AU2008101117A4 - Method of Treatment - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 INNOVATION SPECIFICATION Invention Title: Method and Treatment The following statement is a full description of this invention, including the best method of performing it known to me: -1- 00 C METHOD OF TREATMENT 0 Z The present invention relates to a method of treatment, in particular to a method of treating an effect of glutamate or glutamic acid by administering a dilution or an ultra-high dilution or potentised preparation of glutamic acid or glutamate.

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 oO O 10 general knowledge in Australia or elsewhere.

BACKGROUND

Homoeopathy employs minute doses of usually harmful or toxic agents to stimulate 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 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 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.

-2- 00 CN SUMMARY OF INVENTION 0 SThe inventor has now found that the effects of glutamate within the nervous system or within the organism generally can be treated by administering a dilution or an ultra high dilution or potentised dilution of a stereoisomer of glutamate or glutamic acid.

In the context of the present invention it will be understood by those skilled in the art that 00 Sthe term "glutamic acid or glutamate refers to one or more stereoisomers which induce the oO 0 10 effect to be treated in the organism. The stereoisomer of glutamic acid or glutamate which C may be used to treat the effects of the glutamic acid or glutamate within the organism or nervous system will be the enantiomer of the form of glutamate or glutamic acid naturally occurring within the organism. Therefore, a potency or dilution of D-GA may be used to modulate the toxicity of L-GA. Similarly a potency or dilution of L-GA may be used to modulate the toxicity of D-GA. (Note that D-GA D-Glutamic Acid D-GLU and L-GA L-Glutamic Acid L-GLU. Glutamic Acid GA) Accordingly, there is provided a method of treatment of an organism suffering from the effects of glutamic acid or glutamate, said method comprising the steps of potentising a stereoisomer of glutamic acid or glutamate, and administering said potentised glutamic acid or glutamate to the organism.

Another aspect of the invention provides a method of treatment of an organism suffering from the effects of glutamic acid or glutamate, said method comprising the steps of diluting a stereoisomer of glutamic acid or glutamate, and administering said diluted glutamic acid or glutamate to the organism.

Still yet another aspect of the invention provides a method of treatment of an organism suffering from the effects glutamic acid or glutamate, said method comprising the steps of diluting a stereoisomer of glutamic acid or glutamate to an ultra-high dilution of said stereoisomer, and administering said ultra-high diluted stereoisomer to the organism.

0 However, the same effect may be obtained by using dilutions such as those used in C1 investigations involving hormesis. Such dilutions exist below the toxic range of a given O compound, substance or molecule. Such dilutions below the toxic range are stimulatory Z rather than toxic, but may be bilphasic or multiphasic in their effects, stimulatory or inhibitory in their effects, depending on the concentration below the NOAEL which is being 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'.

0 O 10 The method of the present invention may be used for the treatment of any organism, C including for example, animals, plants, microorganisms and in particular, humans.

Another aspect of the invention is the use of a dilution or an ultra-high dilution or potentised preparation of glutamate or glutamic acid, for the preparation of a medicament for the treatment of the toxic, physiological and/or pathological effects of said glutamate or glutamic acid.

Still yet another aspect of the invention is the use of a dilution or an ultra-high dilution or potentised preparation of glutamate or glutamic acid, for the preparation of a medicament for the treatment of the addictive and other undesirable effects of drugs of addiction.

Still yet another aspect of the invention is the use of a dilution or an ultra-high'dilution or potentised preparation of glutamate or glutamic acid, for the preparation of a medicament for the alleviation of the physical and psychological effects of drugs of addiction.

The present invention may be used for the following non-limiting examples: 1. Increase or decrease neurotransmission 2. decrease apoptosis 3. increase apoptosis, e.g. in the treatment of malignancy 4. treat neuronal degeneration treat Alzheimer's disease 6. treat Parkinson's disease 7. treat Huntington's chorea 8. treat HIV-associated dementia 9. treat multiple sclerosis treat amyotrophic lateral sclerosis 00 S11. treat glaucoma O 12. treat obsessive-compulsive disorder 13. treat stroke O 14. treat dementia S 5 15. treat neuropathic pain S16. treat chronic pain 17. treat anxiety 18. treat depression 19. treat bipolar disorder 20. treat post-traumatic stress disorder 21. treat retinal degeneration of whatever cause 22. treat nicotine addiction or withdrawal 23. treat cocaine addiction or withdrawal 0 24. treat amphetamine addiction or withdrawal 0 15 25. treat morphine or opiate addiction or withdrawal 26. treat addiction or withdrawal due to licit or illicit drugs 27. treat cerebral ischaemia 28. treat neuronal injury in situations like head injury or trauma 29. treat epilepsy 30. treat schizophrenia 31. treat psychosis 32. treat Chronic Fatigue Syndrome CFS has a common physiological mechanism compared with depression, anxiety and stress.

33. treat addiction in general 34. treat alcohol addiction or withdrawal Alternatively, the invention may be used for the preparation of a medicament for the alleviation of the physical and psychological effects of any of the conditions listed above and numbered from 1-34 inclusive.

It has also been found that the enantiomer of the endogenously occurring isomer of glutamate or glutamic acid, used to treat the organism, may be derived from other salts of glutamate or glutamic acid, instead of glutamic acid being used, sodium glutamate may provide a suitable isomer of glutamate.

It should be noted that (-)-glutamate (or L-glutamate) is the naturally occurring isomer in the human. Therefore a preparation of (+)-glutamate (or D-glutamate) would be used to treat the human in the context of this method, when the effects of endogenous, naturally occurring glutamate, are being treated.

Potentization of the stereoisomer, glutamate or glutamic acid, 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 00 Sof the method of preparation of stereoisomers in general, and this includes glutamate, into potency, refer to previous patents by Kuzeff as inventor, especially 'Spacetime modification O of homeopathic medicinal action', 22 23 and also other patents, including AU 2003208170, Z AU 2005100336, AU 2005100337 and AU 2007100082. The description of manufacture of potencies in the following pages applies to any stereoisomer including Glutamic Acid.

Preferably the stereoisomer 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) systems as the standard scales of attenuation, under N which each successive attenuation contains just 1/10, 1/100 or 1/50,000 as much of the 00 0 10 medicinal substance as the preceding attenuation. It is preferred that after each attenuation, C the attenuated medicinal substance is succussed (typically between 10 to 100 times at each stage of attenuation) or triturated. 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, sonication, Lab dancer, etc.

In order to prepare the dilutions or potencies A ml of tincture or B grams of medicinal 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, fourth, fifth, etc. attenuation, the values of A could be AI, 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 (1.0ml) of tincture, 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 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 parts of the vehicle, and represent the following proportions of active principle dried medicinal substance): 00 2X 10- 2 6X 10- 6 O 3X= 10 3 7X= 10 7 4 X 10 4 8X 10 8 5X= 10- 5 (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 N- substance of the one before, nC 10I 2n In the case of fifty millesimal attenuations one 00 O 10 millilitre (1 ml) of the first fifty millesimal attenuation (1LM) represents 4.0x10 9 gram of C dry crude medicinal substance. One millilitre (1 ml) of the second fifty millesimal attenuation (2LM) represents 8.0x 0 14 gram of dry crude medicinal substance. Each 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 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 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.

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 are have been commercially available for many years. Accordingly, one or more potencies of glutamate or GA could be 0 included in such mixtures. It would be possible to add GA potencies to existing available Ci commercially available complexes. Note that the term 'different potencies or attenuations' O also encompasses the situation where the same or different potencies, made from 2 or more Z different medicinal substances, are added or mixed together. Such mixtures are common 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 Scontemporaneously, either simultaneously or within the same course of treatment. This 00 0 10 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 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 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.

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 enantiomer to 2ml of 4th, 12th and 30th potencies enantiomer, or vice versa. Alternatively 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, 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 separately, and mixing could occur in the one container.

