Imaging P-glycoprotein transport activity at the human blood-brain barrier with positron emission tomography

BACKGROUND: Numerous knockout mouse studies have revealed that P-glycoprotein (P-gp) significantly limits drug distribution across the mouse blood-brain barrier (BBB). To determine the importance of P-gp at the human BBB, we developed a state-of-the-art, noninvasive, quantitative imaging technique to measure P-gp activity by use of carbon 11-labeled verapamil as the P-gp substrate and cyclosporine (INN, ciclosporin) as the P-gp inhibitor. METHODS: In brief, 11C-verapamil (approximately 0.2 mCi/kg) was administered to healthy volunteers (n = 12 [6 women and 6 men]) as an intravenous infusion over a period of approximately 1 minute before and after at least a 1-hour infusion of cyclosporine (2.5 mg x kg(-1) x h(-1)). Arterial blood samples and brain positron emission tomography images were obtained at frequent intervals for 45 minutes. Both blood and plasma radioactivity contents were determined in each verapamil sample. The content of verapamil and its metabolites in the 20- and 45-minute plasma samples was determined by a rapid solid-phase extraction method. The brain uptake of 11C-radioactivity (brain area under the curve [AUCbrain ]/blood area under the curve [AUCblood]) was determined in the presence and absence of cyclosporine. RESULTS: The AUCbrain/AUCblood ratio of 11C-radioactivity was increased by 88% +/- 20% (1.02 +/- 0.18 versus 0.55 +/- 0.10, P < .001) in the presence of cyclosporine (mean blood concentration, 2.8 +/- 0.4 micromol/L) without affecting 11C-verapamil metabolism or plasma protein binding. The corresponding increases for the brain white and gray matter were 84% +/- 13% and 84% +/- 18%, respectively. CONCLUSIONS: This is the first time that P-gp activity at the human BBB has been measured. The modest inhibition of human BBB P-gp by cyclosporine has implications for P-gp-based drug interactions at the human BBB. Our method for imaging P-gp activity can be used to identify multidrug-resistant tumors or to determine the contribution of P-gp polymorphism, inhibition, or induction to interindividual variability in drug response.     

Functional analysis of the human variants of breast cancer resistance protein: I206L, N590Y, and D620N.

Previous studies have shown that the V12M and Q141K variants of breast cancer resistance protein (BCRP) can affect expression and function of the transporter. In this study, the effects of the I206L, N590Y, and D620N variants on protein expression, plasma membrane localization, and transport activity of BCRP were investigated. Wild-type BCRP and the three variants were stably expressed in human embryonic kidney (HEK) cells. Confocal microscopy analysis showed that the three variants were predominantly routed to the plasma membrane of HEK cells. The expression level of I206L in the plasma membrane was approximately 45% of that of wild-type protein, whereas the N590Y and D620N levels were increased approximately 3.6-fold and 2.4-fold, respectively, as determined by immunoblotting. All three variants transported mitoxantrone, pheophorbide a, and BODIPY FL-prazosin. After normalization for differences in BCRP expression, I206L, N590Y, and D620N exhibited approximately 2-fold, 0.3-fold, and 0.5-fold wild-type efflux activities, respectively. The variants also conferred resistance to mitoxantrone and topotecan. Mitoxantrone and topotecan resistance by I206L and N590Y was approximately 2-fold and 0.3-fold of the wild-type BCRP resistance levels, respectively. Although D620N conferred a topotecan resistance similar to that of the wild-type protein, its level of mitoxantrone resistance was decreased by 50%. After normalization to BCRP expression levels, ATPase activities of I206L were not significantly different from those of wild-type protein, whereas N590Y and D620N exhibited approximately 30% and 50% of wild-type ATPase activities, respectively. These results suggest that I206L has the lowest protein expression and the highest activity, whereas N590Y and D620N display higher expression and lower activity, relative to wild-type BCRP.     

