Influence of inducers of monooxygenases on cytotoxic efficiency of ellipticine on leukemia L1210 cells.

The effects of some inducers of microsomal cytochrome P450-dependent monooxygenases on the metabolic bioactivation and the cytotoxicity of the antitumoral drug ellipticine (ELPT) were studied. Rate of growth of leukemia L1210 cells was measured in vitro in the absence and presence of ELPT or measured when the ELPT was metabolically transformed by noninbred Sprague-Dawley rat liver microsomes. The animals used were either untreated or pretreated by various inducers such as phenobarbital, 3-methylcholanthrene, beta-naphthoflavone, 2,3,7,8-tetrachlorodibenzo-p-dioxin, Aroclor 1254, or ELPT. The transformation of ELPT into its two main metabolites, 9-hydroxyellipticine (9-OHE) and 7-hydroxyellipticine, was studied and measured by high-pressure liquid chromatography in conjunction with the determination of cytotoxic activity. A large variability was observed in the bioactivation and cytotoxic efficiency of ELPT mediated by the different microsomal preparations: The more P448 and/or P1-450 forms of cytochrome were induced, the more the 9-OHE was produced and the more the cytotoxicity toward L1210 cells was enhanced. These features were compared with those elicited by the activation of cyclophosphamide, which was transformed into cytotoxic metabolites by the cytochrome P450 form specifically induced by phenobarbital-type inducers.

Metabolism of docetaxel by human cytochromes P450: interactions with paclitaxel and other antineoplastic drugs.

The metabolism of docetaxel by human liver microsomes was investigated in vitro and compared with that of paclitaxel. A main docetaxel metabolite was generated by human liver microsomes in the presence of NADPH: retention time in high pressure liquid chromatography and its ion fragmentation in mass spectrometry were identical to those of the authentic derivative hydroxylated at the butyl group of the C13 side chain. Kinetic measurements and chemical and immunological inhibitions demonstrated that CYP3A was implicated in the hydroxylation of docetaxel: Km (2 microM) and Vm values of docetaxel for human liver microsomes were comparable to those calculated for the formation of metabolite p-hydroxy-phenyl C3' paclitaxel (M4). Docetaxel hydroxylation correlated only with the CYP3A content of microsomes and with CYP3A-dependent 6 beta-hydroxylation of testosterone and 16-hydroxylation of dehydroepiandrosterone. The formation of hydroxydocetaxel was strongly reduced by CYP3A inhibitors such as ketoconazole, midazolam, erythromycin, testosterone, orphenadrine, and troleandomycin, whereas quinidine (CYP2D6), hexobarbital, tolbutamide, and mephenytoin (CYP2C) had no or little effect. The hydroxylation of docetaxel exhibited a highly positive correlation with the formation of metabolite M4 of paclitaxel (r = 0.929, P < 0.0001, n = 12), but not with its 6-hydroxylation (r = 0.48, P > 0.15). Docetaxel abolished the hydroxylation of paclitaxel metabolite M4, but was totally inactive on its 6 alpha-hydroxylation. Conversely, paclitaxel reduced significantly the hydroxylation of docetaxel. We examined in vitro the possible interaction among docetaxel, paclitaxel, and drugs which could be associated during chemotherapy. Cisplatin, verapamil, doxorubicin, vinblastine, and vincristine at concentrations usually recommended did not markedly modify taxoid metabolism. Ranitidine and diphenylhydramine had no effect, but 100 microM cimetidine partially inhibited the formation of 6 alpha-hydroxypaclitaxel. Pretreatment of patients with barbiturates strikingly stimulated docetaxel hydroxylation, whereas no acceleration of docetaxel hydroxylation was noticed in a patient receiving steroids.

Taxol metabolism by human liver microsomes: identification of cytochrome P450 isozymes involved in its biotransformation.

