Effects of pharmacological lesion of the nucleus retroambiguus region on the pharyngeal phase of swallowing.

Pharyngeal swallowing is controlled by synaptic interactions within a swallowing central pattern generator (sw-CPG) that is composed of a dorsal and a ventral swallowing group (VSG). Here, we used electrical stimulation (10 s) of the superior laryngeal nerve (SLN; 20 Hz; pulse width: 100 µs) to explore the role of the VSG in an arterially-perfused brainstem preparation of rats. To investigate the effects of pharmacological lesion (local microinjection of an GABA(A)-R agonist) of the nucleus retroambiguus (NRA), a designated component of the VSG, we recorded phrenic (PNA) and vagal nerve (VNA) activities. Control SLN stimulation with stepwise increasing stimulus intensities (from 20 µA to 160 µA) elicited robust suppression of PNA and evoked sequential swallowing activity in the VNA. Lesioning of the NRA had no effect on the pattern of pharyngeal swallowing, but significantly increased the sensory gating of SLN inputs. We conclude that the NRA is not part of the VSG, but appears to have important roles for the central gating of swallowing.

Endogenous Probes for Drug Transporters: Balancing Vision With Reality.

Various endogenous probes have been identified for a number of hepatic and renal drug transporters and available clinical data indicate that they could be leveraged in phase I trials to facilitate subject phenotyping and drug-drug interaction (DDI) assessment. Despite the progress, however, it is recognized that the menu of probes needs expanding, that existing probes need further characterization and validation, and that compound files need to be built in support of probe absorption-metabolism-distribution-excretion-DDI modeling exercises.

Optimization of adeno-associated virus vector-mediated gene transfer to the respiratory tract.

An efficient adeno-associated virus (AAV) vector was constructed for the treatment of respiratory diseases. AAV serotypes, promoters and routes of administration potentially influencing the efficiency of gene transfer to airway cells were examined in the present study. Among the nine AAV serotypes (AAV1-9) screened in vitro and four serotypes (AAV1, 2, 6, 9) evaluated in vivo, AAV6 showed the strongest transgene expression. As for promoters, the cytomegalovirus (CMV) early enhancer/chicken ß-actin (CAG) promoter resulted in more robust transduction than the CMV promoter. Regarding delivery routes, intratracheal administration resulted in strong transgene expression in the lung, whereas the intravenous and intranasal administration routes yielded negligible expression. The combination of the AAV6 capsid and CAG promoter resulted in sustained expression, and the intratracheally administered AAV6-CAG vector transduced bronchial cells and pericytes in the lung. These results suggest that AAV6-CAG vectors are more promising than the previously preferred AAV2 vectors for airway transduction, particularly when administered into the trachea. The present study offers an optimized strategy for AAV-mediated gene therapy for lung diseases, such as cystic fibrosis and pulmonary fibrosis.

Relationships between transepidermal water loss, cutaneous microcirculatory function and autonomic nervous activity.

OBJECTIVE: Several studies have shown that a deterioration of skin properties, an impaired cutaneous microcirculatory function and an imbalance of autonomic nervous activity are observed in smokers and in patients with diabetes mellitus or Raynaud's phenomenon. These observations suggest that skin properties are associated with cutaneous microcirculatory function and autonomic nervous activity in pathological conditions. However, there is no published evidence to support the concept that these two functions have any relationship with skin properties even in healthy subjects. To investigate the hypothesis that these properties are related, we conducted a survey of healthy adult subjects to investigate the relationships between cutaneous microcirculatory function and autonomic nervous activity and skin properties. METHODS: The hydration of the stratum corneum and transepidermal water loss (TEWL) were investigated as skin properties, and the responsiveness of skin blood flow (SkBF) to local warming was examined as an index of cutaneous microcirculatory function in 19 healthy adult male subjects. Electrocardiograms were monitored for 24 h and heart rate variability was analysed considering low-frequency power (LF: 0.04-0.15 Hz), high-frequency power (HF: 0.15-0.40 Hz) and a ratio of low- to high-frequency power (LF/HF) as indices of autonomic nervous activity; HF is an index of parasympathetic activity, whereas LF/HF is an index of sympathovagal balance. The relationships between those indices were then analysed. RESULTS: A moderate negative correlation was found between TEWL and the relative maximum rate of increases in the responsiveness of SkBF on local warming. A moderate positive and a moderate negative correlation were observed between TEWL and LF/HF or HF, respectively. Moreover, a moderate negative and a moderate positive correlation were shown between the responsiveness of SkBF and LF/HF or HF, respectively. The hydration of the stratum corneum showed no correlations with any indices of microcirculation or autonomic nervous activity. CONCLUSION: These results indicate that skin barrier function, cutaneous microcirculatory function and autonomic nervous activity are mutually associated in healthy adults.

