Physiologically-Based Pharmacokinetic-Led Guidance for Patients With Cystic Fibrosis Taking Elexacaftor-Tezacaftor-Ivacaftor With Nirmatrelvir-Ritonavir for the Treatment of COVID-19.

Cystic fibrosis transmembrane conductance regulator (CFTR) modulating therapies, including elexacaftor-tezacaftor-ivacaftor, are primarily eliminated through cytochrome P450 (CYP) 3A-mediated metabolism. This creates a therapeutic challenge to the treatment of coronavirus disease 2019 (COVID-19) with nirmatrelvir-ritonavir in people with cystic fibrosis (CF) due to the potential for significant drug-drug interactions (DDIs). However, the population with CF is more at risk of serious illness following COVID-19 infection and hence it is important to manage the DDI risk and provide treatment options. CYP3A-mediated DDI of elexacaftor-tezacaftor-ivacaftor was evaluated using a physiologically-based pharmacokinetic modeling approach. Modeling was performed incorporating physiological information and drug-dependent parameters of elexacaftor-tezacaftor-ivacaftor to predict the effect of ritonavir (the CYP3A inhibiting component of the combination) on the pharmacokinetics of elexacaftor-tezacaftor-ivacaftor. The elexacaftor-tezacaftor-ivacaftor models were verified using independent clinical pharmacokinetic and DDI data of elexacaftor-tezacaftor-ivacaftor with a range of CYP3A modulators. When ritonavir was administered on Days 1 through 5, the predicted area under the curve (AUC) ratio of ivacaftor (the most sensitive CYP3A substrate) on Day 6 was 9.31, indicating that its metabolism was strongly inhibited. Based on the predicted DDI, the dose of elexacaftor-tezacaftor-ivacaftor should be reduced when coadministered with nirmatrelvir-ritonavir to elexacaftor 200 mg-tezacaftor 100 mg-ivacaftor 150 mg on Days 1 and 5, with delayed resumption of full-dose elexacaftor-tezacaftor-ivacaftor on Day 9, considering the residual inhibitory effect of ritonavir as a mechanism-based inhibitor. The simulation predicts a regimen of elexacaftor-tezacaftor-ivacaftor administered concomitantly with nirmatrelvir-ritonavir in people with CF that will likely decrease the impact of the drug interaction.

The oral protease inhibitor (PF-07321332) protects Syrian hamsters against infection with SARS-CoV-2 variants of concern.

There is an urgent need for potent and selective antivirals against SARS-CoV-2. Pfizer developed PF-07321332 (PF-332), a potent inhibitor of the viral main protease (Mpro, 3CLpro) that can be dosed orally and that is in clinical development. We here report that PF-332 exerts equipotent in vitro activity against the four SARS-CoV-2 variants of concerns (VoC) and that it can completely arrest replication of the alpha variant in primary human airway epithelial cells grown at the air-liquid interface. Treatment of Syrian Golden hamsters with PF-332 (250 mg/kg, twice daily) completely protected the animals against intranasal infection with the beta (B.1.351) and delta (B.1.617.2) SARS-CoV-2 variants. Moreover, treatment of SARS-CoV-2 (B.1.617.2) infected animals with PF-332 completely prevented transmission to untreated co-housed sentinels.

An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

Effect of itraconazole on the pharmacokinetics of faldaprevir in healthy subjects.

Faldaprevir (FDV), a substrate of CYP3A/P-glycoprotein (P-gp), is a selective inhibitor of the hepatitis C virus (HCV) NS3/4 protease. FDV is currently under clinical development for application in interferon-free treatment regimens for patients with chronic HCV infection. Understanding the drug-drug interaction potential of FDV is critical, as certain drug combinations may facilitate the more rapid achievement of steady-state-that is, the ideal drug concentration and balanced metabolic cycle of absorption and elimination that optimize drug efficacy. We thus conducted this study to investigate the effect of itraconazole (ICZ), a strong inhibitor of CYP3A and a moderate inhibitor of P-gp, on the pharmacokinetics (PK) of FDV. Eighteen healthy male and female volunteers participated in this open-label, fixed-sequence study. FDV 120 mg twice daily (BID) was administered on Day 1, followed by 120 mg once daily (QD) from Day 2 until the end of the 10-day study; after 6 days of FDV alone, ICZ 200 mg was added to FDV for an additional 4 days (BID on Day 7 and QD from Day 8 to Day 10). Intensive PK sampling was performed after 6 days of FDV treatment and again after 4 days of combined FDV/ICZ treatment. The adjusted geometric mean (gMean) ratios (%) of area under the concentration curve over dosing interval at steady-state (AUCτ, ss) and maximal concentration at steady-state (Cmax, ss) for combined FDV/ICZ treatment vs. FDV treatment alone were 198.6% and 180.6%, respectively, with 90% confidence intervals (CIs) of 182.4-216.1 and 165.7-196.9. Administration of FDV alone or in combination with ICZ was observed to be safe and well-tolerated. Co-administration with ICZ, however, resulted in an approximately two-fold increase in FDV steady-state exposure. Furthermore, FDV required no dosage adjustment when co-administered with ICZ.

