Heterozygous carriers of germline loss-of-function variants in the tumor suppressor gene checkpoint kinase 2 (CHEK2) are at an increased risk for developing breast and other cancers. While... Show moreHeterozygous carriers of germline loss-of-function variants in the tumor suppressor gene checkpoint kinase 2 (CHEK2) are at an increased risk for developing breast and other cancers. While truncating variants in CHEK2 are known to be pathogenic, the interpretation of missense variants of uncertain significance (VUS) is challenging. Consequently, many VUS remain unclassified both functionally and clinically. Here we describe a mouse embryonic stem (mES) cell-based system to quantitatively determine the functional impact of 50 missense VUS in human CHEK2. By assessing the activity of human CHK2 to phosphorylate one of its main targets, Kap1, in Chek2 knockout mES cells, 31 missense VUS in CHEK2 were found to impair protein function to a similar extent as truncating variants, while 9 CHEK2 missense VUS resulted in intermediate functional defects. Mechanistically, most VUS impaired CHK2 kinase function by causing protein instability or by impairing activation through (auto)phosphorylation. Quantita-tive results showed that the degree of CHK2 kinase dysfunction correlates with an increased risk for breast cancer. Both dam-aging CHEK2 variants as a group [OR 2.23; 95% confidence interval (CI), 1.62-3.07; P < 0.0001] and intermediate variants (OR 1.63; 95% CI, 1.21-2.20; P = 0.0014) were associated with an increased breast cancer risk, while functional variants did not show this association (OR 1.13; 95% CI, 0.87-1.46; P = 0.378). Finally, a damaging VUS in CHEK2, c.486A>G/p.D162G, was also identified, which cosegregated with familial prostate cancer. Alto-gether, these functional assays efficiently and reliably identified VUS in CHEK2 that associate with cancer. Significance: Quantitative assessment of the functional conse-quences of CHEK2 variants of uncertain significance identifies damaging variants associated with increased cancer risk, which may aid in the clinical management of patients and carriers. Show less
In this thesis population pharmacokinetic and physiologically-based pharmacokinetic (PBPK) approaches were applied to investigate the influence of glomerular filtration (GF) and active tubular... Show moreIn this thesis population pharmacokinetic and physiologically-based pharmacokinetic (PBPK) approaches were applied to investigate the influence of glomerular filtration (GF) and active tubular secretion (ATS) on renal clearance in children. For this investigation, the contributions of passive (i.e. GF) and active (i.e. ATS) processes to renal clearance are considered. Both processes contribute to pediatric renal clearance and are expected to be influenced by developmental changes. Hence, the extent to which these developmental changes impact renal clearance is explored in pediatric populations using clinical data of existing drugs, and using a PBPK-based framework for hypothetical drugs with an array of different properties excreted by either GF or both GF and ATS. Show less
Cristea, S.; Krekels, E.H.J.; Allegaert, K.; Paepe, P. de; Jaeger, A. de; Cock, P. de; Knibbe, C.A.J. 2021
To date, information on the ontogeny of renal transporters is limited. Here, we propose to estimate the in vivo functional ontogeny of transporters using a combined population pharmacokinetic ... Show moreTo date, information on the ontogeny of renal transporters is limited. Here, we propose to estimate the in vivo functional ontogeny of transporters using a combined population pharmacokinetic (popPK) and physiology-based pharmacokinetic (PBPK) modeling approach called popPBPK. Clavulanic acid and amoxicillin were used as probes for glomerular filtration, combined glomerular filtration, and active secretion through OAT1,3, respectively. The predictive value of the estimated OAT1,3 ontogeny function was assessed by PBPK predictions of renal clearance (CLR) of other OAT1,3 substrates: cefazolin and piperacillin. Individual CL(R)post-hoc values, obtained from a published popPK model on the concomitant use of clavulanic acid and amoxicillin in critically ill children between 1 month and 15 years, were used as dependent variables in the popPBPK analysis. CLR was re-parameterized according to PBPK principles, resulting in the estimation of OAT1,3-mediated