Plasma RAT.rar
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Results: There was a significant decrease in the femur index value in groups receiving tazarotene and bexarotene on Day 14 (8% and 20% respectively, p=0.0039). On Day 28, 14 days after discontinuation of tazarotene and bexarotene, the difference in femur indexes was still significant (4% for T1-6 and B1-6, p=0.0270). In the bexarotene group an increase in mean plasma osteocalcin level and mean plasma TRACP5b level was detected. In the tazarotene group the mean osteocalcin level remained unchanged and the mean plasma TRACP5b level decreased. An increased yield stress was detected in groups receiving retinoids comparing to controls after 14 days of tazarotene and bexarotene administration.
Hepatocytes and hepatic stellate cells play important roles in retinoid storage and metabolism. Hepatocytes process postprandial retinyl esters and are responsible for secretion of retinol bound to retinol-binding protein (RBP) to maintain plasma retinol levels. Stellate cells are the body's major cellular storage sites for retinoid. We have characterized and utilized an immortalized rat stellate cell line, HSC-T6 cells, to facilitate study of the cellular aspects of hepatic retinoid processing. For comparison, we also carried out parallel studies in Hepa-1 hepatocytes. Like activated primary stellate cells, HSC-T6 express myogenic and neural crest cytoskeletal filaments. HSC-T6 cells take up and esterify retinol in a time- and concentration-dependent manner. Supplementation of HSC-T6 culture medium with free fatty acids (up to 300 micrometer) does not affect retinol uptake but does enhance retinol esterification up to 10-fold. RT-PCR analysis indicates that HSC-T6 cells express all 6 retinoid nuclear receptors (RARalpha, -beta, -gamma, and RXRalpha, -beta, -gamma) and like primary stellate cells, HSC-T6 stellate cells express cellular retinol-binding protein, type I (CRBP) but fail to express either retinol-binding protein (RBP) or transthyretin (TTR). Addition of retinol (10(-8)-10(-5) m) or all-trans-retinoic acid (10(-10)-10(-6) m) rapidly up-regulates CRBP expression. Using RAR-specific agonists and antagonists and an RXR-specific agonist, we show that members of the RAR-receptor family modulate HSC-T6 CRBP expression.Thus, HSC-T6 cells display the same retinoid-related phenotype as primary stellate cells in culture and will be a useful tool for study of hepatic retinoid storage and metabolism.
In the present study, we investigated: (i) whether acute RA infusion alters plasma CORT levels through RAR-α; (ii) whether chronic RA administration modulates HPA axis activity and induces any depression-like behavior via alterations in GR negative feedback; (iii) possible effects of RA on GR-mediated glucocorticoid suppression of CRH expression in vitro; and finally, (iv) whether these parameters can be normalized by treatment with the GR antagonist mifepristone (RU38486).
For the chronic experiment, 36 rats were assigned either to a vehicle (VEH; n=12), RA (n=12), vehicle plus mifepristone (VEH+MIF; n=6) or a RA plus mifepristone group (RA+MIF; n=6). Among the 36 rats, 24 rats (n=6 for each treatment group) were used for plasma corticosterone (CORT) evaluation, behavioral tests and subsequent immunohistochemical study. The other 12 rats (n=6 for each VEH and RA group) were used for western blot analysis of brain hypothalamic samples.