Metabolic flux analysis of branched-chain amino and keto acids (BCAA, BCKA) and ß-hydroxy ß-methylbutyric acid across multiple organs in the pig.

Branched-chain amino acids (BCAA) and their metabolites the branched-chain keto acids (BCKA) and ß-hydroxy ß-methylbutyric acid (HMB) are involved in the regulation of key signaling pathways in the anabolic response to a meal. However, their (inter)organ kinetics remain unclear. Therefore, branched-chain amino acids (BCAA) [leucine (Leu), valine (Val), isoleucine (Ile)], BCKA [α-ketoisocaproic acid (KIC), 3-methyl-2-oxovaleric acid (KMV), 2-oxoisovalerate (KIV)], and HMB across organ net fluxes were measured. In multi-catheterized pigs (n = 12, ±25 kg), net fluxes across liver, portal drained viscera (PDV), kidney, and hindquarter (HQ, muscle compartment) were measured before and 4 h after bolus feeding of a complete meal (30% daily intake) in conscious state. Arterial and venous plasma were collected and concentrations were measured by LC- or GC-MS/MS. Data are expressed as mean [95% CI] and significance (P < 0.05) from zero by the Wilcoxon Signed Rank Test. In the postabsorptive state (in nmol/kg body wt/min), the kidney takes up HMB (3.2[1.3,5.0]) . BCKA is taken up by PDV (144[13,216]) but no release by other organs. In the postprandial state, the total net fluxes over 4 h (in µmol/kg body wt/4 h) showed a release of all BCKA by HQ (46.2[34.2,58.2]), KIC by the PDV (12.3[7.0,17.6]), and KIV by the kidney (10.0[2.3,178]). HMB was released by the liver (0.76[0.49,1.0]). All BCKA were taken up by the liver (200[133,268]). Substantial differences are present in (inter)organ metabolism and transport among the BCAA and its metabolites BCKA and HMB. The presented data in a translation animal model are relevant for the future development of optimized clinical nutrition.NEW & NOTEWORTHY Branched-chain amino acids (BCAA) and their metabolites the branched-chain keto acids (BCKA) and ß-hydroxy ß-methylbutyric acid (HMB) are involved in the regulation of key signaling pathways in the anabolic response to a meal. Substantial differences are present in (inter)organ metabolism and transport among the BCAA and its metabolites BCKA and HMB. The presented data in a translation animal model are relevant for the future development of optimized clinical nutrition.

Experimental Medicine Study to Measure Immune Checkpoint Receptors PD-1 and GITR Turnover Rates In Vivo in Humans.

Development of monoclonal antibodies (mAbs) targeting immune-checkpoint receptors (IMRs) for the treatment of cancer is one of the most active areas of investment in the biopharmaceutical industry. A key decision in the clinical development of anti-IMR mAbs is dose selection. Dose selection can be challenging because the traditional oncology paradigm of administering the maximum tolerated dose is not applicable to anti-IMR mAbs. Instead, dose selection should be informed by the pharmacology of immune signaling. Engaging an IMR is a key initial step to triggering pharmacologic effects, and turnover (i.e., the rate of protein synthesis) of the IMR is a key property to determining the dose level needed to engage the IMR. Here, we applied the stable isotope labeling mass spectrometry technique using 13 C6 -leucine to measure the in vivo turnover rates of IMRs in humans. The 13 C6 -leucine was administered to 10 study participants over 15 hours to measure 13 C6 -leucine enrichment kinetics in 2 IMR targets that have been clinically pursued in oncology: GITR and PD-1. We report the first measurements of GITR and PD-1 median half-lives associated with turnover to be 55.6 and ≥ 49.5 hours, respectively. The approach outlined here can be applied to other IMRs and, more generally, to protein targets.

