Dennis K.J. Gorecki

3,5-Bis(benzylidene)-4-oxo-1-phosphonopiperidines and related diethyl esters: Potent cytotoxins with multi-drug-resistance reverting properties.

A series of 3,5‐bis(benzylidene)‐4‐piperidones 3 were converted into the corresponding 3,5‐bis(benzylidene)‐1‐phosphono‐4‐piperidones 5 via diethyl esters 4. The analogues in series 4 and 5 displayed marked growth inhibitory properties toward human Molt 4/C8 and CEM T‐lymphocytes as well as murine leukemia L1210 cells. In general, the N‐phosphono compounds 5, which are more hydrophilic than the analogues in series 3 and 4, were the most potent cluster of cytotoxins, and, in particular, 3,5‐bis‐(2‐nitrobenzylidene)‐1‐phosphono‐4‐piperidone 5 g had an average IC50 value of 34 nM toward the two T‐lymphocyte cell lines. Four of the compounds displayed potent cytotoxicity toward a panel of nearly 60 human tumor cell lines, and nanomolar IC50 values were observed in a number of cases. The mode of action of 5 g includes the induction of apoptosis and inhibition of cellular respiration. Most of the members of series 4 as well as several analogues in series 5 are potent multi‐drug resistance (MDR) reverting compounds. Various correlations were noted between certain molecular features of series 4 and 5 and cytotoxic properties, affording some guidelines in expanding this study.

Pharmacokinetics and tissue residues of Telazol in free-ranging polar bears.

A pharmacokinetic and tissue residue study was conducted to assess the risks associated with human consumption of polar bears in arctic Canada that have been exposed to the immobilizing drug Telazol®, a mixture of tiletamine hydrochloride and zolazepam hydrochloride. Twenty-two bears were remotely injected with about 10 mg/kg of Telazol®. Following immobilization, serum samples were collected serially at regular intervals until the bears awakened. Sixteen of the bears were relocated and killed under permit by local hunters at various times from 0.5 to 11 days after dosing. Serum, kidney, muscle and adipose tissue samples were collected immediately after death. All samples were stored at -70 C until analysis by HPLC. The concentration-time data of tiletamine and zolazepam in serum during the immobilization period were fitted to curves by computer and the pharmacokinetic parameters assessed. In addition, the serum and tissue samples collected at the time of death were analyzed for both parent drugs, for one metabolite of tiletamine (CI-398), and for three metabolites of zolazepam (metabolites 1, 2 and 4). A one-compartment model with first-order absorption and elimination best fit the time-series data for the drugs in serum during the immobilization period. This model gave half-lives (mean ± SE) for tiletamine and zolazepam of 1.8 ± 0.2 h and 1.2 ± 0.08 h, respectively, clearance values of 2.1 ± 0.3 1 h-1·kg-1 and 1.1 ± 0.1 1·h-1·kg-1, and volumes of distribution of 5.2 ± 0.6 1/kg and 1.8 ± 0.2 1/kg. The concentrations of both drugs and their metabolites declined rapidly to trace levels by 24 h post-dosing, although extremely low concentrations of some metabolites were encountered sporadically over the entire sampling period. In particular, zolazepam metabolite 2, remained detectable in fat and muscle tissue at the end of the study, 11 days after dosing. It was concluded that during immobilization, both tiletamine and zolazepam levels decline rapidly in a monoexponential fashion, and their pharmacokinetic parameters in polar bears are similar to those observed in other species. Tissue levels of the drugs and their metabolites declined sufficiently rapidly that individuals eating meat from exposed bears would be unlikely to experience pharmacological effects from the drugs. Nevertheless, slight exposure to the drugs and/or their metabolites might be possible for an indeterminate time after dosing.

Novel 3,5-bis(arylidene)-4-piperidone dimers: Potent cytotoxins against colon cancer cells

Two novel series of dimeric 3,5-bis(arylidene)-4-piperidones 7 and 8 were prepared as cytotoxic agents. A specific objective of this study was the discovery of novel compounds displaying potent anti-proliferative activities against colon cancers. Most of the compounds demonstrate potent cytotoxicity against HCT116 and HT29 colon cancer cell lines in which the IC50 values range from low micromolar to nanomolar values. In general, the majority of the compounds showed greater cytotoxicity and some degree of selectivity toward HCT116 cells compared to HT29 cells. Compound 9 in which the amidic carbonyl groups were excised was substantially less potent than 8a in both cell lines suggested that the amide groups are important components of the molecules for exhibiting cytotoxicity. Virtually all the compounds were more potent than a reference drug 5-fluorouracil which is used in treating colon cancers as well as a related enone curcumin. QSAR studies were undertaken and some guidelines for amplification of the project have been formulated. Flow cytometry analysis of a representative potent compound 7f revealed that it induces apoptosis in HCT116 cells.