Intrinsic and extrinsic regulatory mechanisms are required to form and maintain a lens of the correct size and shape.

Understanding how tissues and organs acquire and maintain an appropriate size and shape remains one of the most challenging areas in developmental biology. The eye lens represents an excellent system to provide insights into regulatory mechanisms because in addition to its relative simplicity in cellular composition (being made up of only two forms of cells, epithelial and fiber cells), these cells must become organized to generate the precise spheroidal arrangement that delivers normal lens function. Epithelial and fiber cells also represent spatially distinct proliferation and differentiation compartments, respectively, and an ongoing balance between these domains must be tightly regulated so that the lens achieves and maintains appropriate dimensions during growth and ageing. Recent research indicates that reciprocal inductive interactions mediated by Wnt-Frizzled and Notch-Jagged signaling pathways are important for maintaining and organizing these compartments. The Hippo-Yap pathway has also been implicated in maintaining the epithelial progenitor compartment and regulating growth processes. Thus, whilst some molecules and mechanisms have been identified, further work in this important area is needed to provide a clearer understanding of how lens size and shape is regulated.

Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

Quantitative Analyses of Hepatic OATP-Mediated Interactions Between Statins and Inhibitors Using PBPK Modeling With a Parameter Optimization Method.

This study aimed to construct a widely applicable method for quantitative analyses of drug-drug interactions (DDIs) caused by the inhibition of hepatic organic anion transporting polypeptides (OATPs) using physiologically based pharmacokinetic (PBPK) modeling. Models were constructed for pitavastatin, fluvastatin, and pravastatin as substrates and cyclosporin A (CsA) and rifampicin (RIF) as inhibitors, where enterohepatic circulations (EHC) of statins were incorporated. By fitting to clinical data, parameters that described absorption, hepatic elimination, and EHC processes were optimized, and the extent of these DDIs was explained satisfactorily. Similar in vivo inhibition constant (Ki ) values of each inhibitor against OATPs were obtained, regardless of the substrates. Estimated Ki values of CsA were comparable to reported in vitro values with the preincubation of CsA, while those of RIF were smaller than reported in vitro values (coincubation). In conclusion, this study proposes a method to optimize in vivo PBPK parameters in hepatic uptake transporter-mediated DDIs.

Microdosing and Other Phase 0 Clinical Trials: Facilitating Translation in Drug Development.

A number of drivers and developments suggest that microdosing and other phase 0 applications will experience increased utilization in the near-to-medium future. Increasing costs of drug development and ethical concerns about the risks of exposing humans and animals to novel chemical entities are important drivers in favor of these approaches, and can be expected only to increase in their relevance. An increasing body of research supports the validity of extrapolation from the limited drug exposure of phase 0 approaches to the full, therapeutic exposure, with modeling and simulations capable of extrapolating even non-linear scenarios. An increasing number of applications and design options demonstrate the versatility and flexibility these approaches offer to drug developers including the study of PK, bioavailability, DDI, and mechanistic PD effects. PET microdosing allows study of target localization, PK and receptor binding and occupancy, while Intra-Target Microdosing (ITM) allows study of local therapeutic-level acute PD coupled with systemic microdose-level exposure. Applications in vulnerable populations and extreme environments are attractive due to the unique risks of pharmacotherapy and increasing unmet healthcare needs. All phase 0 approaches depend on the validity of extrapolation from the limited-exposure scenario to the full exposure of therapeutic intent, but in the final analysis the potential for controlled human data to reduce uncertainty about drug properties is bound to be a valuable addition to the drug development process.

Thorough debridement and immediate primary wound closure for animal bite injuries of the upper limbs.

