István Sziráki

Genetic determination of mesencephalic tyrosine hydroxylase activity in the mouse

The hereditary factors that affect mesencephalic tyrosine hydroxylase (TH) activity were investigated in highly inbred mouse strains (CXBI/ByJ, C57BL/6ByJ, and BALB/cJ). The progenitor strains and their F1 hybrids, were compared for mesencephalic TH activity with each other and with replicated F2 generations. Quantitative and non-parametric genetic analysis of the data raise the possibility that there is a major gene with robust additive effect that is primarily responsible for the difference between the progenitor strains with intermediate and high mesencephalic TH activity. Strain differences in mesencephalic TH activity have been linked to differences in number of dopamine (DA) neurons in that area. If genetic variation of mesencephalic TH activity is entirely attributable to variation in number of mesencephalic dopamine (DA) neurons, identification of the genetic sources of variation of mesencephalic TH activity may take us a step closer to animal models and preparations that are needed in the study of the physiological and constitutional mechanisms of human disorders in which DA neurotransmission is involved.

Transfer of brain dopamine system-specific quantitative trait loci onto a C57BL/6ByJ background

1Laboratory of Neurobehavioral Genetics, Nathan S. K1ine Institute, 140 Old Orangeburg Road, Orangeburg, New York 10962 2Department of Psychiatry, New York University Medical Center, 550 First Avenue, New York, New York 10016 3Research Genetics, 2130 Memorial Parkway, S.W., Huntsville, Alabama 35801 4Unit on Neurotoxicology & Neuroprotection, Laboratory of Clincal Science, NIMH, Bethesda, Maryland 20892 5Department of Psychiatry, Mt. Sinai School of Medicine, New York, NY 10029 61st Institute of Biochemistry, Semmelweis University Medical School, Budapest, H-1088, Hungary 7Research Department, Meninger Clinic, Box 829, Topeka, Kansas 66601 8Pharmacological Research Center, Chemical Works of Gedeon Richter Ltd., P. O. Box 27, Budapest, H-1475, Hungary 9Energotechno, Budapest, H-1024, Hungary

Efflux transporters in the blood–brain interfaces – in vitro and in vivo methods and correlations

Introduction: Sufficient brain exposure is crucial to the success of CNS drugs. The twofold greater attrition rate in clinical development of CNS drugs over the respective attrition rate of non-CNS drugs is due to lack of efficacy. It is generally thought that poor brain exposure is at least partly responsible for this, as the concentration–time profile at the brain target site is critical for efficacy. Efflux transporters in the blood–brain interfaces play a crucial role in modulation of permeability of drugs across these interfaces. Validation of preclinical tools to correctly predict brain exposure in humans is essential. Areas covered: This review summarizes in vitro and in vivo tools to detect and characterize interactions of drugs with efflux transporters relevant to blood–brain interfaces. Furthermore, the article discusses the strengths and weaknesses of these methods and the limitations of their application, in addition to covering in vitro – in vivo correlations. Expert opinion: A more detailed validation of in vitro efflux transporter assays employing primary brain endothelial cultures is needed. This should go along with mapping uptake transporters expressed in the blood–brain interfaces. With the availability of specific inhibitors, utilization of in vivo methods such as brain microdialysis is increasing. Once transporter-humanized mice are available, we may witness a further increase in applications of in vivo methods.

Verification of brain penetration of the unbound fraction of a novel HER2/EGFR dual kinase inhibitor (TAK-285) by microdialysis in rats