Human intestinal es nucleoside transporter: molecular characterization and nucleoside inhibitory profiles

Purpose: To clone and sequence the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter (es) from the human small intestine and to examine the capacities of nucleosides and nucleoside analogs to inhibit the uptake of uridine by this transporter. Methods: Using PCR, es was cloned from a cDNA library of the human small intestine. The uptake of ³H-uridine (10 μM) by the recombinant es, expressed in Xenopus oocytes, was measured in the presence (2 mM) and absence of nucleosides and nucleoside analogs. Results: The amino acid sequence of this es transporter was identical to that of the human placental es transporter. Uptake of ³H-uridine by this es transporter was inhibitable by 1 μM NBMPR. Removal of the oxygen from the 3′ position or from both the 2′ and 3′ positions, but not from 2′ or 5′ position, resulted in a partial or total loss of the capacity of the nucleosides to inhibit ³H-uridine uptake. No modifications of the adenosine base or of the uridine base (except for 3 and 6 positions on uracil) affected nucleoside inhibitory capacity. Conclusions: The es transporters of the human intestine and placenta are identical in their amino acid sequences. Moreover, the inhibitory profiles of various nucleoside analogs in inhibiting the uptake of uridine by the intestinal es transporter are similar to those obtained with the as-yet-uncloned human erythrocyte es transporter. Collectively, these findings suggest that the es transporter does not appear to be functionally variant in the human placenta, small intestine or erythrocytes. Received 24 March 1999 / Accepted: 4 October 1999     

Structure-inhibitory profiles of nucleosides for the human intestinal N1 and N2 Na+-nucleoside transporters

Purpose: To determine the structure-inhibitory profiles of nucleosides for the N1 and N2 Na⁺-nucleoside transporters of the human intestine. Methods: The uptake of ³H-labeled prototypic substrates of the N1 (inosine) and N2 (thymidine) transporters into human intestinal brush border membrane vesicles was measured by a rapid filtration technique in the presence and absence of various uridine and adenosine analogs and antiviral and anticancer nucleoside drugs (100 and 1000 μM). Results: In the ribose ring, the 3′-oxygen is required for inhibition of uptake of nucleosides by both the N1 and N2 transporters. The structural requirements for such inhibition differ with respect to modifications on the 5′ position of the sugar ring or on the base. The N2 transporter is more tolerant to these substitutions than is the N1 transporter. The 6 position on uracil and the 8 position on adenine are critical for inhibition of uptake of nucleosides by both the N1 and N2 nucleoside transporters. Conclusions: These data are the first evidence that the binding site(s) of the human N1 and N2 transporters differ in their interaction with analogs of their common substrates, uridine and adenosine. Such studies can provide insight into the critical structural determinants of the substrate necessary for recognition by the Na⁺-nucleoside transporters of the human intestine. Received: 16 August 1999 / Accepted: 24 May 2000     

Pharmacokinetics of Azithromycin Administered Alone and with Atovaquone in Human Immunodeficiency Virus-Infected Children

To evaluate if atovaquone (ATQ) interacts pharmacokinetically with azithromycin (AZ) in human immunodeficiency virus-infected children, 10 subjects (ages, 4 to 13 years) were randomized in a crossover study to receive AZ (5 mg/kg/day) alone (ALONE) or AZ (5 mg/kg/day) and ATQ (30 mg/kg/day) simultaneously (SIM) prior to receiving AZ and ATQ staggered by 12 h. Despite a lack of significant difference in the mean AZ pharmacokinetic parameters, the steady-state values of AZ's area under the concentration-time curve from 0 to 24 h and maximum concentration in serum were consistently lower (n = 7 of 7) for the SIM regimen than they were for the ALONE regimen. A larger study will be required to determine if ATQ affects AZ pharmacokinetics and efficacy in a clinically significant manner.     

