Experimental design, development and evaluation of extended release subcutaneous thermo-responsive in situ gels for small molecules in drug discovery.

The objective of this work is to develop extended release subcutaneous thermo-responsive in situ gel-forming delivery systems using the following commercially available triblock polymers: poly (lactic-co-glycolic acid)-poly (ethylene glycol)-poly (lactic-co-glycolic acid) (PLGA-PEG-PLGA, copolymer A & B) and poly (lactide-co-caprolactone)-poly (ethylene glycol)-poly (lactide-co-caprolactone) (PLCL-PEG-PLCL, copolymer C). Performance of two optimized formulations containing ketoprofen as a model compound, was assessed by comparing in vitro drug release profiles with in vivo performance following subcutaneous administration in rats. This work employs a Design of Experiment (DoE) approach to explore first, the relationship between copolymer composition, concentration, and gelation temperature (GT), and second, to identify the optimal copolymer composition and drug loading in the thermo-responsive formulation. Furthermore, this work discusses the disconnect observed between in vitro drug release and in vivo pharmacokinetic (PK) profiles. In vitro, both formulations showed extended-release profiles for 5-9 days, while PK parameters and plasma profiles were similar in vivo without extended release observed. In conclusion, a clear disconnection is observed between in vitro ketoprofen drug release and in vivo performance from the two thermogel formulations tested. This finding highlights a remaining challenge for thermogel formulation development, that is, being able to accurately predict in vivo behavior from in vitro results.

Generation of High-Quality Pharmacokinetic Data From Parallel Tail Vein Dosing And Bleeding in Non-cannulated Rats.

It is common practice to use cannulated rats for pharmacokinetic (PK) in-life studies as it yields high quality PK parameter estimation. While offering many benefits, cannulation requires surgery, post-surgical care, and cannula maintenance. As an alternative approach, the strategy of dosing and bleeding rats via the tail vein in a single experiment is technically feasible and theoretically offers many benefits. Unfortunately, however, as reported by F Tse et al. in 1984 (J Pharm Sci 73: https://doi.org/10.1002/jps.2600731128), parallel tail dosing and bleeding is scientifically flawed and yields inaccurate estimation of PK parameters following intravenous administration. The underlying causality of poor data quality has not been addressed in over 35 years. To overcome the technical flaws associated with parallel tail dosing and bleeding, we have developed a Tail-Dose-Bleed (TDB) method as a substitute for use of cannulated rats. Specifically, the method introduces a flush procedure after dosing, uses separate tail veins for dosing and bleeding, and adjusts dosing and sampling to the proximal and distal portions of the tail, respectively. To demonstrate the proof of principle for this TDB technique, several cassette dosing studies were conducted. The performance of the TDB technique is compared in both stand alone and animal crossover studies employing conventional jugular/femoral bleeding and dosing. The poor data via tail dosing and bleeding previously described by Tse et al. are also recapitulated using their described approach. To ensure broad applicability of the TDB technique, data were generated utilizing compounds of diverse physical chemical properties manifesting a range of clearance and/or volume of distribution characteristics. These data demonstrate that the TDB approach yields comparable PK profiles and parameters as compared to conventional femoral dosing / jugular bleeding. Using this newly described TDB procedure, we demonstrate the ability to overcome documented data quality issues when dosing and bleeding via the tail. The TDB technique has numerous operational advantages of reduced study turnaround time and improved cost effectiveness, but most importantly, addresses key animal welfare concerns relevant to institutional animal care and use committees (IACUC). The notable advantage here is reduced animal stress and discomfort by eliminating the need for surgery and recovery. And by consequence, allows for animals to be group housed and re-used without concern for loss of cannula patency. The tail dose and bleed method is simple and appears readily transferable to other laboratories.

Fragment-Based Design of a Potent MAT2a Inhibitor and in Vivo Evaluation in an MTAP Null Xenograft Model.

MAT2a is a methionine adenosyltransferase that synthesizes the essential metabolite S-adenosylmethionine (SAM) from methionine and ATP. Tumors bearing the co-deletion of p16 and MTAP genes have been shown to be sensitive to MAT2a inhibition, making it an attractive target for treatment of MTAP-deleted cancers. A fragment-based lead generation campaign identified weak but efficient hits binding in a known allosteric site. By use of structure-guided design and systematic SAR exploration, the hits were elaborated through a merging and growing strategy into an arylquinazolinone series of potent MAT2a inhibitors. The selected in vivo tool compound 28 reduced SAM-dependent methylation events in cells and inhibited proliferation of MTAP-null cells in vitro. In vivo studies showed that 28 was able to induce antitumor response in an MTAP knockout HCT116 xenograft model.

