Peter A. Crooks

High performance liquid chromatographic analysis of the pharmacologically active quinones and related compounds in the oil of the black seed (Nigella sativa L.)

An HPLC method for quantifying the putative pharmacologically active constituents: thymoquinone (TQ), dithymoquinone (DTQ), thymohydroquinone (THQ), and thymol (THY), in the oil of Nigella sativa seed is described. Extraction of the constituents from the oil was carried out using C18 PrepSep mini columns followed by quantification of the recovered constituents by HPLC on a reversed-phase muBondapak C18 analytical column, using an isocratic mobile phase of water:methanol:2-propanol (50:45:5% v/v) at a flow rate of 2 ml min(-1). UV detection was at 254 nm for TQ, DTQ, and THY, and at 294 nm for THQ. The above four compounds were separated with good resolution, reproducibility, and sensitivity under these conditions. This analytical method was used to quantify the above four constituents in a commercial sample of N. sativa seed oil, and provides a good quality control methodology for the pharmacologically active components in this widely used natural remedy.

A novel mechanism of action and potential use for lobeline as a treatment for psychostimulant abuse.

Lobeline, an alkaloidal constituent of Lobelia inflata LINN., has a long history of therapeutic usage ranging from emetic and respiratory stimulant to tobacco smoking cessation agent. Although classified as both an agonist and an antagonist at nicotinic receptors, lobeline has no structural resemblance to nicotine, and structure--function relationships do not suggest a common pharmacophore. Lobeline inhibits nicotine-evoked dopamine release and [3H]nicotine binding, thus acting as a potent antagonist at both alpha3beta2(*) and alpha4beta2(*) neuronal nicotinic receptor subtypes. However, lobeline does not release dopamine from its presynaptic terminal, but appears to induce the metabolism of dopamine intraneuronally. Reevaluation of the mechanism by which lobeline alters dopamine function reveals that its primary mechanism is inhibition of dopamine uptake and promotion of dopamine release from the storage vesicles within the presynaptic terminal, via an interaction with the tetrabenazine-binding site on the vesicular monoamine transporter (VMAT2). Thus, lobeline appears to perturb the fundamental mechanisms of dopamine storage and release. Based on its neurochemical mechanism, the ability of lobeline to functionally antagonize the neurochemical and behavioral effects of the psychostimulants amphetamine and methamphetamine was examined. Lobeline was found to inhibit the amphetamine-induced release of dopamine in vitro, and amphetamine-induced hyperactivity, drug discrimination, and self-administration. However, lobeline does not support self-administration in rats, suggesting a lack of addiction liability. Thus, lobeline may reduce the abuse liability of these psychostimulants. The development of lobeline and lobeline analogs with targeted selectivity at VMAT2 represents a novel class of therapeutic agents having good potential as efficacious treatments for methamphetamine abuse.

The NF-κB subunit Rel A is associated with in vitro survival and clinical disease progression in chronic lymphocytic leukemia and represents a promising therapeutic target

In this study, we characterized nuclear factor kappaB (NF-kappaB) subunit DNA binding in chronic lymphocytic leukemia (CLL) samples and demonstrated heterogeneity in basal and inducible NF-kappaB. However, all cases showed higher basal NF-kappaB than normal B cells. Subunit analysis revealed DNA binding of p50, Rel A, and c-Rel in primary CLL cells, and Rel A DNA binding was associated with in vitro survival (P = .01) with high white cell count (P = .01) and shorter lymphocyte doubling time (P = .01). NF-kappaB induction after in vitro stimulation with anti-IgM was associated with increased in vitro survival (P < .001) and expression of the signaling molecule ZAP-70 (P = .003). Prompted by these data, we evaluated the novel parthenolide analog, LC-1, in 54 CLL patient samples. LC-1 induced apoptosis in all the samples tested with a mean LD(50) of 2.8 microM after 24 hours; normal B and T cells were significantly more resistant to its apoptotic effects (P < .001). Apoptosis was preceded by a marked loss of NF-kappaB DNA binding and sensitivity to LC-1 correlated with basal Rel A DNA binding (P = .03, r(2) = 0.15). Furthermore, Rel A DNA binding was inversely correlated with sensitivity to fludarabine (P = .001, r(2) = 0.3), implicating Rel A in fludarabine resistance. Taken together, these data indicate that Rel A represents an excellent therapeutic target for this incurable disease.

