Peter Steinmetzer

Application of cryopreserved precision-cut liver slices in pharmacotoxicology — principles, literature data and own investigations with special reference to CYP1A1-mRNA induction

Principle steps necessary for cryopreservation of precision-cut liver slices as currently applied by different groups are summarized including own results concerning mode of freezing. Now we use rapid freezing by immersion in liquid nitrogen after exposure to 10% DMSO as the cryoprotectant for rat liver slices. The results indicate well-maintained cytochrome P450 (CYP)-dependent deethylation rates in slice homogenate after short-term incubation. ECOD rate in intact thawed slices was even higher than in fresh ones after 2 h incubation. In contrast to fresh slices all parameters except protein content decreased to marginal levels during long-term incubation of thawed slices for 24 h. The first preliminary experiments on albumin secretion by thawed rat liver slices, measured between the 2nd and the 4th hour of incubation, showed partial maintenance of this liver specific differentiated function. Trials to induce CYP1A1 in thawed rat liver slices in vitro by beta-naphthoflavone (BNF) resulted in increased expression of CYP1A1-mRNA within 6 h as shown by RT-PCR and quantified by competitive RT-PCR. The decline of deethylation rates, determined in slice homogenates, and of viability within 24 h incubation was not prevented by exposure to BNF or DMSO. The results derived from one sample of cryopreserved human liver slices indicate a quite acceptable maintenance of function up to 6 h, if the same protocol as developed for rat liver slices was used.

Monooxygenation, cytochrome P450-mRNA expression and other functions in precision-cut rat liver slices

Precision-cut rat liver slices (KRUMDIECK slicer, slice thickness 200-250 microm) were incubated in rollers containing modified Williams medium E at 37 degrees C for 2, 24 and 48 hrs. Protein, DNA, potassium and glutathione concentrations did not decrease during 48 hrs. Lactate dehydrogenase (LDH) leakage into the medium was relatively marked during the first 2 hrs of incubation, from the 2nd to the 48th hr LDH leakage was very low. The same is true of the release of thiobarbituric acid-reactive substances. Albumin synthesis and transport into the medium decreased to about 70% after 48 hrs. Cytochrome P450 (CYP)-dependent 7-ethoxycoumarin O-deethylation rate was relatively stable up to 48 hrs, whereas testosterone hydroxylation decreased significantly without alterations of the proportions of the 7 quantified hydroxylated metabolites. After exposure of the slices to beta-naphthoflavone for 6 hrs CYP1A1-mRNA expression, measured by competitive RT-PCR, was increased by a factor of at least 1000. Precision-cut liver slices are a useful tool for the study of various hepatic functions, drug metabolism and its induction in vitro.

Induction of cytochrome P450 2B1-mRNA and pentoxyresorufin O-depentylation after exposure of precision-cut rat liver slices to phenobarbital

Precision-cut rat liver slices were prepared from male Wistar rats with a Krumdieck slicer and cultured in Williams medium E for up to 24 h. In untreated control slices, CYP2B1-mRNA concentration, which was quantified by competitive RT-PCR, did not decrease during this time. After exposure of the slices to 100 microM phenobarbital, CYP2B1-mRNA increased by about 10- or 60-fold after 6 or 24 h, respectively. The extent of this in vitro induction was similar to that after in vivo administration of 60 mg/kg phenobarbital. Pentoxyresorufin O-depentylation (PROD) was also inducible in vitro after 24 h, but to a lesser extent than the corresponding CYP-mRNA. Precision-cut liver slices proved to be a simple and reliable in vitro system for the sensitive detection of an induction by phenobarbital.

Use of Fresh and Cryopreserved Human Liver Slices in Toxicology with Special Reference to In vitro Induction of Cytochrome P450

Human liver slices were prepared with a Krumdieck slicer from macroscopically healthy surgical waste after partial hepatectomy. They were incubated without or with the addition of the inducer beta-naphthoflavone (BNF) (25 mum) either immediately after preparation (fresh slices) for up to 24 hours or after cryopreservation in liquid nitrogen (thawed slices) for up to 6 hours. Potassium concentration was well maintained in fresh and thawed slices over 24 and 6 hours, respectively, but at lower levels than in rat liver slices. Albumin secretion showed relatively large interindividual differences. Both parameters were lower in thawed slices than in fresh ones, but indicated a certain number of viable cells. In untreated fresh slices CYP1A1-mRNA was not detectable; however, it increased distinctly within 6 hours of exposure to BNF. The amounts of induced CYP1A1-mRNA differed by a factor of more than 100 among six human livers and were lower than in fresh rat liver slices. Even in thawed human slices, CYP1A1-mRNA expression could be induced in vitro by BNF, although at a very low level and preferentially in those specimens with comparably high inducibility already before freezing.

Modification of the N-terminal sulfonyl residue in 3-amidinophenylalanine-based matriptase inhibitors.

Replacement of the N-terminal beta-alanyl-amide moiety in previously identified matriptase inhibitors by non-charged aryl groups caused a slightly decreased potency and partially reduced selectivity, especially towards thrombin. However, some of these analogues are still potent matriptase inhibitors with K(i)-values <10nM. In contrast, improved activity was observed for newly designed tribasic analogues, especially for compound 21, which inhibits matriptase with an K(i)-value of 80pM.