-8- 00 cN Also, in the spirit of the above description, 0.4ml of 3 rd potency, attenuation, or dilution of O the (-)-enantiomer, may be added to 1.2 ml of the 13 th 5.2 ml of the 41 st 4.5 ml of the 200 th Sand 3.7 ml of the 1000th potency, attenuation or dilution. This may then be succussed or not. In turn, this may then be added to a mixture of 5.1 ml of the 5 th potency, attenuation or dilution, 0.3 ml of the 3 7 th and 6.3ml of the 10 5 th 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 Sthe number denoting potencies, attenuations or dilutions can be replaced by any integers 00 0 10 greater than zero. Numbers representing millilitres of potency, attenuation or dilution, can be replaced by any numbers greater than or equal to zero.

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 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 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 mixing could occur in the one container.

Also, in the spirit of the above description, 0.4ml of 3 r d potency, attenuation, or dilution of the (-)-enantiomer, may be added to 1.2g of the 13 th 5.2g of the 41 st 4.5g of the 2 0 0 th and 3.7g of the 1000th potency, attenuation or dilution. This may then be succussed or not. In 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 th and 6.3g of the 10 5 th 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 zero. Numbers representing grams of potency, attenuation or dilution, can be replaced by -9- 00 0 any numbers greater than or equal to zero.

O The same procedures as described above could be used for mixtures of stereoisomers in Z general, including diastereoisomers or mixtures of diastereoisomers and enantiomers.

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 Sparticularly preferable to administer the potentised stereoisomer orally or sublingually.

00 0 10 Specific examples of administration of the potentised stereoisomer include tablets, globuli, N liquid dilutions for injection and liquid external preparations.

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 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.

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 inclusive (or IX 1 OX) and gradually incrementally increasing the potency until the treatment is optimised. Experience may show that 6C, 15C, 30C, 200C are appropriate attenuations in most cases. Attenuation of 1000C, 10,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 water, such as water for injection B.P. or lactose B.P. or sucrose B.P. or U.S.P. ethanol typically in suitable concentrations 15 Also absolute ethanol, purified water, glycerol 85% or other ethanol/water mixture or dilution of glycerol may be 00oO used. Other vehicles will also be apparent to those skilled in the art ofhomoeopathy.

o The methods of preparation of solid or liquid stereoisomers of chemical agents into Z 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 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.

00 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.

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 lactose used for that potency. The process of trituration is typically performed with mortar and pestle for 15 to 20 minutes. The side of 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 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 minutes of trituration. Each successive level of attenuation is called a potency.

The process of the present invention may be used to reverse, or in another embodiment enhance, the effects in vivo, and in vitro, of glutamate or any of its compounds. Another aspect of the invention provides for the use of the method of the present invention to -11 00 0 enhance or reverse the effects in vivo and in vitro of (-)-glutamate or any of its compounds or rC to enhance or reverse the effects in vivo and in vitro of (+)-glutamate or any of its O compounds.

Z It is to be understood that in this specification the terms enantiomer, stereoisomer and optical isomer refer to the relevant isomers of glutamic acid.

EXAMPLES

SThe present invention is further described by the following non-limiting examples.

00 N Use of Enantiomeric Glutamate in the treatment of Depression, Anxiety and Stress a placebo controlled randomized study Introduction Previous papers describe the modulation of toxic effects of optical isomers by use of their stereoisomers.

23 26 To abbreviate the current concept, it could be said that the toxicity of optical isomers can be counteracted by administration of a potency of its enantiomer, however, this can be extended to stereoisomers in general and also can be used to increase isomer activity as well as inhibit it. The present study entails the use of homeopathic potencies of (+)-glutamic acid for the treatment of depression, anxiety and stress as measured by DASS questionnaire.

Background To paraphrase Hyman, major depression has a lifetime prevalence of 1 in 10, 9 attention deficit hyperactivity disorder is attributed to 8.7% of children between 8 and 15,'2 eight million or 1 in 10 children in the United States take stimulants like Ritalin, where the use of such medications increased 400% in the decade from 1986.

53 Autism affects 1 in 166 children, which is an increase of 11-fold in the last 10 years.

5 2 Learning disabilities affect of school age children.

14 Alzheimer's disease affects 30% of people over 85 years and is expected to increase 3 times by 2050, costing tens of billions of dollars annually.

7 Psychotropic medications are the number 2 selling class of prescription drugs.

4 0 With this impressive list of facts it would appear that modern society is in the grip of an epidemic of psychiatric disorders. Hyman describes how such conditions may be viewed as systemic disorders affecting the brain rather than primarily brain disorders, and describes how the -12o00 problem may be approached from the perspective of nutritional and environmental medicine N, using a systems approach, and presents a case history with what was a well-demonstrated o and obviously very satisfying outcome for patient, parents and practitioner.'s It is noted that a number of the above conditions have been associated with increased action of the neurotransmitter glutamate at NMDA receptors. Glutamate is the major excitatory synaptic neurotransmitter in the brain and is found in 80% of neurons and there is increasing evidence that antagonists of glutamate action at NMDA receptors have antidepressant-like action, 32 and anxiolytic action.

1 9;32 Glutamate is characterized by a number of experts to be 00 10 largely responsible for the ability of the nervous system to rapidly transmit information from one part of the body to another, and to be important in thought formation and memories.

27 Excitotoxicity is a term applied to glutamate and is the excessive exposure to the neurotransmitter glutamate or to stimulation of its membrane receptors, and is considered a main contributor to neuronal injury and death in numerous conditions.

27 Excitotoxicity in this context was first described in 1969 35 (in Lipton). In the case of the NMDA receptors these include Alzheimer's disease, Parkinson's disease, Huntington's disease, HIVassociated dementia, multiple sclerosis, amyotrophic lateral sclerosis, and glaucoma, obsessive-compulsive disorder, stroke, dementia and neuropathic pain, 3 and also anxiety and depression.

1 9 Of note is that the role of glutamate in nervous system functioning and its excitotoxic and apoptotic effect draw interesting parallels with comments by Rudolf Steiner, the founder of Waldorf Schools, in the early 2 0 th century, about 1913. He mentions that the process of normal thinking, as opposed to what he calls 'sense free thinking', exerts an effect on the organism in general and the nervous system in particular, contributing gradually to death and cell death. GA is a molecule considered to be central to nervous system functioning, and has an effect leading to cell death. It therefore seems to fulfill important criteria in the context of Rudolf Steiner's claims."' 2 This is really interesting, since the same author, who has a tendency to startle some people with his firm beliefs, nonetheless has a record of being quite prescient.

1. Steiner, R. The Incarnation of Ahriman: The Embodiment of Evil on Earth. Rudolf Steiner Press.

Forest Row. ISBN 1 85584 187 9, pages 101-103, 2006.

2. Steiner, R. Approaching the Mystery of Golgotha, Anthroposophic Press, Massachusetts, 2006.

page 12. ISBN 0-88010-606-9 13- 0 The situation where one specific physiological portal, as represented by GA, may lead to CN wide ranging and multiple pathologies the inventor calls a devils' door phenomenon.

SThe overall impression is that modulation of GA activity may lead to significant CNS Z effects.

Another group of glutamate receptors are the metabotropic glutamate receptors. Evidence suggests that inhibition of some receptors belonging to this category may play a part in counteracting nicotine addiction; other receptors in the same category may assist with Sdepression occurring in early nicotine withdrawal and may be useful in treatment of 00 0 10 depression generally.

31 Metabotropic glutamate receptors may also be promising targets in C the treatment for neurologic disorders derived from abused drugs such as cocaine, morphine and amphetamines, and may also play a part in the regulation of several neurodenerative disorders, epilepsy, and ischemia.

