AU2010203124A1

AU2010203124A1 - Method of Treatment

Info

Publication number: AU2010203124A1
Application number: AU2010203124A
Authority: AU
Inventors: Reinhard Michael Kuzeff
Original assignee: BIRSECK Pty Ltd
Current assignee: BIRSECK Pty Ltd
Priority date: 2003-02-28
Filing date: 2010-07-21
Publication date: 2011-02-03

Description

P/00/0 Il Regulation 3.2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION Invention Title: Method of Treatment The following statement is a full description of this invention, including the best method of performing it known to me: - 1 METHOD OF TREATMENT The present invention relates to a method of treatment, in particular to a method of treating 5 an effect of nicotine by administering a dilution or an ultra-high dilution or potentised preparation of nicotine. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common 10 general knowledge in Australia or elsewhere. BACKGROUND Homoeopathy employs minute doses of usually harmful or toxic agents to stimulate 15 organisms back to health. The agents used in homoeopathy are selected precisely on the basis of their ability to induce disease-like symptoms and signs in healthy people when administered in toxic doses or one or more times in sub-harmful doses. These agents will, in properly diluted form, cure a sick person with similar symptoms. While microdose effects are now well accepted, for example in the phenomenon known as hormesis, some 20 homoeopathic solutions are attenuated beyond Avogadro's constant, i.e. in theory none of the original agent remains. There have been a great number of reported experiments that demonstrate that homoeopathic remedies are effective in treating a variety of symptoms. Many chemical agents induce undesirable effects on organisms such as mammals. The 25 effects which result from chemical agents having one or more chiral centres often result from a specific stereoisomer of the chemical agent. For example, (-)-adrenaline is the isomer which is found in humans and is the chemically active agent. It is about 15 times more active than (+)-adrenaline physiologically. Although chemically hard to differentiate in vitro, in vivo they are readily differentiated by the stereo-specificity of enzymes. 30 -2 SUMMARY OF INVENTION The inventor has now found that the effects or actions of nicotine in the organism generally 5 can be treated by administering a dilution or an ultra high dilution or potentised dilution the enantiomer of the nicotine isomer, the effects of which one desires to treat. Accordingly, there is provided a method of treatment of an organism suffering from the effects of nicotine said method comprising the steps of potentising a stereoisomer of nicotine, and administering said nicotine to the organism. 10 Another aspect of the invention provides a method of treatment of an organism suffering from the effects of nicotine, said method comprising the steps of diluting a stereoisomer of nicotine, and administering said diluted nicotine to the organism. 15 Still yet another aspect of the invention provides a method of treatment of an organism suffering from the effects of nicotine, said method comprising the steps of diluting a stereoisomer of nicotine to an ultra-high dilution of said stereoisomer, and administering said ultra-high diluted stereoisomer to the organism. 20 However, the same effect may be obtained by using dilutions such as those used in investigations involving hormesis. Such dilutions exist below the toxic range of a given compound, substance or molecule. Such dilutions below the toxic range are stimulatory rather than toxic, but may be biphasic or multiphasic in their effects, i.e., stimulatory or inhibitory in their effects, depending on the concentration below the NOAEL which is being 25 used. Usually this phenomenon exists in a narrow range of concentrations just below the toxic range. Put another way, the phenomenon exists just below the 'no observed adverse effect level' or 'NOAEL'. Typically this range is within a factor of 10-20 of the NOAEL dosage, and occasionally the dosage range exceeds a factor of 100. 30 The method of the present invention may be used for the treatment of any organism, including for example, animals, plants, microorganisms and in particular, humans.

-3 Another aspect of the invention is the use of a dilution or an ultra-high dilution or potentised preparation of nicotine, for the preparation of a medicament for the treatment of the toxic, physiological and/or pathological effects of said nicotine. 5 Still yet another aspect of the invention is the use of a dilution or an ultra-high dilution or potentised preparation of nicotine of nicotine, for the preparation of a medicament for the treatment of the addictive and other undesirable effects of nicotine addiction, as occurs for instance in the habit or practice of tobacco smoking. 10 Still yet another aspect of the invention is the use of a dilution or an ultra-high dilution or potentised preparation of nicotine, for the preparation of a medicament for the alleviation of the physical and psychological effects of nicotine. The invention may be used for the preparation of a medicament for the alleviation of the 15 physical and psychological effects of any of nicotine addiction. It has also been found that the enantiomer of the naturally occurring isomer of nicotine (i.e. (-)-nicotine), used to treat the organism, may be derived from other salts of nicotine, e.g., instead of nicotine being used, a toluoyl tartrate salt of nicotine may be used. It should be noted that (-)-nicotine is the naturally occurring isomer in nature. Therefore a 20 preparation of (+)-nicotine would be used to treat the human in the context of this method, when the effects of naturally occurring nicotine, are being treated. Potentization of the stereoisomer, nicotine, may be according to the practices used in homoeopathy, homotoxicology or any other system of medicine or treatment which uses potencies, for example, anthroposophical medicine. For a discussion of the method of 25 preparation of stereoisomers in general, and this includes nicotine , into potency, refer to previous patents by Kuzeff as inventor, especially 'Spacetime modification of homeopathic medicinal action'," and also other patents, including AU 2003208170, AU 2005100336, AU 2005100337 and AU 2007100082. The description of manufacture of potencies in the following pages applies to any stereoisomer including nicotine. Preferably the stereoisomer 30 is potentised by succussion or trituration. Attenuation or dilution of the medicinal substance is, in homoeopathy, usually performed in the decimal, centesimal and fifty millesimal (LM) -4 systems as the standard scales of attenuation, under which each successive attenuation contains just 1/10, 1/100 or 1/50,000 as much of the medicinal substance as the preceding attenuation. It is preferred that after each attenuation, the attenuated medicinal substance is succussed (typically between 10 to 100 times at each stage of attenuation) or triturated. 5 Generally, soluble substances may be subjected to succussion and insoluble or solid substances may be subjected to trituration. Other forms of agitation may be used instead of succussion or trituration, e.g., sonication, Lab dancer, etc. In order to prepare the dilutions or potencies A ml of tincture or B grams of medicinal 10 substance are added to C ml or D grams or E parts of vehicle. Subsequent liquid or solid attenuations are made by serial progression, succussing or triturating one part of the preceding attenuation to C ml, D grams or E parts of the vehicle respectively. A, B, C, D, and E, are any numbers greater than zero. When preparing consecutive attenuations, it is not necessary for A, B, C, D or E, to be kept constant. For example for the first, second, third, 15 fourth, fifth, etc. attenuation, the values of A could be A, A 2 , A 3 , A 4 , A 5 ... etc., where A,

