siRNA-mediated knockdown of P450 oxidoreductase in rats: a tool to reduce metabolism by CYPs and increase exposure of high clearance compounds.

PURPOSE: To develop a tool based on siRNA-mediated knockdown of hepatic P450 oxidoreductase (POR) to decrease the CYP-mediated metabolism of small molecule drugs that suffer from rapid metabolism in vivo, with the aim of improving plasma exposure of these drugs. METHODS: siRNA against the POR gene was delivered using lipid nanoparticles (LNPs) into rats. The time course of POR mRNA knockdown, POR protein knockdown, and loss of POR enzyme activity was monitored. The rat livers were harvested to produce microsomes to determine the impact of POR knockdown on the metabolism of several probe substrates. Midazolam (a CYP3A substrate with high intrinsic clearance) was administered into LNP-treated rats to determine the impact of POR knockdown on midazolam pharmacokinetics. RESULTS: Hepatic POR mRNA and protein levels were significantly reduced by administering siRNA and the maximum POR enzyme activity reduction (~85%) occurred 2 weeks post-dose. In vitro analysis showed significant reductions in metabolism of probe substrates due to POR knockdown in liver, and in vivo POR knockdown resulted in greater than 10-fold increases in midazolam plasma concentrations following oral dosing. CONCLUSIONS: Anti-POR siRNA can be used to significantly reduce hepatic metabolism by various CYPs as well as greatly increase the bioavailability of high clearance compounds following an oral dose, thus enabling it to be used as a tool to increase drug exposure in vivo.

Endosomolytic bioreducible poly(amido amine disulfide) polymer conjugates for the in vivo systemic delivery of siRNA therapeutics.

Efficient siRNA delivery is dependent not only on the ability of the delivery vehicle to target a specific organ but also on its ability to enable siRNA entry into the cytoplasm of the target cells. Polymers with endosomolytic properties are increasingly being used as siRNA delivery vehicles due to their potential to facilitate endosomal escape and intracellular delivery. Addition of disulfide bonds in the backbone of these polymers was expected to provide degradability through reduction by glutathione in cytosol. This paper describes the synthesis of new endosomolytic bioreducible poly(amido amine disulfide) polymers whose lytic potential can be masked at physiological pH, but can be restored at acidic endosomal pH. These polymer conjugates gave good in vitro knockdown (KD) and did not demonstrate cytotoxicity in a MTS assay. Efficient mRNA KD for apolipoprotein B in mouse liver was observed with these polyconjugates following intravenous dosing.

CYP2C75-involved autoinduction of metabolism in rhesus monkeys of methyl 3-chloro-3'-fluoro-4'-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)amino]ethyl}-1,1'-biphenyl-2-carboxylate (MK-0686), a bradykinin B1 receptor antagonist.

After oral treatment (once daily) for 4 weeks with the potent bradykinin B(1) receptor antagonist methyl 3-chloro-3'-fluoro-4'-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)-amino]ethyl}-1,1'-biphenyl-2-carboxylate (MK-0686), rhesus monkeys (Macaca mulatta) exhibited significantly reduced systemic exposure of the compound in a dose-dependent manner, suggesting an occurrence of autoinduction of MK-0686 metabolism. This possibility is supported by two observations. 1) MK-0686 was primarily eliminated via biotransformation in rhesus monkeys, with oxidation on the chlorophenyl ring as one of the major metabolic pathways. This reaction led to appreciable formation of a dihydrodiol (M11) and a hydroxyl (M13) product in rhesus liver microsomes supplemented with NADPH. 2) The formation rate of these two metabolites determined in liver microsomes from MK-0686-treated groups was > or = 2-fold greater than the value for a control group. Studies with recombinant rhesus P450s and monoclonal antibodies against human P450 enzymes suggested that CYP2C75 played an important role in the formation of M11 and M13. The induction of this enzyme by MK-0686 was further confirmed by a concentration-dependent increase of its mRNA in rhesus hepatocytes, and, more convincingly, the enhanced CYP2C proteins and catalytic activities toward CYP2C75 probe substrates in liver microsomes from MK-0686-treated animals. Furthermore, a good correlation was observed between the rates of M11 and M13 formation and hydroxylase activities toward probe substrates determined in a panel of liver microsomal preparations from control and MK-0686-treated animals. Therefore, MK-0686, both a substrate and inducer for CYP2C75, caused autoinduction of its own metabolism in rhesus monkeys by increasing the expression of this enzyme.

