Use of physiologically-based pharmacokinetic modeling to inform dosing of the opioid analgesics fentanyl and methadone in children with obesity.

Obesity is an increasingly alarming public health threat, with nearly 20% of children classified as obese in the United States today. Children with obesity are commonly prescribed the opioids fentanyl and methadone, and accurate dosing is critical to reducing the risk of serious adverse events associated with overexposure. However, pharmacokinetic studies in children with obesity are challenging to conduct, so there is limited information to guide fentanyl and methadone dosing in these children. To address this clinical knowledge gap, physiologically-based pharmacokinetic models of fentanyl and methadone were developed in adults and scaled to children with and without obesity to explore the interplay of obesity, age, and pharmacogenomics. These models included key obesity-induced changes in physiology and pharmacogenomic effects. Model predictions captured observed concentrations in children with obesity well, with an overall average fold error of 0.72 and 1.08 for fentanyl and methadone, respectively. Model simulations support a reduced fentanyl dose (1 vs. 2 µg/kg/h) starting at an earlier age (6 years) in virtual children with obesity, highlighting the importance of considering both age and obesity status when selecting an infusion rate most likely to achieve steady-state concentrations within the target range. Methadone dosing simulations highlight the importance of considering genotype in addition to obesity status when possible, as cytochrome P450 (CYP)2B6*6/*6 virtual children with obesity required half the dose to match the exposure of wildtype children without obesity. This physiologically-based pharmacokinetic modeling approach can be applied to explore dosing of other critical drugs in children with obesity.

The "Super-Child" Approach Is Applied To Estimate Retinol Kinetics and Vitamin A Total Body Stores in Mexican Preschoolers.

BACKGROUND: Retinol isotope dilution (RID) and model-based compartmental analysis are recognized techniques for assessing vitamin A (VA) status. Recent studies have shown that RID predictions of VA total body stores (TBS) can be improved by using modeling and that VA kinetics and TBS in children can be effectively studied by applying population modeling ("super-child" approach) to a composite data set. OBJECTIVES: The objectives were to model whole-body retinol kinetics and predict VA TBS in a group of Mexican preschoolers using the super-child approach and to use model predictions of RID coefficients to estimate TBS by RID in individuals. METHODS: Twenty-four healthy Mexican children (aged 3-6 y) received an oral dose (2.96 µmol) of [13C10]retinyl acetate in corn oil. Blood samples were collected from 8 h to 21 d after dosing, with each child sampled at 4 d and at 1 other time. Composite data for plasma labeled retinol compared with time were analyzed using a 6-component model to obtain group retinol kinetic parameters and pool sizes. Model-predicted TBS was compared with mean RID predictions at 4 d; RID estimates at 4 d were compared with those calculated at 7-21 d. RESULTS: Model-predicted TBS was 1097 µmol, equivalent to ∼2.4 y-worth of VA; using model-derived coefficients, group mean RID-predicted TBS was 1096 µmol (IQR: 836-1492 µmol). TBS at 4 d compared with a later time was similar (P = 0.33). The model predicted that retinol spent 1.5 h in plasma during each transit and recycled to plasma 13 times before utilization. CONCLUSIONS: The super-child modeling approach provides information on whole-body VA kinetics and can be used with RID to estimate TBS at any time between 4 and 21 d postdose. The high TBS predicted for these children suggests positive VA balance, likely due to large-dose VA supplements, and warrants further investigation.

Trafficking of nonesterified fatty acids in insulin resistance and relationship to dysglycemia.

In adipose, insulin functions to suppress intracellular lipolysis and secretion of nonesterified fatty acid (NEFA) into plasma. We applied glucose and NEFA minimal models (MM) following a frequently sampled intravenous glucose tolerance test (FSIVGTT) to assess glucose-specific and NEFA-specific insulin resistance. We used total NEFA and individual fatty acids in the NEFA MM, comparing the model parameters in metabolic syndrome (MetSyn) subjects (n = 52) with optimally healthy controls (OptHC; n = 14). Results are reported as mean difference (95% confidence interval). Using the glucose MM, MetSyn subjects had lower [-73% (-82, -57)] sensitivity to insulin (Si) and higher [138% (44, 293)] acute insulin response to glucose (AIRg). Using the NEFA MM, MetSyn subjects had lower [-24% (-35, -13)] percent suppression, higher [32% (15, 52)] threshold glucose (gs), and a higher [81% (12, 192)] affinity constant altering NEFA secretion (ϕ). Comparing fatty acids, percent suppression was lower in myristic acid (MA) than in all other fatty acids, and the stearic acid (SA) response was so unique that it did not fit the NEFA MM. MA and SA percent of total were increased at 50 min after glucose injection, whereas oleic acid (OA) and palmitic acid (PA) were decreased (P < 0.05). We conclude that the NEFA MM, as well as the response of individual NEFA fatty acids after a FSIVGTT, differ between OptHC and MetSyn subjects and that the NEFA MM parameters differ between individual fatty acids.

Kinetics of trans-10, cis-12-conjugated linoleic acid transfer to plasma and milk following an abomasal bolus in lactating dairy cows.

