Patent Application
20100021567
Throughout the world, various people almost 20% of the whole population enjoy tobacco actions via either smoking [cigarettes, pipes or shisha (nargilla)] or its sublingual intake under the tongue in a semi-dry form moistened with sodium bicarbonate (or as snuff) e.g. in Sudan and a some other African countries (1).
Chronic consumption of tobacco can produce two types of adverse reactions, namely cancer of lungs and throats when it is smoked due to the tar components and a group of diseases such as hypertension, arrhythmias, hyperlipidemias, asthma and gastric and duodenal ulcers together with development of dependence with the consequence maintenance of intake due to its nicotine content (1-3). These disorders usually predispose to death.
Nicotine usually mediates its actions via two types of main receptors located at two sites in the body namely postsynaptically in the neuromuscular junction or the skeletal muscles and in the neuronal tissues peripherally and centrally at the ganglia and neurons. It excites these receptors to release acetylcholine (4). The neuronal nicotinic receptors are ion-gated channels each of which is composed of 17 subunits. Ten of them are α type, 4 are β type together with γ, δ and ε (epsilon) (4). The most important receptor that is involved in nicotine dependence and the support of maintenance of nicotine intake in its various forms is the one containing the subunits α4 β2 (5). Activation of these receptors releases dopamine via release of acetylcholine in the nucleus accumbens and the frontal cortex (6). The release of dopamine plays an important part in nicotine-induced dependence, toxic actions and addiction (7).
Many investigators throughout the world are involved in researches aiming for discovering of drugs to help in cessation of tobacco intake in its various forms. This patent describes for the first time a chemical namely the alkaloid peganine isolated from the aerial parts of Anisotes trisulcus (Forssk.) Vahl family Acanthaceae collected from the Southern region of Saudi Arabia (8) that proved to suppress nicotine consumption and thereby opening the avenue to place itself as one of the limited armenatum available today to help in cessation of tobacco intake.
Peganine is the major quinazoline alkaloid isolated from Anisotes trisulcus (Forssk.) Vahl (represented 23.26% yield from AT-2 fraction). The alkaloid is also previously isolated from different plant species (9-13). Besides peganine, the plant produced other alkaloidal constituents such as anisotine, vasicinone (14), trisulcusine and methoxypeganine (15). Anisotes trisulcus (Forssk.) Vahl is a shrub locally known as Almodh (16) and is a traditional herbal medicine in the Arabian Peninsula and found its way in the folk medicine of Saudi Arabia as an antidiabetic. The plant is also used as treatment for all hepatic conditions including hepatitis, jaundice and other hepatic problems (17-19). The pharmacognostical studies on the leaf of the plant have also been investigated (20). The alkaloid peganine was reported to possess abortifacient and uterine stimulant activity (21-23). In addition, it was shown to produce slight but persistent bronchodilatation, slight hypotension and to inhibit peristalsis of isolated gut (24). Other studies showed bronchoconstriction and a negative inotropic effect of peganine with reduced coronary flow in the isolated heart (24).
Anisotes trisulcus (Forssk.) Vahl was collected in March, 2005 from the way toward Fifa Mountains, Saudi Arabia.
The dried aerial parts (1 kg) of A. trisulcus were exhaustively extracted by cold percolation with 90% EtOH. The combined EtOH extract was concentrated in vacuo at 40° C. to produce a solid residue (75 g). The alcoholic extract was dissolved in 225 ml of 2% tartaric acid and extracted with CHCl3 to remove acidic and neutral components (8 g) (Fraction AT-1). The aqueous acidic solution was then alkalinized with dilute NH4OH to pH 9 and extracted with CH2Cl2 to produce 0.688 g (Fraction AT-2). The aqueous layer was made more alkaline with NH4OH to pH 12 and extracted with EtOAc to get 0.64 (Fraction AT-3). The remaining aqueous phase was made acidic with tartaric acid to pH 4 and then lyophilized to yield 6 g (Fraction AT-4).
The fraction AT-2 (0.688 g) was chromatographed via chromatotron 2 mm plate using EtOH absol.:CHCl3:NH4OH (5:95:4 drops) to afford 160 mg of peganine (23.26% yield) that was crystallized using mixture of CHCl3 and MeOH. The alkaloid was identified as peganine by comparing its physical and spectral data with those reported previously in our laboratory and with the literature (14).
The total extract was suspended in 0.25% aqueous sodium carboxymethylcellulose to give a concentration of 12.5% (w/v) initially and then diluted with the same vehicle as appropriate.
