Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain.

Converging lines of evidence implicate the beta-amyloid peptide (Ass) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce A beta production by functionally inhibiting gamma-secretase, the activity responsible for the carboxy-terminal cleavage required for A beta production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon A beta production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP(V717F) reduces brain levels of Ass in a dose-dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain A beta in vivo. Development of such novel functional gamma-secretase inhibitors will enable a clinical examination of the A beta hypothesis that Ass peptide drives the neuropathology observed in Alzheimer's disease.

Distinct forms of cytochrome P-450 are responsible for 6 beta-hydroxylation of bile acids and of neutral steroids.

Cytochrome P-450-dependent 6 beta-hydroxylation of bile acids in rat liver contributes to the synthesis of the quantitatively important pool of 6-hydroxylated bile acids, as well as to the detoxification of hydrophobic bile acids. The lithocholic acid 6 beta-hydroxylation reaction was investigated and compared with androstenedione 6 beta-hydroxylation. Differential responses of these two activities to inducers and inhibitors of microsomal P-450 enzymes, lack of mutual inhibition by the two substrates and differential inhibition by antibodies raised against several purified hepatic cytochromes P-450 were observed. From these results it was concluded that 6 beta-hydroxylation of lithocholic acid is catalysed by P-450 form(s) different from the subfamily IIIA cytochromes P-450 which are responsible for the bulk of microsomal androstenedione 6 beta-hydroxylation. Similar, but more tentative, results revealed that the 7 alpha-hydroxylation of lithocholic acid and of androstenedione may be also catalysed by distinct P-450 enzymes. The results indicate that cytochromes P-450 hydroxylating bile acids are distinct from analogous enzymes that carry out reactions of the same regio- and stereo-specificity on neutral steroids (steroid hormones). A comparison of pairs of cytochromes P-450 that catalyse the same reaction on closely related steroid molecules will help to define those structural elements in the proteins that determine the recognition of their respective substrates.

Genetic polymorphism of cytochrome P-450-dependent phencyclidine hydroxylation in mice. Comparison of phencyclidine hydroxylation in humans.

A mouse model of P-450 phencyclidine 3-cyclohydroxylase (P-450 PCP 3-cyclohydroxylase) genetic polymorphism is described. Up to a 3-fold difference was observed in the constitutive liver microsomal activity of P-450 PCP 3-cyclohydroxylase between "slow" (A/J and DBA/2J) and "rapid" (C57BL/6J and BALB/CJ) phencyclidine (PCP) metabolizers. The segregation of slow and rapid hydroxylator phenotypes between 17 recombinant inbred mouse strains derived from A/J and C57BL/2J mice suggests control of the activity by a single gene located on the X-chromosome or, less likely, on chromosome 17. A liver deficiency of P-450 PCP 3-cyclohydroxylase was observed in the New Zealand rabbit and Wistar rat, as well as in some human subjects. Any relationship of P-450 PCP 3-cyclohydroxylase polymorphism to other well characterized P-450 polymorphisms in mice (aryl hydrocarbon hydroxylase and coumarin hydroxylase) was excluded on the basis of differences in inducibility and activity distribution among the inbred mouse strains. Lack of relationship to the P-450 debrisoquine hydroxylase was confirmed by direct comparison of both activities in the same mouse and human liver microsomes. The pharmacological consequence of the observed polymorphism in mice appears to be that the rapid PCP metabolizers are more resistant to the effects of PCP compared to the slow metabolizers as based upon its ED50 and duration of action in A/J and C57BL/6J mice. The relevance of this data to humans remains to be determined, but clearly the latter show marked differences in PCP 3-cyclohydroxylase activity, which separate into low, intermediate, and high groups.

Detoxification of lithocholic acid. Elucidation of the pathways of oxidative metabolism in rat liver microsomes.

The hydroxylation of lithocholic acid (3 alpha-hydroxy-5 beta-cholanoic acid) by adult male Sprague-Dawley rat liver microsomes supplemented with NADPH was studied. Metabolites were separated by a combination of thin-layer chromatography and high pressure liquid chromatography, both with and without prior methylation and acetylation of the samples. The resulting products were characterized by thin-layer, gas-liquid, and high pressure liquid chromatography by comparison with authentic bile acid standards; final structure determination was by proton nuclear magnetic resonance spectroscopy and by mass spectrometry. The following reaction products were found: 3 alpha, 6 beta-dihydroxy-5 beta-cholanoic acid (80% of total metabolites) and 3 alpha, 6 alpha-dihydroxy-5 beta-cholanoic, 3 alpha, 7 alpha-dihydroxy-5 beta-cholanoic, 3 alpha, 6 beta,7 beta-trihydroxy-5 beta-cholanoic, and 3 alpha-hydroxy-6-oxo-5 beta-cholanoic acids (less than or equal to 5% each). In addition, one unidentified trihydroxylic bile acid and several minor compounds were present. It is concluded that four different hydroxylation reactions of lithocholic acid, namely the predominant 6 beta as well as the minor 6 alpha, 7 alpha, and 7 beta hydroxylations, are catalyzed by rat hepatic microsomes; 7 beta-hydroxylation may occur only with dihydroxylated bile acids but not with lithocholate itself. The presence of the 6-oxo bile acid can be explained either by direct oxidation of a hydroxyl group by cytochrome P-450, or by the action of microsomal dehydrogenase(s) which could also catalyze the epimerization of hydroxyl groups via their oxidation. The results form the basis of a proposed scheme of the oxidative metabolism of lithocholic acid in rat liver microsomes.