PURPOSE: Animal bite injuries are often encountered in daily practice. In particular, these injuries of the upper limbs can result in severe functional impairment. We have performed early debridement of contaminated tissue and primary closure for these injuries. METHODS: The subjects consisted of 15 patients (6 males and 9 females) aged 1-91 years (mean 53.6 years) who visited our hospital due to animal bite injuries (dog in 9 patients, cat in 6). The bite site was the forearm in 5 patients and the hand in 10. In the operating room, contaminated tissue was removed, and primary wound closure was performed after irrigation. RESULTS: The bite penetrated to the muscle layer in 6 patients, tendon sheath in 5, joint in 1, bone in 1, and involved only the subcutaneous tissue in 3 patients. The mean period until the completion of wound treatment was 19.8 ± 8.4 days. As complications, numbness of finger, metaphalangeal joint contracture and superficial radial nerve injury were observed in each one case. In a patient with bite injury of the palmar and dorsal sides of the thumb reaching the bone, additional debridement was necessary. At the final observation, the visual analog scale was 1.2 ± 1.4, and the Quick Disabilities of the Arm, Shoulder, and Hand score was 9.7 ± 12.2. CONCLUSIONS: Debridement to achieve wound closure is indispensable in patients with animal bite injuries of the upper limbs. The results of our study suggest that thorough debridement allows primary closure, even for animal bite injuries.

Organ Impairment-Drug-Drug Interaction Database: A Tool for Evaluating the Impact of Renal or Hepatic Impairment and Pharmacologic Inhibition on the Systemic Exposure of Drugs.

The organ impairment and drug-drug interaction (OI-DDI) database is the first rigorously assembled database of pharmacokinetic drug exposure data from publicly available renal and hepatic impairment studies presented together with the maximum change in drug exposure from drug interaction inhibition studies. The database was used to conduct a systematic comparison of the effect of renal/hepatic impairment and pharmacologic inhibition on drug exposure. Additional applications are feasible with the public availability of this database.

Kinetic Interpretation of the Importance of OATP1B3 and MRP2 in Docetaxel-Induced Hematopoietic Toxicity.

Neutropenia is a lethal dose-limiting toxicity of docetaxel. Our previous report indicated that the prevalence of severe docetaxel-induced neutropenia is significantly associated with genetic polymorphisms in solute carrier organic anion transporter 1B3 (SLCO1B3) (encoding organic anion-transporting polypeptide 1B3 (OATP1B3)) and ATP-binding cassette subfamily C2 (ABCC2) (encoding multidrug-resistant-associated protein 2 (MRP2)). Therefore, we investigated their significance in docetaxel-induced neutropenia. In vitro experiments suggested their possible involvement in the hepatic uptake of docetaxel and its efflux from bone marrow cells. To further characterize a quantitative impact of OATP1B3 and MRP2 on neutropenia, we used an in silico simulation of the neutrophil count in docetaxel-treated subjects with functional changes in OATP1B3 and MRP2 in a pharmacokinetic/pharmacodynamic model. The clinically reported odds ratios for docetaxel-induced neutropenia risk were explained by the decreased function of OATP1B3 and MRP2 to 41 and 32%, respectively. These results suggest that reduced activities of OATP1B3 and MRP2 associated with systemic exposure and local accumulation in bone marrow cells, respectively, account for the docetaxel-induced neutropenia observed clinically.

Paradoxical downward seizure pattern on amplitude-integrated electroencephalogram.

The use of amplitude-integrated electroencephalography (aEEG) to assess brain function and detect seizures has been increasing worldwide. Results from previous studies have demonstrated that seizure patterns can be recognized as transient rises on aEEG traces. We report here a case of an infant with neonatal seizures that showed paradoxical transient drops on aEEG traces. The ictal EEG showed initial low-amplitude fast rhythmic activity followed by epileptic recruiting rhythms and high-voltage slow waves. Therefore, downward patterns on aEEG traces should be recognized as suspected seizure patterns.

A new objective histological scale for studying human photoaged skin.

BACKGROUND: A quantitative understanding of the histological alteration of the skin is important for assessing the severity of photoaging. METHODS: We performed Elastica-van Gieson staining and immunohistochemistry for decorin on 34 facial skin sections. We evaluated the alteration of collagen fibers and decorin (a modulator for collagen fibrillogenesis), according to the 5 grades of morphological change in elastic fibers that was established by Kligman (1969). The objectivity of a stage (Stages I-VI), which was established in this study, was evaluated using weighted kappa statistical analysis based on the degree of agreement in stage determination by 11 observers using a blind procedure. Correlation between the crow's-feet-area wrinkles grades of another 26 women and stages was also analyzed. RESULTS: The initial alteration of elastic fibers was observed in the deep dermis. Decorin was not detected in very severely altered skin. Based on the combination of changes in the elastic fibers, collagenic fibers, and decorin, skin tissues were categorized into 6 stages according to severity. The statistical analysis showed almost perfect agreement between observers. Significant positive correlation between stages and wrinkle scores was found. CONCLUSIONS: We propose a new objective histological scale that is useful for assessing the severity of photoaging.