Swiss albino mice (25 g body weight) and albino Wistar rats (200 g body weight) were divided each into 5 groups (N=10 animals per group). Animals in the different groups were injected with the extract intraperitoneally to provide doses of 0.4, 0.8, 1.6, 2.4 and 3.2 g/kg in the different groups. The animals were observed for any changes in behavior or death continuously for 2 hours following the injection. Any alive animals were observed for 72 hours thereafter. The percentage of death in each group was calculated. The LD50 values were then determined following the methods of Litchfield and Wilcoxon, (25); Paget (26) and Ghosh (27).
Albino mice were divided into different groups (N=5) and injected with different doses of nicotine dissolved in water. The animals were injected (i.p) with doses 0.8, 1.6, 3.2 and 6.4 mg/kg. The animals were continuously observed for 2 hours for appearance of any changes in behavior such as tremors, convulsions (clonic or tonic), abnormal movements changes in tail position and shapes, changes in locomotor activity and death.
To investigate the effect of the extract on nicotine-induced convulsions and death a sub-effective dose of the extract, (a dose of 1 g/kg) that did not produce any observable changes in behavior was initially administered to a group of 5 naive mice. Then after 5 minutes nicotine LD25 was then administered and the animals were observed continuously for any changes in behavior, convulsions or death. The time of convulsions and death were recorded.
Albino guinea-pigs (450 g body weight) were killed using excessive ether anesthesia. The abdomen was opened and pieces of ileum (2 cm-long) were cut and suspended in an organ bath containing oxygenated Krebs' solution (pH 7.4) at 37° C. One end of the tissue was attached to the bottom of the organ bath and the other end was attached to an isometric transducer (Myopraph F60 with 0.5 and 5 g tension calibrations, supplied by Narco Biosystems, Houston, USA) and attached to a universal coupler No. 7189 fitted into a Narco physiograph. To examine the effect of the extract, the latter was added to the fluid bathing the tissue in different doses and allowed to contact the tissue for 45 seconds. The produced effect was recorded. To examine the effect of the blockers on the induced contraction, the following procedure was used. Hexamethonium (at a dose of 10-100 μg/ml) or atropine (at a dose of 0.1-20 μg/ml) was added to the bathing fluid and allowed to contact the tissue for 5 minutes and then the submaximal dose of the extract was added. The percentage inhibition induced by the blocker on the extract-induced contractions was then calculated.
Male Wistar rats (200 g body weight) were divided at random into various groups (N=6 animals per group). Each animal was placed in a stainless steel cage and provided with a known weight of normal rat chow food pellets and drinking bottle containing a known volume of aqueous 0.025% sodium carboxymethylcellulose containing either nicotine acid tartarate to provide a final concentration of 40 μg/ml (in case of control groups) or the same concentration of nicotine plus the indicated concentrations of the whole extract, one of its fractions or peganine. The latter were added in form of their emulsions in 0.25% aqueous sodium carboxymethylcellulose in volumes that provide a final concentration of 0.025% of sodium carboxymethylcellulose in the drinking fluid. The animals were allowed both food and the drinking liquid ad libitum. The room temperature where the animals were placed was maintained at 23±2° C. The light cycle was kept at 12 h light and 12 h dark. The relative humidity was maintained at 75±5%. Both food and drinking liquid consumptions were measured every 2 days and at the end of the treatment which continued for complete seven consecutive days. The influence of each treatment on food consumption, the drinking fluid intake and hence nicotine consumption was calculated at the end of the treatment.
All results were reported as their means±s. e. mean with N=number of experiments. Statistical significance between control and treated groups were performed using student's t-test.
Symptoms Before Death:
1—Increase in locomotor activity
2—Abdominal respiration (shallow respiration)
3—Shaking of tail
4—Convulsions
Onset of death following administration was 4-5 minutes.
in control rats provided with drinking water containing nicotine in a concentration of (40 μg/ml), the daily consumption of the drinking vehicle per 200 g rat was 26.1±1.3 ml and the daily food consumption was 23.9±0.9 g. Treatment of the animals with the total alcoholic extract in a dose of 600 mg/100 ml drinking vehicle reduced the nicotine consumption and food intake by 87.7 and 84.9%, respectively. The cumulative effects are shown in table 2.