A Thorough QT Study of the Combination Glecaprevir + Pibrentasvir on Cardiac Repolarization in Healthy Subjects.

PURPOSE: Fixed-dose combination glecaprevir (GLE) 300 mg + pibrentasvir (PIB) 120 mg is an orally administered once daily antiviral regimen approved for the treatment of hepatitis C virus (HCV) infection. The objective of this study was to evaluate the potential for cardiac repolarization following GLE + PIB administration in healthy adults. METHODS: This placebo- and active-controlled, randomized, single-dose, 4-period, 4-sequence crossover study enrolled 48 healthy subjects. The doses of GLE 400 mg + PIB 120 mg were selected to provide exposures comparable to those with the doses that are therapeutic in the HCV-infected population, GLE 300 mg + PIB 120 mg. The doses of GLE 600 mg + PIB 240 mg were selected to provide supratherapeutic exposures without exceeding the exposures of the GLE + PIB maximal tolerated doses. Moxifloxacin 400 mg (active control/open label) was used for confirming the sensitivity of the ECG assay in detecting QTc prolongation. Time-matched plasma concentrations and triplicate ECGs were obtained on treatment days -1 and 1. The primary end point was time-matched, placebo-corrected, baseline-adjusted Fridericia-corrected QT interval (ΔΔQTcF). Pharmacokinetic-pharmacodynamic analyses characterized the relationship between GLE and PIB plasma concentrations and ΔΔQTcF using a linear regression model and linear mixed-effects model. Findings from categorical analyses of ECG-interval data were also summarized. Tolerability was evaluated through adverse-events monitoring, physical examination including vital sign measurements, ECGs, and laboratory tests. FINDINGS: A total of 48 subjects (22 women [46%], 26 men [54%]), were enrolled in the study, and 47 subjects completed all 4 periods. None of the subjects had a change from baseline in QTcF interval of >30 msec or an absolute QTcF interval of >450 msec. Peak ΔΔQTcF values observed at 5 h postdose (Tmax) were 2.9 msec (upper 95% confidence limit, 4.9 msec) with the therapeutic dose and 3.1 msec (upper 95% confidence limit, 5.1 msec) with the supratherapeutic dose, with both upper 95% confidence limits well below the 10-msec threshold. Assay sensitivity was confirmed by peak ΔΔQTcF in the positive control (12.8 ms at 2 h postdose). No statistically significant GLE or PIB concentration-dependent effects on ΔΔQTcF were observed. Headache and skin irritation from ECG electrodes were the most commonly reported AEs. No clinically significant vital sign measurements, ECG findings, or laboratory measurements were observed. There were no patterns of T- and U-wave morphologic abnormalities. IMPLICATIONS: The fixed-dose combination regimen of GLE/PIB does not prolong the QTc interval. ClinicalTrials.gov identifier.

Unexpected differences in the pharmacokinetics of N-acetyl-DL-leucine enantiomers after oral dosing and their clinical relevance.

The enantiomers of many chiral drugs not only exhibit different pharmacological effects in regard to targets that dictate therapeutic and toxic effects, but are also handled differently in the body due to pharmacokinetic effects. We investigated the pharmacokinetics of the enantiomers of N-acetyl-leucine after administration of the racemate (N-acetyl-DL-leucine) or purified, pharmacologically active L-enantiomer (N-acetyl-L-leucine). The results suggest that during chronic administration of the racemate, the D-enantiomer would accumulate, which could have negative effects. Compounds were administered orally to mice. Plasma and tissue samples were collected at predetermined time points (0.25 to 8 h), quantified with liquid chromatography/mass spectrometry, and pharmacokinetic constants were calculated using a noncompartmental model. When administered as the racemate, both the maximum plasma concentration (Cmax) and the area under the plasma drug concentration over time curve (AUC) were much greater for the D-enantiomer relative to the L-enantiomer. When administered as the L-enantiomer, the dose proportionality was greater than unity compared to the racemate, suggesting saturable processes affecting uptake and/or metabolism. Elimination (ke and T1/2) was similar for both enantiomers. These results are most readily explained by inhibition of uptake at an intestinal carrier of the L-enantiomer by the D-enantiomer, and by first-pass metabolism of the L-, but not D-enantiomer, likely by deacetylation. In brain and muscle, N-acetyl-L-leucine levels were lower than N-acetyl-D-leucine, consistent with rapid conversion into L-leucine and utilization by normal leucine metabolism. In summary, the enantiomers of N-acetyl-leucine exhibit large, unexpected differences in pharmacokinetics due to both unique handling and/or inhibition of uptake and metabolism of the L-enantiomer by the D-enantiomer. Taken together, these results have clinical implications supporting the use of N-acetyl-L-leucine instead of the racemate or N-acetyl-D-leucine, and support the research and development of only N-acetyl-L-leucine.