The biotransformation of taxol by human liver was investigated in vitro with microsomes isolated from adult and developing human tissues. In vitro, no metabolism was detected with kidney microsomes, whereas two metabolites were generated by liver microsomes. The most prominent metabolite, termed M5, corresponded to an hydroxylation at the C6 position on the taxane ring, while the other metabolite, termed M4, corresponded to an hydroxylation at the para-position on the phenyl ring at the C3'-position of the C13 side chain. These two taxol derivatives have been shown to be the major metabolites recovered in bile from a patient infused with taxol. Several approaches have been used to identify the cytochrome P450 (CYP) isozymes involved in these reactions. No positive correlation was observed between the in vitro synthesis of these two metabolites, suggesting that two cytochrome P450 isozymes could be involved, although they could not be distinguished by their apparent affinities (Km approximately 15 microM). The formation of metabolite M4 was substantially reduced both by antibody directed against CYP3A and by the addition of CYP3A substrates such as orphenadrine, erythromycin, troleandomycin, and testosterone. Conversely, the formation of metabolite M5 remained unaffected by antibodies against CYP3A and by CYP3A substrates but was sensitive to diazepam inhibition, a preferential substrate of CYP2C. Correlation between CYP2C content or diazepam demethylation and the synthesis of metabolite M5 was highly positive. The formation of metabolite M4 developed during the early postnatal period. In contrast, the synthesis of metabolite M5 rose only after 3 months of age. These data clearly implicate CYP3A in the formation of metabolite M4 and CYP2C in the synthesis of metabolite M5. Microsomes from patients treated with barbiturates and benzodiazepines increased the formation of metabolite M4 to the level of metabolite M5, demonstrating that drug interactions could modify the human metabolism of taxol.

Conversion of sterols and triterpenes by mycobacteria. I Formation of progesterone and 1-dehydroprogesterone from Mycobacterium aurum, strain A+.

Side-chain degradation of sterols by bacteria is known to proceed via oxidation of a terminal methyl group followed by a succession of beta-oxidative steps. By this pathway, the pregnane backbone is not produced. However, examination of cholesterol degradation products using a strain of Mycobacterium aurum shows that progesterone and 1-dehydroprogesterone are present at low levels. These pregnane derivatives were identified by gas-liquid chromatography combined with mass spectrometry. This indicates that an alternative pathway for sterol side-chain degradation occurs in bacteria, which could be of great interest for the biological production of corticosteroid precursors.

Peptides derived from the dependence receptor ALK are proapoptotic for ALK-positive tumors.

ALK is a receptor tyrosine kinase with an oncogenic role in various types of human malignancies. Despite constitutive activation of the kinase through gene alterations, such as chromosomal translocation, gene amplification or mutation, treatments with kinase inhibitors invariably lead to the development of resistance. Aiming to develop new tools for ALK targeting, we took advantage of our previous demonstration identifying ALK as a dependence receptor, implying that in the absence of ligand the kinase-inactive ALK triggers or enhances apoptosis. Here, we synthesized peptides mimicking the proapoptotic domain of ALK and investigated their biological effects on tumor cells. We found that an ALK-derived peptide of 36 amino acids (P36) was cytotoxic for ALK-positive anaplastic large-cell lymphoma and neuroblastoma cell lines. In contrast, ALK-negative tumor cells and normal peripheral blood mononuclear cells were insensitive to P36. The cytotoxic effect was due to caspase-dependent apoptosis and required N-myristoylation of the peptide. Two P36-derived shorter peptides as well as a cyclic peptide also induced apoptosis. Surface plasmon resonance and mass spectrometry analysis of P36-interacting proteins from two responsive cell lines, Cost lymphoma and SH-SY5Y neuroblastoma, uncovered partners that could involve p53-dependent signaling and pre-mRNA splicing. Furthermore, siRNA-mediated knockdown of p53 rescued these cells from P36-induced apoptosis. Finally, we observed that a treatment combining P36 with the ALK-specific inhibitor crizotinib resulted in additive cytotoxicity. Therefore, ALK-derived peptides could represent a novel targeted therapy for ALK-positive tumors.

Hepatic metabolism and biliary excretion of Taxol in rats and humans.

To date there have been limited studies of the metabolism and disposition of Taxol in animals and humans. Renal disposition of unmetabolized Taxol has been documented to account for a maximum of 5% to 10% of an administered dose of Taxol in humans, but the principal processes involved in drug disposition, particularly the roles of biliary excretion and drug metabolism, have not been evaluated. Therefore, the biliary excretion of Taxol has been studied in rats and in a human patient receiving Taxol in a phase I trial. Of the total doses administered to rats and the patient, 40% and 20%, respectively, were excreted in the bile in the forms of unmetabolized Taxol and Taxol metabolites until 24 hours posttreatment. Although the biliary excretion of unmetabolized Taxol accounted for 10% and 3% of total drug disposition in the rats and in the patient, respectively, the remaining portion consisted of several metabolites. Nine metabolites were detected in rat bile, and five metabolites were detected in human bile. The chemical structures of four of the rat metabolites and three of the human metabolites have been identified thus far. With the exception of baccatin III, a minor metabolite found only in rat bile that lacks the side chain at C-13 position of the taxane ring, the other metabolites were monohydroxylated or dihydroxylated and had intact taxane rings and side chains at taxane ring positions C-2 and C-13. The taxane ring and both the C-2 and C-13 side chains were susceptible to hydroxylation.(ABSTRACT TRUNCATED AT 250 WORDS)

CSF morphine levels after lumbar intrathecal administration of isobaric and hyperbaric solutions for cancer pain.