Comparison of immortalized Fa2N-4 cells and human hepatocytes as in vitro models for cytochrome P450 induction.

Fa2N-4 cells have been proposed as a tool to identify CYP3A4 inducers. To evaluate whether Fa2N-4 cells are a reliable surrogate for cryopreserved human hepatocytes, we assessed the basal mRNA expression of 64 drug disposition genes in Fa2N-4 cells. Significant differences were found in the expression of major drug-metabolizing enzymes, nuclear receptors, and transporters between both cell types. Importantly, the expression of constitutive androstane receptor (CAR) and several hepatic uptake transporters was significantly lower (>50-fold) in Fa2N-4 cells, whereas the expression of pregnane X-receptor (PXR) and aryl hydrocarbon receptor (AhR) was similar between Fa2N-4 cells and human hepatocytes. By using an optimized induction assay for Fa2N-4 cells, CYP3A4 induction by rifampicin, the prototypical PXR activator, increased from 1.5- to 7-fold at the level of functional activity. With nine selected compounds, which are known inducers of CYP3A4 either via activation of PXR, CAR, or both, we evaluated CYP3A4 and CYP2B6 mRNA induction using Fa2N-4 cells and human hepatocytes. No response was observed in Fa2N-4 cells treated with the selective CAR activators 6-(4-chlorophenyl)imidazo[2,1-b][1,3]-thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime and artemisinin. CYP3A4 and CYP2B6 induction in Fa2N-4 cells were also low for phenytoin, phenobarbital, and efavirenz, which are dual activators of PXR/CAR. This finding was in agreement with the lack of expression of CAR. The EC(50) value for rifampicin-mediated CYP3A4 induction was 10-fold higher than that in human hepatocytes. This result could be attributed to the low expression of hepatic organic anion-transporting polypeptides OATP1B1 and OATP1B3 in Fa2N-4 cells. In summary, our findings identify limitations of Fa2N-4 cells as a predictive induction model.

Diclofenac hydroxylation in monkeys: efficiency, regioselectivity, and response to inhibitors.

The catalytic efficiency, regioselectivity, and response to chemical inhibitors of diclofenac (DF) hydroxylation in three Old World monkey liver microsomes (rhesus, cynomolgus, and African green monkey) are different from those determined with human liver microsomes. In contrast to the high affinity-high capacity (low Km-high Vmax) characteristics of DF 4'-hydroxylation in humans, this reaction proceeded in all monkey species with catalytic efficiencies >20-fold lower. However, DF 5-hydroxylation, a negligible reaction in human liver microsomes, was kinetically favored in monkeys mainly due to the increased Vmax values. Chemical inhibitors (reversible or mechanism-based) selective to human CYP3A4 and CYP2C9 failed to differentiate monkey orthologs involved in DF hydroxylation. Immunoinhibition studies with monoclonal antibodies against human CYPs revealed the major contribution of CYP2C and CYP3A to 4'- and to 5-hydroxylation, respectively, in rhesus and cynomolgus liver microsomes. However, in African green monkeys, in addition to CYP2C, CYP3A also appeared to be involved in 4'-hydroxylation. Further studies with recombinant rhesus and African green monkey CYP2C and CYP3A enzymes (rhesus CYP2C75, 2C74, and 3A64; African green monkey CYP2C9agm and CYP3A4agm) confirmed the major role of CYP enzymes of these two subfamilies in DF 4'- and 5-hydroxylation. Clearly, while monkey CYP2C and 3A enzymes retain the same substrate selectivity towards DF hydroxylation as their human orthologs, their altered catalytic efficiency and response to chemical inhibitors may indicate different structural features of active sites as opposed to human orthologs.

3-methylindole-induced toxicity to human bronchial epithelial cell lines.

Transfected BEAS-2B cells that express different cytochrome P450 enzymes were used to assess whether human bronchial epithelial cell lines are target cells for 3-methylindole (3MI)-induced damage. Four different transfected BEAS-2B lines overexpressing P450s 2A6, 3A4, 2F1, and 2E1 (B-CMV2A6, B-CMV3A4, B-CMV2F1, and B-CMV2E1), respectively, were compared. The B-CMV2F1 and B-CMV3A4 cells were the most susceptible to 3MI-mediated cytotoxicity, measured by leakage of lactate dehydrogenase into the medium after a 48-h incubation. The toxicity was ameliorated by pretreatment with 1-aminobenzotriazole (ABT). Depletion of glutathione with diethylmaleate decreased the onset and increased the extent of cell death with 3MI. Thus, 3MI is cytotoxic to immortalized bronchial epithelial cells overexpressing 2F1 without concomitant depletion of GSH, but depletion of GSH modestly enhances the cytotoxicity of 3MI to human lung cells. Additional studies clearly demonstrated that a low concentration of 3MI (10 micro M) induced apoptosis in BEAS-2B cells that was measured by DNA fragmentation, and apoptosis was inhibited by the presence of ABT. The B-CMV2F1 cells overexpressing 2F1 demonstrated increased apoptosis (measured by Annexin-V binding) at 24 h with 100 micro M 3MI. Therefore, CYP2F1 in human bronchial epithelial lung cells may bioactivate 3MI to 3-methyleneindolenine, which induces programmed cell death at relatively low concentrations. Human lung cells may be susceptible to this prototypical pneumotoxicant.