51 Some metabotropic receptor agonists may be useful in the treatment of psychotic disorders including schizophrenia.

4 5 Note that this sentence refer to agonism on a glutamate receptor function. On the basis of available evidence and consistent with the well-known homeopathic principle of aggravation, it is also possible to increase toxicity of a compound using stereoisomers in potency, and accordingly, future experiments could investigate increase of optical isomer, and in the case of the present paper, glutamate, activity. 13;23;32;50 There are 3 classes of glutamate-gated ion (or ionotropic.) channels, known as AMPA, kainite, and NMDA receptors. Excessive activation of the NMDA receptor leads to production of damaging free radicals and other enzymatic processes contributing to cell death.

28 29 33 In addition the are at least 10 types of metabotropic glutamate receptors. It should be noted that the functions and interactions of receptors in the brain do not act in isolation. GABA receptors for instance balance the actions of glutamate to prevent hyperexcitation of neurons, and abnormal GABA-glutamate tone in the brain may contribute to panic prone or anxiety states.

1 9 Similarly it should be noted that fear extinction involves the new learning of fear inhibition and is considered crucial for effective anti-anxiety treatment.

49 Although the same authors note that NMDA antagonists may have anti-anxiety action, they also note that a partial NMDA receptor agonist may facilitate extinction.

1 3 It should be noted that clinical experience using homeopathic potencies of enantiomeric glutamate indicate a likely 14- 00 0 anxiolytic, antidepressant and anti-stress action measurable after 4-6 weeks treatment, as C1 measured by DASS questionnaire.

34 This has even been noted in patients with severe O psychosocial problems and chronic pain. The effect on pain has not been measured at the Z present time, but it may be a good idea in such patients to record pain scales at baseline and at 2 weekly intervals together with the DASS questionnaires.

6 1 6 r Similarly it could be noted that other glutamate receptors have relevance in other areas.

AMPA potentiators may be of benefit in enhancement of cognitive function.

32 0 0 10 Other agonists of the NMDA receptor such as aspartate and D-serine are known. Glycine is a well known co-agonist of the NMDA receptor with glutamate. Accordingly, potencies of D-serine, aspartate and glycine may be used to modulate the activity of these agonists of the NMDA receptor, and could be utilized in combination either as a complex, or separately as simplexes, to modulate NMDA receptor function.

One of the best opportunities for studying the effects of similar substances, which are almost identical but are in fact not identical, is in the case of optical isomers. Stereoisomers, which are enantiomers of each other, are almost identical chemically. Stereoisomers have identical molecular formulae and structural formulae, but different configurational formulae, the molecules are identical to each other except for their spatial orientation. Enantiomers have the peculiarity that they are mirror images of each other. The 2 mirror images are typically differentiated with the notations and referring to the ability of the compound to rotate polarized light in a polarimeter, and and which refers to the Cahn-Ingold- Prelog convention specifying the orientation of the molecular weights of moieties attached to a chiral centre in the molecule. Their main identifying difference is that they rotate polarized light differently when analysed using a polarimeter. Furthermore, enantiomers rotate polarized light by an approximately equal number of degrees in a polarimeter, but in opposite directions.1;10 The profile of physiological actions of optical isomers and their enantiomers or diastereoisomers are typically very similar, however, the potency of each form of stereoisomer in terms of a given physiological action may vary widely, and the actual physiological actions are very different. For example adrenaline found in humans is the minus isomer and is about 15 times more active than the plus isomer physiologically.

0 Similarly, the 3-receptor blocker (-)-propranolol is about 60-100 times more active than C1 propranolol in blocking the inotropic, chronotropic, and vasodepressor actions of the Po receptor stimulant isoprenaline. However, the (+)-isomer is more effective at inhibiting Z oubain-induced arrhythmias in dogs.

1 7 Previous studies have described how it was sought to treat the toxic or physiological effects of optically active compounds by using their stereoisomers, and more specifically, their enantiomers, in potentized form.

23 26 This is particularly of interest since in nature since a Slarge proportion of biomolecules are stereoisomers. By so doing, the attempt was made to 00 0 10 mimic the simillimum principle of homeopathy. However, rather than focus on the actual C symptoms and signs as is done in homeopathy, the method of the instant application focuses on the profile of physiological actions of stereoisomers and specifically glutamic acid.

Looked at in its simplest manifestation, the simillimum principle says that to treat a sickness one must administer a medicinal preparation which in suitably attenuated form, is capable of producing the same symptoms and signs which are being exhibited and experienced by the patient. Stereoisomer symptoms are different.

Experiments using isopathy have been previously reported 34 56 7 8 9 1 0 11 Isopathy is not the same 3. Cazin Cazin Gaborit Chaoui Boiron Belon Cherruault Y., Papapanayotou C. A study of the effect of decimal and centesimal dilutions of arsenic on the retention and mobilization of arsenic in the rat. Hum Toxicol 6, 315-20 (1987).

4. Fisher, House, Belon, and Turner, P. The influence of the homoeopathic remedy plumbum metallicum on the excretion kinetics of lead in rats. Hum Toxicol 6, 321-4 (1987).

Jonas, Lin, and Tortella, F. Neuroprotection from glutamate toxicity with ultra-low dose glutamate. Neuroreport 12, 335-9 (2001).

6. Jonas, W.B. Do homeopathic nosodes protect against infection? An experimental test. Altern Ther Health Med 5, 36-40 (1999).

7. Aabel, Laerum Dolvic Djupesland, P. Is homeopathic 'immunotherapy' effective? A double-blind, placebo-controlled trial with the isopathic remedy Betula for patients with birch pollen allergy. Br Homeopath J. 2000 Oct;89(4):161-8 8. Aabel, S. No beneficial effect of isopathic prophylactic treatment for birch pollen allergy during a low-pollen season: a double-blind, placebo-controlled clinical trial of homeopathic Betula 30c. Br Homeopath J. 2000 Oct;89(4):159-160.

9. Berchieri A. Jr., Turco Paiva, Oliveira Sterzo, E.V. Evaluation of isopathic treatment of Salmonella enteritidis in poultry. Homeopathy. 2006 Apr;95(2):94-97.

Velkers, te Loo, Madin, F.,van Eck, J.H. Isopathic and pluralist -16- 00 0 as homeopathy. In homeopathy something similar is used and not something identical to the C pathological state (as is the case in isopathy) or to that which causes the pathological state. In O isopathy the effects of a morbific agent are treated by a dilution or potency of the same morbific Z agent. Therefore, to treat the toxicity ofintraperitoneal injections of(-)-U-50488 in an isopathic way, one would use potencies of (-)-U-50488. However, to use a potency of an enantiomer or diastereoisomer, is not isopathy, since we are not administering a potency of the identical substance, but rather a configurationally and functionally different molecule. When the enantiomer is used, we call this enantiomeric treatment or therapy. More generally, it can be Scalled stereoisomeric treatment therapy. Note that the term 'treatment' has been used in a 00 0 10 different context in the bioassay section, describing experiments with microorganisms, which C occupies a later section of this specification.

Although interesting and significant effects have been reported in previous studies involving isopathy, these have tended not to be of the magnitude one seems to observe in the clinic. This suggested that the effects seen with isopathy to date are not representative of true homeopathic effect, where effects seem to be more dramatic.

The basic principle of homeopathy is "like cures like". This is also called the "law ofsimilars".

Note that it is not "the law of identicals", and in homeopathy one does not say "identical cures identical". Isopathy represents the latter approach rather than the former. In homeopathy minute doses of (usually) harmful or toxic agents are used to stimulate a person back to health. The agents used in homeopathy are selected precisely on the basis of their ability to create the noxious symptoms and signs experienced by the patient, if given to healthy people in toxic doses, or repeatedly in sub harmful doses.

1 5 In homeopathy administration of substances in potentized form is believed to accompany the most dramatic effects clinically. It is hypothesized that the enantiomeric treatment of optical isomer effects will demonstrate larger effects than isopathic treatment, if the law of similars is being more faithfully mimicked.

homeopathic treatment of commercial broilers with experimentally induced colibacillosis. Res Vet Sci, 2005, Feb;78(1):77-83.

11 de Almeida, Campos, Herrera, Bonamin, L.V.,da Fonseca, A.H.