Population Pharmacokinetics of Glecaprevir/Pibrentasvir in HCV-infected Japanese Subjects in Phase 3 CERTAIN-1 and CERTAIN-2 Trials.

Glecaprevir (GLE)/pibrentasvir (PIB) 300 mg/120 mg once daily (Mavyret/Maviret) is an all-oral, pangenotypic, interferon- and ribavirin-free combination regimen approved for the treatment of chronic hepatitis C virus (HCV) infection. The objective of the current analyses was to characterize the pharmacokinetics (PK) of GLE/PIB in HCV-infected Japanese patients. Data from 332 subjects enrolled in 2 Japan phase 3 trials, CERTAIN-1 and CERTAIN-2, were used in the analyses. Pharmacokinetics of GLE/PIB were characterized using a nonlinear mixed-effects modeling. The analyses evaluated the impact of covariates (concomitant medications and demographic and clinical covariates such as renal impairment, effect of cirrhotic status) on GLE/PIB PK. GLE and PIB PK were described by 1- and 2-compartment models, respectively. Presence of cirrhosis, age, and body weight were identified as significant covariates on GLE/PIB PK. A trend toward higher GLE and PIB exposures in older patients and higher PIB exposures in heavier patients was observed; however, these increases were not considered clinically meaningful. GLE and PIB exposures were higher in HCV-infected subjects with cirrhosis (Child-Pugh A; GLE area under the plasma concentration-time curve was 160% higher, and PIB area under the plasma concentration-time curve was 21% higher) compared to subjects without cirrhosis. Renal function (including subjects with end-stage renal disease with dialysis) had no impact on GLE or PIB exposures. The GLE/PIB dose was well tolerated in the Japanese population, and no dose adjustment is needed for the evaluated intrinsic and extrinsic factors.

Physicochemical and Pharmacokinetic Evaluation of Spray-Dried Coformulation of Salvia miltiorrhiza Polyphenolic Acid and L-Leucine with Improved Bioavailability.

Background:Salvia miltiorrhiza polyphenolic acid (SMPA) is effective in the treatment of cardiovascular diseases and currently it is administered orally or intravenously. However, SMPA is poorly absorbed orally and quickly eliminated in vivo. A long-term frequent intravenous administration leads to poor patient compliance. Therefore, it is urgently demanded to find a new alternative route of noninjection drug delivery system for SMPA. Methods: Two dry powder inhalation (DPI) formulations of spray-dried SMPA formulation (P1) and spray-dried SMPA-L-leucine formulation (P2) were prepared by spray drying method and their physicochemical properties were assessed by thermogravimetric analysis, X-ray diffraction, scanning electron microscopy, particle size distribution analysis, and in vitro aerodynamic analysis. Moreover, In vitro cytotoxicity of SMPA and P2 was conducted with NR8383 cells. In vivo pharmacokinetics were carried out by Penn-Century endotracheal intubation technique to deliver P2 to the lungs of rats. Results and Conclusions: The moisture content of P1 and P2 were 5.81% ± 0.005%, and 4.08% ± 0.002%, respectively. P1 and P2 were in an amorphous state. Moreover, P1 had slightly corrugated surfaces, whereas P2 exhibited severely corrugated surfaces with invagination due to the presence of L-leucine. In addition, there were more hollow particles with smooth surface in P1 than that in P2. Compared with P1, P2 has shown optimal physical particle size and aerosolization behavior with D (v, 50) of 2.64 ± 0.01 µm and fine particle fraction of 37.55% ± 2.63%. The findings of in vitro cytotoxicity showed that P2 did not inhibit cell viability and could be safe for pulmonary administration. The absolute bioavailability of salvianolic acid B (Sal B) for pulmonary administration was 19.15% ± 7.44%, which is significantly higher than the oral bioavailability of Sal B (<5.56%). In this study, we have shown the feasibleness of pulmonary administration of SMPA in the form of DPIs for systemic delivery to treat cardiovascular diseases.