Trans-10, cis-12-conjugated linoleic acid (CLA) is a potent bioactive fatty acids (FA) that causes milk fat depression in lactating animals. FA are transferred to milk directly through chylomicrons and indirectly by recycling through other tissues. The objective of this study was to characterise the kinetics of trans-10, cis-12 CLA transfer to plasma and milk after a single bolus infusion. Five multiparous mid-lactation cows received a single abomasal bolus infusion of an enriched CLA mixture providing 15 g of trans-10, cis-12 CLA and 15 g of cis-9, trans-11 CLA over a 30-min period. Plasma concentration of trans-10, cis-12 and cis-9, trans-11 CLA peaked 2 h post-bolus, reaching 0·29 and 0·38 % of total plasma FA, respectively, and returned to pre-bolus values at 72 h post-infusion. Milk trans-10, cis-12 CLA yield and concentration peaked 14 h post-bolus (0·25 g/h) and was not detectable in milk after 86 h. Total apparent transfer of trans-10, cis-12 CLA to milk was 41 %, with 73 % transferred to milk through the direct pool (chylomicrons) and the remaining 27 % transferred through the indirect pool (tissue recycling). Compartmental modelling revealed the existence of a transient unavailable pool of trans-10, cis-12 CLA in extravascular tissues represented primarily by the mammary gland, which slowly exchanges with an available pool for secretion in milk fat and transfer to milk. In conclusion, trans-10, cis-12 CLA is predominantly transferred to milk through the direct pathway; however, how this CLA isomer is processed within the mammary gland requires further investigation.

CD44 interacts directly with Lck in a zinc-dependent manner.

CD44 is a widely expressed cell adhesion molecule with functional similarities to the selectin and integrin adhesion molecules. CD44 has a lectin domain that binds hyaluronan, a component of the extracellular matrix. Interactions between CD44 and hyaluronan promote lymphocyte rolling under flow and cell-cell and cell-matrix adhesion. Attachment of lymphocytes to immobilized CD44 antibodies induces cell adhesion and spreading, which is dependent on Src family kinase activity. Both Lck and Fyn associate with CD44 in T cells. CD4 and CD8 associate with Lck via a zinc-dependent interaction that is inhibited by the divalent metal cation chelator, 1,10-phenanthroline. Here we show that both CD4 and CD44-mediated T cell spreading is abolished in the presence of 1,10-phenanthroline and their association with Lck is significantly reduced. In contrast, the co-immunoprecipitation of Fyn by CD44 was unaffected. The cytoplasmic domain of CD44 was required for divalent cation-dependent association of Lck, but not for its association with Fyn. Mutational analysis of CD44 revealed that cysteine residues were not essential for the interaction nor were the carboxy-terminal 41 amino acids. Progressive deletion of the remaining 31 amino acids of the CD44 cytoplasmic domain revealed the importance of this membrane proximal region for its association with Lck. Using purified recombinant proteins, we demonstrated a direct, zinc-inducible interaction between the cytoplasmic domain of CD44 and Lck but not Fyn. The zinc-inducible interaction required the first 13 amino acids of the cytoplasmic domain of CD44 and the non-catalytic regions of Lck. Taken together, we conclude that CD44 directly associates with Lck in a zinc-inducible manner and this is important for the transmission of CD44-mediated signaling events leading to T cell spreading.

Substrate binding determinants of Trypanosoma brucei gamma-glutamylcysteine synthetase.

gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the ATP-dependent ligation of L-Glu and L-Cys, which is the first step in de novo biosynthesis of the tripeptide glutathione. Recently it was demonstrated that gamma-GCS is a structural homologue of glutamine synthetase (GS), providing the basis to build a model for the gamma-GCS active site [Abbott et al. (2001) J. Biol. Chem. 276, 42099-42107]. Substrate binding determinants in the active site of gamma-GCS have been identified and characterized in the enzyme from the parasitic protozoa Trypanosoma brucei using this model as a guide for site-directed mutagenesis. R366 and R491 were identified as key determinants of L-Glu binding. Mutation of R366 to Ala increases the K(d) for L-Glu by 160-fold and eliminates the positive cooperativity observed for the binding of L-Glu and ATP to the wild-type enzyme, based on a rapid equilibrium random mechanism of substrate binding. Unlike the wild-type enzyme, the R366A mutant enzyme was able to form product using the substrate analogue gamma-aminobutyric acid, suggesting that R366 interacts with the alpha-carboxylate of L-Glu. Mutation of R491 to Ala decreased k(cat) for ATP hydrolysis by 70-fold; however, dipeptide product was only formed in 5% of these turnovers. These data suggest that R491 stabilizes the phosphorylated gamma-carboxylate of L-Glu during nucleophilic attack by the L-Cys to form the dipeptide product. T323, R474, and R487 were predicted to be ATP binding determinants. Mutation of each of these residues to Ala increased the apparent K(m) for ATP by 20-100-fold while having only modest effects on k(cat) or the apparent K(m)'s for the other substrates. Finally, mutation of R179, a conserved residue that is present in gamma-GCS, but not in GS, increased the apparent K(m) for both L-Cys and L-Glu.