Fast screening of depression in cancer patients: the effectiveness of the HADS.

In oncology clinics, there is an increasing need for fast and accurate screening scales and procedures in order to evaluate cancer patients for depression. The present study investigated the comparative effectiveness in recognising depressed patients of the Hospital Anxiety and Depression Scale (HADS), a self-report screening scale, and the Montgomery-Asberg Depression Rating Scale (MADRS), a semi-structured clinician-rated scale, in 151 patients affected by mixed cancer pathologies. With the MADRS, 73.5% of the patients were identified as depressed, whereas the HADS identified 36.4% and 58.3% as depressed, using the cut-offs of 11 and 8 respectively. The results suggest moderate agreement between the MADRS and the HADS when a cut-off of 8 is used (K-test: 0.44), while using a HADS cut-off of 11 gave a significantly higher underestimation of depressed patients (K-test: 0.29). In conclusion, the results suggest that the HADS can be useful as a sufficiently accurate first-step screening tool for depression in mixed oncology settings.

Alkylation of a catalytic aspartate group of the SIV protease by an epoxide inhibitor.

Specific irreversible inhibition of the SIV protease by FMOC-protected piperidine epoxide 1 involves alkylation of the protein. Tryptic digestion of the alkylated protein and mass spectrometric analysis of the peptides identify an active site aspartic acid (Asp-25) as the single residue that is alkylated. Computer modeling of 1 bound in the crystal structure of the SIV protease using DOCK 3.5 indicates that 1 has appropriate access to the active site. It is able to align in an orientation that allows a proton to be transferred to the epoxide from one of the catalytic aspartic acid groups in conjunction with nucleophilic attack on the epoxide of the carboxylate moiety of the second catalytic aspartic acid residue. Hydrophobic interactions are not optimal for this process due, in part, to the rigidity of the inhibitor ring system and the planar conformation of the amide. The combination of modeling with protein alkylation can provide insights into structural modifications of the inhibitor that may lead to improved inhibitory activity.

Bile pigments as HIV-1 protease inhibitors and their effects on HIV-1 viral maturation and infectivity in vitro.

Using recently developed molecular-shape description algorithms, we searched the Available Chemical Directory for known compounds similar in shape to the potent HIV-1 protease inhibitor Merck L-700,417; 15 compounds most similar in shape to the inhibitor were selected for testing in vitro. Four of these inhibited the protease at 100 microM or less and the most active of the four were the naturally occurring pigments biliverdin and bilirubin. Biliverdin and bilirubin inhibited recombinant HIV-1 protease in vitro at pH 7.8 with K1 values of approx. 1 microM, and also inhibited HIV-2 and simian immunodeficiency virus proteases. The related pyrrolic pigments stercobilin, urobilin, biliverdin dimethyl ester and xanthobilirubic acid showed similar inhibitory activity at low micromolar concentrations. Biliverdin, bilirubin and xanthobilirubic acid did not inhibit viral polyprotein processing in cultured cells, but they reduced viral infectivity significantly. At 100 microM, xanthobilirubic acid affected viral assembly, resulting in a 50% decrease in the generation of infectious particles. In contrast, at the same concentrations biliverdin and bilirubin exerted little or no effect on viral assembly but blocked infection of HeLaT4 cells by 50%. These results suggest that bile pigments might be a new class of potential lead compounds for developing protease inhibitors and they raise the question of whether hyperbilirubinaemia can influence the course of HIV infection.

In vivo intracerebral microdialysis studies in rats of MPP+ analogues and related charged species.

The in vivo dopaminergic neurotoxic properties of 45 MPTP and MPP+ analogues and related compounds were examined by an intrastriatal microdialysis assay in conscious rats. MPP(+)-like toxicity, as evidenced by the irreversible effects on DA release and enhancement of lactate formation, was observed with a variety of structural types although no compound was more toxic than MPP+. The following global structure-toxicity relationships could be derived: (1) only permanently charged compounds showed neurotoxic effects; (2) with the exception of amino groups, hydrophilic substituents abolished toxicity; (3) activity was enhanced by lipophilic groups although increased steric bulk around the nitrogen atom tended to decrease activity; (4) nonaromatic, quaternary systems (methiodide of MPTP, guanidinium derivatives) were only weakly toxic; and (5) certain bi- and tricyclic systems, including putative metabolites of potential endogenous MPTP-like compounds, were weakly toxic. The lack of toxic effects following perfusions with DA itself confirmed that MPTP dopaminergic neurotoxicity is not likely to be mediated by the MPP(+)-induced release of DA. With some interesting exceptions, these in vivo data correlate reasonably well with in vitro data on the nerve terminal uptake properties and the inhibitory effects on mitochondrial respiration of these compounds.