Capsaicinoids cause inflammation and epithelial cell death through activation of vanilloid receptors.

Capsaicinoids, found in less-than-lethal self-defense weapons, have been associated with respiratory failure and death in exposed animals and people. The studies described herein provide evidence for acute respiratory inflammation and damage to epithelial cells in experimental animals, and provide precise molecular mechanisms that mediate these effects using human bronchiolar and alveolar epithelial cells. Inhalation exposure of rats to pepper sprays (capsaicinoids) produced acute inflammation and damage to nasal, tracheal, bronchiolar, and alveolar cells in a dose-related manner. In vitro cytotoxicity assays demonstrated that cultured human lung cells (BEAS-2B and A549) were more susceptible to necrotic cell death than liver (HepG2) cells. Transcription of the human vanilloid receptor type-1, VR1 or TRPV1, was demonstrated by RT-PCR in all of these cells, and the relative transcript levels were correlated to cellular susceptibility. TRPV1 receptor activation was presumably responsible for cellular cytotoxicity, but prototypical functional antagonists of this receptor were cytotoxic themselves, and did not ameliorate capsaicinoid-induced damage. Conversely, the TRPV1 antagonist capsazepine, as well as calcium chelation by EGTA ablated cytokine (IL-6) production after capsaicin exposure. To address these seemingly contradictory results, recombinant human TRPV1 was cloned and overexpressed in BEAS-2B cells. These cells exhibited dramatically increased cellular susceptibility to capsaicinoids, measured using IL-6 production and cytotoxicity, and an apoptotic mechanism of cell death. Surprisingly, the cytotoxic effects of capsaicin in TRPV1 overexpressing cells were also not inhibited by TRPV1 antagonists or by treatments that modified extracellular calcium. Thus, capsaicin interacted with TRPV1 expressed by BEAS-2B and other airway epithelial cells to cause the calcium-dependent production of cytokines and, conversely, calcium-independent cell death. These results have demonstrated that capsaicinoids contained in pepper spray products produce airway inflammation and cause respiratory epithelial cell death. The mechanisms of these cellular responses to capsaicinoids appear to proceed via distinct cellular pathways, but both pathways are initiated by TRPV1.

An in vivo evaluation of amphiphilic, biodegradable peptide copolymers as siRNA delivery agents.

A series of amphiphilic, biodegradable polypeptide copolymers were prepared for the delivery of siRNA (short interfering ribonucleic acid). The molecular weight (or polymer chain length) of the linear polymer was controlled by reaction stoichiometry for the 11.5, 17.2, and 24.6 kDa polypeptides, and the highest molecular weight polypeptide was prepared using a sequential addition method to obtain a polypeptide having a molecular weight of 38.6 kDa. These polymers were used to prepare polymer conjugate systems designed to target and deliver an apolipoprotein B (ApoB) siRNA to hepatocyte cells and to help delineate the effect of polymer molecular weight or polymer chain length on siRNA delivery in vivo. A clear trend in increasing potency was found with increasing molecular weight of the polymers examined (at a constant polymer:siRNA (w/w) ratio), with minimal toxicity found. Furthermore, the biodegradability of these polymer conjugates was examined and demonstrates the potential of these systems as siRNA delivery vectors.

Development of a liver-targeted siRNA delivery platform with a broad therapeutic window utilizing biodegradable polypeptide-based polymer conjugates.