Fine-Needle Aspiration for the Evaluation of Hepatic Pharmacokinetics of Vaniprevir: A Randomized Trial in Patients With Hepatitis C Virus Infection.

Fine-needle aspiration (FNA) for serial hepatic sampling may be an efficient and less invasive alternative to core needle biopsy (CNB), the current standard for liver tissue sampling. In this randomized, open-label trial in 31 participants with hepatitis C virus genotype 1 infection (NCT01678131/Merck protocol PN048), we evaluated the feasibility of using FNA to obtain human liver tissue samples appropriate for measuring hepatic pharmacokinetics (PK), using vaniprevir as a tool compound. The primary end point was successful retrieval of liver tissue specimens with measurable vaniprevir concentrations at two of three specified FNA time points. Twenty-nine patients met the primary end point and, therefore, were included in the PK analyses. Hepatic vaniprevir concentrations obtained with FNA were consistent with known vaniprevir PK properties. The shape of liver FNA and CNB concentration-time profiles were comparable. In conclusion, FNA may be effective for serial tissue sampling to assess hepatic drug exposure in patients with liver disease.

Nanoporous CD-MOF particles with uniform and inhalable size for pulmonary delivery of budesonide.

It is essential to optimize a carrier of dry powder inhalation (DPI) for the aerodynamic deposition in vitro to achieve pulmonary delivery of drug molecules in vivo. In this study, neutralized nanoporous γ-cyclodextrin metal-organic framework (CD-MOF) crystals with cubic morphology and uniform inhalation size were developed and modified as a DPI carrier for budesonide (BUD). Cholesterol (CHO) and leucine (LEU)-poloxamer were used to modify the CD-MOF powder for the improvement of flowability and particle aerodynamic behaviour, for which the particle size distribution, Carr's index and in vitro pulmonary deposition were assessed. Compared to CD-MOF or LEU-CD-MOF-BUD, CHO-CD-MOF had a superior mass median aerodynamic diameter (4.35 ±â€¯0.04 µm) and inhalable performance (fine particle fraction of 30.60 ±â€¯0.76%), which were maintained after budesonide loading (4.47 ±â€¯0.30 µm, 24.95 ±â€¯4.33%). The crystallinity, cytotoxicity and in vivo deposition of drug loaded samples (CHO-CD-MOF-BUD) were then investigated by powder X-ray diffraction (PXRD), cell viability study, in vivo fluorescence imaging and pharmacokinetic studies in rats. The characteristic PXRD crystallinity peaks of budesonide disappeared after being loaded into CHO-CD-MOF, potentially indicating the molecular incorporation of budesonide into the pores of CD-MOF. The cell viability of A549 cell was more than 90% for CHO-CD-MOF-BUD as a result of the good biocompatibility of CD-MOF. When Rhodamine B was carried by the DPI particles, the fluorescence signal at the lung tissue was markedly improved after cholesterol modification compared with CD-MOF, whilst the bioavailability of CHO-CD-MOF-BUD in rat was equivalent with that of the commercial product of Pulmicort Turbuhaler. Therefore, the CD-MOF powders modified by cholesterol can be used as a promising inhalable carrier for pulmonary delivery of drugs with small dose.

Discovery of NV-5138, the first selective Brain mTORC1 activator.

The mechanistic target of rapamycin complex 1 (mTORC1) has been linked to several important chronic medical conditions many of which are associated with advancing age. A variety of inputs including the amino acid leucine are required for full mTORC1 activation. The cytoplasmic proteins Sestrin1 and Sestrin2 specifically bind to the multiprotein complex GATOR2 and communicate leucine sufficiency to the mTORC1 pathway activation complex. Herein, we report NV-5138, a novel orally bioavailable compound that binds to Sestrin2 and activates mTORC1 both in vitro and in vivo. NV-5138 like leucine transiently activates mTORC1 in several peripheral tissues, but in contrast to leucine uniquely activates this complex in the brain due lack of metabolism and utilization in protein synthesis. As such, NV-5138 will permit the exploration in areas of unmet medical need including neuropsychiatric conditions and cognition which have been linked to the activation status of mTORC1.

