Ontogeny of Human Liver Aldehyde Oxidase: Developmental Changes and Implications for Drug Metabolism.

Despite the increasing importance of aldehyde oxidase (AO) in the drug metabolism of clinical candidates, ontogeny data for AO are limited. The objective of our study was to characterize the age-dependent AO content and activity in the human liver cytosolic fraction (HLC) and human hepatocytes (HH). HLC (n = 121 donors) and HH (n = 50 donors) were analyzed for (1) AO protein content by quantitative proteomics and (2) enzyme activity using carbazeran as a probe substrate. AO activity showed high technical variability and poor correlation with the content in HLC samples, whereas hepatocyte samples showed a strong correlation between the content and activity. Similarly, AO content and activity showed no significant age-dependent differences in HLC samples, whereas the average AO content and activity in hepatocytes increased significantly (∼20-40-fold) from the neonatal levels (0-28 days). Based on the hepatocyte data, the age at which 50% of the adult AO content is reached (age50) was 3.15 years (0.32-13.97 years, 95% CI). Metabolite profiling of carbazeran revealed age-dependent metabolic switching and the role of non-AO mechanisms (glucuronidation and desmethylation) in carbazeran elimination. The content-activity correlation in hepatocytes improved significantly (R2 = 0.95; p < 0.0001) in samples showing <10% contribution of glucuronidation toward the overall metabolism, confirming that AO-mediated oxidation and glucuronidation are the key routes of carbazeran metabolism. Considering the confounding effect of glucuronidation on AO activity, AO content-based ontogeny data are a more direct reflection of developmental changes in protein expression. The comprehensive ontogeny data of AO in HH samples are more reliable than HLC data, which are important for developing robust physiologically based pharmacokinetic models for predicting AO-mediated metabolism in children.

Effect of probenecid on blood levels and renal elimination of furosemide and endogenous compounds in rats: Discovery of putative organic anion transporter biomarkers.

Transporter-mediated drug-drug interactions (DDIs) are assessed using probe drugs and in vitro and in vivo models during drug development. The utility of endogenous metabolites as transporter biomarkers is emerging for prediction of DDIs during early phases of clinical trials. Endogenous metabolites such as pyridoxic acid and kynurenic acid have shown potential to predict DDIs mediated by organic anion transporters (OAT1 and OAT3). However, these metabolites have not been assessed in rats as potential transporter biomarkers. We carried out a rat pharmacokinetic DDI study using probenecid and furosemide as OAT inhibitor and substrate, respectively. Probenecid administration led to a 3.8-fold increase in the blood concentrations and a 3-fold decrease in renal clearance of furosemide. High inter-individual and intra-day variability in pyridoxic acid and kynurenic acid, and no or moderate effect of probenecid administration on these metabolites suggest their limited utility for prediction of Oat-mediated DDI in rats. Therefore, rat blood and urine samples were further analysed using untargeted metabolomics. Twenty-one m/z features (out of >8000 detected features) were identified as putative biomarkers of rat Oat1 and Oat3 using a robust biomarker qualification approach. These m/z features belong to metabolic pathways such as fatty acid analogues, peptides, prostaglandin analogues, bile acid derivatives, flavonoids, phytoconstituents, and steroids, and can be used as a panel to decrease variability caused by processes other than Oats. When validated, these putative biomarkers will be useful in predicting DDIs caused by Oats in rats.

Dissecting Parameters Contributing to the Underprediction of Aldehyde Oxidase-Mediated Metabolic Clearance of Drugs.