Treatment of rats with each of the different fractions of the total alcoholic extract mixed with nicotine in the drinking vehicle revealed that only fraction AT-2 possessed some ability to suppress both nicotine and food intake. Table 3 shows the cumulative results. When higher concentrations of the different fractions were used, here again fraction AT-2 proved to be the most effective and produced significant reductions in both actions (see table 4).
As peganine was found to be the major constituent from the fraction AT-2 in this study, it was tested in rats. Treatment of the animals with peganine 1.4 mg/ml in the drinking vehicle mixed with nicotine (40 μg/ml) produced significant reduction in both nicotine and food consumptions. It decreased the nicotine consumption and food intake by 79 and 77.8%, respectively. The details are shown in table 5.
The results of this study revealed that the extract of Anisotes trisulcus (Forssk.) Vahl induced significant inhibition in nicotine consumption, reduced the lethal dose of nicotine by 75% and mimicked nicotine in inducing intestinal contractions (probably via activation of a subtype of nicotine receptors in the guinea-pig intestinal ganglia-albeit not blocked by the ganglionic blocker hexamethonium). Furthermore, it induced significant suppression of food intake. Its active constituent peganine (vasicine or linarine) which is known chemically as 1,2,3,9-tetrahydropyrolo[2,1-b]quinazolin-3-ol produced potent significant inhibitions of nicotine intake and food consumption. It exerted a potent anorexigenic action. In fact this constituent acted as a nicotine substitute.
Peganine actions are probably mediated via activation of α4 β2 nicotinic receptors as does nicotine leading to release of dopamine in the mesolimbic sites of the brain. Thus, it probably acted as a nicotine substitute leading to satisfaction of the rewarding system leading to an explanation of the reduced nicotine intake (28).
Generally, speaking an agonist at a certain receptor may either be a potent, low potent partial or potent partial agonist. Previous pharmacological studies revealed that potent nicotinic agonists or low potency partial agonists would not be expected to antagonize the effects of nicotine but agents that act as potent partial agonists can antagonize nicotine (29-30). For instance vareniciline, the potent partial nicotinic α4 β2 agonist, in a dose of 5.6 mg/kg significantly blocked nicotine (1 mg/kg)-induced increase in dopamine turnover in the mesolimbic system of rats (30). Thus, since the extract containing peganine did not antagonize nicotine-induced convulsions and death but instead mimicked nicotine and reduced its LD50 by >50% and in addition it substituted for nicotine in the nicotine drinking experiments it is plausible to suggest that peganine acted as does nicotine at the α4 β2 nicotinic receptor. Indeed, the types of convulsions observed before death following peganine were reminiscent to those of nicotine.
Thus, it is highly likely that peganine can act to limit craving and withdrawal from nicotine in chronic tobacco consumers (smokers or snuff intakers). Thus, we claim that peganine has the potential of a novel treatment for tobacco dependence. In this aspect, it is hoped to be more fruitful than the partial nicotinic agonist (−)-cytisine (31-32) that has poor blood brain barrier penetrability (33). It is hoped to be a new addition to the armenatum of tobacco cessation campaigns that include the skin patch containing both nicotine and its antagonist mecamylamine (34). The soonly expected varenicline, the partial α4 β2 nicotinic agonist (30), the antidepressant bupropion (or amfebutamine) (35) and the nicotine conjugate vaccine (Nicvax®) (36).
Considering food intake and appetite, we can recall that the two most important brain neurotransmitters that suppress food intake are dopamine and serotonin (37). The significant inhibitions induced by both extract and its constituent peganine in food intake incline us to believe that the anorexigenic effects were mediated by dopamine release via initial activation α4 β2 nicotinic receptors. Indeed activation of these receptors releases dopamine (6, 7, 28).
In conclusion, this patent showed that both of the extract of Anisotes trisulcus (Forssk.) Vahl family Acanthaceae and its constituent peganine possessed properties that enabled them to suppress nicotine consumption and food intake. Thus, we claim:
We would like to thank Professor Farouk S. El-Feraly for his kind suggestions and encouragements. We would also like to thank the College of Pharmacy, King Saud University for providing the facilities to carry out this work.
aCalculated from the daily consumed liquid
aCalculated and chosen in relation to the % of the fraction in the total extract.
bCalculated with reference to the daily consumption.
450a
aFrom table 3, fraction AT-2 seemed to contain the highest activity. Thus, a dose equivalent to 75% of the effective dose of the total extract was chosen and tried.
bQuantity was calculated by reference to the daily drinking liquid consumption.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2006/002059 | 11/7/2006 | WO | 00 | 3/27/2008 |