The greatest challenge standing in the way of effective in vivo siRNA delivery is creating a delivery vehicle that mediates a high degree of efficacy with a broad therapeutic window. Key structure-activity relationships of a poly(amide) polymer conjugate siRNA delivery platform were explored to discover the optimized polymer parameters that yield the highest activity of mRNA knockdown in the liver. At the same time, the poly(amide) backbone of the polymers allowed for the metabolism and clearance of the polymer from the body very quickly, which was established using radiolabeled polymers to demonstrate the time course of biodistribution and excretion from the body. The fast degradation and clearance of the polymers provided for very low toxicity at efficacious doses, and the therapeutic window of this poly(amide)-based siRNA delivery platform was shown to be much broader than a comparable polymer platform. The results of this work illustrate that the poly(amide) platform has a promising future in the development of a siRNA-based drug approved for human use.

Cytotoxicity of a Quinone-containing Cockroach Sex Pheromone in Human Lung Adenocarcinoma Cells.

The cytotoxic effects of blattellaquinone (BTQ), a sex pheromone produced by adult female German cockroaches, have been studied using human lung adenocarcinoma A549 cells. 1,4-Benzoquinone (BQ), a toxic chemical implicated in benzene toxicity, was used as a reference compound. Both BQ and BTQ showed comparable toxicity toward A549 cells, with LD50 values estimated to be 14 and 19 microM, respectively. These two compounds increased the formation of an oxidized fluorescent probe, 2',7'-dichlorofluorescein, but had no effect on the cellular GSSG level. Interestingly, BTQ increased the level of 8-epi-prostaglandin F2alpha and was 4-fold more efficient in depleting cellular GSH content than BQ. Of the five GSH adducts of BTQ isolated, three were identified as mono-GSH conjugates, and the other two were di-conjugates. Mass spectrometric and NMR analyses of the di-conjugates showed that the second GSH molecule displaced the isovaleric acid moiety, potentially via a nucleophilic substitution reaction. The ability of BTQ to conjugate a second GSH molecule without quinone regeneration indicated that it may be a more effective cross-linking agent than BQ. Future experiments may be needed to evaluate the overall safety of BTQ before the commercialization of the compound as a cockroach attractant.

Sp1 and Sp3 regulate basal transcription of the human CYP2F1 gene.

Selective transcription of the human CYP2F1 gene in lung tissues may control the susceptibilities of this organ to diverse pneumotoxicants and lung carcinogens. However, the mechanisms responsible for CYP2F1 organ-selective transcription have not been elucidated. The objectives of the current studies were to identify and characterize basal transcription elements within the TATA-less promoter region of CYP2F1. Four putative Sp1-like sites were identified in the CYP2F1 promoter. Competitive electrophoretic mobility shift assay analysis with mutated oligonucleotide probes and lung A549 cell nuclear extract, along with supershift studies using antibodies to either Sp1 or Sp3 proteins, demonstrated that all four sites formed three specific protein-DNA complexes. Mutations in any of the four core Sp1-like motifs abolished protein-DNA binding. Western blot analysis of both human tissues and cells showed that Sp1 was considerably higher in lung than liver and that Sp3 was much higher in liver than lung. Promoter activation of a luciferase reporter construct was sequentially increased by addition of each of the four Sp1-like motifs in lung A549 cells but not in liver HepG2 cells. Cotransfection of a Sp1 expression vector with the reporter construct dramatically increased luciferase activity in either A549 cells or Sp1-deficient Drosophila Schneider line 2 (SL-2) cells. However, similar cotransfections with an Sp3 expression vector failed to increase activity. Cotransfection of both the Sp1 and Sp3 expression vectors considerably decreased Sp1-mediated activity in A549 cells and abolished activity in SL-2 cells. Thus, these studies demonstrated that four Sp1-dependent proximal promoter elements drive organ-selective CYP2F1 gene transcription, and that Sp1 and Sp3 factors interact to modulate constitutive CYP2F1 transcription in lung cells.

Selective inhibition of dog hepatic CYP2B11 and CYP3A12.