We investigated the effect of variability and instability in aldehyde oxidase (AO) content and activity on the scaling of in vitro metabolism data. AO content and activity in human liver cytosol (HLC) and five recombinant human AO preparations (rAO) were determined using targeted proteomics and carbazeran oxidation assay, respectively. AO content was highly variable as indicated by the relative expression factor (REF; i.e., HLC to rAO content) ranging from 0.001 to 1.7 across different in vitro systems. The activity of AO in HLC degrades at a 10-fold higher rate in the presence of the substrate as compared with the activity performed after preincubation without substrate. To scale the metabolic activity from rAO to HLC, a protein-normalized activity factor (pnAF) was proposed wherein the activity was corrected by AO content, which revealed up to sixfold higher AO activity in HLC versus rAO systems. A similar value of pnAF was observed for another substrate, ripasudil. Physiologically based pharmacokinetic (PBPK) modeling revealed a significant additional clearance (CL; 66%), which allowed for the successful prediction of in vivo CL of four other substrates, i.e., O-benzyl guanine, BIBX1382, zaleplon, and zoniporide. For carbazeran, the metabolite identification study showed that the direct glucuronidation may be contributing to around 12% elimination. Taken together, this study identified differential protein content, instability of in vitro activity, role of additional AO clearance, and unaccounted metabolic pathways as plausible reasons for the underprediction of AO-mediated drug metabolism. Consideration of these factors and integration of REF and pnAF in PBPK models will allow better prediction of AO metabolism. SIGNIFICANCE STATEMENT: This study elucidated the plausible reasons for the underprediction of aldehyde oxidase (AO)-mediated drug metabolism and provided recommendations to address them. It demonstrated that integrating protein content and activity differences and accounting for the loss of AO activity, as well as consideration of extrahepatic clearance and additional pathways, would improve the in vitro to in vivo extrapolation of AO-mediated drug metabolism using physiologically based pharmacokinetic modeling.

Continuous Large Area Monolayered Molybdenum Disulfide Growth Using Atmospheric Pressure Chemical Vapor Deposition.

The growth of large crystallite continuous monolayer materials like molybdenum disulfide (MoS2) with the desired morphology via chemical vapor deposition (CVD) remains a challenge. In CVD, the complex interplay of various factors like growth temperatures, precursors, and nature of the substrate decides the crystallinity, crystallite size, and coverage area of the grown MoS2 monolayer. In the present work, we report about the role of weight fraction of molybdenum trioxide (MoO3), sulfur, and carrier gas flow rate toward nucleation and monolayer growth. The concentration of MoO3 weight fraction has been found to govern the self-seeding process and decides the density of nucleation sites affecting the morphology and coverage area. A carrier gas flow of 100 sccm argon results in large crystallite continuous films with a lower coverage area (70%), while a flow rate of 150 sccm results in 92% coverage area with a reduced crystallite size. Through a systematic variation of experimental parameters, we have established the recipe for the growth of large crystallite atomically thin MoS2 suitable for optoelectronic devices.

Quantitative Characterization of Clinically Relevant Drug-Metabolizing Enzymes and Transporters in Rat Liver and Intestinal Segments for Applications in PBPK Modeling.

Rats are extensively used as a preclinical model for assessing drug pharmacokinetics (PK) and tissue distribution; however, successful translation of the rat data requires information on the differences in drug metabolism and transport mechanisms between rats and humans. To partly fill this knowledge gap, we quantified clinically relevant drug-metabolizing enzymes and transporters (DMETs) in the liver and different intestinal segments of Sprague-Dawley rats. The levels of DMET proteins in rats were quantified using the global proteomics-based total protein approach (TPA) and targeted proteomics. The abundance of the major DMET proteins was largely comparable using quantitative global and targeted proteomics. However, global proteomics-based TPA was able to detect and quantify a comprehensive list of 66 DMET proteins in the liver and 37 DMET proteins in the intestinal segments of SD rats without the need for peptide standards. Cytochrome P450 (Cyp) and UDP-glycosyltransferase (Ugt) enzymes were mainly detected in the liver with the abundance ranging from 8 to 6502 and 74 to 2558 pmol/g tissue. P-gp abundance was higher in the intestine (124.1 pmol/g) as compared to that in the liver (26.6 pmol/g) using the targeted analysis. Breast cancer resistance protein (Bcrp) was most abundant in the intestinal segments, whereas organic anion transporting polypeptides (Oatp) 1a1, 1a4, 1b2, and 2a1 and multidrug resistance proteins (Mrp) 2 and 6 were predominantly detected in the liver. To demonstrate the utility of these data, we modeled digoxin PK by integrating protein abundance of P-gp and Cyp3a2 into a physiologically based PK (PBPK) model constructed using PK-Sim software. The model was able to reliably predict the systemic as well as tissue concentrations of digoxin in rats. These findings suggest that proteomics-informed PBPK models in preclinical species can allow mechanistic PK predictions in animal models including tissue drug concentrations.