Effects of homeopathy in mice experimentally infected with Trypanosoma cruzi.

Homeopathy. 2008 Apr;97(2):65-9.

-17- 0 In previous experiments we used enantiomers ofpropranolol, U-50488, isocyanate to provide an N1 experimental model which attempted to mimic the homeopathic law of similars, rather than O some notion of a "law ofidenticals" 26 In this study we use the amino acid glutamic acid. However, it should be noted that other amino acids could also be of use. These include aspartate and glycine. In fact aspartate, glutamate and glycine isomers could be used to perform experiments to modify plant metabolism, carbon fixation. Glutamine isomers could be used potentially to influence nitrogen fixation in plants.

oO 0 10 Materials and Methods This study compares the ability of a (+)-Glutamic acid homeopathic complex with placebo, in terms of their respective abilities to improve DASS questionnaire scores over a 6-week period, in patients scoring an average of 14 or more points on the DASS questionnaire. Patients eligible for admission would be those with depression, anxiety or stress. Patients with bipolar disorder, PTSD, OCD and panic attacks would also be admissible. Patients with psychotic disorders or a history of psychosis would not be eligible.

(+)-Glutamic acid homeopathic complex or simplex will be administered to 90 patients in the course of their normal treatment in a prospective randomized placebo controlled study. patients will be in each arm. This sample is sufficient to detect a difference of 10 points in the DASS questionnaire score with power 0.95 at the 2.5% level.

Patients must have average DASS questionnaire scores above 14 will be suitable for admission to the study. Randomization and blinding of Glutamic Acid potencies and indistinguishable succussed placebos will be performed by Brauer Biotherapies, Tanunda, S.A. Bottles will be coded 1A and 1B up to 82A and 82B or further depending on sample size. Either A or B will be medicine or placebo according to randomization and blinding. Consecutive patients will receive bottles IA,B up to 82A,B. Patient will take the bottles for 6 weeks. Alternatively, bottles will be randomized by coin toss to medicine or placebo groups and numbered consecutively This latter method will be more practical in the performance of the study in view of the next paragraph, since each sequential bottle is randomized and has a 50% chance of being medicine or placebo.

-18- 00 c1 When data for each patient is collected, results will be sent to Brauer who will record the result o and then break the blinding for that patient. If the patient received placebo he will then be Z offered treatment with the active bottle if he so chooses. If the patient received the active treatment in bottle A, then he will be offered the chance of continuing treatment if he is favorably disposed. The minimal sample size needed for a plausible study would be 31 in each arm see below.

The DASS questionnaire consists of 42 questions divided into 3 groups of 14 questions relating 00 S 10 to 3 areas: Depression Anxiety and Stress The investigator can score for the individual items or take an average of the entire score. Scores for each of the D, A and S data range from a minimum of zero, to a maximum of 42. Up to approximately 14 is considered a normal score for all 3 items depression, anxiety and stress. It is proposed to take an average score of all the items as the primary end-points in the study at baseline, 2 weeks, 4 weeks and 6 weeks, however, at the end of the study analyses will be performed on each of the 3 areas of Depression, anxiety and stress at the baseline, 2 week, 4 week and 6 week milestones. Therefore this will be a repeated measures study. The study will not be stratified to give a definitive answer for each individual outcome D, A and S since this will require a trebling of the sample size. Also, we will not restrict the study to just D, A or S, since this will limit the number of eligible patients for admission to the study, since only one clinic is involved in the recruitment.

To detect a difference of 5 points in the DASS questionnaire we require 82 patients in each arm for power of 0.95 and p=0.05. To detect a difference of 10 points in the DASS questionnaire we require 45 patients in each arm for power of 0.95 and p=0.025. For power of 0.8 and p=0.05 and to detect a difference of 10 points on the DASS questionnaire we require a sample size of 31 in each arm.

8 Normative data available for the DASS questionnaire were used in the generation of the table below: 6 8;30 -19-

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Table A, below, shows values for power calculation: O Table A. Power calculation table for Glutamic Acid study using DASS questionnaire Z based on published normative data.

6 8 Average (Pre Test Post Treatment Stress Score) VAR(D) Alpha Z-Alpha Power Z-Beta 0.050 1.64 0.80 0.84 0.025 1.96 0.80 0.84 125.14 0.050 1.64 0.90 1.28 S0.025 1.96 0.90 1.28 0 0.050 1.64 0.95 1.64 0.025 0.025 1.96 0.95 1.64

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1 2 3 4 5 6 7 8 9 311 156 104 78 62 52 44 39 35 31 351 175 117 88 70 58 50 44 39 366 183 122 92 73 61 52 46 41 37 406 203 135 101 81 68 58 51 45 41 412 206 137 103 82 69 59 51 46 41 451 226 150 113 90 75 64 56 50 These calculations give the sample size required to detect significant differences between Pre and Post test stress scores. They test the null hypothesis that expected Pre Test Scores expected Post Test score. For example, under the null hypothesis a difference of 5 is significant at the level (one sided) with a power of 0.8 with a sample size of 62. These calculations assume that the SD for high Stress scores is the same for low scores (specifically I've used the Normative value of 7.91 throughout).

Furthermore, I have assumed that the pairs of scores are uncorrelated; this gives an upper bound for the sample size for a given Alpha level and power.

Clinical Cases Persons presenting to the inventor with chronic depression, anxiety or stress according to DASS questionnaire, were offered treatment, free of charge, with potentized D-Glutamic Acid (D-Glutamic Acid D-GA, L-Glutamic Acid L-GA). Chronic in the context of the following case data means more than 6 months duration, but most cases had a history of years of distress not adequately responding to treatment with usual medical therapy. Note that prior to days on which patients were recruited which are reported below, the invention had not been discussed with any of the patients. Patients were recruited and given D- Glutamic acid potencies after giving verbal informed consent.

The first person to be recruited and to receive potentized glutamic acid was case 1. The second was case 2 and so on. No cases are omitted. Data was extracted from the computerized records between approximately 17/7/2008 and 27/7/2008. The first case was recruited and commenced on potentized D-Glutamic acid therapy on 9 th November 2007.

00 Patients were asked to perform DASS scores at a two weekly intervals for 6-8 weeks at least.

Failing this, the data was collected opportunistically upon representation for consultation.

SFor each patient, the data collected is included in the graphs below, with intervening Z measurements not omitted. Measurements were supposed to be collected every 2 weeks preferably and not sooner. Advice for taking potencies is below after Case 21 of the clinical cases.

Potencies were manufactured according to the Homeopathic Pharmacopeia of the United States, HPUS Revision Service General Pharmacy, 2004. 100 succussions occurred at each 00oO stage of potency preparation. Adding equal volumes of the individual constituent potencies N, together made final potencies, to make the LM 4/12/30 potency, equal volumes of the LM 4, 12 and 30 potencies were added together. No succession occurred. Potencies were prepared in 30% ethanol. All potencies given to patients were in drop form in 30% ethanol, unless otherwise stated.

Case 1 A patient presented to the clinic, which he/she had attended since August 2007, who was taking regular large doses of intramuscular narcotic for pain control. Chronic pain has been a problem for years. Patient was recruited on 9 h November 2007 (9/11/07 or 9.11.07 according to Australian notation) and commenced treatment with D-Glutamic acid potencies. The patient was asked to alternate a potency chord of D-Glutamic Acid LM 4/12/30 daily with a potency chord of D-Glutamic Acid 4/12/30 C every 2 days. Thus the first potency chord referred to is a mixture of LM 4, LM 12 and LM 30 potencies of D- Glutamic acid. The LM notation refers to the 50 th millesimal scale of attenuation in homeopathy. The second potency referred to is a mixture of 4 th, 12 th and 30 th centesimal potencies. 'C chord' and 'LM chord' in the following text means the 4/12/30 C potency chord of D-Glutamic acid and the LM 4/12/30 potency chord of D-Glutamic acid respectively. The reason for alternating potencies, which is unusual if not unknown in homeopathic practice, was in order for the practitioner to obtain an impression of whether the two potency chords differed in their clinical efficacy, or indeed if they would work synergistically. If it transpired that one of the potency chords was more efficacious, then it was anticipated putting forward a proposal to conduct a double blind randomized controlled trial using the more efficacious potency chord. If both of the chords were not effective in -21 clinical use, then another potency or combination would be suggested after a period of clinical use. The full 42 question DASS questionnaire was used for this patient.

