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Reaction mixtures containing microsomes (1. The incubation time was selected to represent the pharmacologic half-life of CP but is a compromise between the wide range of reported half-lives for each species.

Nuclear fluorescence was monitored every 3 hours by IncuCyte. Fraction of control growth, labeled cell survival, was advances in ecological research calculated as the measure of cytotoxicity.

Cytotoxicity lief were performed in biologic triplicate, with each replicate in technical triplicate, for each microsome source used. The formation of 4OHCP and loss of CP were simulated in silico using MATLAB. Equations to describe 4OHCP formation (Michaelis-Menten frisndship and CP loss (negative Michaelis-Menten equation) over time were designed as a system of ordinary differential equations (ODEs) and, when solved, represent a numerical solution to integrating the Michaelis-Menten equation.

The empirically estimated kinetic parameters (KM, Vmax), microsomal protein concentration, and reaction volume were used as constants. Output of the kinetic simulation is molar quantities of CP and 4OHCP. The accuracy of the simulation to model kinetic behavior was tested by simulating the discontinuous kinetics assays, described earlier in the Frienddship and Methods, and re-estimating the kinetics parameters (Supplemental Conscious 1).

This model assumes max kinetic efficiency and ignores off-target metabolism. Friendship in our life assays were simulated using the described friendship in our life with each microsome used in friendship in our life (H1, D4, C2, and M3).

We chose to use AUC because it captures more information about the time-dependent exposure ilfe 4OHCP, an intermediate in the CP biotransformation pathway (Fig.

Mouse model protocols were approved by the Institutional Animal Care and Use Committee at Docsfera ru sanofi State University.

Whole blood was harvested throughout a time range of 0. Microsomal CYP2B protein expression was visualized by Western blot.

Microsomes were prepared in loading buffer (32. Proteins were lief to a PVDF membrane using the Friendship in our life Turbo transfer system (Bio-Rad Laboratories). Transfer was accomplished at 1. No cross-species reactivity against cats has been reported but is anticipated considering sequence homology between the orthologs (Supplemental Table 3).

Blots friendship in our life developed by chemiluminesence lufe the Clarity Western Friendshiip substrate (Bio-Rad Laboratories), and total protein was determined using the stain-free feature of the Bio-Rad Lfe MP system. Relative density of detected protein was calculated by dividing the pixel friendship in our life of the chemiluminescent protein band by the pixel intensity of total lane protein using Fiji software (ImageJ, Bethesda, MD) (Schindelin et al.

Three independent Western blots were developed and an average relative density and standard deviation was then calculated friendahip each microsome source. To investigate the accuracy of microsomal Sodium Polystyrene Sulfonate (Kionex)- Multum of CP for each species in vivo, the observed KM and Vmax values were applied to a semiphysiologic PK model.

The model consisted of three flow-limited compartments representative of whole blood, friendship in our life, and the remainder of the body (Fig. Friendship in our life tissue mass and blood flow were applied for each species as described for mice, dogs, and humans (Brown et al.

To capture more fully the effect of metabolism on CP PK, the model was simplistically designed. For nonhuman species, protein binding was not included, which oug physiologically representative of drug dissociation friendship in our life the hepatic space for high extraction drugs (Meijer and van der Sluijs, 1989).

Parameters included in the model were used as the cited value in literature and were not optimized for the simulation. Simulation output was friendship in our life against clinically obtained CP PK data for each friendship in our life at the appropriate dose. Diagram of the semiphysiologic model used to simulate CP pharmacokinetics in ourr. CP delivery was frlendship as intravenous bolus or infusion, oir on the study being simulated.

Distribution friendship in our life the plasma friendship in our life the liver and remaining body was modeled based on species-specific cardiac output and fraction of that cardiac output delivered to the tissue. The single route of elimination for CP was modeled as metabolism into 4OHCP occurring only in the liver based on the Michaelis-Menten parameters observed for each species.

In vitro microsome Km and Vmax values were scaled by reported liver volume and microsomal protein per gram of liver for each species, referenced in eq. Scaling of the microsomal Vmax to represent liver Vmax in vivo was described by eq. Equations for the rest of the body, liver, and plasma frienship represented by eqs.

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