Rituximab, bendamustine, and lenalidomide in patients with aggressive B cell lymphoma not eligible for high-dose chemotherapy or anthracycline-based therapy: phase I results of the SAKK 38/08 trial.

This phase I trial was designed to develop a new effective and well-tolerated regimen for patients with aggressive B cell lymphoma not eligible for front-line anthracycline-based chemotherapy or aggressive second-line treatment strategies. The combination of rituximab (375 mg/m(2) on day 1), bendamustine (70 mg/m(2) on days 1 and 2), and lenalidomide was tested with a dose escalation of lenalidomide at three dose levels (10, 15, or 20 mg/day) using a 3 + 3 design. Courses were repeated every 4 weeks. The recommended dose was defined as one level below the dose level identifying ≥2/6 patients with a dose-limiting toxicity (DLT) during the first cycle. Thirteen patients were eligible for analysis. Median age was 77 years. WHO performance status was 0 or 1 in 12 patients. The Charlson Comorbidity Index showed relevant comorbidities in all patients. Two DLTs occurred at the second dose level (15 mg/day) within the first cycle: one patient had prolonged grade 3 neutropenia, and one patient experienced grade 4 cardiac adverse event (myocardial infarction). Additional grade 3 and 4 toxicities were as follows: neutropenia (31 %), thrombocytopenia (23 %), cardiac toxicity (31 %), fatigue (15 %), and rash (15 %). The dose of lenalidomide of 10 mg/day was recommended for a subsequent phase II in combination with rituximab 375 mg/m(2) on day 1 and bendamustine 70 mg/m(2) on days 1 and 2.

Cellular localization and regional distribution of CYP2D6 mRNA and protein expression in human brain.

The cytochrome P4502D6 (CYP2D6) is involved in the biotransformation of many drugs which predominantly act in the central nervous system (CNS), including opioids, various psychotrophic drugs and neurotoxins. Until now, however, only controversial information is available regarding the presence of CYP2D6 in CNS. In this study, the regional and cellular expression of CYP2D6 transcripts and proteins in postmortem brain tissues of three individuals was analysed. A combination of in-situ hybridization coupled with immunohistochemistry on adjacent sections allowed simultaneous detection of CYP2D6 mRNA and protein. However, discrepancies existed in the results such that the mRNA was more widely distributed in the brain areas analysed compared to the protein. Neuronal cells, as well as glial cells, showed labelling for mRNA in brain regions such as the neocortex, caudate nucleus, putamen, globus pallidus, hippocampus, hypothalamus, thalamus, substantia nigra and cerebellum. In contrast, CYP2D6 protein was primarily localized in large principal neurons such as pyramidal cells of the cortex, pyramidal cells of the hippocampus, and Purkinje cells of the cerebellum. In glial cells, CYP2D6 protein was absent. These results provide clear evidence of CYP2D6 expression in certain regions of the CNS and may indicate the role CYP2D6 plays in a number of drug interactions that are of potential clinical importance for neurological diseases.

Morphine-related metabolites differentially activate adenylyl cyclase isozymes after acute and chronic administration.

Morphine-3- and morphine-6-glucuronide are morphine's major metabolites. As morphine-6-glucuronide produces stronger analgesia than morphine, we investigated the effects of acute and chronic morphine glucuronides on adenylyl cyclase (AC) activity. Using COS-7 cells cotransfected with representatives of the nine cloned AC isozymes, we show that AC-I and V are inhibited by acute morphine and morphine-6-glucuronide, and undergo superactivation upon chronic exposure, while AC-II is stimulated by acute and inhibited by chronic treatment. Morphine-3-glucuronide had no effect. The weak opiate agonists codeine and dihydrocodeine are also addictive. These opiates, in contrast to their 3-O-demethylated metabolites morphine and dihydromorphine (formed by cytochrome P450 2D6), demonstrated neither acute inhibition nor chronic-induced superactivation. These results suggest that metabolites of morphine (morphine-6-glucuronide) and codeine/dihydrocodeine (morphine/dihydromorphine) may contribute to the development of opiate addiction.

Loss of analgesic effect of morphine due to coadministration of rifampin.