In the present study, N-(alpha-methylbenzyl-)-1-aminobenzotriazole (MBA) and ketoconazole (KET) were identified as the inhibitors with selectivity toward dog CYP2B11 and CYP3A12, respectively. Their selectivity was evaluated using phenacetin O-deethylation (CYP1A), diazepam (DZ) N1-demethylation (CYP2B11), diclofenac 4'-hydrxylation (CYP2C21), bufuralol 1'-hydroxylation (CYP2D11), and DZ C3-hydroxylation (CYP3A12) activities in dog liver microsomes (DLM). MBA exhibited potent mechanism-based inhibition of DZ N1-demethylase activity catalyzed by both baculovirus-expressed CYP2B11 and DLM. In both cases, inhibition was characterized by a low K(I) (0.35 and 0.46 microM, respectively) and high k(inact) (1.5 and 0.56 min(-1), respectively). Despite complete loss of DZ N1-demethylase activity in the presence of MBA, there was no significant loss of cytochrome P450 (P450) CO-binding spectrum. These data suggest that the inactivation involved covalent modification of P450 apoprotein, instead of the prosthetic heme moiety. A homology model of CYP2B11 was constructed, based on the crystal structure of rabbit CYP2C5, for docking the substrate (DZ) and the inhibitor (MBA), respectively. The model, within the limits of our approximations, helped explain the substrate specificity and inhibitor selectivity of CYP2B11. In contrast to MBA, KET was identified as a potent and selective reversible (competitive) inhibitor of CYP3A12 (K(I) = 0.13-0.33 microM). In fact, complete inhibition of CYP3A12-dependent DZ C3-hydroxylation was possible at a low KET concentration (1 microM). Therefore, it is concluded that one can attempt to conduct P450 reaction phenotype studies with DLM using MBA and KET as selective inhibitors of CYP2B11 and CYP3A12, respectively.

Characterization of the human lung CYP2F1 gene and identification of a novel lung-specific binding motif.

The CYP2F1 gene encodes a cytochrome P450 enzyme capable of bioactivating a number of pulmonary-selective toxicants. The expression of CYP2F1 is highly tissue-selective; the highest expression is observed in the lung with little or no hepatic expression. The objective of these studies was to elucidate the mechanisms that govern the unique tissue-specific regulation of CYP2F1. Cosmid and bacterial artificial chromosome clones were screened and sequenced to identify a gene that spanned 14 kbp containing 10 exons, including an untranslated exon 1. Primer extension analysis and 5'-rapid amplification of cDNA ends were used to identify the transcription start site. Several sequences homologous to known cis-elements were identified in the 5'-upstream region of the CYP2F1 promoter. Transient transfection studies with luciferase reporter constructs demonstrated a significant functional lung cell-specific CYP2F1 promoter region (from position -129 to +115). DNase footprinting analysis of 1.6 kbp of the upstream sequence with nuclear extracts from human lung tissues revealed one strong DNA-protein complex at -152 to -182. This nuclear protein (called lung-specific factor, LSF) was present only in lung but not liver or heart tissues. Competitive electrophoretic mobility shift assays characterized a DNA consensus site, within the LSF-binding domain, that was highly similar to two E box motifs, but no known "E box" trans-factors were identified. These studies identified a novel LSF and its consensus sequence that may control tissue-specific expression of CYP2F1.

Characterization of pulmonary CYP4B2, specific catalyst of methyl oxidation of 3-methylindole.

The selective toxicity of chemicals to lung tissues is predominantly mediated by the selective expression of certain pulmonary cytochrome P450 enzymes. This report describes the purification, cloning, and characterization of a unique enzyme, CYP4B2, from goat lung. The purified P450 enzyme was isolated by multistep ion exchange chromatography to electrophoretic homogeneity with an apparent molecular mass of 55,000 Da. Western blotting studies demonstrated that CYP4B enzymes were selectively expressed in lung tissues of rabbits, rats, and mice. Two cDNAs, CYP4B2 and CYP4B2v, were cloned from goat lung tissue. CYP4B2 was predicted to be 511 amino acids and approximately 82% similar to the four known CYP4B1 proteins. Concurrently, a variant of the known human CYP4B1 cDNA, that contained a S207 insertion, was cloned from human lung tissue. The modified recombinant goat CYP4B2 was expressed in Escherichia coli and the enzyme catalyzed the N-hydroxylation of the prototypical substrate 2AF. CYP4B2 preferentially dehydrogenated, rather than hydroxylated, the pneumotoxicant 3-methylindole (3MI) (V(max) = 4.61 versus 0.83 nmol/nmol of P450/min, respectively). To investigate the relevance of covalent heme binding of CYP4 enzymes in CYP4B2-mediated metabolism of 3MI, a site-directed mutant (CYP4B2/A315E) was evaluated. The mutation had little effect on the V(max) of either dehydrogenation or hydroxylation but increased the K(m), which decreased the catalytic efficiency (V/K) for 3MI. The A315E mutation shifted the absorbance maximum of the enzyme from 448 to 451 nm, suggesting that the electron density of the heme was altered. These results demonstrate that CYP4B2 is highly specific for methyl group oxidation of 3MI, without formation of ring-oxidized metabolites, and seems to be predominately responsible for the highly organ-specific toxicity of 3MI in goats.