The results for case one, collected opportunistically, are in Table 1.

Table 1 Case 1 I DASS Score vs date depression anxiety stress 1.11.2007 6.3.2008 e depression 19 0 anxiety 14 stress 24 date Patient reported a marked improvement in DASS score and cessation of intramuscular narcotic use. MK has it on good authority that the patent is not a drug seeker. Patient recommenced narcotic use for pain, but at a much lower dose of methadone 10mg twice daily orally.

Unfortunately the DASS question sheet for 9/11/07, which is the date of recruitment to the study, has been misplaced in the files, or perhaps the patient did not hand one in on the day.

However, a DASS questionnaire completed on 1/11/07 prior to recruitment produced the scores used in the graph above.

These data are consistent with efficacy of alternating LM and C glutamic acid potency chords.

Case 2 New patient presents for consultation for first time on 9 1h November 2007. Usually attends another practitioner. Also attends a psychiatrist for anxiety. Recruited and commenced on -22- 00 D-Glutamic Acid LM 4/12/30 and D-Glutamic Acid 4/12/30 C potency chords, alternating CI them every 2 days.

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Z Score of first page of 42 question DASS questionnaire was: Depression 24, Anxiety 34 and Stress 30. Patient did not represent. Thus, no conclusion is possible.

Case 3 Patient with long history of recurrent depression who had been attending the practice since

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S5/11/07. Recruited to the study on 16 November 2007 with LM 4/12/30 and 4/12/30 C 00 O 10 potency chords of D-Glutamic Acid alternating every day, instead of every 2 days, LM C chord taken on day one, and C chord taken on day 2 and so on, with the chords alternating each other on a daily basis. The full 42 question DASS questionnaire was used for this case.

Graph of response for case 3 in table 3: Table 3 case 3.

DASS score vs date 0 depression anxiety stress 0- 16.11.2 7.12.07 3.1.08 31.1.08 21.2.08 21.4.08 007 depression 27 23 18 22 15 21 anxiety 19 27 23 18 17 stress 32 24 18 8 18 date 7.12.07: Patient had already been taking long-term antidepressant at presentation.

Complained of neurological symptoms. Pre-existing hypertensive. Neurological examination normal. CT brain normal. A new antihypertensive medication was started in view of poorly controlled hypertension. Increased anxiety score on this date consistent with presentation. Other scores lower. Patient stopped his antidepressant medication at his/her own insistence. Patient is reluctant to take antidepressant medication.

17.1.08: Patient presented to locum. Patient reported feeling better on treatment.

-23- 00 0 31.1.08: Patient stopped glutamic acid treatment. Patient is reluctant to take any CN antidepressant treatment including D-Glutamic acid. Scores noted to gradually deteriorate O over the next 3 months. It is noted that at the time of cessation of glutamic acid treatment, all Z readings were less than baseline. Therefore, the results are consistent with beneficial effect of alternating LM and C glutamic acid potency chords.

NOTE: For the following cases, DASS 21 scores were used: Case 4 00 O 10 Case 4 has many years history of panic attacks and takes regular medication for this.

Given full 42-question DASS questionnaire on 7/11/08, recruited to glutamic acid treatment on 16/11/07, and commenced D-Glutamic acid alternating LM 4/12/30 potency chord with 4/12/30 C potency chord thereof on 16/11/07. Potency chords were alternated daily. Results were collected opportunistically for this patient.

Table 4 Case 4 DASS score vs date P 30 depression anxiety S---stress 10 0 7.11.07 4.3.08 depression 34 27 anxiety 16 13 stress 31 date Results are consistent with beneficial effect of alternating LM and C glutamic acid potency chords.

Case A narcotic and anxiolytic using patient with many years history of fluctuating mood and anxiety was recruited on 16/11/07 at a time when he/she was relatively well, to see if -24- 00 0 alternating D-Glutamic potency chords could help maintain his/her low scores.

CN Measurements given by the patient were far between. Also, patient did not have enough O potency to last 6 months. Alternating drops daily, the supply would last at most 4 months.

Z Results are shown in table 5 below: Table 5 Case DASS score vs date 00 2 0 -depression U 20 -U-anxiety m 10 stress 0 16.11.07 27.5.08 depression 12 34 anxiety 10 28 stress 16 36 date Results are not consistent with an effect of D-Glutamic Acid potency chords, but in the context are difficult to interpret. Also, compliance is an issue. If the patient was not improving, then the usual practice would have been to modify the treatment after about 1 month from recruitment and commencement of potencies, change to exclusive use of the LM potency chord.

Case 6 Case 6 was recruited on 16/11/07. DASS was not done on that day. A questionnaire had been completed on 8/11/07 and this was used as baseline. Long term chronic back pain, anxiety and depression. Commenced on daily alternating LM 4/12/30 and 4/12/30 C D- Glutamic Acid potency chords.

00 0 Table 6 Case 6 DASS Score vs date 4 Z p depression anxiety 10- stress 0 S8.11.07 6.12.07 20.12.07 24.2.08 depression 37 35 32 37 anxiety 35 26 22 24 00 stress 27 31 24 24 date 5.12.07 Administration of alternating potency chords increased from every 2 days to daily alternation. Date on question sheet was 6/12/08, but patient presented it on 5/12/08.

2.1.08 Patient's opinion is that LM potency chord helps the most. Changed to daily LM 4/12/30 potency chord. 4/12/30 C potency chord stopped.

12.2.08 Patient deteriorated after changing to daily LM potency from alternating LM/C potencies, however, anxiety and stress scores remained lower than at baseline.

Not a big change overall, but results are consistent with beneficial effect of the LM and C glutamic acid potency chords.

Case 7 Patient was recruited on 12/12/07. Many years anxiety and panic attacks. Severe 'white coat hypertension'. Commenced on D-Glutamic acid LM 4/12/30 potency chord daily.

Patient had relatively low scores to start with, however, it was determined to see if the stress score could be decreased further. Results of DASS 21 questionnaire are in Table 7. Relevant comments from the notes are included below the graph, but are not necessarily made on the same dates as the questionnaires.

-26- 00 0 Table 7 Case 7 DASS Score vs date 0e u0 8 30 -depression _a 20 anxiety 10 stress 0 0 12.12. 27.12. 10.1.0 27.2.0 29.3.0 18.4.0 30.5.0 16.6.0 21.7.0 18.7.0 *-depression 12 12 8 2 8 12 20 30 22 anxiety 2 2 2 2 2 4 10 16 12 12 stress 22 10 10 10 10 10 16 26 20 24 date 00 14.3.08A11 scores had decreased. LM potency chord was stopped. Patient changed to 4/12/30 C potency chord daily. DASS sheet not available for this day. Patient was changed to C chord to see if lower scores would be maintained.

1.4.08 Scores virtually identical since changing to 4/12/30 C potency. Improvement in all scores since baseline. Patient changed to daily LM 4/12/30 potency. 4/12/30 C potency stopped.

18.4.08 From this time patient's scores gradually deteriorated. It transpired that contrary to instructions the patient was taking the potency at night and was having difficulty with sleep. Results from 18.4.08 are consistent with homeopathic aggravation. Patient needed to stop potency. It would be suggested to restart with a potency chord composed of lower D-Glutamic acid potencies, e.g, LM 2/3/6, after a period of abstinence from Dglutamic acid potency, or perhaps to restart potency at less frequent rate of administration.

Potency should then be taken during the day and not at bedtime.