Methadone withdrawal symptoms have been reported in drug addicts treated with the tuberculostatic rifampin. Whereas this interaction can be explained by induction of phase I drug metabolism (CYP3A4), knowledge about induction of phase II metabolism (e.g., UDP-glucuronosyltransferases = UGTs) and its influence on drug effects in man, however, is very limited. The potent analgesic morphine is metabolized by more than one UGT to the active metabolite morphine-6-glucuronide and to morphine-3-glucuronide, which is devoid of analgesic activity. Thus, differential induction of UGTs involved in metabolism of morphine might lead to decreased or increased analgesic effects, depending on which UGT is preferentially induced. We therefore investigated the influence of the potent enzyme inducer rifampin on analgesic effects and pharmacokinetics of morphine, which is primarily eliminated by phase II metabolism. Ten healthy male volunteers participated in this double-blind, placebo-controlled study with double crossover design. Morphine (10 mg p.o.) and placebo were administered on two separate occasions before and near the end of 13 days of treatment with rifampin (600 mg/day). Blood samples were collected for 31 h. Morphine effects on pain sensation were determined using the cold pressor test. When morphine was given alone, the opioid elicited a significant increase in pain threshold and pain tolerance in comparison to placebo (P < or = 0.05). However, following administration of rifampin no analgesic effect of morphine was observed. In agreement, the area under the serum concentration-time curve (AUC) of morphine and the maximum serum concentration of morphine were considerably reduced during coadministration of rifampin (-27.7 +/- 19.3% and -40.7 +/- 27.1%; P < or = 0.01). Moreover, during treatment with rifampin a proportional reduction of AUCs of morphine-3-glucuronide (P < or = 0.01), morphine-6-glucuronide (P < or = 0.05) and morphine was observed. Since urinary recoveries of both morphine-3-glucuronide and morphine-6-glucuronide were also reduced during administration of rifampin, there is no evidence for a contribution of UGT induction to the observed interaction. In summary, a major drug interaction was observed between morphine and rifampin, which could not be attributed to induction of UGTs, but resulted in a complete loss of analgesic effects of the opioid.

Same incidence of adverse drug events after codeine administration irrespective of the genetically determined differences in morphine formation.

The analgesic effect and adverse events of the weak opioid codeine is assumed to be mediated by its metabolite morphine. The cytochrome P-450 enzyme CYP2D6 catalysing the formation of morphine exhibits a genetic polymorphism. Two distinct phenotypes, the extensive (EMs) and poor metabolisers (PMs), are present in the population. The prevalence of PMs in the Caucasian population is 7% to 10%. Since PMs do not express functional CYP2D6, they have a severely impaired capacity to metabolise drugs which are substrates of this enzyme. Provided the analgesic effect and the adverse events of codeine are mediated by its metabolite morphine, large phenotype-related differences are to be expected and PMs, as they form only trace amounts of morphine, can serve as a model to test the hypothesis whether the analgesia and adverse events of codeine are mediated by the parent drug or its metabolite morphine. Therefore we have studied in a randomised placebo-controlled double-blind trial the analgesic effect of 170 mg codeine (p.o.) compared to 20 mg morphine (p.o.) and placebo in 9 EMs and 9 PMs using the cold pressor test. The duration and intensity of the side effects were assessed using visual analogue scales (VAS). Codeine and morphine concentrations were measured in serum and urine. Compared to placebo, 20 mg morphine caused a significant increase in pain tolerance in both phenotypes, EMs and PMs (16.2+/-27.4 vs. -0.66+/-27.4 s x h, n=18). However, following administration of codeine, analgesia was only observed in EMs but not in PMs (EMs: 54.9+/-42.2 vs. 1.7+/-4.2 s x h, P < 0.01; PMs: 9.6+/-10.9 vs. 3.3+/-23.7 s x h, not significant). Adverse events were significantly more pronounced after morphine and codeine compared to placebo in both EMs and PMs. In contrast to the phenotype-related differences in the analgesic effect of codeine, however, no difference in adverse events between the phenotypes could be observed. In the pharmacokinetic studies, significant differences between the two phenotypes in the formation of morphine after codeine administration could be observed. Whereas morphine plasma concentrations were similar in PMs (Cmax: 44+/-13 nmol/l: AUC: 199+/-45 nmol x h/l) and EMs (Cmax: 48+/-17 nmol/l); AUC: 210+/-65 nmol x h/l) after morphine administration, following 170 mg codeine, morphine plasma concentrations comparable to those after morphine application were only observed in EMs (Cmax: 38+/-16 nmol/l; AUC: 173+/-90 nmol x h/l). In PMs only traces of morphine could be detected in plasma (Cmax: 2+/-1 nmol/l; AUC: 10+/-7 nmol x h/l). The percentage of the codeine dose converted to morphine and its metabolites was 3.9% in EMs and 0.17% in PMs. The interindividual variability in analgesia of codeine which is related to genetically determined differences in the formation of morphine clearly indicate that this metabolite is responsible for the analgesic effect of codeine. In contrast to the analgesic effect, frequency and intensity of the adverse events did not present significant differences between the two phenotypes. These findings have implications for the clinical use of codeine. Since side effects occurred in both EM and PM subjects, the use of codeine as an analgesic will expose 7% to 10% of patients who are PMs to the side effects of the drug without providing any beneficial analgesic effects.

