Applicability of free drug hypothesis to drugs with good membrane permeability that are not efflux transporter substrates: A microdialysis study in rats

Abstract

This is an open access article under the terms of the Creat ive Commo ns Attri butio n-NonCo mmerc ial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.Abbreviations: ACD, acid citrate dextrose; aCSF, artificial cerebrospinal fluids; ATP, adenosine triphosphate; BBB, blood-brain barrier; BCRP, breast cancer resistance protein; C csf , concentration in cerebrospinal fluids; CL, clearance; C m,blood/brain , unbound concentration in blood/brain measured by microdialysis; CNS, central nervous system; CSF, cerebrospinal fluids; C ss , steady-state concentration; C u,brain , unbound concentration in brain measured by equilibrium dialysis; DMEM, Dulbecco's Modified Eagle's Medium; DMSO, dimethyl sulfoxide; EDTA-K 2 , ethylenediaminetetraacetic acid dipotassium; ER, efflux ratio; FBS, Fetal bovine serum; f u,brain , unbound fraction in brain; HBSS, Hank's balanced salt solution;HPLC/MS/MS, high-performance liquid chromatography combined with tandem mass spectrometry; HP-β-CD, hydroxypropyl-β-cyclodextrin; IACUC, institutional animal care and use committee; ISF, interstitial fluids; K p,uu,brain , ratio of unbound brain concentration to unbound blood AbstractIn clinical pharmacology, the free drug hypothesis has been widely applied in the interpretation of the relationship between pharmacokinetics and pharmacodynamics (PK/ PD). The free drug hypothesis assumes that the unbound drug concentration in blood is the same as that in the site of action at steady state. The objective of this study is to demonstrate whether the free drug hypothesis is universally applicable for all drugs.The unbound concentrations of the 18 compounds in blood and in brain interstitial fluids (ISF) at steady state following constant intravenous infusion were simultaneously monitored up to 6 hours via in vivo microdialysis technique. Based on the permeability and efflux ratio (ER), the test compounds can be divided into two classes.Class I includes the compounds with good membrane permeability that are not substrates of efflux transporters (eg, P-gp, BCRP, and MRPs), whereas Class II includes the compounds that are substrates of efflux transporters. The steady-state unbound drug concentrations in blood, brain, and CSF are quantitatively very similar for Class I compounds, whereas the steady-state unbound concentrations in the brain and CSF are significantly lower than those in blood for Class II compounds. These results strongly suggest that the free drug hypothesis is not universal for all drugs but is only applicable for drugs with good permeability that are not substrates of efflux transporters. K E Y W O R D SBBB, efflux transporter, microdialysis, permeability, unbound concentration

Effects of membrane transport activity and cell metabolism on the unbound drug concentrations in the skeletal muscle and liver of drugs: A microdialysis study in rats

“…Note: T-test was performed for C m, liver versus C m, blood and C m, muscle versus C m, blood . Steady-state unbound drug concentrations in liver, muscle, and blood of Class II compounds that are efflux transporter substrates (n = 3, mean ± SD)As demonstrated in our previous study,6 all of the Class II compounds, ofloxacin, atenolol, quinidine, fexofenadine and digoxin are substrates of efflux transporters. Ofloxacin, atenolol, quinidine, and fexofenadine showed moderate to high plasma CL, while digoxin had a low CL in rats.…”

“…16,17 The mean values of plasma CL were 97. and 15.0 ml/min/kg, respectively). [24][25][26][27] As shown in our previous study, 6 With the exception of propranolol unbound concentrations in liver, the unbound drug concentrations of all compounds reached a steady state in blood, liver and skeletal muscle within 2 h after the start of infusion. Thus, the steady state unbound concentrations were calculated as the average of unbound concentrations from 2 h up to 6 h time points.…”

“…The unbound drug concentration was simultaneously measured by microdialysis in liver, muscle, and blood 31.1, 27.2, 27.5, 11.0, and 18.4 × 10 −6 cm/s, respectively, based on the parental MDCK cell assay. 6 Given the fact that these five Class I compounds are lipophilic and have good membrane permeability, the unbound drug concentrations of these compounds in the liver and skeletal muscle are expected to be quantitatively similar to that in blood, and the values of K puu,liver and K puu,muscle are expected to be close to 1.…”

“…As demonstrated in our previous study, the free drug hypothesis is not universally applicable for all drugs, but only for drugs with good permeability that are not substrates of efflux transporters. 6 The unbound concentration of an efflux transporter drug in tissues where efflux transporters are highly expressed is expected to be lower than that in blood. Similarly, the unbound concentration of a drug in an eliminating organ (such as liver) is also expected to be lower than that in blood when the drug is subject to an extensive cell metabolism.…”

Use of Intravenous Infusion Study Design to Simultaneously Determine Brain Penetration and Systemic Pharmacokinetic Parameters in Rats

“…f Data were obtained from Belpaire et al (1990). g Data were obtained from Chen et al (2020). h Data were obtained from Lemmer et al (1985).…”