A

2 , A 3 , A 4 , A 5 ... etc. represent any numbers greater than zero. The same principle applies for values of B, C, D, and E. In the decimal scale of attenuation is generally practised one millilitre (1.Oml) of tincture, 20 one millilitre of IX aqueous solution, or one gram (1.0g) of IX trituration represents 0.10 gram of dry crude medicinal substance. One millilitre of 2X attenuation, or one-gram (1.0g) of 2nd trituration contains 0.01 gram of the dry crude medicinal substance. Subsequent liquid or solid attenuations are made by serial progression, succussing or triturating one (1) part of the preceding attenuation to nine (9) parts of the vehicle, and represent the following 25 proportions of active principle (i.e. dried medicinal substance): 2X = 10- 2 6X = 10 3X= 10- 7X = 10 4X = 10 4 8X = 10 30 5X = 10-' (and so on .... ) In decimal attenuations nX = 10~ where n is an integer greater than 0. In the case of centesimal attenuations, each attenuation contains just one hundredth of the medicinal -5 substance of the one before, nC = 1 0 ".2 In the case of fifty millesimal attenuations one millilitre (1 ml) of the first fifty millesimal attenuation (ILM) represents 4.0x10- 9 gram of dry crude medicinal substance. One millilitre (1 ml) of the second fifty millesimal attenuation (2LM) represents 8.Ox 10- gram of dry crude medicinal substance. Each 5 subsequent attenuation represents a further decrease in concentration of dry crude medicinal substance by a factor of 2x10 5 . Each attenuation such as 2X, 3X or nX (2C, 3C or nC) (2LM, 3LM etc) is generally referred to as a potency. In order to prepare the solid or liquid stereoisomers of chemical agents, it is effective to add 10 2 or more different potencies or attenuations together. A potency refers to a solution, which has undergone serial dilution and succussion and/or agitation whereas attenuation refers to a process of dilution, which may or may not involve succussion or agitation. The term potency also refers to solid attenuations as described herein. The term 'different potencies or attenuations' encompasses 2 potencies or attenuations of different dilutions as well as 15 solutions which have undergone a different number of steps of serial dilution or attenuation with succussion, or, in the case of solid attenuations, a different number of steps of serial trituration as described herein. For example, a person skilled in the art could add the fourth and twelfth potencies or attenuations together in equal or unequal quantities. The solution may then be succussed (shaken) N times, where N is any integer greater than zero. 20 Alternatively, the solution is not succussed. Similarly, one could add the fourth, twelfth, or thirtieth potencies or attenuations together, or any number of combinations of potencies or attenuations. It is also common practice in homeopathy to make mixtures of different potencies of the same medicinal substance or mixtures of potencies of different substances in various combinations. Such mixtures are called complexes and have been commercially 25 available for many years. Accordingly, one or more potencies of nicotine could be included in such mixtures. It would be possible to add nicotine potencies to existing available commercially available complexes. Note that the term 'different potencies or attenuations' also encompasses the situation where the same or different potencies, made from 2 or more different medicinal substances, are added or mixed together. Such mixtures are common 30 practice in homeopathy and are called 'complexes'. Also, there is the situation where one could treat the effects of a racemic mixture or of specific stereoisomers (or optical isomers) with both the (+)- and (-)- enantiomers -6 contemporaneously, either simultaneously or within the same course of treatment. This could be with a mixture of equal or unequal volumes of potencies or attenuations or dilutions or a combination of one or more of same or different potencies or attenuations of each enantiomer. This is illustrated in the discussion and non-limiting examples which 5 follow: For instance, one could mix one, two, three, four, five or more potencies, attenuations or dilutions of the (+)- enantiomer to one, two, three, four, five or more potencies of the (-) enantiomer or vice versa. One or more potencies, attenuations or dilutions of an enantiomer 10 may be prepared in one mixture, and added to an equal or unequal number of the same or different or any combination of potencies or attenuations of the other enantiomer prepared in a separate mixture. Alternatively, all potencies, attenuations or dilutions could be added to the same mixture. 15 (+)- and (-)- enantiomers may be mixed 50:50 or 25:75 or in any proportion. Thus, a mixture could be prepared by adding 2ml of 4th, 12th and 30th potencies of (+)- enantiomer to 2ml of 4th, 12th and 30th potencies of (-)- enantiomer, or vice versa. Alternatively 0.5ml of 4th, 2ml of 12th and 3.4ml of 30th potencies could be used, and in fact the numbers 0.5, 2 and 3.4 could be replaced by any numbers greater than zero or equal to zero. Also, the 4th, 20 12th and 30th potencies could be replaced by any potencies represented by integers greater than or equal to 1. The mixtures of (+)- and (-)-enantiomers prepared separately, could then be administered separately or mixed together. If mixed together, the resulting solutions could be succussed or not succussed, and subsequently serially diluted or attenuated or potentised or not. Alternatively, the (+)- and (-)-enantiomers may not be prepared 25 separately, and mixing could occur in the one container. Also, in the spirit of the above description, 0.4ml of 3'd potency, attenuation, or dilution of the (-)-enantiomer, may be added to 1.2 ml of the 13 th, 5.2 ml of the 41 ", 4.5 ml of the 2 0 0 th, and 3.7 ml of the 1000th potency, attenuation or dilution. This may then be succussed or 30 not. In turn, this may then be added to a mixture of 5.1 ml of the 5 'h potency, attenuation or dilution, 0.3 ml of the 3 7 'h, and 6.3ml of the 10 5 h potency, attenuation or dilution of the (+) enantiomer. This latter mixture may have been succussed or not. The resulting combination of mixtures may then be serially diluted, attenuated or potentised or not. In this paragraph -7 the number denoting potencies, attenuations or dilutions can be replaced by any integers greater than zero. Numbers representing millilitres of potency, attenuation or dilution, can be replaced by any numbers greater than or equal to zero. 5 (+)- and (-)- enantiomers may be mixed 50:50 or 25:75 or in any proportion. Thus, a mixture could be prepared by adding 2g of 4th, 12th and 30th potencies of (+)- enantiomer to 2g of 4th, 12th and 30th potencies of (-)- enantiomer, or vice versa. Alternatively 0.5g of 4th, 2g of 12th and 3.4g of 30th potencies could be used, and in fact the numbers 0.5, 2 and 3.4 could be replaced by any numbers greater than zero or equal to zero. Also, the 4th, 12th 10 and 30th potencies could be replaced by any potencies represented by integers greater than or equal to 1. The mixtures of (+)- and (-)-enantiomers prepared separately, could then be administered separately or mixed together. If mixed together, the resulting solutions could be succussed or not succussed, and subsequently serially diluted or attenuated or potentised or not. Alternatively, the (+)- and (-)-enantiomers may not be prepared separately, and 15 mixing could occur in the one container. Also, in the spirit of the above description, 0.4ml of 3 rd potency, attenuation, or dilution of the (-)-enantiomer, may be added to 1.2g of the 13 th, 5.2g of the 4 1, 4.5g of the 2 0 0 th, and 3.7g of the I000th potency, attenuation or dilution. This may then be succussed or not. In 20 turn, this may then be added to a mixture of 5.1g of the 5 th potency, attenuation or dilution, 0.3g of the 3 7 1h, and 6.3g of the 105t potency, attenuation or dilution of the (+)-enantiomer. This latter mixture may have been succussed or not. The resulting combination of mixtures may then be serially diluted, attenuated or potentised or not. In this paragraph the number denoting potencies, attenuations or dilutions can be replaced by any integers greater than 25 zero. Numbers representing grams of potency, attenuation or dilution, can be replaced by any numbers greater than or equal to zero. The same procedures as described above could be used for mixtures of stereoisomers in general, including diastereoisomers or mixtures of diastereoisomers and enantiomers. 30 The administration of the potentised stereoisomer is typically by an oral route but may be administered intravenously, intramuscularly, transdermally, subcutaneously, topically, intrathecally, intraperitoneally or via any mucous membrane (typically sublingually). It is -8 particularly preferable to administer the potentised stereoisomer orally or sublingually. Specific examples of administration of the potentised stereoisomer include tablets, globuli, liquid dilutions for injection and liquid external preparations. Administering potencies via different routes is part of routine optimization of therapy in homeopathy; i.e., one uses 5 whichever route is most expedient. Another method of administering the potentised or attenuated stereoisomer is to use devices such as the MORA machine, Listen Machine or Vega Select Machine or other bioresonance or electrodermal testing devices to detect an electromagnetic or bioresonance signal from the 10 potency or attenuation and then administer the signal in an unchanged, modified or inverted form to the organism to be treated. These devices which are commercially available, claim to be able to copy the effects of medicines, dilution or potency and pass the attributes of a medicine, dilution or potency onto a heretofore placebo or medicinally inactive vehicle. The terms "modification" or "inversion" of the signal includes changing the polarity of the signal. 15 In order to determine the appropriate potency of the stereoisomer in order to achieve the most effective treatment of the undesirable effect of the chemical agent it is usually preferred to commence by administering a low potency of the stereoisomer, say between 1 C-I OC inclusive (or I X - I OX) and gradually incrementally increasing the potency until the 20 treatment is optimised. Experience may show that 6C, 15C, 30C, 200C are appropriate attenuations in most cases. Attenuation of I OOOC, I O,OOOC or higher may also produce desirable results. This also applies for non-decimal and non-centesimal potencies. The vehicles used to attenuate the stereoisomer may be selected from the group consisting of 25 water, such as water for injection B.P. or U.S.P., lactose B.P. or U.S.P., sucrose B.P. or U.S.P. ethanol typically in suitable concentrations (e.g. 15 - 95%). Also absolute ethanol, purified water, glycerol 85% or other ethanol/water mixture or dilution of glycerol may be used. Other vehicles will also be apparent to those skilled in the art of homoeopathy. 30 The methods of preparation of solid or liquid stereoisomers of chemical agents into potentised attenuations include where water-soluble or alcohol-soluble isomers are to be prepared into potencies the use of water B.P. or purified water alone, or in a mixture of water and ethanol, say, 30-45% ethanol. Ethanol-soluble stereoisomers may be prepared using -9 higher concentration ethanol solutions, say 55-95% ethanol or absolute ethanol. It is possible to start using lower and incrementally lower ethanol concentrations as the potency reaches 3 to 5X or 3 to 5C. Final homeopathic liquids often contain 30-40% ethanol. 5 The stereoisomer may be prepared by a process of trituration. The process of trituration is particularly advantageous when the stereoisomer is not readily soluble in water, ethanol or water/ethanol mixes. At potencies beyond 3C or 6X it is possible to convert from trituration to liquid dilutions or vice versa. For example, liquid dilutions may be prepared by first making a trituration and then diluting it in liquid such as water for injection. 10 In trituration one part of the medicinal substance, when preparing the first potency, or one part of the preceding attenuation when preparing the second or subsequent potencies, is added to one third of the total vehicle (e.g. lactose B.P.) used for that potency. The process of trituration is typically performed with mortar and pestle for 15 to 20 minutes. The side of 15 the mortar is then scraped for five minutes to dislodge any attenuated substance with the pestle or with a spatula. Then the second third of the vehicle for attenuation is added to the mortar and the contents subjected to a further 15 to 20 minutes trituration prior to scraping the sides of the mortar for a further five minutes to dislodge attenuated substance. The remaining one third of the vehicle is added to the mortar and the combined mixture is 20 subjected to trituration for a further 15 to 20 minutes to complete the trituration for that potency. Alternatively, the total vehicle may be added to the medicinal substance or preceding attenuation at each successive stage of potency preparation and subjected to 60 minutes of trituration. Each successive level of attenuation is called a potency. 25 The process of the present invention may be used to reverse, or in another embodiment enhance, the effects in vivo, and in vitro, of nicotine or any of its compounds. Another aspect of the invention provides for the use of the method of the present invention to enhance or reverse the effects in vivo and in vitro of (-)-nicotine or any of its compounds or to enhance or reverse the effects in vivo and in vitro of (+)-nicotine or any of its compounds. 30 It is to be understood that in this specification the terms enantiomer, stereoisomer and optical isomer refer to the relevant isomers of nicotine.