Modulation of K+-evoked [3H]-noradrenaline release from rat and human brain slices by gabapentin: involvement of KATP channels.

To elucidate the mechanism of action of the anticonvulsant gabapentin (GBP), we compared its effects on K+-evoked [3H]-noradrenaline ([3H]-NA) release from rat hippocampal and human neocortical slices with those of the KATP channel opener pinacidil and the Na+ channel blockers phenytoin, carbamazepine and lamotrigine. Rat hippocampal and human neocortical slices were loaded with [3H]-NA and superfused. [3H]-NA release was evoked by increasing the extracellular [K+] from 3 to 15 mM. GBP decreased [3H]-NA release from rat hippocampal with a pIC50 of 5.59 and a maximum inhibition of 44%. Concentration-dependent inhibition was also seen in human neocortical slices (39% inhibition with 100 microM GBP). These inhibitory effects were antagonized by the KATP channel antagonist glibenclamide, yielding a pA2 of 7.50 in the rat. The KATP channel opener pinacidil (10 microM), like GBP, decreased [3H]-NA release from rat hippocampal slices by 27% and this effect was also antagonized by glibenclamide. In human neocortical slices the inhibition by pinacidil (10 microM) was 31%. Although phenytoin (10 microM), carbamazepine (100 microM) and lamotrigine (10 microM) also decreased [3H]-NA release (by 25%, 57% and 22%, respectively), glibenclamide did not antagonize the effects of these classical Na+ channel blockers. These findings suggest that GBP inhibits K+-evoked [3H]-NA release through activation of KATP channels. To establish whether the KATP channels under investigation were located on noradrenergic nerve terminals or on other neuronal elements, the effects of GBP were compared in the absence and in the presence of tetrodotoxin (TTX 0.32 microM) throughout superfusion. Since the functional elimination of the perikarya of interneurons by TTX reduced the inhibitory effect of GBP, the KATP channels mediating the effect of GBP may be located on nerve terminals, probably on both noradrenergic and glutamatergic nerve endings.

Isolation and characterisation of five neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima.

Five toxins (APE 1 to APE 5) of the sea anemone species Anthopleura elegantissima (Brandt) have been isolated from a toxic by-product fraction of its concentrated crude watery-methanolic extract, prepared previously for the isolation of a neuropeptide (the head-activator) by Schaller and Bodenmüller (Proc. Natl. Acad. Sci. USA 78 (1981) 7000) from 200kg sea anemones. Toxin purification was performed by desalting of the starting material by dialysis (MWCO 3500) against distilled water, anion exchange chromatography on QAE-Sephadex A25 at pH 8, twice gel filtration on Sephadex G50 m, repeated chromatography on QAE-Sephadex at pH 10 and chromatography on the cation exchanger Fractogel EMD SO(3)(-)-650 M.Final purification of the toxins was achieved by HPLC on MN SP 250/10 Nucleosil 500-5 C(18) PPN and MN SP 250/21 Nucleosil 300-7 C(18). Each toxin was composed of at least two isotoxins of which APE 1-1, APE 1-2, APE 2-1, APE 2-2 and APE 5-3 were isolated in preparative scale. With exception of APE 5-3 the sequences of the isotoxins have been elucidated. They resemble the 47 residue type-I long polypeptide toxins native to Anemonia sulcata (Pennant). All isotoxins paralyse the shore crab (Carcinus maenas) by tetanic contractions after i.m. application. The toxins modify current passing through the fast Na(+) channel in neuroblastoma cells, leading to delayed and incomplete inactivation. APE 1-1, APE 2-1 and APE 5-3 produce a positive inotropic effect in mammalian heart muscle, although they differ in potency. The order of potency is APE 2-1>APE 1-1>APE 5-3 (i.e. threshold concentrations are 1, 10 and 300nM, respectively). In addition, they enhance the spontaneous beating frequency in isolated right atria (guinea pig). The most potent cardiotoxic isotoxin is APE 2-1, its sequence is identical with that of AP-C, a toxin isolated and characterised previously by Norton et al. (Drugs and Foods from the Sea, 1978, University of Oklahoma Press, p. 37-50).LD50 APE 2-1:1 micro g/kg b.w. C. maenas (i.m.). LD50 APE 1-1:10 microg/kg b.w. C. maenas (i. m.). LD50 APE 5-3:50 microg/kg b.w. C. maenas (i.m.).