Aminoparthenolides as novel anti-leukemic agents: Discovery of the NF-κB inhibitor, DMAPT (LC-1)

A series of aminoparthenolide analogs (6-37) were synthesized and evaluated for their anti-leukemic activity. Eight compounds exhibited good anti-leukemic activity with LD(50)s in the low microM range (1.5-3.0microM). Compounds 16, 24 and 30 were the most potent compounds in the series, causing greater than 90% cell death at 10microM concentration against primary AML cells in culture, with LD(50) values of 1.7, 1.8 and 1.6microM.

Molecular pathway for thymoquinone-induced cell-cycle arrest and apoptosis in neoplastic keratinocytes

Thymoquinone (TQ), the most abundant constituent in black seed, was shown to possess potent chemopreventive activities against DMBA-initiated TPA-promoted skin tumors in mice. Despite the potential interest in TQ as a skin antineoplastic agent, its mechanism of action has not been examined yet. Using primary mouse keratinocytes, papilloma (SP-1) and spindle (I7) carcinoma cells, we studied the cellular and molecular events involved in TQs antineoplastic activity. We show that non-cytotoxic concentrations of TQ reduce the proliferation of neoplastic keratinocytes by 50%. The sensitivity of cells to TQ treatment appears to be stage dependent such that papilloma cells are twice as sensitive to the growth inhibitory effects of TQ as the spindle cancer cells. TQ treatment of SP-1 cells induced G0/G1 cell-cycle arrest, which correlated with sharp increases in the expression of the cyclin-dependent kinase inhibitor p16 and a decrease in cyclin D1 protein expression. TQ-induced growth inhibition in I7 cells by inducing G2/M cell-cycle arrest, which was associated with an increase in the expression of the tumor suppressor protein p53 and a decrease in cyclin B1 protein. At longer times of incubation, TQ induced apoptosis in both cell lines by remarkably increasing the ratio of Bax/Bcl-2 protein expression and decreasing Bcl-xL protein. The apoptotic effects of TQ were more pronounced in SP-1 than in I7 cells. Collectively, these findings support a potential role for TQ as a chemopreventive agent, particularly at the early stages of skin tumorigenesis.

Unexpected pehnomenon in the high-performance liquid chromatographic anlaysis of racemic 14C-labelled nicotine: Separation of enantiomers in a totally achiral system

Abstract Two different forms of racemic 14 C-labelled nicotine exhibit unusual behavior on Partisil-ODS and Partisil-SCX high-performance liquid chromatography systems, when co-injected with unlabelled nicotine of varying enantiomeric composition. Alone, either radiolabelled compound elutes as a single rdioactive peak. In the precense of unlabelled ( S )-(—)-nicotine or its antipode as standard, the 14 C activity is divided into two equal peaks. If unlabelled racemic nicotineis used as standard, only one 14 C peak is produced. Experiments were performed which demonstrate that the two 14 C peaks observed are due to the separated enantiomers of the radiolabelled material. The phenomenon responsible for this separation is not known, but may be due to differential enantiomeric association between like and unlike optical isomers of nicotine. The resulting effect provides a facile method for the isolation of the pure enantiomers of 14 C-labelled nicotine, on a totally achiral system. This method may also be applicale to other optically active radiolabelled compounds.

Nornicotine is self-administered intravenously by rats

Abstract  Rationale: Nicotine is a tobacco alkaloid known to be important in the acquisition and maintenance of tobacco smoking. However, other constituents in tobacco may contribute to the dependence liability. Objective: The present report sought to determine whether nornicotine, a tobacco alkaloid and metabolite of nicotine, has a reinforcing effect. Methods: Rats were prepared with a jugular catheter, then were allowed to self-administer intravenously either S(–)-nicotine (0.03 mg/kg/infusion), RS(±)-nornicotine (0.3 mg/kg/infusion) or saline using a two-lever operant procedure. The response requirement for each infusion was incremented gradually from a fixed ratio 1 (FR1) to FR5. When responding stabilized on the FR5, other doses of nicotine (0.01 mg/kg/infusion and 0.06 mg/kg/infusion) and nornicotine (0.075, 0.15, and 0.6 mg/kg/infusion) were tested for their ability to control responding. Results: Similar to nicotine, rats self-administered nornicotine significantly above saline control levels. Within the dose ranges tested, both nicotine and nornicotine yielded relatively flat dose–response functions. Extinction of responding was evident when saline was substituted for nornicotine, and responding was reinstated when nornicotine again was available. The rate of nornicotine self-administration was similar between rats tested with either 24-h or 48-h inter-session intervals. Conclusion: These results indicate that nornicotine contributes to the dependence liability associated with tobacco use.