- 10 EXAMPLES The present invention is further described by the following non-limiting examples. 5 Example 1. A case series of smokers is given a potency or potency chord of (+)-nicotine in order to encourage cessation of smoking. Smokers report their success rate after 3 months and this is compared with norms for other methods of smoking cessation. Smokers are given a potency 10 chord consisting of a mixture of the 4 , 12 "h and 3 0 "h decimal, centesimal or fifty millesimal potencies prepared according to the methods of a national pharmacopoeia such as the Homeopathic Pharmacopoeia of the United States 2004. Alternatively a mixture of the 6*, 15 th and 30t decimal, centesimal or fifty millesimal potencies are administered. Other combinations of potencies may be used. Alternatively a simplex may be used, e.g., 30 'h fify 15 millesimal potency. Example 2 Smokers are admitted to a prospective double blind randomized placebo controlled study. 20 The end point is the rate of cessation of smoking after 3 months. Also of interest is the total number of cigarettes being smoked daily at the end of 3 months in the 2 arms of the study. One of the potency chords mentioned in Example I is used and compared with the effect of identically manufactured placebo or succussed placebo. 25 Example 3 The patients in examples 1 and 2 are administered a potency of (-)-glutamic acid (D Glutamic acid) either in the form of a potency chord or as a simplex. The reason for this is that inhibition of endogenous L-Glutamic acid in the nervous system at glutamate receptors 30 such as the NMDA receptor for example, may facilitate recovery from addiction in general and nicotine addiction in particular. The D-Glutamic acid potency may be administered separately from the (+)-nicotine potency, or they may be mixed in combination in the same container.

- 11 Example 4 The ability of potencies or dilutions of nicotine isomers to modulate toxic doses of their 5 enantiomers are tested in bioassay or other toxicity tests.