16.6.08 LM 4/12/30 administration increased to twice daily, morning and mid afternoon. Contrary to instructions patient continued taking second dose at bedtime.

Improvement after increased rate of administration of LM potency was noted.

For the first 4 months to 18.4.08 results were consistent with action of D-glutamic acid potencies. For the last 3 months results were consistent with homeopathic aggravation and/or incorrect time of dosing contrary to instructions. Scores improved after increased potency administration to twice daily.

-27- 0 Case 8 ri Patient with years of chronic pain and narcotic use was recruited on 2/1/08 and commenced O on LM 4/12/30 and 4/12/30 C D-Glutamic Acid potencies alternating every 2 days. Results Z are shown in Table 8.

Table 8 case 8 DASS Score vs date 0- 4 depression 00 20 A anxiety stress 2.1.0 16.1. 30.1. 2.2.0 19.2. 4.3.0 27.3. 9.4.0 23.4. 7.5.0 21.5.

depression 25 16 2 22 6 0 0 6 8 22 2 anxiety 25 22 22 28 28 10 18 16 28 8 8 stress 25 24 24 26 28 18 22 22 30 18 16 date 18.2.08: Patient changed to D-Glutamic acid 4/12/30 C potency daily, in view of deterioration in scores on 2.2.08 compared with previously.

27.3.08Improved scores but some deterioration since last time. Patient changed to LM 4/12/30 potency. C potency chord stopped.

9.4.08.1 Deterioration since last visit. Today this patient was given LM 4/12/30 D- Glutamic acid potency in globule (round pill) form. To do this 5 drops (20 drops lml) of the LM 4/12/30 D-Glutamic acid potency was added to 30 gm sucrose placebo globules available from Brauer Biotherapies, Tanunda, South Australia, product code D3770 in a Brown tinted bottle with plastic screw top. The contents were immediately given forceful downward succussions at approximately 45 succussions per 60 seconds or 1-2 Hz.

2.5.08 Deterioration compared with last visit but still improved since baseline.

Has been placing pills under the tongue contrary to instructions. Suggest put in water first and mix before placing contents under the tongue.

16.5.08 Overall improvement. LM pill dose increased to twice daily, morning and mid afternoon.

27.5.08 Marked improvement over the course of treatment. Average DASS score decreased from 25 to 5.3. The trend of the 3 graphs has been downward throughout the course of treatment. Results are consistent with action of D-Glutamic acid.

-28- 00 O Case 9 C1 Patient with long history of depression over years was recruited on 5/2/08 and started D- O Glutamic acid LM 4/12/30 and 4/12/30 C potency chords alternating daily doses. Patient Shad been using anti-depressant medication since at least 2003.

Table 9 Case 9 I DASS 21 Scores vs date 2 00 P 30 depression anxiety u 1stress 010 0 5.2.2008 25.3.2008 9.4.2008 30.4.2008 depression 26 24 10 6 anxiety 36 36 22 18 stress 38 38 24 22 date 25.3.08: There had been no change over 6 weeks of treatment with alternating potency, therefore, C potency was stopped and the patient continued with daily D-Glutamic acid LM 4/12/30 potency.

3.4.08: Patient was changed from the drops to globules to see if there were any effects on scores subsequently. Globules were prepared as described above in Case 8.

Results are consistent with effect of D-Glutamic acid potency, in particular the LM potency.

-29- Case Long history anxiety. Commenced on daily alternating 4/12/30 C D-Glutamic Acid and LM 4/12/30 D-Glutamic acid potency chords. Changed to daily LM potency chord on 18/4/08.

Table 10 Case DASS 21 Score vs date

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depression anxiety stress 22.2.0 18.4.0 21.5.0 23.5.0 8.2.08 5.5.08 6.6.08 8 8 8 8 20.6.0 8 11.7.0 8 depression 14 10 8 7 7 8 4 2 3 anxiety 20 12 16 12 12 8 8 6 stress 28 18 26 22 22 18 15 16 17 date Consistent with action of D-Glutamic acid potencies.

Case 11 Patient with long history of chronic pain was recruited on 11.2.08 and commenced on daily alternating D-Glutamic acid potencies alternating 4/12/30 C and LM 4/12/30 potency chords on a daily basis.

Table 11 Case 11 DASS Score vs date -depression anxiety stress date o00 10.3.08: Scores had not improved. Alternating doses were ceased and patient was C changed to daily LM potency chord.

o 10.4.08: Scores improved.

Z 9.5.08: Ran out of glutamic acid LM drops 4 days previously. Changed to LM potency chord globules produced as described above 7 globules daily mixed for 10-20 seconds with 1/2-1 teaspoon water in a glass and placed under the tongue for 10-20 seconds before swallowing the liquid remainder of pills were left under the tongue to dissolve.

22.5.08: Patient perception is that globules are better than drops, and indeed scores did drop further compared with the previous visit when globules were started.

00 S 10 29.5.08: Slight deterioration in scores. LM globules increased to twice daily.

N 9.6.08: Increase in scores noted on this day, but had decreased again by one month later.

Consistent with action of D-Glutamic acid potency.

Case 12 Patient admitted to study on 19 March 2008 and was given D-Glutamic Acid 4/12/30 C potency chord. DASS2 1: D 10, A= 6, S 26. Patient did not complete subsequent DASS questionnaires. However, at presentation on 30/6/08 the patient was noted to have a low (Kessler Psychological Distress Scale) score of 12. No conclusion can be drawn.

Case 13 Patient admitted on 19 March 2008 who usually attended another practitioner in the practice.

DASS 21 scores: D=14, A=12, S=34. Patient did not represent. No conclusion can be drawn.

Case 14 Admitted 3 April 2008. Years of psychological distress. Past diagnoses include Autism and Schizophrenia. Started on LM 4/12/30 D-Glutamic Acid potency chord. Compliance was an issue and a 3 d party undertook to administer the D-GA.

-31 Table 14 Case 14 DASS Score vs date 4 0 o 30 -*-depression anxiety S10stress 0 3.4.08 16.4.08 depression 28 28 anxiety 38 22 stress 36 24 date Results consistent with effect of D-Glutamic Acid. Data only available for 2 weeks after recruitment.

Case Years of depression and anxiety recruited on 8/4/08. Started on LM D-Glutamic acid (D- GA) 4/12/30 potency chord, 10 globules daily.

Table 15 Case 21/5/08: Patient presented for the first time since recruitment and presented all DASS 21 questionnaires simultaneously, the preferred procedure being to complete and forward a questionnaire to the investigator every 2 weeks, thereby allowing dose and potency adjustment. No discernible effect D-GA at this stage. D-GA potency was increased to twice daily morning and mid afternoon preferred.

-32- 5/6/08: Substantial improvement in DASS 21 scores.

3/7/08: Further improvement in DASS 21 scores.

17/7/08: Some deterioration in DASS 21 score, but still substantially improved compared with baseline. Consistent with effect of D-GA potency.

Case 16 Several months depression and anxiety. Recruited 9/4/08. Started on D-GA 4/12/30 C potency 7-10 drops daily.

Case 16 table 16 DASS Score vs date M= depression anxiety stress 9.4.08 23.4.08 depression 42 42 i- anxiety 42 42 stress 22 date No improvement in this patient. This patient did not respond to concurrent treatment with a substantial dose of a common anti-depressant either, and was subsequently admitted for psychiatric evaluation and management. Not consistent with efficacy of D-GA.

-33 Case 17 Recruited on 22/5/08 and started on D-GA LM4/12/30 potency chord drops, 7-10 drops daily.

Case 17 -table 17 DASS Score vs date depression anxiety stress 23.5.08 30.5.08 13.6.08 depression 20 4 26 anxiety 8 2 4 stress 14 2 6 date 30/5/08: Substantial improvement in scores consistent with efficacy of D-GA.

13/6/08: Slight deterioration in anxiety and stress score since last record and substantial deterioration in depression score. Patient reported that family member had experienced a misfortune since the last DASS questionnaire result. Anxiety and stress score remain less than half of baseline.