Discharge Against Medical Advice of Elderly Inpatients in the United States

Discharge against medical advice (DAMA) is associated with greater risk of hospital readmission and higher morbidity, mortality, and costs, but with a rapidly increasing elderly inpatient population, there is a lack of national data on DAMA in this subgroup. The National Inpatient Sample (2003–2013 for trends, 2013 for multivariable analysis, n = 29,290,852) was used to describe trends in DAMA in elderly inpatients, to study diagnosis codes associated with admission, and to assess factors associated with DAMA using multivariable logistic regression. Odds ratios (ORs) and 95% confidence intervals (CIs) are reported for risk factors of interest. Although DAMA rates in individuals aged 65 and older were one fourth of those found in individuals aged 18 to 64, an increasing trend was found in both groups. From 2003 to 2013, rates increased in individuals aged 18 to 64 (from 1.44% to 1.78%) and in those aged 65 and older (from 0.37% to 0.42% (both P < .001). In both age groups, individuals admitted for mental illness had the highest risk of DAMA. Factors associated with higher adjusted odds of DAMA were generally similar between age groups, although risk of DAMA was higher in elderly adults than in those aged 18 to 64 for blacks (OR 1.65, 95% CI 1.49–1.82 vs OR 1.16, 95% CI 1.12–1.20), Hispanics (OR 1.58, 95% CI 1.41–1.77 vs OR 0.83, 95% CI 0.79–0.87), and those in the lowest income quartile (OR 1.57, 95% CI 1.43–1.72 vs OR 1.12, 95% CI 1.08–1.17), suggesting that race/ethnicity and poverty are more pronounced as risk factors for DAMA in elderly inpatients.     

Cost of Care for Patients With Pre-Existing Comorbidities Undergoing Total Joint Arthroplasty: A Retrospective Cohort Study Evaluating Disease-Specific Perioperative Care

BackgroundInvestigations suggest a relationship between increased resource utilization with disease burden and advanced age. However, it remains unknown the degree increased resource utilization is associated with pre-existing conditions, before complications occur.MethodsThis retrospective study identified total hip/knee arthroplasty cases in the Premier Database from 2006 to 2016 (N = 1,613,744), with hospitalization cost as the primary outcome. With a variable combining the conditions and complication, generalized linear models measured associations between condition/complication interaction groups and hospitalization cost. Estimates of percent cost increase by variable were obtained.ResultsAcross all conditions, an increase in cost ranging from 0.38% to 4.28% was found in the absence of a complication. The “Condition = No, Complication = Yes” group was associated with a range of 11.50%-12.40% increase in average hospitalization cost, and the range was 14.43%-30.85% for the “Condition = Yes, Complication = Yes” group.ConclusionWe found that having a high-risk condition without a complication accounted only for a modest hospitalization cost increase.     

National Institute on Drug Abuse Conference report on placental proteins, drug transport, and fetal development

The use of illicit and licit drugs during pregnancy is a major public health concern because of potential adverse effects on the fetus and the risk to maternal health. Because the placenta is the primary link between the mother and the conceptus and is essential for the growth and survival of the fetus, abnormalities in placental formation and function resulting from drug use could have a major influence on pregnancy outcome. At present, little information is available on the impact of abused drugs on placental biology alone or in combination with other "host" factors (eg, stress, infections). This prompted the National Institute on Drug Abuse (NIDA) to convene a meeting of experts in placental biology to review cutting-edge research with the mission to translate existing information to new clinical and research initiatives in the drug abuse field. This report summarizes the presentations and research recommendations resulting from the workshop discussions.     

Intestinal Absorption of Ribavirin Is Preferentially Mediated by the Na+-Nucleoside Purine (Nl) Transporter

Pharmaceutical Research -     

Simultaneous measurement of in vivo P-glycoprotein and cytochrome P450 3A activities

Digoxin and midazolam are routinely used as probe drugs to measure in vivo activity of P-glycoprotein (P-gp) and cytochrome P450 3A4/5 (CYP3A), respectively. We investigated whether digoxin and midazolam could be coadministered to simultaneously determine P-gp and CYP3A activity without a significant pharmacokinetic interaction. In a randomized crossover design, digoxin (0.5 mg oral) or midazolam (2.0 mg oral) was administered individually or in combination (digoxin 1 hour after midazolam) to 14 healthy volunteers. Blood and urine samples were collected for up to 48 hours. Pharmacokinetic parameters of digoxin, midazolam and 1'-OH midazolam were evaluated to determine the presence of an interaction. The geometric mean ratios of all measured pharmacokinetic parameters of digoxin and midazolam were not significantly affected by coadministration. Coadministration of digoxin and midazolam can be used to simultaneously phenotype P-gp and CYP3A activity without a significant pharmacokinetic interaction.