Highly Potent and Selective Substrate Analogue Factor Xa Inhibitors Containing D‐Homophenylalanine Analogues as P3 Residue: Part 2

A series of highly potent substrate‐analogue factor Xa inhibitors containing D‐homophenylalanine analogues as the P3 residue has been identified by systematic optimization of a previously described inhibitor structure. An initial lead, benzylsulfonyl‐d‐hPhe‐Gly‐4‐amidinobenzylamide (3), inhibits fXa with an inhibition constant of 6.0 nM. Most modifications of the P2 amino acid and P4 benzylsulfonyl group did not improve the affinity and selectivity of the compounds as fXa inhibitors. In contrast, further variation at the P3 position led to inhibitors with significantly enhanced potency and selectivity. Inhibitor 27, benzylsulfonyl‐D‐homo‐2‐pyridylalanyl(N‐oxide)‐Gly‐4‐amidinobenzylamide, inhibits fXa with a Ki value of 0.32 nM. The inhibitor has strong anticoagulant activity in plasma and doubles the activated partial thromboplastin time and prothrombin time at concentrations of 280 nM and 170 nM, respectively. Compound 27 inhibits the prothrombinase complex with an IC50 value of 5 nM and is approximately 50 times more potent than the reference inhibitor DX‐9065a in this assay.

RT-PCR-based evidence for the in vivo stimulation of renal tubular p-aminohippurate (PAH) transport by triiodothyronine (T3) or dexamethasone (DEXA) in kidney tissue of immature and adult rats

Our previous studies have shown that a pre-treatment of rats with triiodothyronine (T3) or dexamethasone (DEXA) increases renal PAH excretion significantly. This stimulation was accompanied by an enhanced protein synthesis within the renal cortex. To explore the molecular basis for this sub-chronic induction process, we investigated the stimulation of PAH accumulation in renal cortical slices as well as the expression level of organic anion transporter 1 (OAT1), the recently cloned renal basolateral PAH-transporter, using RT-PCR techniques under the applied conditions. 10- and 55-day-old Han:WIST rats were treated in vivo with T3 (20 microg/100 g b.wt.) or DEXA (60 microg/100 g b.wt.), both for 3 days, once daily. Renal cortical slices were incubated for 2 hours in Cross-Taggart medium and PAH uptake into kidney tissue was measured time dependently (slice to medium ratio, QS/M). The accumulation capacity is comparable between immature and mature rats (control-QS/M: 6.7 +/- 0.1 vs. 6.9 +/- 0.2, respectively). Both age groups showed a significant increase of PAH accumulation capacity after T3 treatment (10-day-old rats: 15.0 +/- 0.2; 55-day-old rats: 11.7 +/- 1.3). After DEXA pre-treatment, PAH accumulation was only slightly changed (10-day-old rats: 5.9 +/- 0.2; 55-day-old rats: 8.2 +/- 1.3). Semi-quantitative measurements of OAT1 mRNA expression level showed a significant increase of OAT1 mRNA after pre-treatment with both T3 and DEXA in the two age groups. Thus, this is the first evidence that T3 and DEXA pre-treatment induces the expression of OAT1.

Incorporation of neutral C-terminal residues in 3-amidinophenylalanine-derived matriptase inhibitors

A novel series of matriptase inhibitors based on previously identified tribasic 3-amidinophenylalanine derivatives was prepared. The C-terminal basic group was replaced by neutral residues to reduce the hydrophilicity of the inhibitors. The most potent compound 22 inhibits matriptase with a K(i) value of 0.43 nM, but lacks selectivity towards factor Xa. By combination with neutral N-terminal sulfonyl residues several potent thrombin inhibitors were identified, which had reduced matriptase affinity.

In vitro induction of cytochrome P450 2B1- and 3A1-mRNA and enzyme immunostaining in cryopreserved precision-cut rat liver slices

With the exception of cytochrome P450 (CYP) 1A1 and its mRNA, in vitro induction of other CYP forms has not been demonstrated in cryopreserved liver slices until now. Therefore precision-cut rat liver slices were cultured after cryopreservation and thawing in Williams medium E for up to 24 h in the presence of inducers to demonstrate CYP2B1- and CYP3A1-mRNA induction. CYP-mRNA expression was determined by competitive RT-PCR. Exposure to 100 microM phenobarbital caused a more than 20-fold increase in CYP2B1-mRNA expression within 24 h, reaching concentrations comparable with those of PB-exposed fresh rat liver slices. Exposure to 1 microM pregnenolone 16 alpha-carbonitrile enhanced CYP3A1-mRNA expression by more than 30-fold within 24 h. This is in the same range, although with higher variability, as detected with fresh liver slices. In spite of considerable variability among the thawed slices, the induction factors are high enough for a sensitive detection of an induction at mRNA level. Additionally, immunostaining of respective CYP-forms was performed in sections of few samples, indicating CYP increase in viable cells of cryopreserved slices.

Correlating structure and ligand affinity in drug discovery: a cautionary tale involving second shell residues

Abstract A high-resolution crystallographic structure determination of a protein–ligand complex is generally accepted as the ‘gold standard’ for structure-based drug design, yet the relationship between structure and affinity is neither obvious nor straightforward. Here we analyze the interactions of a series of serine proteinase inhibitors with trypsin variants onto which the ligand-binding site of factor Xa has been grafted. Despite conservative mutations of only two residues not immediately in contact with ligands (second shell residues), significant differences in the affinity profiles of the variants are observed. Structural analyses demonstrate that these are due to multiple effects, including differences in the structure of the binding site, differences in target flexibility and differences in inhibitor binding modes. The data presented here highlight the myriad competing microscopic processes that contribute to protein–ligand interactions and emphasize the difficulties in predicting affinity from structure.