- 12 HOMEOPATHIC TREATMENT OF THE (-)-NICOTINE TOXICITY EXPERIMENTAL STUDIES IN DAPHNIA MA GNA TESTING TESTING MOBILITY INHIBITION 5 Introduction Concept of testing Daphnia magna was used as testorganism to measure the mobility inhibitory effect of (-) nicotine in the presence of (+)-nicotine potencies and placebo. A mixture of 4th, 12 th 30 th potencies from (+)-nicotine (H2) and placebo were added to the 10 Daphnia culture. The (-)-nicotine were applied after the addition of H2 potency and placebo. The toxic effect on mobility of Daphnia magna was measured. Daphnia magna bioassay The test is an acute toxicity test with the Daphnia magna water flea. The adult Daphnia magna is usually five to six millimetres in length, reproduce only by cyclic parthenogenesis in which 15 the males contribute to the genetic make up of the young during the sexual stage of reproduction. Daphnia magna are used in freshwater acute toxicity testing at 24 hours old (approx. 1-2 millimetres length) and survival rates are recorded after they have been challenged by a substance or environmental sample. The Daphnia culture for the test is prepared under standardised conditions in our laboratory and 20 reproduced only by parthenogenesis passing the sexual stage in order to be genetically same and comparable. Materials and methods General conditions - preparation of potencies 25 The potencies were prepared in glass tubes, taking care not to contact or being close to metal objects or electric wires. Breeding of the Daphnis, feeding, aeration and other manipulation was executed in an alternative way compared to usual, excluding contact with metal tools, surfaces and equipments. Storage of potencies in ethanol was performed in wooden cupboard (far from electric cables) in dark. 30 Materials " (-)-nicotine, liquid, 99 % (CAS: 54-11-5) 1425810 22009185 Cat.: N3876 (SIGMA®, Sigma-Aldrich Chemie GmbH, Germany) molecular weight 162.23 " (+)-nicotine (+)-di-p-toluoyltartarate salt, powder, minimum 98 % TLC 35 (CAS:68935-27-3) 090K 1644 Cat: N9765 SIGMA®, Sigma-Aldrich Chemie GmbH, Germany, molecular weight: 548.58 e Ethanol 96 % puriss. (Spektrum 3D® 64-17-5), e Testorganism: Daphnia magna 40 Daphnia magna medium - 13 Spring water from a public well used for cultivation and testing of Daphnia magna Requirements for spring-water: Temperature: 20-25 'C Conductivity: 450-500 [pS 5 pH: 7.6-8.0 Total hardness in CaCO 3 : 225-275 mg/i Solved oxygen concentration: at least 7 mg/l, can be achieved by aeration for 30 minutes. 10 EXPERIMENTAL Preparation of potencies Thirty round bottom 10 ml of volume calibrated glass test tubes with glass stoppers (CLS842450 Aldrich Pyrex@ round-bottom centrifuge tube) were used for the (+)-nicotine potencies preparation and were numbered from 1 to 30. Potencies were 15 all prepared and stored in 35 % ethanol, except the final potencies, which were administered to organisms, because they were prepared in distilled water. Into the first tube 10 mg (+)-nicotine was placed and then 1 ml 35 % ethanol. The test tube was sealed and given 20 forceful downward succussions at 0.75-1 Hz. The same ethanol stock bottle was used for all of the test tubes described below. The next 29 tubes, i.e. tube numbers 2-30, 20 each contained 5 ml 35 % ethanol in water. One drop (20 p) was removed from the Is' test tube using automatic pipette, and was added to the second test tube. This was given 20 succussions as shown by the video. 20 pl from the second test tube was removed and added to the third test tube and so on until the 30 th test tube. All potencies were succussed by hand. In the experiments the mixture of 4 'h + 12'+ 301 25 potencies were tested and the placebos. To make the separate 4 h, 12 'h and 3 0 th potencies, 20 pl (one drop) of tube 3, 11 and 29 was added to 5 ml distilled water in a 100 ml glass flask with plastic screw stopper (SIMAX Reagent bottles with screw GL 45 acc. to DIN 100 ml Code: 414 321 100). These were given 20 successions to produce the potencies in distilled water. To prepare the mixture of 4th + 2 +30th potencies (H2 potency) the contents of test tubes 4,12 30 and 30 were added to a 100 ml glass flask with plastic screw stopper (SIMAX Reagent bottles with screw GL 45 acc. to DIN 100 ml Code: 4 14 321 100). These were given 20 forceful downward successions. 20 pl (one drop) of the content was added to 5 ml distilled water in a 100 ml glass flask. This test flask was the succussed 20 times as already mentioned. The placebo was prepared on the same way but without (+)-nicotine. These final homeopathic 35 potencies in distilled water were prepared immediately before application. These were applied within 5-10 minutes after preparation. Breeding of Daphnia magna colony The colony is maintained in natural spring water, what is obtained from a public well, before 40 use, the spring water's main parameters (pH, conductivity, total hardness and solved oxygen concentration) are checked. The colony is kept in a glass aquarium, with 14:10 light: dark cycle with light intensity of 35 lux (source: Hagen Aqua-Glo aquarium lamp, 15 W, T8, length: 43.74 cm, light temperature 18.000 K) and temperature regulated at 23'C and fed every two days with of concentrated - 14 Scenedesmus subspicatus or Chlorella vulgaris suspension, the required algae concentration in the holding water: 5* 10 3 cells/ml. Preparation of test colony 5 The colonies of testorganism (Daphnia) were prepared by separation of 20 adult animals (approx. 7 days old) with eggs in the brood pouch. After 24 h hour the released young water fleas are separated from the adult ones with a classifying net and finally, used in the test. The animals picked up with a small spoon-shaped planktonic-net very quickly and put 10 animals into each glass bakery (Duran Bakery, Cat. Nr.: 21 106 36, capacity: 250 ml, diameter 0 70 mm, 10 height 95 mm). Preparation of (-)-nicotine solution (-)-nicotine solution (500 mg/l, 20 C) was prepared to test the effect of (-)-nicotine on the immobilization of Daphnia magna. The final concentration was 40 times diluted during test. Test protocol 15 Daphnia magna not more than 24 hours old at the beginning of the test, laboratory bred, apparently healthy and with a known history (breeding method, pre-treatment) are used in this test. Testing sensitivity of testorganism to (-)-nicotine Screening of a dilution series of (-)-nicotine: 5 member dilution series with distilled water was 20 prepared from 1000 mg/l (-)-nicotine stock-solution, to test the sensitivity of Daphnia magna. From the 1000 mg/l stock solution a five member, ten-fold dilution series was made. 5 ml of each solution was added to 195 ml of (24 hours old) 10 Daphnis containing spring water. The final (-)-nicotine concentrations in the Daphnis solutions were: 2.5; 25; 250; 2 500 and 25 000 pig/l. After 24, 48, 72, 96 hours of contact time immobilized Daphnis were 25 counted. The observation of immobility was according to the OECD 2021 standard: The immobile animals are not able to swim within 15 seconds after gentle agitation of the test container are considered to be immobile. Testing the effect of (+)-nicotine potencies and placebo on testorganism in the presence of (-)-nicotine 30 1. Pick up 10 (24 hour old) Daphnis and put into 190 ml natural spring water with required conditions 2. 5 ml of freshly prepared (+)-nicotine potencies and placebos were added tol90 ml of Daphnis colony 3. 5 ml of each solution was added to the water flea colony 35 4. After 24-48-72-96 hours of contact time the mobility of the Daphnis was observed by visual observations Evaluating measured data Primary (measured) data were placed into an excel table and the comparative evaluation was 40 executed with them. At the end of the testing we have 24, 48, 72, 96 hours data for each concentration and for the control (no containing (-)-nicotine). From these data we calculated the H %, the relative mobility inhibition of luminescence compared to control. The endpoints used for the sensitivity testing were EC20 / EC 50 values, where EC 20 and EC 50 mean concentrations 1 202 OECD Guidelines for testing of chemicals 4 April 1984, "Daphnia sp., Acute Immobilisation Test and Reproduction Test": Part I - the 24h EC 50 acute immobilisation test; Part 11 - the reproduction test (at least 14 days). http://wwv.oecd.org/dataoecd/ 17/2 1/l 948249.pdf - 15 caused 20 % and 50 % inhibition. EC 20 and EC5o values were determined from concentration response curve (inhibition percent values of concentrations) after sigmoidal fitting by ORIGIN 8.0* software. RESULTS AND DISCUSSION 5 First we measured the sensitivity of the testorganism on nicotine, and draw up the concentration-response curve. Than the potencies were tested in comparison with placebo. 1. Sensitivity of Daphnis to (-)-nicotine The objective of this experiment was to study the sensitivity of the testorganism to the (-) 10 nicotine (-)-nicotine was applied in the concentration range: 0; 2.5; 25; 250; 2 500 and 25 000 ptg/l (ppb). The sensitivity of Daphnia magna was proved in the investigated concentration range (Figure 1). Concentration-response curve of Daphnia magna with (-)-nicotine 100 - 24 hour _ e48 hourU A72 hourU 80 - V 96 ho __ 40 E 0 E -C40 0,01 0,1 1 10 100 1000 10000 log (-)-nicotine [ppb] 15 Figure 1. Concentration-response curve of Daphnia magna with (-)-nicotine EC2 0 and EC 50 values were determined from concentration-response curve (Figure 1) after sigmoidal fitting by ORIGIN 8.0® software.