A Beginning Guide for Dental Photography: A Simplified Introduction for Esthetic Dentistry.

Photography is one of the most important skills dentists need to master in order to perform esthetic dentistry at a high level. Today, digital single-lens reflex cameras are commonplace. Young dentists have grown up with Internet, smartphones, and online platforms exposing them, and their patients, to cases that other dentists have shared, increasing the awareness and popularity of esthetic-focused treatment. This article provides readers with a simplified and attainable approach to begin the dental photography journey, as well as increase skill level, depending on practice style and desired investment.

Branding Dynamics for the Esthetic Dentist: Building Your Brand to Build Your Practice.

The objective of this article is to introduce the concept of branding to dentists interested in implementing elective esthetic treatment into their practice. For many, this will serve as an introduction to begin; for others, it can provide a road map for revising and reinforcing a branding program already in place.

Characterization of Meta-Iodobenzylguanidine (mIBG) Transport by Polyspecific Organic Cation Transporters: Implication for mIBG Therapy.

Radiolabeled meta-iodobenzylguanidine (mIBG) is an important radiopharmaceutical used in the diagnosis and treatment of neuroendocrine cancers. mIBG is known to enter tumor cells through the norepinephrine transporter. Whole-body scintigraphy has shown rapid mIBG elimination through the kidney and high accumulation in several normal tissues, but the underlying molecular mechanisms are unclear. Using transporter-expressing cell lines, we show that mIBG is an excellent substrate for human organic cation transporters 1-3 (hOCT1-3) and the multidrug and toxin extrusion proteins 1 and 2-K (hMATE1/2-K), but not for the renal organic anion transporter 1 and 3 (hOAT1/3). Kinetic analysis revealed that hOCT1, hOCT2, hOCT3, hMATE1, and hMATE2-K transport mIBG with similar apparent affinities (K m of 19.5 ± 6.9, 17.2 ± 2.8, 14.5 ± 7.1, 17.7 ± 10.9, 12.6 ± 5.6 µM, respectively). Transwell studies in hOCT2/hMATE1 double-transfected Madin-Darby canine kidney cells showed that mIBG transport in the basal (B)-to-apical (A) direction is much greater than in the A-to-B direction. Compared with control cells, the B-to-A permeability of mIBG increased by 20-fold in hOCT2/hMATE1 double-transfected cells. Screening of 23 drugs used in the treatment of neuroblastoma identified several drugs with the potential to inhibit hOCT- or hMATE-mediated mIBG uptake. Interestingly, irinotecan selectively inhibited hOCT1, whereas crizotinib potently inhibited hOCT3-mediated mIBG uptake. Our results suggest that mIBG undergoes renal tubular secretion mediated by hOCT2 and hMATE1/2-K, and hOCT1 and hOCT3 may play important roles in mIBG uptake into normal tissues. SIGNIFICANCE STATEMENT: mIBG is eliminated by the kidney and extensively accumulates in several tissues known to express hOCT1 and hOCT3. Our results suggest that hOCT2 and human multidrug and toxin extrusion proteins 1 and 2-K are involved in mIBG renal elimination, whereas hOCT1 and hOCT3 may play important roles in mIBG uptake into normal tissues. These findings may help to predict and prevent adverse drug interaction with therapeutic [131I]mIBG and develop clinical strategies to reduce [131I]mIBG accumulation and toxicity in normal tissues and organs.

A candidate drug administered subcutaneously to rodents as drug particles showing hepatic recirculation which influenced the sustained release process.

The aim of the present study was to evaluate and interpret the pharmacokinetic profiles after subcutaneous (s.c.) administration of crystalline AZ'72 nano- and microsuspensions to rodents. Both formulations were injected at 1.5 and 150 mg/kg to rats. For the lower dose, the profiles were similar after s.c. injection but extended as compared to oral administration. The overall exposure was higher for nanoparticles compared with microparticles during the investigated period. For the higher dose, injection of both suspensions resulted in maintained plateaus caused by the drug depots but, unexpectedly, at similar exposure levels. After addition of a further stabilizer, pluronic F127, nanosuspensions showed improved exposure with dose and higher exposure compared to larger particles in mice. Obviously, a stabilizer mixture that suits one delivery route is not necessarily optimal for another one. The differences in peak concentration (Cmax) between nano- and microparticles were mainly ascribed to differences in dissolution rate. Plasma profiles in mice showed curves with secondary absorption peaks after intravenous and oral administration, suggesting hepatic recirculation following both administration routes. This process, together with the depot formulation, complicates the analysis of absorption from s.c. administration, i.e. multiple processes were driving the plasma profile of AZ'72.