Dimethandrolone, a Potential Male Contraceptive Pill, is Primarily Metabolized by the Highly Polymorphic UDP-Glucuronosyltransferase 2B17 Enzyme in Human Intestine and Liver.

Dimethandrolone undecanoate (DMAU), an oral investigational male hormonal contraceptive, is a prodrug that is rapidly converted to its active metabolite, dimethandrolone (DMA). Poor and variable oral bioavailability of DMA after DMAU dosing is a critical challenge to develop it as an oral drug. The objective of our study was to elucidate the mechanisms of variable pharmacokinetics of DMA. We first identified DMA metabolites formed in vitro and in vivo in human hepatocyte incubation and serum samples following oral DMAU administration in men, respectively. The metabolite identification study revealed two metabolites, DMA-glucuronide (DMA-G; major) and the androstenedione analog of DMA (minor), in the hepatocyte incubations. After oral DMAU administration, only DMA-G was detected in serum, which was >100-fold compared with DMA levels, supporting glucuronidation as the major elimination mechanism for DMA. Next, 13 clinically relevant UDP-glucuronosyltransferase (UGT) enzymes were tested for their involvement in DMA-G formation, which revealed a major role of UDP-glucuronosyltransferase 2B17 (UGT2B17) isoform with a smaller contribution of UGT1A9 in DMA-G formation. These data were confirmed by dramatically higher DMA glucuronidation rates (>200- and sevenfold) in the high versus the null UGT2B17-expressing human intestinal and liver microsomes, respectively. Since human UGT2B17 is a highly variable enzyme with a 20%-80% gene deletion frequency, the in vitro data suggest a major role of UGT2B17 polymorphism on the first-pass metabolism of DMA. Further, considering DMA is a selective and sensitive UGT2B17 substrate, it could be used as a clinical probe of UGT2B17 activity. SIGNIFICANCE STATEMENT: Dimethandrolone (DMA) is an active metabolite of dimethandrolone undecanoate (DMAU), an investigational male hormonal contraceptive. Previous studies have indicated poor and inconsistent bioavailability of DMAU following oral administration. This study found that UDP-glucuronosyltransferase 2B17-mediated high intestinal first-pass metabolism is the key mechanism of variable DMA bioavailability.

A Tribute to Professor Saranjit Singh - A Critical Thinker, Innovator, Mentor, and Educator.

This commentary presents contributions and accomplishments of Professor Saranjit Singh, National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, India, to pharmaceutical research and education. Prof. Singh completed his successful tenure in October 2021. Over his 40+ years of illustrious academic career, he trained 147 Masters and 15 PhD students in the fields of drug stability testing, degradation chemistry, impurity and metabolite characterization, and advanced analytical technologies. He has published ∼250 research articles, reviews, editorials, patent, book, and book chapters, and received numerous awards, including the Professor M.L. Khorana Memorial Lecture Award from the Indian Pharmaceutical Association (IPA) and the Outstanding Analyst and Eminent Analyst awards from the Indian Drug Manufacturers' Association (IDMA). This commentary highlights Prof. Singh's inspiring personal and renowned professional journey, including early life, education, career, accomplishments, as well as his services to academia, industry, and regulatory. By sharing the contributions and accomplishments of Prof. Singh, we strongly believe that his story will inspire the next generation of scientists to continue his legacy to advance the field.

Role of limited hydrogen and flow interval on the growth of single crystal to continuous graphene by low-pressure chemical vapor deposition.