Bis[3,5‐bis(benzylidene)‐4‐oxo‐1‐piperidinyl]amides: A Novel Class of Potent Cytotoxins

The principal objective of this study was the examination of the theory of cytotoxic synergism. In this exploratory study, we tested the hypothesis that doubling the number of sites available for thiol alkylation in a series of candidate cytotoxins increases potency more than two‐fold. This concept was verified in one‐third of our comparisons using human Molt 4/C8 and CEM T‐lymphocytes and murine L1210 cells. In addition, the significant potencies of various members of our compound series justified further studies. Molecular modeling revealed that relative locations of the amidic groups correlate with cytotoxicity. A potent cytotoxic compound, 1,2‐bis(3,5‐dibenzylidene‐4‐oxo‐piperidin‐1‐yl)ethane‐1,2‐dione (1 a) inhibited the growth of a large number of human tumor cell lines and displayed greater toxicity toward certain non‐adherent cells than toward adherent neoplasms or fibroblasts. The mode of action of 1 a includes induction of apoptosis and necrosis.

Depression of the electroretinogram in rats deficient in zinc and taurine during prenatal and postnatal life

The objective of this study was to investigate whether zinc interacts with taurine to influence the development of the electroretinogram. Virgin female Sprague-Dawley rats were bred overnight and assigned to 1 of 4 treatments in a 2 × 2 factorial design with two levels of zinc (50 μg/g through gestation and 50 μg/g after parturition; 15 μg/g through gestation and 7.5 μg/g after parturition) and two levels of taurine (2 or 0 μmol/g). Guanidinoethyl sulfonate (10 g/L), a structural analogue of taurine, was added to the drinking water of the animals receiving 0 μmol/g taurine. At postnatal day 23, male pups (n = 10) were weaned onto their respective diets. Dark-adapted electroretinograms were recorded as a function of stimulus intensity on 7 1/2–8 1/2-week-old anesthetized pups. Two-factor analysis of variance demonstrated no interaction between zinc and taurine for a- or b-wave amplitudes or latencies (P < 0.05). Zinc and taurine deficiencies each independently depressed electroretinogram a-wave and b-wave amplitudes but not latencies. The amplitude of the b-wave was plotted as a function of log stimulus intensity, and an iterative curve-fitting procedure was used to determine the maximum response, slope, and half-saturation constant. No interaction was noted. A significant treatment effect on maximum response was demonstrated for zinc (P = 0.0498) and taurine (P = 0.0014). No treatment effects were evident for the half-saturation constant or slope. These findings indicate that zinc and taurine deficiencies are not synergistic in their depressing effects on the electroretinogram in this model.

Postnatal deficiencies of zinc and taurine alter electroretinograms, oscillatory potentials and morphology of the rat retina

The study objective was to evaluate the retinal response to deficiencies of zinc and taurine present throughout the period of postnatal retinal development. At parturition, Sprague-Dawley dams were assigned to one of four treatments in a 2 × 2 factorial design with two levels of zinc (4.5 and 50 μg/g) and two levels of taurine (0 and 2 μmol/g). Guanidinoethyl sulfonate, a taurine transport inhibitor, was added to the drinking water of the rats receiving 0 μmol/g taurine. Male pups (n = 10) were weaned on to their respective diets at postnatal day 22. Dark adapted electroretinograms and oscillatory potentials (OP) were recorded in the pups at 48-57 days of age. At maximal light intensity, the amplitudes of the a- and b-waves were depressed by deficiency of either nutrient, but the influence of combining these treatments was less than additive; the same pattern was evident for Vmax, the maximum amplitude obtained when the b-wave was plotted as a function of light intensity. This type of interaction was also evident for the amplitudes of OP1, OP3 and OP4. Zinc deficiency independently decreased the amplitude and increased the latency of OP5, and increased the latencies of OP3 and OP4. Light and transmitting electron microscopic examination revealed the most pronounced retinal degeneration in the rats deficient in both zinc and taurine. Tibia zinc and liver taurine concentrations provide evidence that these nutrients also interact in other tissues. The findings of this study demonstrate retinal damage with deficiencies of zinc and taurine during postnatal life. These nutrients interact in at least some of their functions in the retina through an as yet unidentified mechanism.