Contribution of CNS nicotine metabolites to the neuropharmacological effects of nicotine and tobacco smoking

Nicotine, the principal alkaloid in tobacco products, is generally accepted to be the active pharmacological agent responsible for CNS effects resulting from tobacco use. Arguments are presented in this commentary which take issue with this popular dogma, by providing evidence that nicotine metabolites may also be responsible for the CNS effects commonly attributed to nicotine. CNS effects attributed to nicotine include reinforcing effects, mood elevation, arousal, locomotor stimulant effects, and learning and memory enhancement. The reinforcing and locomotor stimulant effects of nicotine have been suggested to be the result of activation of CNS dopaminergic systems, and nicotine-induced modulation of dopaminergic neurotransmission has been studied in detail. Nicotine acts at a family of nicotinic receptor subtypes composed of multiple subunits; however, the exact composition of the subunits in native nicotinic receptors and the functional significance of the receptor subtype diversity are currently unknown. This nicotinic subtype diversity increases the complexity of the potential mechanisms of action of nicotine and its metabolites. Although peripheral metabolism of nicotine has been studied extensively, metabolism in the CNS has not been investigated to any great extent. Recently, studies from our laboratory have demonstrated that several nicotine metabolites are present in the CNS after acute nicotine administration. Moreover, nicotine metabolites are pharmacologically active in neurochemical and behavioral assays. Thus, CNS effects resulting from nicotine exposure may not be due solely to nicotine, but may result, at least in part, from the actions of nicotine metabolites.

Vesicular monoamine transporter 2: Role as a novel target for drug development

In the central nervous, system, vesicular monoamine transporter 2 (VMAT2) is the only transporter that moves cytoplasmic dopamine (DA) into synaptic vesicles for storage and subsequent exocytotic release. Pharmacologically enhancing DA sequenstration by VMAT2, and thus preventing the oxidation of DA in the cytoplasm, may be a strategy for treating diseases such as Parkinsons disease. VMAT2 may also be a novel target for the development of treatments for psychostimulant abuse. This review summarizes the possible role of VMAT2 as a therapeutic target, VMAT2 ligands reported in the literature, and the structure-activity relationship of these ligands, including tetrabenazine analogs, ketanserin analogs, lobeline analogs, and 3-amine-2-phenylpropene analogs. The molecular structure of VMAT2 and its relevance to ligand binding are briefly discussed.

Lobeline Displaces [3H]Dihydrotetrabenazine Binding and Releases [3H]Dopamine from Rat Striatal Synaptic Vesicles: Comparison with d‐Amphetamine

Abstract: Lobeline, an alkaloid from Indian tobacco (Lobelia inflata), is classified as a nicotinic agonist and is currently used as a smoking cessation agent. However, our previous in vitro studies demonstrate that lobeline does not act as a nicotinic agonist but alters presynaptic dopamine (DA) storage by potently inhibiting DA uptake into synaptic vesicles. Recently, d‐amphetamine has been reported to act at the level of the synaptic vesicle to alter presynaptic function. The present in vitro studies further elucidate the mechanism of lobelines action and compare its effects with those of d‐amphetamine. [3H]Dihydrotetrabenazine ([3H]DTBZ), used routinely to probe a high‐affinity binding site on the vesicular monoamine transporter (VMAT2), bound to vesicle membranes from rat striatum with a KD of 1.67 nM and Bmax of 8.68 pmol/mg of protein. Lobeline inhibited [3H]DTBZ binding with an IC50 of 0.90 µM, consistent with its previously reported IC50 of 0.88 µM for inhibition of [3H]DA uptake into vesicles. These results suggest that lobeline specifically interacts with DTBZ sites on VMAT2 to inhibit DA uptake into synaptic vesicles. Interestingly, d‐amphetamine inhibited [3H]DTBZ binding to vesicle membranes with an IC50 of 39.4 µM, a concentration 20 times greater than reported for inhibition of VMAT2 function, suggesting that d‐amphetamine interacts with a different site than lobeline on VMAT2 to inhibit monoamine uptake. Kinetic analysis of [3H]DA release from [3H]DA‐preloaded synaptic vesicles in the absence of drug revealed a t1/2 of 2.12 min. Lobeline and d‐amphetamine evoked [3H]DA release with EC50 values of 25.3 and 2.22 µM, respectively. At a concentration 10 times the EC50, lobeline and d‐amphetamine significantly decreased the t1/2 of [3H]DA release to 1.58 and 1.48 min, respectively. Thus, in contrast to d‐amphetamine, which is equipotent in inhibiting DA uptake and promoting release from the synaptic vesicles, lobeline more potently (28‐fold) inhibits DA uptake (via an interaction with the DTBZ site on VMAT2) than it evokes DA release to redistribute presynaptic DA storage.