Gene conversion and differential regulation in the rat P-450 IIA gene subfamily. Purification, catalytic activity, cDNA and deduced amino acid sequence, and regulation of an adult male-specific hepatic testosterone 15 alpha-hydroxylase.

Previous studies on regulation of the rat hepatic P-450 IIA1 cDNA have provided evidence for a second gene closely related to but regulated in a manner quite distinct from P-450 IIA1. Experiments were carried out to isolate the cDNA for this second P-450 gene, designated IIA2, in order to study more directly its regulation and relationship to IIA1. A full length cDNA to IIA2 was isolated from an adult male rat liver lambda gt11 library and sequenced completely. The IIA2 cDNA shared 93% nucleotide and 88% deduced amino acid similarities with the previously characterized IIA1 cDNA (Nagata, K., Matsunaga, T., Gillette, J., Gelboin, H. V., and Gonzalez, F. J. (1987) J. Biol. Chem. 262, 2787-2793). The protein, deduced from the cDNA, contained 492 amino acids and a calculated Mr of 56,352. Comparison of the IIA1 and IIA2 cDNAs revealed areas of low nucleotide similarity interspersed with areas of absolute identity, suggesting that gene conversions have played a role in the evolution of the IIA subfamily. Expression of IIA1 and IIA2 mRNAs in rat liver during development was studied with use of specific oligonucleotide probes. IIA1 mRNA was increased within 1 week after birth in both male and female rats; however, its postpubertal expression was decreased in males yet remained elevated in females. In contrast, IIA2 mRNA was markedly induced in male rat liver at puberty but was not detectable in females at any age examined. Furthermore, only IIA1 mRNA was induced by treatment of rats with 3-methylcholanthrene. Although IIA1 and IIA2 mRNAs were actively expressed in hepatic tissue, no evidence for their expression was found in lung, kidney, or intestine, suggesting that the IIA genes have tissue-specific promoters. Reconstituted enzyme assays on the purified protein products P-450 IIA1 and P-450 IIA2 showed that, although both enzymes share considerable sequence similarity, their positional specificities toward the prototype substrate testosterone are strikingly different.

Cytochrome P-450 cholesterol 7 alpha-hydroxylase: inhibition of enzyme deactivation by structurally diverse calmodulin antagonists and phosphatase inhibitors.

Cytochrome P-450 cholesterol 7 alpha-hydroxylase (P-450Ch7 alpha) catalyzes the first and rate-limiting step in the conversion of cholesterol to bile acids. Incubation of rat liver microsomes in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer resulted in a time-dependent deactivation of P-450Ch7 alpha which was markedly accelerated by the nonionic detergent Tween 80. Microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450-dependent 7-ethoxycoumarin O-deethylase activities were unaffected under these conditions, evidencing the selectivity of the deactivation process for P-450Ch7 alpha. The rate (t 1/2 = 15-19 min at 37 degrees C) and maximal extent of P-450Ch7 alpha deactivation (greater than or equal to 90%) were both unaffected by the presence of cytosolic proteins and were also not dependent on the initial enzyme level, as shown using liver microsomes isolated from untreated, cholestyramine-fed, and xenobiotic-induced rats exhibiting an eight-fold range in P-450Ch7 alpha activity. Scavengers for reduced oxygen species were also without effect. P-450Ch7 alpha was stabilized some six- to sevenfold (t 1/2 = 94-143 min) by the phosphatase inhibitor NaF. Of a series of other phosphatase inhibitors examined, including, among others, EDTA, vanadate, and molybdate, only phosphate-containing compounds and the calmodulin antagonist trifluoperazine, and inhibitor of the Ca2+-calmodulin-dependent phosphatase calcineurin, effectively stabilized P-450Ch7 alpha. Modulation of P-450Ch7 alpha deactivation by these inhibitors generally paralleled their effects on isolated calcineurin. A variety of structurally diverse calmodulin antagonists examined were also found to effectively protect P-450Ch7 alpha from deactivation; these include calmidazolium and tamoxifen (IC50 = 25 to 50 microM), chlorpromazine, thioridazine, amitriptyline, imipramine, and the naphthalene sulfonamide compound W-7 (IC50 = 50 to 300 microM). Structure-activity analysis of several phenothiazines and their derivatives indicated that although little activity was exhibited by the sulfoxides, some protection was provided by the corresponding sulfones. On the basis of these observations, various models for the molecular basis of enzyme deactivation are considered, including the hypothesis that a calcineurin-like microsomal phosphatase mediates deactivation of this cytochrome P-450 enzyme.