Plasma corticosterone (CORT) concentration and depression/anxiety-related behavioral changes in the chronic all-trans retinoic acid (RA) treatment experiment. (a) Experimental design of chronic drug treatment experiment. Animals were chronically exposed to RA or vehicle (VEH) via intracerebroventricular (i.c.v.) injection for 19 days, in combination with or without mifepristone (MIF) injection during the last 5 days. This resulted in four chronic treatment groups: VEH group, RA group, VEH plus MIF (VEH+MIF) group and an RA plus MIF (RA+MIF) group (n=6 animals per group). After chronic treatment, animals were subject to four sequential sets of behavioral tests and evaluated for depression/anxiety-related behavior: dexamethasone suppression test (DST; at day 20), sucrose preference test (starting adaptation at day 20 after DST, and tested at day 21), elevated plus maze (EPM) test (at day 22) and open-field test (at day 23). At day of 24, animals were killed. (b) Basal plasma CORT concentrations compared among the four chronic treatment groups. (c) Acute stress-induced CORT concentration changes compared between the chronic RA group and the VEH group. Animals were subject to a 10-min acute forced swimming stress. Note that the plasma CORT values before stress in these two groups are the same as the basal CORT concentration as shown in panel b. (d) Sucrose preference percentage compared among the four chronic treatment groups. (e) The frequencies of open arm entry and (f) the percentage of duration in the open arms measured in the EPM experiment compared among the four chronic treatment groups. All data are presented as means.e.m. (n=6 animals per group). *P
(a) Correlations between nuclear receptor retinoic acid receptor-α (RAR-α)- and glucocorticoid receptor (GR)-immunoreactive (IR) cell number in the hypothalamic paraventricular nucleus (PVN); and (b) correlations between sucrose preference percentage and the corresponding plasma corticosterone (CORT) concentration. Note that the number of RAR-α-IR and GR-IR cells is based on pooled data from the four chronic treatment groups analyzed throughout the entire PVN as shown in Figures 3g and h; the value of sucrose preference percentage and corresponding plasma CORT is also based on pooled data from the four chronic treatment groups as shown in Figures 1b and d. *P
According to the free drug hypothesis only the unbound drug is available to act at physiological sites of action, and as such the importance of plasma protein binding primarily resides in its impact on pharmacokinetics and pharmacodynamics. Of the major plasma proteins, alpha-1-acid glycoprotein (AAG) represents an intriguing one primarily due to the high affinity, low capacity properties of this protein. In addition, there are marked species and age differences in protein expression, homology and drug binding affinity. As such, a thorough understanding of drug binding to AAG can help aid and improve the translation of pharmacokinetic/pharmacodynamic (PK/PD) relationships from preclinical species to human as well as adults to neonates. This review provides a comprehensive overview of our current understanding of the biochemistry of AAG; endogenous function, impact of disease, utility as a biomarker, and impact on PK/PD. Experimental considerations are discussed as well as recommendations for understanding the potential impact of AAG on PK through drug discovery and early development.
Given the striking differences between fetal, newborn, and adult AAG levels, it may be important to understand placental transfer and the milk to plasma ratio (M/P) for drugs that bind to AAG. Fleishaker and McNamara (61) described a diffusional model to assess drug distribution in milk, showing that the in vitro drug binding to serum and milk protein reasonably predict M/P drug ratio in vivo. The same authors tested the model in lactating rabbits using propranolol, a compound known to bind with high affinity to AAG. To mimic the disease setting, rabbits were dosed with bovine AAG and propranolol PK parameters were evaluated. The diffusional model was able to accurately predict the decrease in propranolol M/P from 2.13 to 1.23 before and after AAG administration. Importantly, a roughly proportional reduction in total plasma CL (35%) counteracted the decrease in fu (22%), maintaining consistent CLu rate and total drug levels in milk.
As described above, albumin, AAG, and lipoproteins are considered the most important plasma proteins involved with drug binding. While albumin has a higher drug capacity due to its relative abundance in plasma, AAG levels are lower and with high affinity drugs saturation may occur. The saturation of AAG may or may not be buffered by albumin depending on the drug binding affinity for albumin. Plasma protein levels can change with disease state, a decrease in albumin and an increase in AAG are generally observed, which may impact protein binding and PK. Both albumin and AAG levels are significantly lower in the newborn, with newborn:adult ratios of about 0.81 and 0.38, respectively (47), a factor that should be considered when predicting PK in the very young pediatric population.
As with any assay it is good practice to include control compounds that are assessed along with test compounds to ensure a properly functioning assay. If data for control compounds are not available for a given assay/species one can still monitor the value for the control over time to ensure its consistency. When conducting definitive assays the use of multiple control compounds is advised since multiple factors can influence binding and some are compound specific. Literature values for propranolol and warfarin are summarized across species in Table VII. The intent here is to provide references for acceptable free fraction values for control compounds across species. Propranolol was selected because it has moderately high binding to human plasma proteins, however the affinity is higher for AAG relative to albumin by approximately two orders of magnitude (125), therefore, propranolol can serve as a control for compounds that preferentially bind to AAG. The reported propranolol fu values range from 0.10 to 0.29 in human plasma, a 3-fold difference, and outside what is typically deemed normal assay variability. The acceptable assay fu value for propranolol should be within 0.10 to 0.20 in healthy human plasma. Warfarin was selected because it is highly bound to both AAG and albumin (126, GE life sciences application note 29263246AA). It is helpful to include a highly bound control compound since they generally require longer incubation time to achieve equilibrium. While the reported warfarin fu values range from 0.005 to 0.022 in human plasma, more than a 4-fold difference, the absolute difference is low. The majority of references indicate a narrower range, therefore the acceptable assay fu value for warfarin should be within 0.005 to 0.015, a range similar to that reported in the recently published white paper (116). It is not advised to select a compound with moderate or low protein binding to serve as a control because the fu values are generally more variable. 59ce067264
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