigs to serve as surrogates for children during preclinical PK testing of selected rifampin dosage forms. Pigs were surgically modified with jugular vein catheters that were externalized in the dorsal scapular region and connected to an automated blood sampling system (PigTurn-Culex-L). Commercially available rifampin capsules were administered to both 20 and 40 kg pigs to determine relevant PK parameters. Orally disintegrating tablet formulations of rifampin were also developed and administered to 20 kg pigs. Plasma samples were prepared from whole blood by centrifugation and analyzed for rifampin content by liquid chromatography-tandem mass spectrometry. Porcine PK parameters were determined from the resultant plasma-concentration time profiles and contrasted with published rifampin PK data in human adults and children. Results indicated significant similarities in dose-normalized absorption and elimination parameters between pigs and humans. Moreover, ontogenic changes observed in porcine PK parameters were consistent with ontogenic changes reported for human PK. These results demonstrate the potential utility of the juvenile porcine model for predicting human pediatric PK for rifampin. Furthermore, utilization of juvenile pigs during formulation testing may provide an alternative approach to expedite reformulation efforts during pediatric drug development.