A method for defect-free large crystallite graphene growth remains unknown despite much research effort. In this work, we discuss the role of flow duration of H2 gas for the production of graphene as per requirement and production at a minimum flow rate considering the safety issue of hydrogen utilization. The copper substrate used for growth was treated for different time intervals (0 to 35 min) in H2 flow prior to growth. Structural and chemical changes occurring in the copper substrate surface were probed by grazing incidence x-ray diffraction and x-ray photoelectron spectroscopy. The results were correlated with the Raman spectroscopy data, which can quantify the quality of graphene. With increasing H2 flow interval, secondary nucleation sites were observed and growth favored few-layer graphene structures. The surface-adsorbed oxygen molecules and its conversion to an OH terminated surface with increasing hydrogen flow interval was found to be a key factor in enhancing nucleation density. The Stranski-Krastanov type of nucleation was observed for samples grown with different time intervals of H2 treatment, except 5 min of H2 flow prior to growth for which the Volmer-Weber type of growth favored monolayer graphene crystallite growth.

Selective electric and magnetic sensitivity of aperture probes.

We report the effect of geometrical factors governing the polarization profiles of near-field scanning optical microscope (NSOM) probes. The most important physical parameter controlling the selective electric or magnetic field sensitivity is found to be the width of the metal rim surrounding aperture. Probes with metal rim width w < λ/2 selectively senses the optical electric field, while those with w > λ/2 selectively senses the optical magnetic field. Intensity variation of optical Hertz standing wave formed upon reflection at oblique incidence shows a phase difference of π/2 between electric and magnetic probes: an analogue of the classical Wiener's experiment. Our work paves way towards electromagnetic engineering of nanostructures.

Optical field enhancement of nanometer-sized gaps at near-infrared frequencies.

We report near-field and far-field measurements of transmission through nanometer-sized gaps at near-infrared frequencies with varying the gap size from 1 nm to 10 nm. In the far-field measurements, we excluded direct transmission on the metal film surface via interferometric method. Kirchhoff integral formalism was used to relate the far-field intensity to the electric field at the nanogaps. In near-field measurements, field enhancement factors of the nanogaps were quantified by measuring transmission of the nanogaps using near-field scanning optical microscopy. All the measurements produce similar field enhancements of about ten, which we put in the context of comparing with the giant field enhancements in the terahertz regime.

Maxillary palatal ramp prosthesis: A prosthodontic solution to manage mandibular deviation following surgery.

Mandibular resection following surgical treatment for neoplastic lesions of the oral cavity leads to numerous complications including altered mandibular movements, disfigurement, difficult in swallowing, impaired speech and articulation, and deviation of the mandible towards the resected site. Various prosthetic methods are employed to reduce or minimize mandibular deviation and improve and restore the lost functions and esthetic, like maxillomandibular fixation, implant supported prosthesis, removable mandibular guide flange prosthesis, and palatal based guidance restoration. This clinical report describes the rehabilitation of a patient following segmental mandibulectomy using palatal ramp prosthesis.

Ultrasensitive and fast detection of denaturation of milk by coherent backscattering of light.

In this work, Coherence backscattering (CBS) of light has been used to detect the onset of denaturation of milk. The CBS cone shape and its enhancement factor are found to be highly sensitive to the physical state of the milk particles. The onset of denaturing of milk not visible to the naked eye, can be easily detected from changes in the CBS cone shape. The onset of denaturation is confirmed by spectral changes in Raman spectra from these milk samples. Further, the possibility to estimate the dilution of milk by water as an adulterant is demonstrated. The method reported has a broad scope in industry for making an inline ultrafast cost effective sensor for milk quality monitoring during production and before consumption.

High-yield chemical synthesis of hexagonal ZnO nanoparticles and nanorods with excellent optical properties.

Large yield and low temperature growth of nanostructures are key requirements for fulfilling the demand of large scale applications of nanomaterials. Here, we report a highly efficient chemical method to synthesize high quality hexagonal ZnO nanoparticle and nanorods utilizing the low temperature oxidation of metallic zinc powder in the presence of an appropriate catalyst. This one-step method has advantages such as low temperature (90 degrees C) and atmospheric pressure synthesis and a high yield (> 90%). Microstructure and optical properties of the as-synthesized ZnO nanoparticles are found to be identical or better than those of the commercial ZnO nanopower (Sigma-Aldrich). In particular, in comparison to the commercial nanopowder the as-grown ZnO nanorods and nanoparticles exhibit stronger UV absorption at 376 nm and intense UV photoluminescence emission at -382 nm, with negligible defect emission band. This method is suitable for large-scale production of nanosized ZnO and could be extended for the synthesis of other metal oxides.