A retinoic acid receptor alpha antagonist counteracts retinoid teratogenicity in vitro and reduced incidence and/or severity of malformations in vivo.

The role of retinoic acid receptors (RAR) in retinoid-induced teratogenesis is mainly unknown. The aim of the present studies was to demonstrate the effect of a RAR alpha antagonist on retinoid-induced teratogenic effects in vitro and in vivo. In micromass cultures of rat limb bud cells a RAR alpha antagonist was able to counteract differentiation inhibiting effects of a RAR alpha agonist. In mouse studies, the selective RAR alpha antagonist reduced frequency and/or severity of major malformations. Our observations indicate the potentiality of selective RAR agonists and antagonists in dissecting the function of nuclear receptors and in particular cases of retinoid teratogenesis, to assign to the different receptors a primary role in determining one or another of the multiple malformations.

Mutagenicity study of Remsen-Fahlberg saccharin and contaminants.

Saccharin and contaminants of commercial Remsen-Fahlberg saccharin were studied for mutagenic potential with the use of the Salmonella/microsome test, Basc-test in Drosophila melanogaster and micronucleus test in mice. In none of these tests were mutagenic effects of saccharin observed. Likewise, the ortho- and para-sulfamoylbenzoic acids (OSBA and PSBA) were ineffective. Para-toluenesulfonamide (PTS) and the major contaminant ortho-toluene-sulfonamide (OTS) exhibited weak mutagenic effects in a modified Salmonella/microsome test and in Drosophila. These results do not indicate mutagenic and therewith correlated carcinogenic potential of saccharin, but they emphasize the possible activity of contaminants.

Mutagenicity studies with x-ray-contrast media, analgesics, antipyretics, antirheumatics and some other pharmaceutical drugs in bacterial, Drosophila and mammalian test systems.

As part of our investigation into mutagenic effects of environmental compounds, we studied 21 pharmaceuticals most frequently sold in West Germany: 6 X-ray-contrast media, 13 analgesics, antipyretics and antirheumatics, 1 central stimulant, and 1 antidepressant. They were studied in different bacterial, Drosophila and mammalian test systems. 4 of these 21 compounds could be detected as mutagens in one of the test systems. namely: 1,2-dichloroethane induced an increase in the frequency of recessive sex-linked lethal mutations in Drosophila melanogaster, quinine dihydrochloride and dimethylaminophenazone were mutagenic in the Salmonella typhimurium tester strain TA98 in the presence of S-9 liver fraction derived from Aroclor-induced rats, and trilithium citrate caused a significant effect in the micronucleus test on bone marrow of NMRI mice.

Mutagenicity of cosmetics ingredients licensed by the European Communities.

As part of our investigation into mutagenic effects of environmental compounds, we studied chemicals allowed as ingredients of cosmetics according to the guidelines of the Council of the European Communities (27 July 1976). We used three systems, the Salmonella/microsome test, the Basc test on Drosophila and the micronucleus test on mouse bone marrow. Of the 31 chemicals tested, 15 were mutagenic in the Ames test; and of these, 5 were also mutagenic in the Basc test and 2 in the micronucleus test.

Autoradiographic detection of 6-thioguanine-resistant lymphocytes of mice. A novel system in somatic mutagenesis testing.

After treatment of mice with ethylnitrosourea in vivo, induction of 6-thioguanine-resistant splenic lymphocytes was measured in vitro. The lymphocytes were mitogen-stimulated in presence of thioguanine. After a culture period of 30 h, TG resistance was determined by the ability of cells to incorporate [3H]thymidine. The results suggest that at this selection condition the majority of resistant cells stem from somatic mutations. Thus, the system is potentially useful as a screening assay for somatic mutagenesis testing. The problem of misleading results due to the presence of phenocopies was investigated by treatment in vitro with UV radiation or ethylnitrosourea.

Mutagenicity study of fried sausages in Salmonella, Drosophila and mammalian cells in vitro and in vivo.

The basic extract of pan-fried sausages was studied for mutagenic potential in seven test systems. Mutagenic activity was high in the standard Ames assay in the Salmonella typhimurium strains TA1538 and TA98 in presence of S9 mix. In vivo, in the intrasanguine host-mediated assay with strain TA98 on Aroclor-pretreated mice, the mutagenic activity of the extract was low. A borderline activity was seen in the SCE assay in vitro with V79 Chinese hamster cells in presence of S9 mix. No significant mutagenic action was found in the gene-mutation assay for thioguanine resistance with V79 cells, the Drosophila sex-linked recessive lethal test, the micronucleus test and the mammalian spot test.