-34- Case 18 Recruited on 30/4/08.

Table 18 Case 18 DASS 21 score vs date depression anxiety stress 30.4.08 21.7.08 depression 26 -U anxiety 22 stress 26 22 Dass score 1/5/08: Patient started D-GA LM pills 7 daily. Consistent with effect of D-GA potency Case 19 Recruited 7/5/08. History of depression and fibromyalgia.

Table 19 Case 19 DASS 21 Scores vs date depression S anxiety stress 0 7.5.08 25.6.08 7.7.08 depression 26 0 2 anxiety 14 6 4 stress 16 6 2 Dass scores Consistent with effect of D-GA potency Case Recruited 6/5/08. Did not take D-GA potency daily and hand in all completed DASS questionnaires at one time on 11/6/08, without any consideration of the 2 weekly dates written on the sheets. Order of completion of questionnaires not known. Hence, it was impossible to know when or in what order the DASS 21 questionnaires were completed.

Poor compliance.

Unable to interpret data.

Case 21 Many years of anxiety and depression. Long term major tranquilizer and antidepressant medication use.

Table 21 Case 21 DASS Score vs date -4 depression anxiety stress 13.6.08 8.7.08 22.7.08 depression 30 24 26 anxiety 38 28 22 stress 32 38 24 date 13/06/08: Recruited to study. Consistent with effect of D-GA potency -36- 00 Instructions for drops and pills: o Drops: z Place one-teaspoon water in a clean glass. Add about 7 drops of the homeopathy bottle, which has a dripulator (20 drops Iml). Put the liquid under your tongue and leave it there for about 10 seconds before swallowing. Take the drops every day. Take the drops at a time of day when you feel relatively well. For example, if you tend to feel worse in the morning, then take the drops later in the day. Taking drops at night may interfere with sleep.

00 N STORAGE: Store on a shelf away from direct sunlight. Do not store near electrical appliances, computers, TV etc. Do not store in the pantry, medicine cabinet or fridge. Do not store near strong smelling things like perfume, vicks, camphor, tiger balm etc.

Pills (globules): Place one-teaspoon water in a clean glass. Add about 7 pills to the water. Swirl the contents in the glass for about 20 seconds backwards and forwards. The pills will not completely dissolve. Put the liquid and any undissolved pills under you tongue and leave it there for about 20 seconds before swallowing. Allow the pills to dissolve under the tongue.

Take this dose of pills every day. The best time to take them is at a time of day when you feel relatively well. For example, if you tend to feel worse in the morning, then take the drops later in the afternoon, or if you feel worse in the evening, then take them in the morning. Taking drops at night may interfere with sleep.

STORAGE: Store on a shelf away from direct sunlight. Do not store near electrical appliances, computers, TV etc. Do not store in the pantry, medicine cabinet or fridge. Do not store near strong smelling things like perfume, Vicks, camphor, tiger balm etc.

1 -37- Use of Tetrahymena pyriformis as a Test Organism for the purpose of detecting effects of D-Glutamic acid potency on toxicity of L-Glutamic acid.

Tetrahymena Growth Inhibition The growth curve was taken up and the specific growth rate was calculated and used for the calculation of the The results are summarised in Table 5. The calculated inhibition rate values, expressed as H% (Icontrol Isample) Icontrol) are shown. (Icontrol Inhibition by Control; Isample= inhibition by GA sample.) Table 5. The growth of Tetrahymena on the effect of increasing L-glutamic acid concentration L-GA/ in the L-GA Specific test tube conc. Cell concentration (*1000 db/mL) growth Inhibition mg/mL 0 hour 20 hours 44 hours 68 hours 92 hours rate H% Control 0 2.91 4.125 10.875 25.500 335.75 3.01 Control 1/2 0 2.91 6.125 14.875 37.250 111.50 1.08 0 0.01 2 2.91 3.625 1.000 0.500 1.375 -0.03 102 0.025 5 2.91 1.875 1.250 0.250 2.875 -0.01 101 0.05 10 2.91 1.875 1.875 1.500 2.000 -0.01 101 0.1 20 2.91 1.750 1.125 1.375 0.875 -0.04 103 0.25 50 2.91 1.625 0.500 0.125 0.625 -0.03 102 100 2.91 0.875 0.000 0.500 0.125 -0.03 102 The effect testing was carried out in a nutrient medium, which is a 2-fold dilution of the normal one (Control to see better the acute effect of the GA on the cells propagation. 2 mg/mL GA concentration caused 100% growth inhibition and depletion in the cell number after two days, 5 mg/mL caused depletion already after 20 hours. The concentration range between 0 and 2 mg/mL should be used for potency testing.

Figure 5. L-GA growth inhibition effect measured on the protozoon Tetrahymena The results show that the single cell animal test-organism is highly sensitive to the L-GA, the concentration of 0.01 g/litre (=mg/mL) caused 100% inhibition in the growth.

-38oO 0 Tetrahymena could be used as test-organism for testing potency.

Tests With Tetrahymena

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S 5 Materials and methods General conditions The potencies were prepared in glass tubes, taking care not to contact or being close to metal objects or electric wires. (This is possibly being over cautious in the case of electric wires, since it must be admitted that it has not been the experience of homeopaths in the clinic, that potencies lose efficacy, when they are stored in quite strong electric fields near power sockets,) 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. Potencies in ethanol were stored in wooden cupboard (far from electric cables) O0 in dark.

0 15 Materials L-Glutamic acid, 99 (CAS:56-86-0) Cat.: 12,843-0 (ALDRICH®, Sigma-Aldrich Chemie GmbH, Germany) Lot.: S45561-108, molecular weight 147.13 D-Glutamic acid, minimum 99 TLC (CAS:6893-26-1) 085K1083 SIGMA®, Sigma-Aldrich Chemie GmbH, Germany Ethanol 96 puriss. (Spektrum 3D® 64-17-5), molecular weight 147.13 Micro-organism: Tetrahymena pyriformis 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 D-GA potency preparation and were numbered from 1 to Potencies were 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 D-glutamic acid was placed 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 2each contained 5 ml 35% ethanol in water.

One drop (20 .pl) was removed from the 1 st test tube using automatic pipette, and was added to the second test tube. This was given 20 succussions as shown by the video. 20 1 from the second test tube was removed and added to the third test tube and so on until the 3 0 th potency.

All potencies were succussed by hand.

In the experiments with tetrahymena the 13 th potency of D-Glutamic acid was used and its effect on tetrahymena growth was compared with indistinguishable placebo.

To make the separate 5 th 13 th and 31st potencies, 20 .1t (one drop) of the 4 th 12 th and 30 th potencies respectively, were individually added to 5 ml distilled water in each of three identical 100 ml glass flasks with plastic screw stopper (SIMAX Reagent bottles with screw GL 45 ace.

to DIN 100 ml Code: 414 321 100). 5 t h and 31 st potencies were used in other experiments.

These flasks were each given 20 succussions by agitating the freshly diluted solutions by rapping the glass reagent bottles hard against a hard and elastic object such as a leather-bound book) to produce the potencies in distilled water. Succussion of each flask occurred immediately after addition of potency to the 5 ml of distilled water in each flask.

The placebo was prepared on the same way but without D-glutamic acid.

-39- These final homeopathic D-GA potencies in distilled water were prepared immediately before application to tetrahymena cultures; within 5-10 minutes after preparation.

In these tests we investigated the effect of D-Glutamic Acid 13 th potency prepared as described above on inhibition of L-Glutamic acid toxicity in tetrahymena. Note that the 13 th potency was used for both pre-treatment and treatment. In the case of the tetrahymena experiments, 'treatment' means that D-Glu 13 th potency was added 10 minutes after addition of L-Glu (as toxin) to cultures. 'Pre-treatment' means that D-Glu 13 th potency was added 24 hours prior to addition of L-Glu to cultures.