A Combination of Tri-Leucine and Angiopep-2 Drives a Polyanionic Polymalic Acid Nanodrug Platform Across the Blood-Brain Barrier.

One of the major problems facing the treatment of neurological disorders is the poor delivery of therapeutic agents into the brain. Our goal is to develop a multifunctional and biodegradable nanodrug delivery system that crosses the blood-brain barrier (BBB) to access brain tissues affected by neurological disease. In this study, we synthesized a biodegradable nontoxic ß-poly(l-malic acid) (PMLA or P) as a scaffold to chemically bind the BBB crossing peptides Angiopep-2 (AP2), MiniAp-4 (M4), and the transferrin receptor ligands cTfRL and B6. In addition, a trileucine endosome escape unit (LLL) and a fluorescent marker (rhodamine or rh) were attached to the PMLA backbone. The pharmacokinetics, BBB penetration, and biodistribution of nanoconjugates were studied in different brain regions and at multiple time points via optical imaging. The optimal nanoconjugate, P/LLL/AP2/rh, produced significant fluorescence in the parenchyma of cortical layers II/III, the midbrain colliculi, and the hippocampal CA1-3 cellular layers 30 min after a single intravenous injection; clearance was observed after 4 h. The nanoconjugate variant P/LLL/rh lacking AP2, or the variant P/AP2/rh lacking LLL, showed significantly less BBB penetration. The LLL moiety appeared to stabilize the nanoconjugate, while AP2 enhanced BBB penetration. Finally, nanoconjugates containing the peptides M4, cTfRL, and B6 displayed comparably little and/or inconsistent infiltration of brain parenchyma, likely due to reduced trans-BBB movement. P/LLL/AP2/rh can now be functionalized with intra-brain targeting and drug treatment moieties that are aimed at molecular pathways implicated in neurological disorders.

D-leucine microparticles as an excipient to improve the aerosolization performances of dry powders for inhalation.

The objective of the work was to produce a new excipient based on D-leucine to improve the aerosolization properties of the poorly flowable micronized budesonide. The D-leucine powders produced by a nano spray-dryer were characterized in terms of dimensions, morphology, thermal behavior, X-ray powder diffraction and infrared spectroscopy. Then, micronized budesonide was mixed with the different leucine powders obtained or commercial D-leucine at different weight ratios (1:1, 5:1, 10:1) to investigate their aerodynamic characteristics using a glass twin-stage impinger. Commercial D-leucine powder is composed of large flattened crystals of about 30 µm with ~3 µm thickness, while micronized budesonide appeared as small irregular crystals. After spray-drying, D-leucine particles appeared wrinkled and porous. Particle sizes were mainly influenced by the amino acid concentration. Aerodynamic assessment showed that D-leucine was able to improve the aerodynamic behavior of micronized budesonide from about 28 to 45% emitted fraction. The best aerodynamic properties were obtained with D-leucine powdered from a water:ethanol (1:1, v:v) solution and using micronized budesonide:leucine weight ratio of 5:1. The direct physical mixing of the atomized D-leucine with the micronized active pharmaceutical ingredient is a valid and economic alternative for the production of carrier-free dry powders for inhalation.

Alterations in branched-chain amino acid kinetics in nonobese but insulin-resistant Asian men.

Background: Branched-chain amino acids (BCAAs) are elevated in the insulin-resistant (IR) state. The reasons for this increase remain unclear, but it may be related to abnormalities in BCAA metabolism and free fatty acid (FFA) metabolism. Objective: In this study, we quantified BCAA and FFA kinetics of IR and insulin-sensitive (IS) nonobese Asian men with the use of stable-isotope tracers. We hypothesized that in addition to greater substrate flux, the BCAA oxidative pathway is also impaired to account for the higher plasma BCAA concentration in the IR state. Design: We recruited 12 IR and 14 IS nonobese and healthy Asian men. Oral-glucose-tolerance tests (OGTTs) were performed to quantify insulin sensitivity, and subjects underwent 2 stable-isotope infusion studies. [U-13C6]Leucine was infused to measure leucine flux and oxidation as indexes of BCAA metabolism, whereas [U-13C16]palmitate was infused to measure palmitate flux and oxidation to represent FFA metabolism, The 2H2O dilution method was used to estimate body composition. Results: IR subjects had greater adiposity and significantly higher fasting and post-OGTT glucose and insulin concentrations compared with the IS group. However, none of the subjects were diabetic. Despite similar dietary protein intake, IR subjects had a significantly higher plasma BCAA concentration and greater leucine flux. Leucine oxidation was also greater in the IR group, but the relation between leucine oxidation and flux was significantly weaker in the IR group than in the IS group (r = 0.530 compared with 0.695, P < 0.0388 for differences between slope). FFA oxidation was, however, unaffected despite higher FFA flux in the IR group. Conclusion: The higher plasma BCAA concentration in healthy nonobese individuals with IR is associated with a weaker relation between BCAA oxidation and BCAA flux and this occurs in the presence of accelerated FFA flux and oxidation.