The objectives of this study were to compare the pharmacokinetic properties and the duration of analgesia following intrathecal administration (L5-S1) of 2 mg morphine in 2 forms: (1) an isobaric (NaCl 0.9%) and (2) a hyperbaric solution (7% dextrose). The study was carried out on 5 cancer patients with severe, intractable pain in the lower half of the body. Samples of CSF were collected at the level of the 10th thoracic vertebra at regular intervals for 15 h after administration. Morphine concentrations were determined by HPLC. The pharmacokinetic properties of the solutions (I and II) were quite different. Peak levels (I) were reached in 5-15 min (30 and 60 micrograms/ml); they then fell rapidly during the 1st hour (7 and 11 micrograms/ml) with an elimination half-life of 10 and 15 min, followed by a change in slope (elimination half-life of 108 and 140 min). Peak levels (II) were reached in 4-5 h (0.8-3.3 micrograms/ml); they then fell progressively according to a single exponential function (elimination half-life: 144-246 min). The duration of analgesia for a dose of 2 mg was 30 h for solution 2 and 24 h for solution 1. The hyperbaric solution, which produced the same degree of analgesia as the isobaric solution, limited the cephalad diffusion of morphine and reduced or abolished the central depressant effects of the drug.

Synthesis and cytotoxic activity of hydroxylated derivatives of olivacine in relation with their biotransformation.

The chemical synthesis of 9-hydroxyolivacine and 7-hydroxyolivacine based on a biomimetic approach is described. These two hydroxylated derivatives have been found as main in vitro metabolites of olivacine after incubation with rat hepatic microsomes. The pretreatment of animals with benzo[a]pyrene caused a large increase in both microsomal hydroxylations, whereas the pretreatment with phenobarbital caused a weak increase, with a preservation of 9-hydroxylation/7-hydroxylation ratio greater than 1 in both cases. The two hydroxyolivacines have been also found as principal in vivo metabolites of olivacine in rat bile as glucuronide and sulfate conjugates. The pretreatment of animals with benzo[a]pyrene reverses the 9-hydroxyolivacine/7-hydroxyolivacine ratio excretion in bile to a value that is less than 1. In both in vitro and in vivo experiments, the free metabolites were identified by HPLC and UV-visible, MS, and 1H NMR spectra. Hydroxylation at position 9 increases the in vitro cytotoxicity against leukemia L1210 cells (ID50 = 0.06 microM compared to 2.03 microM for olivacine) and an opposite effect is observed for hydroxylation at position 7 (ID50 = 12.8 microM). On the other hand, hydroxylation at position 9 has no effect on the in vivo antitumor activity against L1210. This might be related to the oxidative and conjugative metabolic pathways that play an important role in antitumor activity and deactivation of olivacine and its hydroxy metabolites.

Identification of neuropeptide FF-related peptides in rodent spinal cord.

Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in mouse and rat spinal cord, by using reverse phase high pressure liquid chromatography with radioimmunoassay and electrospray mass spectrometry detection. In both species, two octapeptides, NPFF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and NPSF (Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-amide) were identified but a longer peptide NPA-NPFF (Asn-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was present at the highest concentration in rat spinal cord. In mouse, the homologous peptide, SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was not detected. Both peptides NPFF and NPSF reverse morphine-induced analgesia in the tail flick test. Our data reveal species differences in the maturation of NPFF precursor.

Evidence for electrophilic properties of N2-methyl-9-hydroxy ellipticinium acetate (Celiptium) from human biliary metabolites.

The human biliary metabolism of the antitumor agent N2-methyl-9-hydroxyellipticinium acetate is described. Three major compounds have been identified by high-performance liquid chromatography and comparison with synthetic reference derivatives: the unchanged drug, the O-glucuronide conjugate and the cysteinyl-ellipticinium adduct. The latter one is the expected detoxification compound of an intermediate electrophilic quinone-imine derivative generated in vivo. This result provides a further evidence that hydroxylated forms of ellipticine derivatives might be activated by a biooxidation route.

Vincristine degradation by serum from leukemic patients: role of myeloperoxidase.