TAK-285, an investigational, orally active HER2/EGRF inhibitor is in clinical development for potential use in HER2 over-expressing metastatic breast cancer. The objective of the present work was to verify the presence of unbound TAK-285 in the rat brain after oral administration by a microdialysis technique with simultaneous sampling of blood and brain. In a pilot microdialysis experiment no detectable amount of TAK-285 was found in the brain dialysate samples after oral administration of the drug (50 mg/kg). A conventional pharmacokinetic study was performed simultaneously with the pilot microdialysis study using the same dosing suspension. TAK-285 was detected in the brain even at the last time point when the samples were taken from the animal at the end-point of the microdialysis experiment. The apparent absence of TAK-285 in blood and brain dialysate samples might be explained by a very low recovery of microdialysis probes for TAK-285 and/or by the adsorption of the compound to the outflow tubing of the microdialysis probes. Results of an in vitro recovery study with TAK-285 were indicative of the strong adsorption of the compound to the microdialysis tubings. Adding bovine serum albumin (4%, w/v) in perfusion fluids and reducing perfusion flow rate (from 1.0 μL/min to 0.5 μL/min) in in vitro experiments substantially improved the detectability of TAK-285 in dialysate samples. Application of new perfusion conditions resulted in a manifold increase of the relative recovery of the microdialysis set-up for TAK-285 (from 1.6% to 47%). Subsequent in vivo microdialysis experiments were performed using the modified perfusion conditions in animals dosed with TAK-285 (75 mg/kg, p.o.). Detectable level of unbound TAK-285 was found in the extracellular space in the brain as long as 24-28 h after administration of the drug. The brain-to-blood ratios of the unbound TAK-285 were 0.18 and 0.24 (calculated from the C(max) values or from the area under the curve [AUC] values) similarly to the brain-to-blood ratios of total TAK-285. On the basis of substantial brain penetration of unbound TAK-285, it is concluded that TAK-285 might have the potential in the treatment of brain metastases of HER2 over-expressing metastatic breast cancer. The methodological approach described here might help to solve similar problems in determination of brain penetration of other substances with strong adsorption to the tubing of microdialysis setups.

Genomic characterization of two introgression strains (B6.Cb4i5) for the analysis of QTLs

The effect of an individual locus that affects complex traits is difficult to detect because it is responsible for only a fraction of the total environmental, genetic, and interaction variation, and its contribution is therefore usually masked. In 1983, to diminish the above-mentioned difficulties, we began the development of a novel approach to QTL mapping, by providing the same standardized genetic background for each QTL that affects a particular trait, and by distributing the QTLs individually in recombinant quasi-congenic lines (Vadasz 1990; Vadasz et al. 1987, 1994a, 1994b). We assumed that the relatively low genetic signal of the distributed QTLs could be amplified in these lines by measuring a number of isogenic animals. To test this hypothesis, we constructed a series of inbred quasicongenic recombinant QTL introgression strains by repeated backcross-intercross cycles with concomitant selection for the extreme expressions of a quantitative trait, and fixation of the genes by strict brother-sister mating (Vadasz et al., 1994a). In the present pilot study, in order to assess the amount and distribution of the introduced donor genome, we tested the B6 background strain, the BALB/cJ (C) donor strain, and two strains of the B6.Cb4i5 series, which had been developed by four backcross-intercross cycles with concomitant selection for high midbrain tyrosine hydroxylase (TH) activity, followed by at least 19 generations of brother-sister matings. Currently, not including sublines, we have 14 strains of the B6.Cb4i5 series (Fn>20). We present evidence that the strains can be considered quasicongenic, inbred, and recombinant, and that the method has the potential for QTL mapping of both the differential trait and other quantitative traits affected by passenger genes. Also, we present phenotypic data with respect to the trait being selected for, and demonstrate that the two tested introgression strains have higher TH/MES than the background strain. Microsatellite markers were selected from the Whitehead Institute/MIT database (Dietrich et al. 1992; Whitehead Institute/ MIT 1994) on the basis of previously detected allelic differences between the B6 background and the C donor strain. A 10-cM (average) resolution map was created for two representative QTL introgression strains by testing 169 polymorphic markers in the B6.Cb4i5a-12A (a-12A) and B6.Cb4i5b-13 (b-13) strains and in their B6 background partner strain. PCR products were analyzed on 6% Nusieve agarose gels (Love et al. 1990). Because in the b-13 strain no PCR product was obtained for the most distal marker D19Mit6, we could not estimate the length of a segment on Chromosome (Chr) 19 marked by three consecutively positioned C-type alleles (D19Mit37, D19Mit36, D19Mit34). No heterozygous loci were found in the QTL introgression strains. The mapping results indicated that the maximum length of an introgressed donor segment, estimated by the distances of flanking background type markers from the centromere, varied from 3.3 cM to 22.5 cM on the Whitehead Institute/MIT map. The segments are shorter than the estimated average length of an introgressed chromosome segment that carries the differential locus after four backcrosses with concomitant selection for one donor-strain gene (39 cM; Ln 4 200 × (1–2)/n; n1 4 F1; Flaherty 1981), presumably because (i) during the development of the b4i5 series each backcross was followed by intercrosses (Vadasz et al. 1994a), and (ii) there were additional chances for recombination during fixation (Taylor 1978). Assuming one differential locus, the theoretically expected proportion of the fixed, nonselected, nonlinked donor genes was estimated as 3.0%, while the proportion of the linked donor genes is about 1.2% in inbred QTL introgression strains of the b4i5 series. Our results suggest that the total of the introduced donor genome carried by seven segments in each strain was about 73.7 cM (4.6%) in the a-12A strain, and (assuming that the problematic D19Mit6 marker was of B6-type) was about 117.3 cM (7.3%) in the b-13 strain. The a-12A strain carried C-type chromosome segments on Chrs 2, 8, 9 (2 segments), 13 (2 segments), and 18. The beta-13 strain carried C-type chromosome segments on Chrs 1, 2, 7 (2 segments), 15, 18, and 19. If the length of an introgressed chromosome segment after four backcrosses is estimated as 39 cM, and if during inbreeding four crossovers per 100 cM occurred (Taylor 1978), then the introgressed segment had a 1.5 chance on the average for meiotic recombination before fixation. Therefore, if the length of the differential segment is approximately 26 cM after fixation, and there are two unlinked QTLs with major effects (Vadasz et al. 1994a), the total length of the donor material (including the nonselected, nonlinked donor genes estimated as 3% of 1600 cM) would be about 100 cM. Assuming that the 169 microsatellite markers are randomly distributed in the genome of 1600 cM, the proportion (P) of the genome lying within ±10 cM from the markers is 88% (P 4 1 − e; Jacob et al. 1991). The above estimates indicate that about 12% of the donor segments have not been detected, and that about 240 markers will be needed to ensure that 95% of the genome will lie within ±10 cM of a marker. A detailed summary of the genotyping results of the informative polymorphic markers is provided in Fig. 1 and Fig. 2. Two loci were of identical C-type in the two strains: D2Nds1 (50 cM from the centromere, on a maximum 3.3 cM long segment) and D18Mit107 (26.2 cM from the centromere, on a maximum 9 cM [a-12A] and on a maximum 12.4 cM [b-13] long segment). If the probability of detecting a fixed, nonselected, nonlinked donor gene in one of the b4i5 strains is about 3.05% (p 4 0.0305), then their joint probability of occurrence is p 4 0.00093, while the probability of retaining in two strains the same passenger gene residing 10 cM from the differential gene is p > 0.3 (Flaherty 1981). Thus, it is possible that a maximum 3.3-cM region about 50 cM from the Correspondence to: C. Vadasz Mammalian Genome 7, 545–548 (1996).