EC

2 0 (ppb) EC 50 (ppb) 24h 1200 1960 48 h 4 20 72 h 0.15 2 96 h 0.5 4 20 -16 The curves show clearly, how the sensitivity changes in time. The 24 h old culture showed very low sensitivity, while the 72 hours old culture had the highest sensitivity, with the smallest effective concentrations of (-)-nicotine (EC 20 =0. 15 ppb; EC 50 =2 ppb). The experiments carried out by (+)-nicotine H2 potency and placebo-treated bacteria on the 5 toxicity testing of (-)-nicotine were planned on the base of these results.

- 17 2. The effect of (+)-nicotine H2 potency and placebo on Daphna in the presence of (+) nicotine The main objective was the evaluation of the effect of H2 (+)-nicotine potency-treatment with 5 regard to their usefulness for curing the toxicity of (-)-nicotine. Daphnia magna mobility test was applied to study and evaluate the effect of addition of H2 potencies and H2 placebos. The inhibition of mobility was observed after 24 h; 48 h; 72 h and 96 h contact time with H2 potentised and placebo-treated Daphnia magna with and without (-)-nicotine. The curing effect of the H2 potency was significant after 72 hours between 250 and 2500 10 ppb of (-)-nicotine concentration. Table I Number of mobile Daphnis in H2 (+)-nicotine potency and H2 placebo treated (-) nicotine 15 (-)-nicotine Number of mobile Daphnia concentration 24 h 48 h 72 h 96 h (ppb) H2 potency placebo H2 potency placebo H2 potency placebo H2 potency placebo 2500 10 10 2 1 2 0 0 0 250 10 10 7 6 5 4 2 2 25 10 10 10 10 9 9 6 5 2,5 10 10 10 10 10 9 9 10 0 10 10 10 10 10 10 10 10 potency 2 500 ppb (-)-nicotine 10- placebo 2 500 ppb (-)-nicotine -C Q. 0 0 E 0 .o E 01 0 40 80 exposure time (hours) Figure 1. The effect of H2 potency and H2 placebo treatment on Daphnia mobility in 2 500 ppb 20 (-)-nicotine - 18 Significant difference was measured after 72 hours in the 2500 ppb concentration (-) nicotine on the effect of (+)-nicotine potency treatment. 10- - potency 250 ppb (-)-nicotine - placebo 250 ppb (-)-nicotine C: -C E ' 5 ~0 E C 0 40 80 exposure time (hours) 5 Figure 2. The effect of H2 potency and H2 placebo treatment on Daphnis mobility in 250 ppb (-)-nicotine Significant difference was measured between 48 and 72 hours in the 250 ppb concentration 10 (-)-nicotine on the effect of (+)-nicotine potency treatment.

- 19 10 -E 8 -C O CU 6 0 E 4 potency 25 ppb (-)-nicotine (D -0 2- - placebo 25 ppb (-)-nicotine E 0 0 40 80 exposure time (hours) Figure 3 Effect of H2 potency and H2 placebo treatment on Daphnis mobility in 25 ppb (-) nicotine 10 ecn C, C. -o _ - potency 2.5 ppb (-)-nicotine placebo 2.5 ppb (-)-nicotine 0 E 0 .o E c 0 I 'I 0 40 80 exposure time (hours) 5 Figure 4 Effect of H2 potency and H2 placebo treatment on Daphnis mobility in 2.5 ppb (-)-nicotine No significant difference was measured for the low concentration (-)-nicotine on the effect 10 of (+)-nicotine potency treatment.