Interactions between lens epithelial and fiber cells reveal an intrinsic self-assembly mechanism.

How tissues and organs develop and maintain their characteristic three-dimensional cellular architecture is often a poorly understood part of their developmental program; yet, as is clearly the case for the eye lens, precise regulation of these features can be critical for function. During lens morphogenesis cells become organized into a polarized, spheroidal structure with a monolayer of epithelial cells overlying the apical tips of elongated fiber cells. Epithelial cells proliferate and progeny that shift below the lens equator differentiate into new fibers that are progressively added to the fiber mass. It is now known that FGF induces epithelial to fiber differentiation; however, it is not fully understood how these two forms of cells assemble into their characteristic polarized arrangement. Here we show that in FGF-treated epithelial explants, elongating fibers become polarized/oriented towards islands of epithelial cells and mimic their polarized arrangement in vivo. Epithelial explants secrete Wnt5 into the culture medium and we show that Wnt5 can promote directed behavior of lens cells. We also show that these explants replicate aspects of the Notch/Jagged signaling activity that has been shown to regulate proliferation of epithelial cells in vivo. Thus, our in vitro study identifies a novel mechanism, intrinsic to the two forms of lens cells, that facilitates self-assembly into the polarized arrangement characteristic of the lens in vivo. In this way the lens, with its relatively simple cellular composition, serves as a useful model to highlight the importance of such intrinsic self-assembly mechanisms in tissue developmental and regenerative processes.

International Transporter Consortium commentary on clinically important transporter polymorphisms.

This Commentary focuses on genetic polymorphisms in membrane transporters. We present two polymorphisms for which there is a compelling body of literature supporting their clinical relevance: OATP1B1 (c.521T>C, p.V174A, rs4149056) and BCRP (c.421C>A, p.Q141K, rs2231142). The clinical evidence demonstrating their role in variation in pharmacokinetics and pharmacodynamics is described along with their allele frequencies in ethnic populations. Recommendations for incorporating studies of transporter polymorphisms in drug development are provided, along with the regulatory implications.

Intracellular drug concentrations and transporters: measurement, modeling, and implications for the liver.

Intracellular concentrations of drugs and metabolites are often important determinants of efficacy, toxicity, and drug interactions. Hepatic drug distribution can be affected by many factors, including physicochemical properties, uptake/efflux transporters, protein binding, organelle sequestration, and metabolism. This white paper highlights determinants of hepatocyte drug/metabolite concentrations and provides an update on model systems, methods, and modeling/simulation approaches used to quantitatively assess hepatocellular concentrations of molecules. The critical scientific gaps and future research directions in this field are discussed.

Ethnic variability in the plasma exposures of OATP1B1 substrates such as HMG-CoA reductase inhibitors: a kinetic consideration of its mechanism.

Because the plasma exposure levels of rosuvastatin in Asians are generally twice those in Caucasians, the starting dose for Asians in the United States is set to half of that for non-Asians. However, the precise role of ethnicity in the clearance of rosuvastatin has not yet been clarified. This review focuses on ethnic variability in the clinical pharmacokinetics of 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase inhibitors (statins) and angiotensin II receptor antagonists. The mechanisms of such variability are discussed quantitatively, with building a hypothetical model for pravastatin, and validated against other statins. Our analyses suggest that the ethnic variability in the plasma exposure of statins cannot be explained only by the difference in the allele frequencies of organic anion-transporting polypeptide (OATP)1B1 and breast cancer resistance protein (BCRP), and the intrinsic ethnic variability in the activity of OATP1B1 (the ratio of Japanese/Caucasians is 0.584) must be considered. Further work and validation with additional data will clarify the applicability of this model to other OATP1B1 substrates.

Activation of TRPV4 strengthens the tight-junction barrier in human epidermal keratinocytes.