Analysis of the Mesotelencephalic Dopamine System by Quantitative-Trait Locus Introgression

One of the significant factors that affect brain dopamine function is the activity of tyrosine hydroxylase (TH), the first and rate-limiting enzyme in catecholamine biosynthesis. For the analysis of the genetically determined role of dopamine function and TH in behavior and in the regulatory mechanisms of the mesotelencephalic dopamine system we devised a novel genetic strategy (Vadasz; Mouse Genome 88:16–18; 1990). We hypothesized that phenotypic introgression and recombinant fixation could ensure the transfer of Quantitative Trait Loci (QTL) from one strain onto the genetic background of another strain, and new, genetically very similar quasi-congenic strains could be created that would carry individual QTLs, or QTLs in various combinations. Here we summarize the construction of the first set of QTL Introgression strains, and present evidence that QTLs that are responsible for the continuous variation of mesencephalic tyrosine hydroxylase activity (TH/MES), have been transferred onto the C57BL/6By (B6) strain background from BALB/cJ (C) and CXBI (I) donor strains with high and low TH/MES, respectively. The QTL transfer was carried out in two directions by repeated backcross-intercross cycles with concomitant selection for the extreme high and low expressions of TH/MES in replicates, resulting in four QTL Introgression lines. Analysis of regional brain TH activities in the course of the QTL introgression indicated that (a) TH activity in B6.I lines showed quite limited heritability, (b) TH/MES was not highly correlated with striatal TH, and (c) the control of hypothalamic and olfactory tubercle TH activities was largely independent from that of TH/MES. Examination of the open-field (OF) behavior data demonstrated that TH activity did not correlate significantly with OF behavior. After 5 backcross-intercross cycles, TH/MES in each replicate line was still significantly different from that of the B6 background strain. A genomewide scanning of microsatellite markers in the QTL introgression lines demonstrated that about 96% of the markers were of background (B6) type. These results indicate the successful transfer of TH/MES QTLs. After the QTL transfer phase of the experiment altogether more than 100 new RQI strains were initiated in the QTL Introgression lines by strict brother × sister mating. After fixing the introgressed QTLs, ten of the inbred RQI strains were tested for TH/MES. The results showed that in one of the new RQI strains TH/MES was restored to a level that is characteristic to the C donor strain, while TH/MES values in some other strains were between those of the background and donor strains, confirming our hypothesis that phenotypic introgression and recombinant fixation can ensure a virtually complete transfer of QTLs. We conclude from this study that complex, continuously distributed neural traits can successfully be subjected to QTL introgression, and the results raise the possibility that the RQI method can be efficiently applied for gene mapping of complex neural and behavioral traits even if their phenotypic expression is sensitive to confounding developmental and environmental variations, genetic interactions, and genotype-environment interactions.