- 20 Table I The effect of treatment with H2 (+)-nicotine potency and placebo on the toxicity of (-)-nicotine (-)-nicotine H24h ( %) H48h (%) H72h (%) H96h (N) concentration (ppb) H2 potency placebo H2 potency placebo H2 potency placebo H2 potency placebo 2500 0 0 80 90 80 100 100 100 250 0 0 30 40 50 60 80 80 25 0 0 0 0 10 10 40 50 2,5 0 0 0 0 0 10 10 0 0 0 0 0 0 0 0 0 0 5 Concentration-response curve Effect of treatment with H2 potency and placebo 100 m H2 potency 80- 0 H2 placebo 60[ WO 40 20 IE EC 20~~~~2 $0______ ___ 1E -3 0,01 0,1 1 10 100 1000 10000 log (-)-nicotine [ppb] Figure 1 Effect of H2 potency and H2 placebo treatment on Daphnis mobility inhibition after 48 hours of exposure time -21 Concentration-response curve Effect of treatment with H2 potency and placebo 100 0 H2 potency 80 - * H2 placebo 60_ S40 - _ 20 _ _ EC 0_EC -Y,,-0.Tj 1. . . . . . . . . . 1E-3 0,01 01 1 10 100 1000 10000 log (-)-nicotine [ppb] Figure 2 Effect of H2 potency and H2 placebo treatment on Daphnis mobility inhibition after 72 hours of exposure time Concentration-response curve Effect of treatment with H2 potency and placebo 100 m n H2 potency 0 H2placebo 80 601 1 401 20_7 EC - I EK E 50 0 __ _ _I201 _ _ _ _ _ _ 1E-3 0,01 0,1 1 10 100 1000 10000 log (-)-nicotine [ppb] 5 Figure 3 Effect of H2 potency and H2 placebo treatment on Daphnis mobility inhibition after 96 hours of exposure time 10 - 22 Table 2 EC 2 0 and EC 5 0 values After 48 hours of exposure After 72 hours of exposure After 96 hours of exposure time time time H2 potency placebo H2 potency placebo H2 potency placebo

EC

2 0 [ppb] 240 240 150 100 20 20 ECso[ppbl 270 270 250 210 130 130 -23 HOMEOPATHIC TREATMENT OF (-)-NICOTINE TOXICITY EXPERIMENTAL STUDIES ON VIBRIO FISCHERJ BY TESTING BIOLUMINESCENCE INHIBITION 5 Introduction Concept of testing Vibriofischeri was used as testorganism to measure the growth inhibitory effect of(-) nicotine in the presence of (+)-nicotine potencies and placebo. 10 Potencies of 4 th 12 th 3 th from (+)-nicotine (separately) and the mixture of 4' + 12 th + 30"e potencies (H2) and placebo were added to the bacterial cell culture. The (-)-nicotine was applied immediately after the addition of potencies and placebo. The toxic effect on bioluminescence of Vibriofischeri was measured. 15 Vibrio fisheri bioassay The test is an acute toxicity test with the marine luminescent bacterium Vibriofischeri (formerly known as Photobacterium phosphoreum). Vibriofischeri is a facultative anaerobic, rod-shaped bacterium disposed with a polar whip-like filament called flagella. The test system measures the light emission of the luminescent bacteria after they have 20 been challenged by a substance or environmental sample and compares it to the light output of a blank control. The difference in intensity of luminescent light emission (between the sample and the control) is attributed to the effect of the substance/environmental sample on the organisms. The Vibriofischeri bioassay uses a suspension of V. fischeri bacteria in saline water and measures the reduction in light intensity of its natural luminescence on exposure to the 25 toxicant/toxic sample of interest. The bacterial inoculum for the test-suspension is prepared under standardised conditions in our laboratory. Materials and methods General conditions - preparation of potencies 30 The potencies were prepared in glass tubes, taking care not to contact or being close to metal objects or electric wires. The propagation of the microbes, inoculation and other microbiological manipulation was executed in an alternative way compared to usual, excluding contact with metal tools, surfaces and equipments. Storage of potencies in ethanol was performed in wooden cupboard (far from electric cables) in dark. 35 Materials e (-)-nicotine, > 99 % (GC, liquid) (CAS:54-11-5) Cat.: N3876-25 ml, molecular weight 162.23; ALDRICH@, Sigma-Aldrich Chemie GmbH, Germany) 40 e (+)-nicotine, (+)-di-p-toluoyltartrate salt > 98 % (TLC, powder) (CAS:68935-27-3) - 24 Cat.: N9765, molecular weight 548.58; ALDRICH@, Sigma-Aldrich Chemie GmbH, Germany) 0 Ethanol 96 % puriss. (Spektrum 3D® 64-17-5), molecular weight 147.13 5 Microorganism: Vibrio fisheri NRRL-B- 11177 strain Vibrio fischeri medium I. Liquid media used for cultivation and testing of Vibriofischeri - I. 30 g NaCl (REANAL@ 24640-1-01, CAS:7647-14-5) 10 6.lg NaH 2

PO

4 x H 2 0 (REANAL@ 24430-1-08, CAS: 10049-21-5) 2.75 g K 2

HPO

4 (REANAL@ 08790-1-08, CAS: 7758-11-4) 0.204g MgSO 4 x7H 2 O (REANAL@ 20341-1-08, CAS: 10034-99-8) 0.5 g (NH 4

)

2 HP0 4 (REANAL@ 08400-1-08, CAS: 7783-28-0) 5 g peptone form meat (trypsin digest) (REANAL@ 25 773-1-99-33, CAS: 73049-73-7) 15 0.5 g yeast extract (OXOID@ L21) 3 cm 3 glycerine (REANAL@ 11290-0-01, CAS: 56-81-5) 1000 cm 3 distilled water pH=7.2 Sterilised by autoclaving at 121 *C, for 10 minutes. Vibrio fischeri medium II. 20 Agar media used for cultivation of Vibriofischeri - II. 30 g NaCl (REANAL@ 24640-1-01, CAS: 7647-14-5) 6.1g NaH 2

PO

4 x H 2 0 (REANAL® 24430-1-08, CAS: 10049-21-5) 2.75 g K 2

HPO

4 (REANAL@ 08790-1-08, CAS: 7758-11-4) 0.204g MgSO 4 x7H20 (REANAL@ 20341-1-08, CAS: 10034-99-8) 25 0.5 g (NH 4

)

2

HPO

4 (REANAL@ 08400-1-08, CAS: 7783-28-0) 5 g peptone form meat (trypsin digest) (REANAL@ 25773-1-99-33, CAS:73049-73-7) 0.5 g yeast extract (OXO1D@ L21) 3 cm 3 glycerine (REANAL@ 11290-0-01, CAS: 56-81-5) 17 g agar-agar OXOID@ III CAS:9002-18-0 30 1000 cm 3 distilled water pH=7.2 Sterilised by autoclaving at 121 "C, for 10 minutes. Sterile Erlenmeyer flasks were used for the propagation of the bacterial cells. EXPERIMENTAL 35 Preparation of potencies Thirty round bottom 10 ml of volume calibrated glass test tubes with glass stoppers (CLS842450 Aldrich Pyrex® round-bottom centrifuge tube) were used for the (+)-nicotine potencies preparation and were numbered from I to 30. Potencies were all prepared and stored in 35 % ethanol, except the final potencies, which were administered to organisms, because they 40 were prepared in distilled water. Into the first tube 10 mg (+)-nicotine was measured and then 1 ml 35 % ethanol. The test tube was sealed and given 20 forceful downward successions at 0.75-1 Hz. The same ethanol stock bottle was used for all of the test tubes described below. The next 29 tubes, i.e. tube numbers 2-30, each contained 5 ml 35 % ethanol in water. One drop (20 pl) was removed from 45 the 1s' test tube using automatic pipette, and was added to the second test tube. This was given 20 succussions. 20 p1 from the second test tube was removed and added to the third test tube and so on until the 30 h test tube.