Renal secretion of hydrochlorothiazide involves organic anion transporter 1/3, organic cation transporter 2, and multidrug and toxin extrusion protein 2-K.

Hydrochlorothiazide (HCTZ) is the most widely used thiazide diuretic for the treatment of hypertension either alone or in combination with other antihypertensives. HCTZ is mainly cleared by the kidney via tubular secretion, but the underlying molecular mechanisms are unclear. Using cells stably expressing major renal organic anion and cation transporters [human organic anion transporter 1 (hOAT1), human organic anion transporter 3 (hOAT3), human organic cation transporter 2 (hOCT2), human multidrug and toxin extrusion 1 (hMATE1), and human multidrug and toxin extrusion 2-K (hMATE2-K)], we found that HCTZ interacted with both organic cation and anion transporters. Uptake experiments further showed that HCTZ is transported by hOAT1, hOAT3, hOCT2, and hMATE2-K but not by hMATE1. Detailed kinetic analysis coupled with quantification of membrane transporter proteins by targeted proteomics revealed that HCTZ is an excellent substrate for hOAT1 and hOAT3. The apparent affinities (Km) for hOAT1 and hOAT3 were 112 ± 8 and 134 ± 13 µM, respectively, and the calculated turnover numbers (kcat) were 2.48 and 0.79 s-1, respectively. On the other hand, hOCT2 and hMATE2-K showed much lower affinity for HCTZ. The calculated transport efficiency (kcat/Km) at the single transporter level followed the rank order of hOAT1> hOAT3 > hOCT2 and hMATE2-K, suggesting a major role of organic anion transporters in tubular secretion of HCTZ. In vitro inhibition experiments further suggested that HCTZ is not a clinically relevant inhibitor for hOAT1 or hOAT3. However, strong in vivo inhibitors of hOAT1/3 may alter renal secretion of HCTZ. Together, our study elucidated the molecular mechanisms underlying renal handling of HCTZ and revealed potential pathways involved in the disposition and drug-drug interactions for this important antihypertensive drug in the kidney.

Organic Cation Transporter 3 Facilitates Fetal Exposure to Metformin during Pregnancy.

Metformin, an oral antihyperglycemic, is increasingly being prescribed to pregnant women with gestational diabetes. Metformin is a hydrophilic cation and relies on organic cation transporters to move across cell membranes. We previously demonstrated that human and mouse placentas predominantly express organic cation transporter 3 (OCT3), but the impact of this transporter on maternal and fetal disposition of metformin is unknown. Using immunofluorescence colocalization studies in term human placenta, we showed that OCT3 is localized to the basal (fetal-facing) membrane of syncytiotrophoblast cells with no expression on the apical (maternal-facing) membrane. OCT3 positive staining was also observed in fetal capillaries. To determine the in vivo role of OCT3 in maternal and fetal disposition of metformin, we determined metformin maternal pharmacokinetics and overall fetal exposure in wild-type and Oct3-null pregnant mice. After oral dosing of [14C]metformin at gestational day 19, the systemic drug exposure (AUC0-∞) in maternal plasma was slightly reduced by ∼16% in the Oct3-/- pregnant mice. In contrast, overall fetal AUC0-∞ was reduced by 47% in the Oct3-/- pregnant mice. Consistent with our previous findings in nonpregnant mice, metformin tissue distribution was respectively reduced by 70% and 52% in the salivary glands and heart in Oct3-/- pregnant mice. Our in vivo data in mice clearly demonstrated a significant role of Oct3 in facilitating metformin fetal distribution and exposure during pregnancy. Modulation of placental OCT3 expression or activity by gestational age, genetic polymorphism, or pharmacological inhibitors may alter fetal exposure to metformin or other drugs transported by OCT3.

Disposition of Methamphetamine and Major Metabolites in Mice: Role of Organic Cation Transporter 3 in Tissue-Selective Accumulation of Para-Hydroxymethamphetamine.