Myeloperoxidase (MPO) has been shown to catalyze the in vitro degradation of vincristine (VCR). Given that MPO is a lysosomal enzyme that can be released into the circulation by both normal activated and leukemic myeloid cells, we investigated the possibility that sera from patients with acute myeloblastic leukemia (AML) might exhibit an increased capacity to degrade VCR. 31 serum samples (23 from patients with acute myeloblastic leukemia and 8 from patients with other conditions) were analyzed after incubation with ((3)H)VCR by using HPLC. Sera from patients with AML demonstrated an increased ability to breakdown VCR when compared to either normal sera or to sera from patients with lymphoid leukemias. VCR degradation was significantly increased by adding hydrogen peroxide, an electron donor for MPO, to the sera and was almost completely inhibited by adding 1 mM acetaminophen, an inhibitor of MPO. VCR peroxidation in the presence of hydrogen peroxide correlated both with the number of leukemic blasts in the circulation at the time the sera were obtained and with serum MPO concentrations determined by an immunoassay. These data suggest that the inactivity of VCR in AML may be due in part to its rapid peroxidation to inactive species by the MPO of leukemic myeloblasts.

Morphine metabolism in the naturally morphine-tolerant afghan pika: a preliminary study.

The afghan pika (Ochotona rufescens), a lagomorph which is naturally tolerant to the analgesic action of morphine, metabolizes morphine into morphine 3-glucuronide apparently faster than does the rabbit, another lagomorph which is however normally responsive to morphine. In the two species, following morphine administration, another unidentified component appears very soon (5 min) in pika blood plasma and much later (60 min) in rabbit blood plasma. This unknown component which appears not to be morphine derived might be involved in the natural resistance of the Afghan pika to morphine.

Ellipticines and human liver microsomes: spectral interaction with cytochrome P-450 and hydroxylation. Inhibition of aryl hydrocarbon metabolism and mutagenicity.

Some pharmacological properties of ellipticine (E) and its derivatives linked to their interaction with cytochrome P-450 have been investigated with human liver microsomes. 9-Hydroxyellipticine (9-OHE) interacts with human liver cytochrome P-450 exhibiting a type II spectrum (lambda max: 428 nm, Ks = 1.1 microM). After incubation with human liver microsomes the E was converted to 9-OHE; 7-hydroxyellipticine was not produced. The cytotoxic effect of this biotransformation has been evaluated on leukemic L1210 cells, in vitro, and found to be equal to those elicited by liver microsomes of control or phenobarbital (PB) pretreated rats. Moreover, 9-OHE and 9-fluoroellipticine (9-FE) strongly inhibit the benzo[alpha]pyrene hydroxylase (AHH) activity of human liver microsomes (I50 = 2.6 microM and 1.6 microM, respectively) as well as the mutagenesis induced by the polycyclic aromatic hydrocarbon 2-acetylaminofluorene (AAF); 1 microgram/plate of each of these compounds is able to inhibit by more than 50% the mutagenicity of 5 microgram/plate AAF.

Oxidative biotransformation of the antitumour agent elliptinium acetate: Structural characterization of its human and rat urinary metabolites.

The electrophilic properties of the antitumour drug N(2)-methyl-9-hydroxyellipticinium acetate (Celiptium) are revealed by the detection of thiol-conjugate metabolites in man and rat urine. Besides the unchanged drug and its glucuronide, the cysteinyl- (in man) and the N-acetylcysteinyl- (in man and rat) conjugates have been unambiguously characterized using NMR, UV and mass spectral data. The urinary excretion profile exhibits total excreted products of 21% (in man) and 9% (in rat) with respect to the administered dose. The unchanged drug is found to be the major excreted compound from urine in both species (17% in man, 6.3% in rat); whereas the glucuronide (2.6% in man, 1.5% in rat), cysteinyl- (1.3% in man) and N-acetylcysteinyl- (0.2% in man, 1.2% in rat) conjugates represent the minor excreted compounds. The presence of the latter thio-conjugates provides an indirect proof of the in vivo generation of an oxidized intermediate form of the administered drug.

The stability of morphine in isobaric and hyperbaric solutions in a drug delivery system.

Intrathecal preparations of morphine for use in intractable pain must contain no preservatives. They are generally formulated in saline (isobaric) or dextrose (hyperbaric) which raises questions of stability. The behaviour of morphine in hyperbaric and isobaric solutions stored in a reservoir for implantation has been examined and the effect of temperature and the time of contact of morphine with the different components of the reservoir as well as the sterilization procedure have been investigated. The best stability was observed with a hyperbaric solution in which there was 15 to 20 times less pseudomorphine than in the isobaric solution, which was found to contain 1% pseudomorphine after 1 month of storage at 37 degrees C in the reservoir. Similar solutions stored in ampoules did not degrade.