Enhancing the photostability of poly(3-hexylthiophene) by preparing composites with multiwalled carbon nanotubes.

Poly(3-hexylthiophene) (P3HT) degrades in organic solvents containing dissolved molecular oxygen when irradiated with ultraviolet light. Hence, it is important to develop strategies that can enhance the photostability of P3HT and enhance the device performance. In this work, we report that preparing composites of P3HT with appropriate amounts of multiwalled carbon nanotube (MWCNT) results in superior photostability of P3HT. UV-visible and fluorescence spectroscopy have been used as primary tools to study the photostability of P3HT and its composites. Scanning electron microscopy (SEM) images of the composites display dispersed CNTs being well coated by P3HT. Transmission electron microscopy (TEM) micrographs along with SAED patterns reveal that P3HT coats the CNTs, which is the reason for superior dispersion of the composite. ESR spectroscopy was also performed to pursue and follow the degradation of P3HT. Ten weight percent of MWCNTs in P3HT was found to be the optimum loading amount that results in maximum photostability of the P3HT as compared to the other ratios. This enhanced photostability of P3HT on preparing composites with MWCNT in addition to its easy processability directly from solution makes these composites immensely important for optoelectronic applications.

Strain anisotropy in freestanding germanium nanoparticles synthesized by ball milling.

Although embedded Ge nanocrystals (NCs) have been grown by variety of techniques and its properties have been studied extensively, intrinsic properties of isolated Ge NCs have not been studied properly due to lack of proper synthesis technique. Here we report on the synthesis of freestanding Ge nanoparticles (NPs) down to approximately 7 nm using ball milling technique and study its structural evolution as a function of milling time. Morphology and microstructure of the freestanding Ge NPs are studied using atomic force microscopy, transmission electron microscopy and X-ray diffraction (XRD) analysis. A systematic study of the XRD line profile using Williamson-Hall method reveals presence of anisotropic strain in the milled NPs. Strain anisotropy factor is calculated using a modified Williamson-Hall method by taking into consideration a dislocation contrast factor, assuming that dislocations are main contributors to the strain in these NPs. Our calculations suggest that screw type dislocations are main contributors to the strain anisotropy in the Ge NPs. We find that for milling time up to 40 hrs, NPs size monotonically goes down to approximately 7.3 nm and then almost saturates, while the dislocation density first increases from 1.64 x 10(16) m(-2) to 11.62 x 10(17) m(-2) for milling time up to 20 hrs and then decreases drastically during further milling. We have monitored a low temperature heat release at approximately 310 degrees C from the milled NPs using differential scanning calorimetry, clearly indicating a kind of structural relaxation of the strained NPs.

Optical signature of structural defects in single walled and multiwalled carbon nanotubes.

Though defects are invariably present in as-grown and purified carbon nanotubes (NTs), spectroscopic properties of defects in NTs have not been established yet. In this work, single walled (SW) and multiwalled (MW) carbon nanotubes (NTs) grown by chemical vapor deposition have been studied by high resolution transmission electron microscopy (HRTEM), Raman scattering and photoluminescence (PL), electron spin resonance (ESR) and thermo gravometric (TGA) analysis. Raman spectra of both SWNT and MWNT show additional features in the frequency range intermediate between 600-1300 cm(-1) and 1700-2600 cm(-1), in addition to well-known radial breathing modes, D- and G-bands. Room temperature PL studies show two broad but distinct peaks centered at approximately 2.05 eV and approximately 2.33 eV, for both SWNT and MWNT samples. TGA analysis shows very low impurity content in MWNT sample as compared to the SWNT sample. HRTEM analysis reveals various kinds of structural defects in nanotube wall. With the help of HRTEM and ESR studies, we argue that the intermediate frequency Raman modes and the visible PL from the pristine NTs are definite signatures of structural defects in the nanotubes.