Methamphetamine is one of the most widely abused illicit drugs. Although human intoxication and multiple tissue toxicities frequently occur in abusers, little is known about the distribution of methamphetamine or its primary metabolites, amphetamine and para-hydroxymethamphetamine (p-OHMA), to their sites of toxicity. This study determined the pharmacokinetics, tissue exposure, and partition ratios of methamphetamine and major metabolites in various mouse tissues and investigated the impact of organic cation transporter 3 (Oct3) following i.v. injection of methamphetamine to male Oct3+/+ and Oct3-/- mice. Methamphetamine, amphetamine, and p-OHMA were readily detectable in plasma with Oct3+/+ and Oct3-/- mice displaying similar plasma pharmacokinetic profiles for all three analytes. In addition to kidney and liver, salivary glands highly accumulated methamphetamine, amphetamine, and p-OHMA with total exposure 3.3- to 9.4-fold higher than plasma area under the concentration-time curve (AUC). Consistent with being an Oct3 substrate, p-OHMA AUC in salivary glands is reduced by 50% in Oct3-/- mice. p-OHMA AUC in skeletal muscle is also significantly reduced in Oct3-/- mice. Our data identified salivary glands as a novel site of high accumulation of methamphetamine and metabolites, which may underlie methamphetamine toxicity in this tissue. Furthermore, our study identified Oct3 as an important determinant of tissue uptake and exposure to p-OHMA in salivary glands and skeletal muscle. Our findings suggest that local tissue accumulation of methamphetamine and/or its metabolites may play a role in several of the reported peripheral toxicities of methamphetamine, and Oct3 can significantly impact tissue exposure to its substrates without affecting systemic elimination.

Interaction and Transport of Methamphetamine and its Primary Metabolites by Organic Cation and Multidrug and Toxin Extrusion Transporters.

Methamphetamine is one of the most abused illicit drugs with roughly 1.2 million users in the United States alone. A large portion of methamphetamine and its metabolites is eliminated by the kidney with renal clearance larger than glomerular filtration clearance. Yet the mechanism of active renal secretion is poorly understood. The goals of this study were to characterize the interaction of methamphetamine and its major metabolites with organic cation transporters (OCTs) and multidrug and toxin extrusion (MATE) transporters and to identify the major transporters involved in the disposition of methamphetamine and its major metabolites, amphetamine and para-hydroxymethamphetamine (p-OHMA). We used cell lines stably expressing relevant transporters to show that methamphetamine and its metabolites inhibit human OCTs 1-3 (hOCT1-3) and hMATE1/2-K with the greatest potencies against hOCT1 and hOCT2. Methamphetamine and amphetamine are substrates of hOCT2, hMATE1, and hMATE2-K, but not hOCT1 and hOCT3. p-OHMA is transported by hOCT1-3 and hMATE1, but not hMATE2-K. In contrast, organic anion transporters 1 and 3 do not interact with or transport these compounds. Methamphetamine and its metabolites exhibited complex interactions with hOCT1 and hOCT2, suggesting the existence of multiple binding sites. Our studies suggest the involvement of the renal OCT2/MATE pathway in tubular secretion of methamphetamine and its major metabolites and the potential of drug-drug interactions with substrates or inhibitors of the OCTs. This information may be considered when prescribing medications to suspected or known abusers of methamphetamine to mitigate the risk of increased toxicity or reduced therapeutic efficacy.

Potent inhibition of human organic cation transporter 2 (hOCT2) by ß-carboline alkaloids.

1. Beta-carbolines are indole alkaloids with a wide range of pharmacological and toxicological activities. Beta-carbolines are structurally related to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), a known substrate of organic cation transporters (OCTs). The goal of this study is to determine the interaction of ß-carbolines with human OCT1, 2, and 3 (SLC22A1-3). 2. Dose-dependent inhibition studies were performed for five commercially available ß-carbolines using a fluorescent substrate assay in HEK293 cells stably expressing hOCT1-3. The substrate potential was evaluated by uptake assays and the impact of active transport on cellular toxicity examined. 3. All tested ß-carbolines potently inhibited hOCT2 with IC50 values in the sub- or low micromolar range. Harmaline is the most potent hOCT2 inhibitor (IC50 = 0.50 ± 0.08 µM). hOCT1 and hOCT3 are less sensitive to ß-carboline inhibition. Harmaline, norharmanium, and 2,9-dimethyl-4,9-dihydro-3H-ß-carbolinium accumulated 2- to 7-fold higher in cells expressing hOCT1-3. HEK293 cells expressing hOCT1-3 were 6.5- to 13-fold more sensitive to harmane and norharmanium toxicity. 4. Our data support a significant role of hOCT1-3 in tissue uptake and disposition of ß-carbolines. Importantly, the potent inhibition of hOCT2 by ß-carbolines also raises the concern of potential drug interactions between naturally occurring bioactive alkaloids and drugs eliminated by hOCT2.