Biotransformation of taxoids by human cytochromes P450: structure-activity relationship.

The metabolism of paclitaxel and docetaxel by human liver microsomes was investigated in vitro. The main metabolite of paclitaxel formed in vitro was the 6 alpha-hydroxypaclitaxel: its formation largely exceeded the formation of other metabolites hydroxylated on the lateral chain by rat liver microsomes and initially characterized in rat bile. In contrast, in vitro studied showed that the initial metabolite of docetaxel resulted from the hydroxylation of the tert-butyl of the lateral chain at C13 and that the same metabolites were formed in human and animal models. Comparison of individual CYP protein content of human microsomes and catalytic activities with taxoid biotransformation, showed that 2 distinct isoforms were assigned to the 6 alpha-hydroxylation (CYP2C) and to the hydroxylation of the lateral chain (CYP3A4). Chemical and immunological inhibitions confirmed these assumptions. The effect of antineoplastic drugs potentially associated with taxoids during chemotherapy has been tested in vitro on paclitaxel and docetaxel biotransformations. In the therapeutic range, vincristine, vinblastine, doxorubicine and cisplatin elicited a moderate or no inhibition of paclitaxel and docetaxel metabolism, as well as cimetidine, ranitidine and diphenylhydramine used to prevent major side effects associated with taxoid therapy. In patients given barbiturates, the hydroxylation on the lateral chain of paclitaxel and docetaxel was markedly stimulated and resulted from the induction of CYP3A isoforms. These results clearly demonstrated that the biotransformation of paclitaxel and docetaxel by human liver microsomes was supported by 2 distinct CYP proteins and that drug interactions could modify the therapeutic efficiency of taxoids during chemotherapy.

Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA.

Agents that interfere with DNA replication in Escherichia coli induce physiological adaptations that increase the probability of survival after DNA damage and the frequency of mutants among the survivors (the SOS response). Such agents also increase the survival rate and mutation frequency of irradiated bacteriophage after infection of treated bacteria, a phenomenon known as Weigle reactivation. In UV-irradiated single-stranded DNA phage, Weigle reactivation is thought to occur via induced, error-prone replication through template lesions (translesion synthesis [P. Caillet-Fauquet, M: Defais, and M. Radman, J. Mol. Biol. 117:95-112, 1977]). Weigle reactivation occurs with higher efficiency in double-stranded DNA phages such as lambda, and we therefore asked if another process, recombination between partially replicated daughter molecules, plays a major role in this case. To distinguish between translesion synthesis and recombinational repair, we studied the early replication of UV-irradiated bacteriophage lambda in SOS-induced and uninduced bacteria. To avoid complications arising from excision of UV lesions, we used bacterial uvrA mutants, in which such excision does not occur. Our evidence suggests that translesion synthesis is the primary component of Weigle reactivation of lambda phage in the absence of excision repair. The greater efficiency in Weigle reactivation of double-stranded DNA phage could thus be attributed to some inducible excision repair unable to occur on single-stranded DNA. In addition, after irradiation, lambda phage replication seems to switch prematurely from the theta mode to the rolling circle mode.

Structural characterization of Mycobacterium tuberculosis lipoarabinomannans by the combination of capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) associated with capillary electrophoresis (CE) has been used for structural characterization of mannooligosaccharide caps from Mycobacterium tuberculosis H37rv mannosylated lipoarabinomannans (ManLAMs). The mannooligosaccharide caps were released by mild acid hydrolysis, labeled with 1-aminopyrene-3,6,8-trisulfonate (APTS) prior to being separated by CE, collected, and analyzed by MALDI-TOF-MS and post-source decay experiments. This approach was optimized using standard APTS-labeled oligosaccharides. With the selected (9:1) mixture of 2,5-dihydroxybenzoic acid (DHB) and 5-methoxysalicylic acid (MSA) as matrix and the on-probe sample cleanup procedure with cation-exchange resin, standard APTS-maltotriose was successfully detected down to 50 fmol using linear-mode negative MALDI-TOF-MS. Moreover, using extraction delay time, only 100 and 500 fmol of this standard were required, respectively, to obtain accurate reflectron mass measurements and sequence determination through post-source decay experiments. Applied to only 5 microg (294 pmol) of M. tuberculosis ManLAMs, this analytical approach allowed successful mass characterization of the mannooligosaccharide cap structures from the deprotonated molecular ions [M - H]- and the y-type ion fragments obtained in post-source decay experiments. This powerful analytical approach opens new insights into both the characterization of oligosaccharides and the capping motifs displayed by ManLAMs purified from mycobacteria isolated from tubercular patients without in vitro culturing.