Interaction between leucine and palmitate catabolism in 3T3-L1 adipocytes and primary adipocytes from control and obese rats.

Metabolic profiling studies have highlighted increases in the plasma free fatty acid (FFA) and branched-chain amino acid (BCAA) concentrations, which are hallmarks of the obese and insulin-resistant phenotype. However, little is known about how the increase of the BCAA concentration modifies the metabolic fate of FFA, and vice versa, in adipocytes. Therefore, we incubated differentiated 3T3-L1 adipocytes or primary adipocytes from rats fed a control or a high-fat diet with: (1) 0, 250, 500 and 1000 µM of leucine and determined the oxidation and incorporation of [1-14C]-palmitate into lipids or proteins or (2) 0, 250, 500 or 1000 µM of palmitate and evaluated the oxidation and incorporation of [U-14C]-leucine into lipids or proteins. Leucine decreased palmitate oxidation and increased its incorporation into the lipid fraction in adipocytes; the latter was reduced in adipocytes from obese rats. However, palmitate increased leucine oxidation in adipocytes as well as reduced leucine incorporation into the protein and lipid fractions in adipocytes from obese rats. These results demonstrate that leucine modifies the metabolic fate of palmitate, and vice versa, in adipocytes and that the metabolic interaction between leucine and palmitate catabolism is altered in adipocytes from obese rats.

Consumption of whole eggs promotes greater stimulation of postexercise muscle protein synthesis than consumption of isonitrogenous amounts of egg whites in young men.

Background: Protein in the diet is commonly ingested from whole foods that contain various macro- and micronutrients. However, the effect of consuming protein within its natural whole-food matrix on postprandial protein metabolism remains understudied in humans.Objective: We aimed to compare the whole-body and muscle protein metabolic responses after the consumption of whole eggs with egg whites during exercise recovery in young men.Design: In crossover trials, 10 resistance-trained men [aged 21 ± 1 y; 88 ± 3 kg; body fat: 16% ± 1% (means ± SEMs)] received primed continuous l-[ring-2H5]phenylalanine and l-[1-13C]leucine infusions and performed a single bout of resistance exercise. After exercise, participants consumed intrinsically l-[5,5,5-2H3]leucine-labeled whole eggs (18 g protein, 17 g fat) or egg whites (18 g protein, 0 g fat). Repeated blood and muscle biopsy samples were collected to assess whole-body leucine kinetics, intramuscular signaling, and myofibrillar protein synthesis.Results: Plasma appearance rates of protein-derived leucine were more rapid after the consumption of egg whites than after whole eggs (P = 0.01). Total plasma availability of leucine over the 300-min postprandial period was similar (P= 0.75) between the ingestion of whole eggs (68% ± 1%) and egg whites (66% ± 2%), with no difference in whole-body net leucine balance (P = 0.27). Both whole-egg and egg white conditions increased the phosphorylation of mammalian target of rapamycin complex 1, ribosomal protein S6 kinase 1, and eukaryotic translation initiation factor 4E-binding protein 1 during postexercise recovery (all P < 0.05). However, whole-egg ingestion increased the postexercise myofibrillar protein synthetic response to a greater extent than did the ingestion of egg whites (P= 0.04).Conclusions: We show that the ingestion of whole eggs immediately after resistance exercise resulted in greater stimulation of myofibrillar protein synthesis than did the ingestion of egg whites, despite being matched for protein content in young men. Our data indicate that the ingestion of nutrient- and protein-dense foods differentially stimulates muscle anabolism compared with protein-dense foods. This trial was registered at clinicaltrials.gov as NCT03117127.