- 25 All potencies were successed by hand. In the experiments the 40', 12'h and 30 h potencies of (+)-nicotine separately and the mixture of 4 '+ 12 h+30h potencies were tested and the placebos. To make the separate 4'h, 12th and 3 0 th potencies, 20 pl (one drop) of tube 3, II and 29 was added to 5 ml distilled water in a 100 ml glass flask with plastic screw stopper (SIMAX 5 Reagent bottles with screw GL 45 acc. to DIN 100 ml Code: 414 321 100). These were given 20 successions to produce the potencies in distilled water. To prepare the mixture of 4 th + + 30' potencies (H2 potency) the contents of test tubes 4,12 and 30 were added to a 100 ml glass flask with plastic screw stopper (SIMAX Reagent bottles with screw GL 45 acc. to DIN 100 ml Code: 4 14 321 100). These were given 10 20 forceful downward successions. 20 pl (one drop) of the content was added to 5 ml distilled water in a 100 ml glass flask. This test flask was the succussed 20 times as already mentioned. The placebo was prepared on the same way but without (+)-nicotine. These final homeopathic potencies in distilled water were prepared immediately before application. These were applied within 5-10 minutes after preparation. 15 Preparation of bacterial inoculum The suspensions of testorganisms (bacteria) were prepared in two subsequent steps. 1) One loop of Vibriofischeri maintained on agar media (II) was cultured in 25 ml of liquid medium I (in a shaken flask) for 24 hours at 25 *C. 20 2) 100 ld of step 1) cell suspensions was added to 30 ml fresh liquid medium I., and was cultured for 20 hours at 25 'C in a shaken flask. Preparation of (-)-nicotine solution The naturally occurring nicotine isomer ((-)-nicotine), in liquid form was diluted with steril 25 distilled water. Test protocol Instrumental LUMAC Biocounter M1500 P luminometer was used to measure the emitted light 30 (luminescence). Testing the sensitivity of test bacterium to (- )-nicotine Screening of a dilution series of (-)-nicotine: 8 member dilution series with distilled water was prepared from the saturated L-GA solution, to test the sensitivity of Vibrio fischeri. Different dilution of 20 000ppm (-)-nicotine solution (2x, 4x, 8x, 16x, 32x, 64x) was prepared 35 to test the effect of the (-)-nicotine on the bioluminescence of Vibriofischeri. 50 pl of different dilutions of (-)-nicotine (0, 312.5, 625, 1250, 2500, 5000, 10 000, 20 000 ppm) solution was added to 200 pl of (24 hours old) bacterial cell-suspension. Luminescence was measured before addition of nicotine and after 30 minutes contact time. Testing the effect of (+)-nicotine potencies and placebo on test-bacterium in the 40 presence of (-)-nicotine 5. 500 pl of freshly prepared potencies and placebos were added to 5 ml of (24 hours old) Vibrio fisheri cell suspension. 6. 200-200 pl of potentised or placebo-treated bacterial inoculum was pipetted into cuvettes of luminometer and the intensity of the emitted light was measured (la).

-26 7. 50 pl of (-)-nicotine solution of 0, 312.5, 625, 1250, 2500, 5000, 10 000, 20 000 ppm were added to the bacterial cell-suspensions (in cuvettes), after which the intensity of the emitted light was immediately measured (11). 8. After 30 minutes of contact time the intensity of the emitted light was measured 5 again (I30). Evaluating measured data Primary (measured) data were placed into an excel table and the comparative evaluation was executed with them. At the end of the testing we have I, I, and 130 toxicity data. From these 10 data we calculated the H %, the relative inhibition of luminescence compared to I and adjusted with Io. The endpoints used for the sensitivity testing were effect concentration EC 20 / EC 5 0 values, where EC 20 and EC 50 mean concentrations caused 20 % and 50 % inhibition. EC 20 and EC 50 values were determined from concentration-response curve (inhibition percent values of 15 concentrations) after sigmoidal fitting by ORIGIN 6.0* software.

- 27 RESULTS 1. Measuring the sensitivity of test-bacteria to (-)-nicotine Table 1. Effect of (-)-nicotine on luminescence light emission of Vibriofischeri Concentration of nicotine solution Inhibition of luminescence {ppm (% 0 0 312.5 6 625 21 1250 49 2500 76 5000 100 10000 100 20000 100 5 Concentration-response curve E 60 0 o -U 0 - ----- -- 100 EC 1000 EC 10000 log (-)nicotine [ppm] Figure 1. The effect of (-)-nicotine solution on the bioluminescence of Vibriofischeri Concentration-response curve

EC

20 and EC 50 values were determined from concentration-response curve (Figure 1) after 10 sigmoidal fitting by ORIGIN 6.0* software. EC2 0 = 622 ppm

EC

50 = 1284 ppm -28 2. The effect of (+)-nicotine potencies and placebo on test-bacteria in the presence of (-)-nicotine 2.1. The effect of treatment with 4 'h (+)-nicotine potency and placebo 5 The main objective was the evaluation of the effect of (+)-nicotine potency-treatment with regard to their usefulness for curing the toxicity of (-)-nicotine. Vibriofischeri bioluminescence test was applied to study and evaluate the effect of addition of potencies and placebos. The inhibition of bioluminescence was measured after 30 minutes contact time with potentised and placebo-treated Vibriofischeri with and without L-GA. 10 The curing effect of the 4" potency was observed only at lower concentrations of (-) nicotine (Table 2, Figure 2-3), compared to placebo treatment. Table 2. The effect of treatment with 4 th (+)-nicotine potency and placebo Treatment (-)-nicotine 4 potency 4 placebo [ppm tnLuminescence inhibition [H%] 0 0 0 312.5 0 1 625 3 28 1250 52 52 2500 86 78 5000 98 100 10000 100 100 120 - _ 100 M 4th potency 80 -4th placebo 40 -E 40 20 0 0 312.5 625 250 2500 5000 10000 (-)-nicotine concentration [ppm] 15 Figure 2. The effect of treatment with 4 th (+)-nicotine potency and placebo on luminescence 20 25 - 29 5 Treatment with 4 (+) nicotine potency and placebo 100 80_ 8 m Potency C 60P oR 10 E o 40__ _ o2 20,F 0 10 100 1000 10000 log (-) nicotine [ppm] Figure 3. The effect of treatment with 4 h potency and placebo on the toxicity The 4 th potency is effective in the low concentration range of 312-1250 ppm of nicotine. 10 EC 2 0 and EC 50 values were determined from concentration-response curve (Figure 3) after sigmoidal fitting by ORIG[N 6.0* software.

EC

2 o [ppmJ

EC

5 o (ppmJ 4' potency 838 1218 4' placebo 650 1218 Potency of (+)-nicotine decreased the toxic effect of (-)-nicotine with 23%, based on the EC20 values. There were no significant differences in the higher concentration ranges.