From the USDA: Educating the Next Generation: Funding Opportunities in Food, Agricultural, Natural Resources, and Social Sciences Education.

The National Institute of Food and Agriculture within the U.S. Department of Agriculture provides leadership, capacity, and funds to support the continuing development of a safe and competitive agricultural system. Many of the agency's educational programs are led by the Division of Community and Education (DOCE). These programs span agricultural education, enhancing agricultural literacy through both formal and nonformal education. Here, we have highlighted funding opportunities within DOCE that enhance agricultural education and literacy by supporting the improvement of students' critical communication, leadership skills, and experiential learning opportunities. Some of these programs include opportunities for which students can apply, while others focus on faculty applications. Opportunities faculty can apply for may support student-recruitment and student-retention techniques, curriculum development, innovative teaching methods, and institutional capacity-building programs. Overall, these programs foster a diverse workforce in agricultural science that matches the increasing diversity of the country.

Polyspecific organic cation transporters and their impact on drug intracellular levels and pharmacodynamics.

Most drugs are intended to act on molecular targets residing within a specific tissue or cell type. Therefore, the drug concentration within the target tissue or cells is most relevant to its pharmacological effect. Increasing evidences suggest that drug transporters not only play a significant role in governing systemic drug levels, but are also an important gate keeper for intra-tissue and intracellular drug concentrations. This review focuses on polyspecific organic cation transporters, which include the organic cation transporters 1-3 (OCT1-3), the multidrug and toxin extrusion proteins 1-2 (MATE1-2) and the plasma membrane monoamine transporter (PMAT). Following an overview of the tissue distribution, transport mechanisms, and functional characteristics of these transporters, we highlight the studies demonstrating the ability of locally expressed OCTs to impact intracellular drug concentrations and directly influence their pharmacological and toxicological activities. Specifically, OCT1-mediated metformin access to its site of action in the liver is impacted by genetic polymorphisms and chemical inhibition of OCT1. The impact of renal OCT2 and MATE1/2-K in cisplatin intrarenal accumulation and nephrotoxicity is reviewed. New data demonstrating the role of OCT3 in salivary drug accumulation and secretion is discussed. Whenever possible, the pharmacodynamic response and toxicological effects is presented and discussed in light of intra-tissue and intracellular drug exposure. Current challenges, knowledge gaps, and future research directions are discussed. Understanding the impact of transporters on intra-tissue and intracellular drug concentrations has important implications for rational-based optimization of drug efficacy and safety.

Lysophosphatidic acid modulates the healing responses of human periodontal ligament fibroblasts and enhances the actions of platelet-derived growth factor.

BACKGROUND: Platelet-derived growth factor (PDGF) has been used to promote healing in many in vitro and in vivo models of periodontal regeneration. PDGF interacts extensively with lysophosphatidic acid (LPA). We recently showed that LPA modulates the responses of human gingival fibroblasts to PDGF. The objectives of this study were as follows: 1) to evaluate the basic interactions of LPA with primary human periodontal ligament fibroblasts (PDLFs) alone and with PDGF-BB for promoting PDLF growth and migration; 2) to determine the effects in an in vitro oral wound-healing model; and 3) to identify the LPA receptors (LPARs) expressed by PDLF. METHODS: PDLF regenerative responses were measured using 1 and 10 microM LPA in the absence or presence of 1 or 10 ng/ml PDGF. Cell proliferation was determined by 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry and by cell counting. Migration responses were measured using a microchemotaxis chamber. PDLFs were grown to confluence on glass slides, a 3-mm-wide wound was mechanically inflicted, and wound fill on days 4, 6, and 9 was reported. PDLF LPAR expression was determined using Western blotting. RESULTS: PDLFs exhibited proliferative and chemotactic responses to LPA; these responses were enhanced when LPA and PDGF were present together. LPA plus PDGF elicited complete wound fill. PDLFs express the LPARs LPA(1), LPA(2), and LPA(3). CONCLUSIONS: To our knowledge, this study provides the first evidence that LPA stimulates human PDLF wound healing responses and interacts positively with PDGF to regulate these actions. These results suggest that LPA and its receptors play important modulatory roles in PDLF regenerative biology.