Deuterium isotope effect measurements on the interactions of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine with monoamine oxidase B.

Kinetic deuterium isotope effects for the noncompetitive, intermolecular monoamine oxidase B-catalyzed oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to the corresponding 1-methyl-4-phenyl-2,3-dihydropyridinium species MPDP+ were found to be 3.55 on Vmax and 8.01 on Vmax/Km with MPTP-6,6-d2 as the deuterated substrate. Similar values were obtained with MPTP-2,2,6-d4 and MPTP-CD3-2,2,6,6-d4. The deuterium isotope effect for the electrochemical oxidation of 1 mM MPTP-2,2,6,6-d4 was only 1.35. These results indicate that the monoamine oxidase B-catalyzed oxidation of this substrate may not proceed via a reaction pathway involving alpha-carbon deprotonation of an aminium radical intermediate. Isotope effect measurements also established that the rate of inactivation of monoamine oxidase B by MPTP is unaffected by replacement of the C-6 methylene protons with deuterons, but is retarded by replacement of the C-2 methylene protons (DKi = 1.9). The mechanism-based inactivation of monoamine oxidase B by MPTP, therefore, is likely to mediated by a species derived from the enzyme-generated 2,3-dihydropyridinium oxidation product.

Degradation of rat hepatic cytochrome P-450 heme by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine to irreversibly bound protein adducts.

Administration of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) (a structural analog of the dihydropyridine Ca2+ antagonists) to untreated, phenobarbital-, or dexamethasone-pretreated rats results in time-dependent losses of hepatic cytochrome P-450 content. Functional markers for various cytochrome P-450 isozymes have permitted the identification of P-450h, P-450 PB-1/k, and P-450p as the isozymes inactivated preferentially by the drug. DDEP-mediated cytochrome P-450 destruction may be reproduced in vitro, is most prominent after pretreatment of rats with dexamethasone, pregnenolone 16 alpha-carbonitrile or phenobarbital, and is blocked by triacetyloleandomycin. These findings together with the observation that DDEP markedly inactivates hepatic 2 beta- and 6 beta-testosterone hydroxylase and erythromycin N-demethylase tend to indict the steroid-inducible P-450p isozyme as a key protagonist in this event. The precise mechanism of such DDEP-mediated P-450p heme destruction is unclear, but involves prosthetic heme alkylation of the apocytochrome at its active site in what appears to be a novel mechanism-based "suicide" inactivation. Such inactivation appears to involve fragmentation of the heme to reactive metabolites that irreversibly bind to the protein, but the chemical structure of the heme-protein adducts is yet to be established. Intriguingly, such DDEP-mediated P-450p destruction in vivo also results in accelerated loss of immunochemically detectable apocytochrome P-450p. It remains to be determined whether or not this loss is due to enhanced proteolysis triggered by the structural modification of the apocytochrome.

Inactivation of multiple hepatic cytochrome P-450 isozymes in rats by allylisopropylacetamide: mechanistic implications.

In vivo administration of the porphyrogenic agent allylisopropylacetamide (AIA) to phenobarbital-pretreated rats results in marked loss of hepatic cytochrome P-450 content. Using isozyme-selective functional markers, we now show that such loss reflects inactivation of several phenobarbital-inducible and constitutive isozymes. Some of the isozymes (P-450a,b,h and PB-1) are largely reparable by reconstitution with exogenous hemin, indicating that after AIA-mediated loss of their prosthetic heme, their apoprotein moieties are essentially intact and functionally reconstitutable with hemin. On the other hand, after AIA-mediated inactivation, isozymes such as cytochrome P-450p remain refractory to such repair. The cause for such intractability remains somewhat elusive since AIA-mediated alkylation of the apocytochrome, proteolytic loss of the hemoprotein, or even irreversible binding of prosthetic heme catabolites to the apocytochrome does not appear to be responsible.