- 30 2.2. The effect of treatment with 12' (+)-nicotine potency and placebo In the case of 12'h-potentised bacteria the toxic effect of (-)-nicotine was counteracted only at the concentration of 312.5 ppm, compared with placebo-treatment. 5 Table 3. The effect of treatment with 12*h (+)-nicotine potency and placebo Treatment (-)-nicotine 1 2 ' potency 11 placebo &pm/ Luminescence inhibition [H%] 0 0 0 312.5 3 16 625 39 37 1250 80 61 2500 96 83 5000 100 100 10000 100 100 120 100 U 12th potency 80 .

i 12th placebo 60 .E 40. 20 0 0 31 . 5 1250 2500 5000 10000 (-)-nicotine concentration [ppm] 10 Figure 4. The effect of treatment with 12 h (+)-nicotine potency and placebo on luminescence The 12" potency is active in the low nicotine concentration range between 312 and 625. In higher concentration range potency increased the adverse effect of (+)-nicotine. 15 20 25 -31 Treatment with 1 2 (+) nicotine potency and placebo 100 80 0 60 0 / Potency 40 0 Placebo 0 .2 0 10 100 1000 10000 log (-) nicotine [ppm] Figure 5. The effect of treatment with 12 h potency and placebo on the toxicity 5

EC

20 and EC 50 values were determined from concentration-response curves (Figure 5) after sigmoidal fitting by ORIGIN 6.0* software.

EC

2 0 [ppm[ ECso {ppm[ 1 2 'F potency 519 734 1 2 'h placebo 366 908 The decrease in inhibition is significant in EC 20 values, in connection with the low 10 concentration range, where potency is able to compensate adverse effect of (-)-nicotine. The decrease in toxicity based on EC20 is 30%, it means that inhibition is the 70% of the untreated. The same cannot be stated in higher concentration ranges, that it is not true for the EC50 values. Effect of potency evaluated based on EC50: potency is 23% more toxic than placebo.

- 32 2.3. The effect of treatment with 30 'b (+)-nicotine potency and placebo The curing effect of the 30 'h potency was considerable in a wider concentration range of applied nicotine, compared to 12'h potency-treatment (Table 4, Figure 6-7). The applied 3 0 th potency of (+)-nicotine was able to decrease the toxic effect of naturally occurring isomer at 5 312.5, 625 and 1250 ppm concentrations. Table 4. The effect of treatment with 3Oth (+)-nicotine potency and placebo Treatment (-)-nicotine 3 0h potency 3 jM placebo fppm Luminescence inhibition [H% 0 0 0 312.5 0 11 625 0 31 1250 43 52 2500 87 81 5000 99 100 10000 100 100 120 100 U 30th potency 80 - 30th placebo 40 20 0. 0 312.5 625 1250 2500 5000 10000 (-)-nicotine concentration [ppm] 10 Figure 6. The effect of treatment with 30 th (+)-nicotine potency and placebo on luminescence The 3 0 t' potency is very active in compensating toxicity of (-)-nicotine between 312 and 1250 ppm. 15 20 25 -33 Treatment with 30' (+) nicotine potency and placebo 100 80 60 Potency 2 - 0Placebo 0 0 10 10 1,0 ,00 1000 log (-)nicotine [ppm] Figure 7. The effect of treatment with 30th potency and placebo on the toxicity 5 EC20 and EC50 values were determined from concentration-response curves (Figure 7) after sigmoidal fitting by ORIGIN 6.0*0 software. EC2o ppm[ ECso {Rppm 30' potency 958 1399 30 placebo 580 1088 Toxicity decreased to the 60% (with 40%) in case of EC20 and with 22% in case of EC50 on the 10 effect of the treatment with (-)-nicotine. Both results of decrease are significant.

- 34 2.4. The effect of treatment with H2 (+)-nicotine potency and placebo The curing effect of the H2 potency (4 +12 h+ 30) was the most significant compared with other treatments shown previously (Table 5, Figure 8-9). 5 Table 5. The effect of treatment with H2 (+)-nicotine potency and placebo Treatment (-)-nicotine H2 potency H2 placebo fppmf Luminescence inhibition [H%] 0 0 0 312.5 0 16 625 2 38 1250 41 62 2500 86 87 5000 99 100 10000 100 100 The luminescence-inhibition was lower almost in all cases of H2 potency-treatment. 10 120 100 E H2 potency 80 E H2 placebo 0 S60 -40 20 0 0 312.5 25 125 2500 5000 10000 (-)-nicotine concentration [ppm] Figure 8. The effect of treatment with H2 (+)-nicotine potency and placebo on luminescence 15 20 - 35 Treatment with H2 (+) nicotine potency and placebo 100 80 60 C/ o Potency S Placebo 0 10 100 1000 10000 log (-)nicotine [ppm] Figure 9. The effect of treatment with H2 potency and placebo on the toxicity H2 potency mixture showed the highest effectiveness in healing the adverse effect of (-) 5 nicotine, it worked in a wide concentration range of 312-2500 ppm. Higher EC 20 and EC 50 values in case of H2 potency-treatment also demonstrated the curing effect of the applied potency.

EC

20 and EC 5 0 values were determined from concentration-response curves (Figure 5) 10 after sigmoidal fitting by ORIGIN 6.0* software.

EC

2 o {ppmL ECo {ppmJ H2potency 958 1423 H2 placebo 382 864 Toxicity decreased more than to the half (with 60%) in case of EC20 and with 40% in case of EC50 on the effect of the treatment with (-)-nicotine. Both decreases are highly significant. 15 3. Summary The results of the present study confirmed that the 30 th and H2 potencies of enantiomer of (+)-nicotine are able to significantly counteract the toxicity of naturally occurring (-) nicotine. Although these potencies could not decrease the inhibition of the higher concentrations of (-)-nicotine, which are extremely high and not relevant for any realistic 20 situations.

Claims

1. A method of treatment of an organism suffering from the effects of nicotine said method comprising the steps of potentising a stereoisomer of nicotine, and administering 5 said nicotine to the organism.

2. A method of treatment of an organism suffering from the effects of nicotine, said method comprising the steps of diluting a stereoisomer of nicotine, and administering said diluted nicotine to the organism. 10

3. A method of treatment of an organism suffering from the effects of nicotine, said method comprising the steps of diluting a stereoisomer of nicotine to an ultra-high dilution of said stereoisomer, and administering said ultra-high diluted stereoisomer to the organism. 15

4. The use of a dilution or an ultra-high dilution or potentised preparation of nicotine, for the preparation of a medicament for the alleviation of the physical and psychological effects of nicotine.

5. The use of a dilution or an ultra-high dilution or potentised preparation of nicotine of 20 nicotine, for the preparation of a medicament for the treatment of the addictive and other undesirable effects of nicotine addiction, as occurs for instance in the habit or practice of tobacco smoking.

6. The use of a dilution or an ultra-high dilution or potentised preparation of nicotine, 25 for the preparation of a medicament for the alleviation of the physical and psychological effects of nicotine.