intrinsic clearance (CLint,OAT1,3,invivo) and its ontogeny. CLint,OAT1,3,invivo ontogeny was described by a sigmoidal function, reaching half of adult level around 7 months of age, comparable to findings based on renal transporter-specific protein expression data. PBPK-based CLR predictions including this ontogeny function were reasonably accurate for piperacillin in a similar age range (2.5 months-15 years) as well as for cefazolin in neonates as compared to published data (%RMSPE of 21.2 and 22.8%, respectively and %PE within +/- 50%). Using this novel approach, we estimated an in vivo functional ontogeny profile for CLint,OAT1,3,invivo that yields accurate CLR predictions for different OAT1,3 substrates across different ages. This approach deserves further study on functional ontogeny of other transporters. Show less
Glomerular filtration (GF) and active tubular secretion (ATS) contribute to renal drug elimination, with the latter remaining understudied across the pediatric age range. Therefore, we... Show moreGlomerular filtration (GF) and active tubular secretion (ATS) contribute to renal drug elimination, with the latter remaining understudied across the pediatric age range. Therefore, we systematically analyzed the influence of transporter ontogeny on the relative contribution of GF and ATS to renal clearance CL(R)for drugs with different properties in children. A physiology-based model for CL(R)in adults was extrapolated to the pediatric population by including maturation functions for the system-specific parameters. This model was used to predict GF and ATS for hypothetical drugs with a range of drug-specific properties, including transporter-mediated intrinsic clearance (CLint,T) values, that are substrates for renal secretion transporters with different ontogeny patterns. To assess the impact of transporter ontogeny on ATS and total CLR, a percentage prediction difference (%PD) was calculated between the predicted CL(R)in the presence and absence of transporter ontogeny. The contribution of ATS to CL(R)ranges between 41 and 90% in children depending on fraction unbound and CL(int,T)values. If ontogeny of renal transporters is < 0.2 of adult values, CL(R)predictions are unacceptable (%PD > 50%) for the majority of drugs regardless of the pediatric age. Ignoring ontogeny patterns of secretion transporters increasing with age in children younger than 2 years results in CL(R)predictions that are not systematically acceptable for all hypothetical drugs (%PD > 50% for some drugs). This analysis identified for what drug-specific properties and at what ages the contribution of ATS on total pediatric CL(R)cannot be ignored. Drugs with these properties may be sensitivein vivoprobes to investigate transporter ontogeny. Show less
To understand the drivers in the biological system response to dopamine D2 receptor antagonists, a mechanistic semiphysiologically based (PB) pharmacokinetic-pharmacodymanic (PKPD) model was... Show moreTo understand the drivers in the biological system response to dopamine D2 receptor antagonists, a mechanistic semiphysiologically based (PB) pharmacokinetic-pharmacodymanic (PKPD) model was developed to describe prolactin responses to risperidone (RIS) and its active metabolite paliperidone (PAL). We performed a microdialysis study in rats to obtain detailed plasma, brain extracellular fluid (ECF), and cerebrospinal fluid (CSF) concentrations of PAL and RIS. To assess the impact of P-glycoprotein (P-gp) functioning on brain distribution, we performed experiments in the absence or presence of the P-gp inhibitor tariquidar (TQD). PK and PKPD modeling was performed by nonlinear mixed-effect modeling. Plasma, brain ECF, and CSF PK values of RIS and PAL were well described by a 12-compartmental semi-PBPK model, including metabolic conversion of RIS to PAL. P-gp efflux functionality was identified on brain ECF for RIS and PAL and on CSF only for PAL. In the PKPD analysis, the plasma drug concentrations were more relevant than brain ECF or CSF concentrations to explain the prolactin response; the estimated EC50 was in accordance with reports in the literature for both RIS and PAL. We conclude that for RIS and PAL, the plasma concentrations better explain the prolactin response than do brain ECF or CSF concentrations. This research shows that PKPD modeling is of high value to delineate the target site of drugs. Show less