Intravenous Clomipramine and Obsessive-Compulsive Disorder

A 62 year old woman presented with a long history of obsessive-compulsive disorder, the symptoms of which were not adequately controlled by oral clomipramine. The patient was started on intravenous clomipramine. Drug levels of both clomipramine and its active metabolite desmethylclomipramine were measured. Symptoms were controlled during her hospital stay. A three year follow-up report indicated the patient is doing well with no evidence of obsessive-compulsive or depressive symptoms. A discussion of the blood levels obtained and the caution to be exercised during intravenous administration is presented.

E-2-[3-(3,4-dichlorophenyl)-1-oxo-2-propenyl]-3-methylquinoxaline-1,4-dioxide: a lead antitubercular agent which alters mitochondrial respiration in rat liver.

A series of 2-(3-aryl-1-oxo-2-propenyl)-3-methylquinoxaline-1,4-dioxides 1a-l and 2-acetyl-3-methylquinoxaline-1,4-dioxide 2 were evaluated against Mycobacterium tuberculosis H(37)Rv. With the exception of the 4-nitro analog 1k, significant antitubercular potencies were observed in series 1 and 2 which have IC(50) values in the range of 1-23 microM. Negative correlations were noted between the IC(50) values of 1a-j, l towards M. tuberculosis and both the sigma and pi constants of the substituents in the benzylidene aryl ring. In particular, 1h emerged as a lead compound having IC(50) and IC(90) figures of 1.03 microM and 1.53 microM, respectively. This molecule affected respiration in rat liver mitochondria which is likely one way that 1h and the bioactive analogs exert their antitubercular properties. The quinoxaline 2, which lacks an alpha,beta-unsaturated group, has no effect on mitochondrial respiration using concentrations which inhibit the growth of M. tuberculosis.

Vitamin E deficiency and erythrocyte deformability in the rat

It has been suggested that erythrocyte deformability is decreased in vitamin E deficiency due to oxidative damage to the cell membrane. Male Wistar rats (66 to 88 g) were fed ad libitum an AIN76-based diet containing tocopherol-stripped corn oil without added vitamin E for 8 weeks (−E; n = 8). Control animals were fed ad libitum the same diet containing 50 IU/kg dl-α-tocopheryl acetate (+E; n = 7). Vitamin E deficiency was confirmed by depressed mean (±SEM) plasma α-tocopherol levels (μmol/L), as measured by high performance liquid chromatography [−E: 0.5 ± 0.1; +E: 20.3 ± 1.8] and elevated hydrogen peroxide-induced hemolysis (%) [−E: 92.6 ± 2.4; +E: 4.2 ± 1.6; P < 0.05 by Students t test]. The only alteration in a complete blood count was a depression in reticulocyte number (× 1012/L) [−E: 0.19 ± 0.02; +E: 0.46 ± 0.03; P < 0.05]. Erythrocyte deformability was measured at standard shear stress under conditions of increasing osmolality in the ektacytometer. Elongation index (the ratio of length to width of the diffraction pattern of the deformed cells) was plotted against osmolality to generate an osmotic deformability profile. EImax (the maximum elongation index) and Ohyper (the osmolality at which the elongation index is half of EImax on the hypertonic arm of the curve) were significantly increased in samples from the −E group (P < 0.05 by Students t test). In summary, erythrocyte deformability as measured by the ektacytometer was not decreased by a subclinical vitamin E deficiency in the rat. In fact, a small but significant increase in maximum deformability was observed in erythrocytes from vitamin E-deficient rats.

3-Isoxazolidone—An inhibitor of GABA metabolism

Abstract 3-Isoxazolidone, a cyclic analogue of aminooxypropionic acid, was prepared and evaluated for its effect on γ-aminobutyric acid (GABA) metabolism. Its administration to animals resulted in potent inhibitions of brain glutamic acid decarboxylase (GAD) and GABA-α-oxoglutarate aminotransferase (GABA-T) activities. A small but significant increase in brain GABA content was observed. The inhibiting effects varied with the experimental species being greatest in chicks and least in mice. The enzyme inhibitions were not prevented by the concurrent administration of pyridoxine to the animals nor by the addition of pyridoxal phosphate to the assay system. Additions of 3-isoxazolidone directly to brain homogenates inhibited the activities of both GAD and GABA-T but not to the extent that might be expected from the studies in vivo .