5-Bromodeoxyuridine tablets with improved depot effect for analysis in vivo of sister-chromatid exchanges in bone-marrow and spermatogonial cells.

An improved 5-bromodeoxyuridine (BrdU) tablet technique for observation in vivo of SCE in mouse bone-marrow and spermatogonial cells is described. BrdU tablets were coated with agar as protecting barrier before subcutaneous implantation into mice. In comparison with the original tablets, the agar-coated tablets provided a slower and more uniform delivery of BrdU to the animals. This was corroborated (1) by recovering the undissolved portion of tablets at 1-2-h intervals, and (2) by quantitative determination of the BrdU levels in blood with the help of an analytical HPLC technique. The time required for complete dissolution of the coated tablets was considerably longer than that for the original tablets. This means that the dose of BrdU required for observation of SCE in mouse bone-marrow cells can be reduced accordingly. By using these modified tablets, therefore, undesired effects of high doses of BrdU on mutation (base-line SCE frequency) as well as on cellular replication and proliferation can be diminished. Moreover, the improved depot effect of the modified tablets facilitates the differential labeling of sister chromatids in mouse spermatogonia, a tissue containing cells with a relatively long DNA synthesis period.

Mutagenicity studies with the mouse spot test.

The mammalian spot test, which detects somatic gene mutations in mouse embryos, was investigated with selected chemicals to (a) further validate this test system (ENU, EMS, 2AAF, colchicine) and (b) evaluate the mutagenic potential, in a whole-mammal system, of environmental compounds that had been previously recognized as mutagens in other mammalian or submammalian test systems (1,2-dichloroethane, hydroquinone, nitrofurantoin, o-phenylenediamine, fried sausage extract). Of these substances, ENU, EMS and 2AAF were significantly mutagenic, 1,2-dichloroethane was probably weakly mutagenic. The ENU data were used to estimate the number of pigment precursor cells present at the time of treatment (day 9.25). We also describe in this report the use of a fluorescence microscope for classification of hairs from spots on the coat of C57BL/6JHan X T hybrids.

Study of artificial flavouring substances for mutagenicity in the Salmonella/microsome, Basc and micronucleus tests.

Seventy-six compounds used as artificial flavouring substances in food products were studied for mutagenic properties by the use of the Salmonella/mammalian microsome test (Ames test), Basc test on Drosophila melanogaster and micronucleus test on mouse bone marrow. The following four compounds were mutagenic in Ames tests: ethyl nitrite, ethyl 3-phenylglycidate, 6-methylquinoline and musk ambrette. Of these, ethyl nitrite and musk ambrette also induced a significant (P less than or equal to 0.01) increase in sex-linked recessive lethal mutations in Drosophila. Two further compounds, ethyl 3-methyl-3-phenylglycidate and 4-n-propylanisole, appeared weakly mutagenic in Drosophila only. The result with 4-n-propylanisole was judged to be of equivocal biological significance. None of the flavouring substances induced micronuclei, i.e. cytogenetic damage in the bone marrow of mice.

Analysis of the reversal in breast feeding trends in the early 1970s.

The long downward trend in the practice of breast feeding was reversed during the 1972-73 period. Data from the National Survey of Family Growth conducted by the National Center for Health Statistics were used to investigate the social correlates of breast feeding during the periods 1970-72 and 1973-75 to determine if these factors were related to the reversal in the breast feeding trend. A multivariate log linear modeling technique was used to test hypotheses regarding the direct and indirect effects of education, race, employment status, and source of prenatal care. While education, race, and employment status were directly related to the breast feeding decision, the analysis showed that the trend in breast feeding was unrelated to these correlates. Two alternate conclusions may be drawn from these findings: first, it is possible that changes in infant feeding practices occur earlier in some groups than in others, but the characteristics that distinguish such groups are not included in conventional social demographic data. Alternately, it is possible that the practice of breast feeding appeals equally to all social groups, and changes in the practice occur in response to broad social forces which affect society as a whole.

An intravenous potassium policy with concurrent physician evaluation.

Hospitals have an ethical, as well as a legal, duty to provide safe care to patients. Responsibility for providing care involving medications is distributed to practitioners within the institution including physicians, nurses, and pharmacists. Each practitioner plays an essential role in the provision of safe intravenous potassium supplementation. A procedure is described which incorporates drug usage evaluation into a safe, simple, intravenous potassium policy.