Bioactivation of MPTP: reactive metabolites and possible biochemical sequelae.

Expression of the selective nigrostriatal neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP] requires its bioactivation by MAO B which leads to the formation of potentially reactive metabolites including the 2-electron oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium species [MPDP+] and the 4-electron oxidation product, the 1-methyl-4-phenyl pyridinium species [MPP+]. The latter metabolite accumulates in brain striatal tissues, is a substrate for dopaminergic active uptake systems and is an inhibitor of mitochondrial NADH dehydrogenase, a respiratory chain enzyme located in the inner mitochondrial membrane. In intact mitochondria this inhibition of respiration may be facilitated by active uptake of MPP+, a process dependent on the membrane electrical gradient. In considering possible mechanisms involved in the biochemical effects of MPP+, its redox cycling potential appears to be much lower than its chemical congener paraquat, based on attempted radical formation by chemical or enzymic reduction. Theoretically, a carbon-centered radical intermediate could be formed by 1-electron reduction of MPP+, or by 1-electron oxidation of 1-methyl-4-phenyl-1,2-dihydropyridine, the free base form of MPDP+. The 1-electron reduction of such a radical could form 1-methyl-4-phenyl-1,4-dihydropyridine [DHP]. Synthetic DHP is neurotoxic in C57B mice, and its administration leads to the formation of MPP+ in the brain, presumably through rapid auto-oxidation. The hydrolysis of DHP would yield 3-phenylglutaraldehyde and methylamine. Recent studies demonstrating the formation of methylamine in brain mitochondrial preparations containing MPTP support our suggestion that DHP may be a brain metabolite of MPTP.

Studies on the 1-methyl-4-phenyl-2,3-dihydropyridinium species 2,3-MPDP+, the monoamine oxidase catalyzed oxidation product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

The nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is biotransformed by brain monoamine oxidase (MAO) to an unstable dihydropyridinium intermediate that reacts with cyanide ion to form an alpha-cyano-tetrahydropyridine adduct and, in the absence of cyanide ion, undergoes disproportionation to the 1-methyl-4-phenylpyridinium species MPP+ and MPTP. Comparison of the HPLC retention times, diode array UV, and chemical ion mass spectral characteristics of these products with those of synthetic standards led us to propose the 1-methyl-4-phenyl-2,3-dihydropyridinium species 2,3-MPDP+ and 6-cyano-1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as tentative structure assignments for the dihydropyridinium metabolite and the cyano adduct, respectively. Results presented in this paper confirm the first assignment and establish that, although the proposed 6-cyano adduct is initially formed, the product that was isolated from the mitochondrial incubation mixtures of MPTP and sodium cyanide actually is the isomeric 2-cyano-1-methyl-4-phenyl-1,2,3,6 -tetrahydropyridine. On the basis of the selective incorporation of deuterium into these products, we provide rational mechanistic interpretations of the disproportionation reaction and the rearrangement of the cyano adducts. These results establish that the MAO-catalyzed bioactivation of MPTP leads to the formation of a variety of reactive molecules that are potentially cytotoxic to nigrostriatal cells.

Metabolism of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by liver homogenate fractions.

The metabolic fate of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been examined in rat and rabbit liver mitochondrial and rabbit liver microsomal preparations. The mitochondrial preparations rapidly oxidized MPTP, in a pargyline-sensitive reaction, to a polar material that was shown to contain the 1-methyl-4-phenylpyridinium species as the principal product. NADPH-supplemented microsomal preparations converted MPTP to two principal products: 4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine N-oxide. Carbon monoxide and SKF 525A selectively inhibited the oxidation of MPTP to the nor compound, indicating that this N-demethylation reaction is cytochrome P-450 catalyzed. Attempts to trap possible unstable iminium metabolites of MPTP in microsomal incubation mixtures with sodium cyanide led to the isolation of a monocyano adduct that proved to be the N-cyanomethyl derivative. Thus, hepatic mitochondrial and microsomal enzyme systems catalyze the oxidation of MPTP by different pathways, the former leading to the generation of species that may possess neurotoxic properties.