Quantitation of phencyclidine, its metabolites, and derivatives by gas chromatography with nitrogen-phosphorus detection: application for in vivo and in vitro biotransformation studies.

A sensitive and specific assay for the quantitation of phencyclidine (PCP), its several metabolites, and derivatives in biological samples is described. The method is based on the extraction of PCP and related compounds with organic solvents followed by gas chromatographic (GC) separation with nitrogen-phosphorous (NP) detection of the extract derivatized with heptafluorobutyric anhydride. The detection limit was about 5 pmol per injection with a linear standard curve to 16 nmol in the initial sample. The recovery of different compounds ranged from 80 to 98%. Precision of the method was within 3 to 5%, while day-to-day variations did not exceed 5%. The present procedure permits the identification and quantitation of PCP and its major primary and secondary metabolites. In addition, several PCP analogs and derivatives that are not recognized as PCP metabolites can be quantitated using this procedure. The assay was applied for the quantitation of PCP and its metabolites in tissue homogenates and body fluids of PCP-dosed animals as well as in the urine of PCP-intoxicated humans. An in vitro system for PCP biotransformation studies by liver and placental microsomes was also developed.

Effects of metaphit, a proposed phencyclidine receptor acylator, on catalepsy in pigeons.

Metaphit, a derivative of phencyclidine (PCP), irreversibly binds to PCP sites in rat brain homogenates. PCP-induced catalepsy in pigeons, which is a pharmacologically specific and stereoselective phenomenon, was used to study pharmacological consequences of the proposed covalent bonding of metaphit to PCP sites. Metaphit pretreatment increased the cataleptic effects induced by cumulative doses of PCP-type drugs (i.e., PCP, ketamine and m-amino PCP) and of drugs that have PCP-like actions (i.e., dexoxadrol, LY 154716 and cyclazocine). Metaphit did not affect pentobarbital-induced loss of righting, head-drop and eye closure. Metaphit itself induced a PCP-like catalepsy. Isobolographic analysis of the interactions between metaphit and PCP-like drugs suggested that metaphit potentiated the catalepsy-inducing effects of these drugs. The possibility that metaphit exerts its potentiating effects by inhibition of PCP biotransformation was evaluated by measuring plasma and brain concentrations of PCP after pretreatment with either metaphit or SKF-525A, an inhibitor of the enzyme systems involved in PCP biotransformation. SKF-525A, but not metaphit, increased brain levels of PCP. The results suggest that metaphit acts not as an antagonist of PCP but as a less-potent, long-acting, specific PCP-like agonist. Potentiation by metaphit of the cataleptic effects of chemically diverse drugs with PCP-like actions does not appear to be based on inhibition of the enzyme systems involved in metabolism of those drugs.

Biotransformation of phencyclidine.

PCP is metabolized extensively in the body via a variety of metabolic routes. Biotransformation is a major mechanism of PCP elimination in humans and termination of PCP action in mice. In general, PCP metabolites are less active pharmacologically than PCP itself. Primary metabolism involves hydroxylation of the alicyclic rings at several carbon atoms by cytochrome P-450-mediated monooxygenase. Hydroxylation of the aromatic ring seems to be less likely and has not been conclusively demonstrated. Hydroxylation of PCP at carbon 2 of the piperidine ring to form the unstable carbinolamine leads to formation of a series of polar, open-ring compounds. Monohydroxylated metabolites are conjugated with glucuronic or sulfuric acid, or are further hydroxylated to dihydroxy derivatives that can also be subject to conjugation. Formation of highly reactive electrophilic metabolites of PCP have been demonstrated in vitro in microsomal preparations. Covalent modification of tissue macromolecules by reactive intermediates can be responsible for suicide inactivation of cytochrome P-450 and can possibly mediate some long-term toxic effects of PCP. PCP inhaled by cigarette smoking is metabolized via similar routes. About 50% of the PCP in cigarette smoke is converted to PC, a major product of thermal degradation of PCP. PC and its hydroxylated and conjugated metabolites appear to contribute little to the pharmacology or acute toxicity of PCP.

N-allyl analogues of phencyclidine: chemical synthesis and pharmacological properties.

Several N-allyl derivatives of 1-phenylcyclohexylamine (PCA) were prepared, and their pharmacology was briefly characterized. The mono- and diallyl derivatives had phencyclidine-like activities in mice but were less potent behaviorally than phencyclidine (PCP). None were PCP antagonists. In vitro these compounds were competitive inhibitors of butyrylcholinesterase (BChE) and protected against inhibition by DFP. In addition, these agents displaced tritiated N-methyl-4-piperidyl benzilate from mouse-brain homogenates and inhibited the effects of acetylcholine on isolated guinea pig ileum. None of these in vitro effects correlated with their PCP-like behavioral activity in vivo in mice.