The transient receptor potential cation channel, subfamily V (TRPV), is expressed in the epidermis and considered to be a sensor of extrinsic stimuli such as temperature and other physical or chemical factors. In this study, we examined whether or not the activation of TRPVs by their agonists alters the epidermal tight junction (TJ) function in cultured human epidermal keratinocytes. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that mRNA for TRPV1, 3 and 4 were expressed in differentiated keratinocytes in which TJs had formed. Stimulation of the keratinocytes with a TRPV4 agonist (4α-phorbol 12, 13-didecanoate, 4α-PDD) strengthened the TJ-associated barrier, analyzed by means of transepithelial electric resistance measurements and flux measurements of the paracellular tracer. Stimulation with TRPV1 and TRPV3 agonists did not have the same result. Simultaneously, the 4α-PDD-stimulated keratinocytes showed an upregulation of TJ structural proteins, occludin and claudin-4, and TJ regulatory factors, phospho-atypical PKCζ/ι. It was also observed that the amounts of occludin and phospho-atypical PKCζ/ι complex were higher in 4α-PDD stimulated keratinocytes. In conclusion, we demonstrated that the activation of TRPV4 strengthened the TJ-associated barrier of epidermal cells. It was also suggested that the upregulation of TJ structural proteins and/or the posttranslational modification of TJ structural proteins by phospho-atypical PKCζ/ι are responsible for the enhancement of TJ function. Our study supports the hypothesis that TJs change their function in response to a change in the external environment sensed through TRPVs.

N-methylnicotinamide is an endogenous probe for evaluation of drug-drug interactions involving multidrug and toxin extrusions (MATE1 and MATE2-K).

Multidrug and toxin extrusion 1 (MATE1) and MATE2-K are H(+)/organic cation exchangers mediating the efflux of cationic drugs into the urine. N-methylnicotinamide (NMN) was found to be an endogenous substrate of MATE1 (Michaelis constant (K(m)) 301 ± 18 µmol/l) and MATE2-K (K(m) 422 ± 63 µmol/l) as well as a basolateral influx transporter, organic cation transporter 2 (K(m) 318 ± 29 µmol/l). A potent MATE inhibitor, pyrimethamine, competitively inhibited the uptake by MATE1 and MATE2-K with inhibition constant (K(i)) values of 83 ± 15 and 56 ± 11 nmol/l, respectively. The uptake of NMN by human kidney brush border membrane vesicles with a H(+) gradient was saturable (K(m) 360 ± 55 µmol/l) and completely inhibited by pyrimethamine. The renal clearance of endogenous NMN was 403 ± 61 in healthy male subjects, and it was significantly decreased to 119 ± 16 ml/min/kg by an oral dose of pyrimethamine (50 mg). These results support the utility of NMN as an endogenous in vivo probe for investigating MATE1 and MATE2-K in humans.

Analysis of regulatory review times of new drugs in Japan: association with characteristics of new drug applications, regulatory agency, and pharmaceutical companies.

WHAT IS KNOWN AND OBJECTIVE: Various factors have been reported to be associated with the duration of regulatory review of new drug applications (NDAs). We investigated potential links between the review times in Japan and the attributes of NDAs, the regulatory agency and pharmaceutical companies. METHODS: We analysed new drugs approved in 2000-2009 in Japan using a proprietary database collected through annual surveys to pharmaceutical companies. Regression models in which individual firms were treated as either a fixed effect or a random effect were applied to examine factors associated with the overall review time and the duration of each step of the review. RESULTS AND DISCUSSION: The fixed effect model analysis using variations within each firm indicated that new molecular entities that were submitted to the Pharmaceuticals and Medical Devices Agency (PMDA), priority reviews and pre-NDA consultations were associated with a shorter overall review time, whereas additional studies during the review resulted in a longer review. In the random effect model analysis using both within- and between-firm variations, use of end-of-phase 2 consultations and foreign clinical data also had negative coefficients, suggesting the effect of these two vary among firms. Analysis of each step of the review process revealed NDAs reviewed by the Committee on Drugs under the Ministry of Health, Labour and Welfare, and the number of NDAs assigned to a review team were significantly linked with their duration, whereas consultation services and the number of reviewers had no relation. WHAT IS NEW AND CONCLUSION: Factors associated with each step of the review process as well as the differences in attributes and strategies among pharmaceutical companies should be considered to further improve the speed, quality and efficiency of the regulatory review.