Genetic determination of striatal tyrosine hydroxylase activity in mice

An additive major gene effect is described for tyrosine hydroxylase activity in mouse corpus striatum (CS). Quantitative genetic analysis indicated the presence of a segregating Mendelian factor with robust additive effect in F2 generations derived from crossing two highly inbred mouse strains, C57BL/6ByJ and BALB/ cJ, with intermediate (INT) and high (HI) TH activity in CS. Significant positive correlation was found between striatal and mesencephalic TH activity in the segregating generations, raising the possiblity that a common single gene may express its effect through pleiotropy or linkage. Genetic preparations taking advantage of the major gene effect should serve well as animal models of DA-mediated neuropsychiatric disorders.

Genetic determination of hypothalamic tyrosine hydroxylase activity in mice

Tyrosine hydroxylase (TH) activity data obtained from hypothalamic tissue samples of highly inbred mouse strains with known differences in their mesencephalic TH activity (BALB/cJ, C57BL/6ByJ, CXBI/ByJ), F1 hybrids and F2 generations were subjected to quantitative genetic analysis. No differences were observed between C57BL/6ByJ and CXBI/ByJ strains, but highly significant differences were found in hypothalamic TH activity between BALB/cJ and C57BL/6ByJ strains. Segregating genetic factors could not be detected in the replicate (C57BL/6ByJ X CXBI/ByJ) F2 generations, while the presence of segregating genetic units was indicated in the (C57BL/6ByJ X BALB/cJ)F2 population. Estimation of minimum number of genes and Elstons non-parametric one-locus test reveal that more genes are responsible for strain differences of TH activity in the hypothalamus compared to the dopaminergic areas of the mesotelencephalon. The results indicate that the heterogeneity of the catecholamine neuronal populations and terminal fields in the hypothalamus is reflected by the complex nature of the genetic control of TH activity in this brain region.

The use of microdialysis techniques in mice to study P-gp function at the blood-brain barrier

An integrated assay system involving dual/triple-probe microdialysis techniques in rats was developed earlier for testing interactions with P-glycoprotein (P-gp) at the blood-brain barrier using quinidine/PSC-833 as a P-gp substrate/inhibitor combination. The aim of the present study was to expand our assay system to mice using microdialysis with simultaneous sampling of blood and brain and to compare the result with a primary mouse brain endothelial cell monolayer (pMBMEC) assay. Brain penetration of quinidine was dose dependent in both anesthetized and awake mice after intraperitoneal drug administration. PSC-833 pretreatment caused a 2.5- to 3.4-fold increase in quinidine levels of brain dialysate samples in anesthetized or awake animals, after single or repeated administration of PSC-833. In pMBMEC, a 2.0- to 2.5-fold efflux ratio was observed in the transcellular transport of quinidine. The P-gp–mediated vectorial transport of quinidine was eliminated by PSC-833. These results indicate that quinidine with PSC-833 is a good probe substrate-reference inhibitor combination for testing drug-drug interactions with P-gp in the in vivo and in vitro mouse systems. With increasing number of humanized transgenic mice, a test system with mouse microdialysis experimentation becomes more important to predict drug-drug interactions in humans.