0 First trial with Tetrahymena: propagation in test-tubes; curing type treatment by 1/10 volume ratio of 13 t h potency or placebo, 1 part potency or placebo in 10 parts culture: In the following graphs the notation '15 Glu'means that the particular arm of the study, indicated by the line to the left of the '15 Glu'notation, in the key on each graph, received L- Glu added to the culture as a toxin, at a concentration of 0.5 mg/ml or 15mg/30ml in each test. The absence of 15 Glu next to the description of a line in the key on each graph, indicates that L-Glu was not added to the culture.

1 2 Hence, 'Graph 1- tetrahymena' shows that the 13 th potency of D-Glu added to culture resulted in substantially less inhibition in tetrahymena growth after 60 hours of incubation.

Graph 1 tetrahymena 12th potency or placebo treatment potency, 15 Glu 4 placebo, 15 GLu 0 3

E

E 2 o2 0 40 60 80 100 1 0 -1 time (h) Growth vs time note that 13 th potency was used and not 12 th potency of D-Glu 12 Note that '15 Glu' is depicted in the keys belonging to each of the 5 graphs titled 'Graph 1 tetrahymena' up to 'Graph 5 tetrahymena' inclusive, in various synonymous forms: 15 Glu 0.5mg/ml Glu 0.5 Glu.

2 nd trial: propagation in 30 ml flasks, prevention type pre-treatment with 13 t h potency in 1/10 volume ratio, curative treatment with 13 th potency in 1/60 volume ratio: all combinations.

1 3 Graph 2 tetrahymena (Note that 13 't potency was administered and not 12 th potency.) Comparison of 13th potency and placebo pre-treatments+treatments potency pre-treated+potency, 0.5 mg/ml Glu 180 placebo pre-treated+placebo, 0.5 mg/ml Glu 160 potency pre-treated+potency x- placebo pre-treated+placebo E 140 untreated E 120 100 0 2 S 20 40 60 80 100 120 110 time [h] In 'graph 2 tetrahymena' the first 2 lines in the key on the graph, going from top to bottom, have 0.5mg/ml Glu next to them. This means that these 2 arms had L-Glu added to them as a toxin. The other 3 arms in the key did not have addition of L-Glu. Line 1 in the key of this graph shows the arm that received 13 th potency D-Glu 10 minutes after addition of L-Glu, and also at 24 hours before addition of L-Glu. This showed less inhibition of growth due to L-Glu compared with the identical placebo shown in the second line, and depicted with smaller squares (or diamonds, depending on the angle at which the reader looks at them) along the growth curve. Line 1 is shown with the larger squares (or diamonds). The 4 th and th lines on this graph show 2 types of placebos with very similar growth curves. They show the growth curve unaffected by L-Glu toxicity. They represent parallel cultures to those shown in lines 1 and 2.

The 3 rd line shows the growth curve of parallel culture in the absence of L-Glu toxicity, but with pre-treatment with 13 th potency administered 24 hours prior to addition of L-Glu toxin to the results of cultures shown in lines 1 and 2.

The same as in graph 2, but without potency pre-treatment, only curative treatment, is shown in 'Graph 3-tetrahymena' "Note that 1 in 10 volume ratio means 1 part potency or placebo by volume was added to 10 parts culture by volume. I in 60 volume ratio means that 1 part potency or placebo by volume was added to 60 parts culture by volume.

-41 Graph 3 tetrahymena (Note that 1 3 t h potency was administered and not 1 2 th potency.) 'Graph 3 tetrahymena' shows a similar trend to the 'Graph 2-tetrahymena' in parallel cultures. However, in these cultures there was no pre-treatment with potency or placebo.

Potency or placebo was only added to the cultures 10 minutes after addition of L-GA to the cultures depicted by lines 1 and 2. It is emphasized that L-GA was added only to the cultures represented by lines 1 and 2 in this graph.

Third trial: repetition of the 2 nd: Tetrahymena was not co-operative in this case: its behaviour was not normal: much slower growth, etc. But still can be seen the effect of the potency compared to placebo.

In the context of the descriptions given above, the relevant interpretation of 'Graphs 4 and tetraymena' below are obvious. They also show action of D-Glu potency inhibiting the toxic effect of L-Glu on growth of tetrahymena, as represented by cell concentrations. In 'graph 4 tetrahymena' and 'graph 5 tetrahymena', the notation '15 Glu', or rather its synonym '0.5 Glu', appears next to all the arms depicted by lines in the key contained in each graph. Hence, L-Glu was added as toxin to all the cultures represented by these graphs.

-42 Graph 4 tetrahymena (note that 13 1hand not 12 1h potency D-Glu was used) 12th potency and placebo treatment potency, E C 0 0 0 -U-placebo, 15 Glu time Nh The effect of the preventive pre-treatment with 13 1h potency is shown in graph 5, below: Graph 5 tetrahymena.

13th potency and placebo pre Are atme nts+treatme nts potency pre-treated+potency, 0.5 Glu -U-placebo pre-treated+piacebo, 0.5 Glu -lK- potency pre-treated+piacebo,0.5 Glu potency pre-treatedy +untreated, 0.5 Glu 815 0 .210 0 r 0 0 40 60 80 100 120 140 1 0 time (h) Pre-treatment was not effective, but curative treatment was e ffective, in spite of the abnormnally slow growth. (Slow growth is not associated with the potency or placebo treatment: growth was slow without L-GLU treatment as well). Note that all graph lines in 'Graph 5 tetrahymena' arc clustered along the x-axis except for 'potency pre-treated potency, 15 Glu'. In other words, line 1 shows the effect of culture with pre-treatment and also treatment with 13 thpotency D-GA. Line 2 shows the effect of placebo pre-treatment -43- 00 0 and placebo treatment. Line 3 shows the effect of potency pre-treatment followed by Splacebo treatment. Line 4 shows the effect of potency pre-treatment only, with no addition of placebo or potency later.

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S 5 Note that bigger effects may be obtained by more frequent administration of potency than occurred in these experiments; daily, twice daily, four times daily, etc.

In addition to the cell numbers, we prepared some photos about the cells, because we observed morphological differences between potency treated and placebo cultures. To demonstrate it, we show some of microscopic pictures: 00 Normal cells in optimal culture medium in the above 2 pictures, without any treatment: The cells are big and healthy. Moving intensively with their hair-like filaments. The size can be seen from the lines under the cells, which are the engravings of a Buerker-chamber, and shown on most of the pictures.

Figure 2 tetrahymena poisoned with L-GA L-Glu placebo: the L-Glu toxicated cells are not only fewer in number but the still living ones are much smaller than the healthy ones. Their inner structure is also different, less structured morphology.

-44- Figure 3. Tetrahymena poisoned with L-GA. Pre-treatment treatment upper picture.

Lower picture received treatment but no pre-treatment L-Glu+pre-treatment treatment with potency (upper picture) L-Glu potency (lower picture) The cells poisoned with L-GLU and treated with potency appear healthy and are present in greater numbers, than the cells which were not treated with potency. Only curative and preventive+curative are both very effective from the morphological point of view.

You can compare the pictures according to the size: the same magnification was used for each. The size of the pictures are comparable by the engraving of the chamber. If the two lines are an identical distance apart, the magnification of the picture is the same (when no engraving can be seen, it is, because the cells were not co-operative in being fixed on a certain scratch..

The observation of the morphology is also a good tool, but not quantitative like the number of cells. But sometimes it is better evidence, although the best is to consider the two together.

Bacteria are too small to use this method on. The difference can be seen in the flasks visually.

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o below: Isopathic versus Enantiomeric Inhibition of (-)-trans-(S,2S)-U-50488 Hydrochloride blind randomized studies (Note that all references for this example are given as footnotes.)

ABSTRACT

Background: Previous studies have investigated toxicity inhibition of optically active compounds by potentized preparations of their enantiomers. This paper presents 2 studies investigating identically prepared and (+)-stereoisomer potencies in terms of their ability to counteract toxicity of the (-)-stereoisomer. The stereoisomers used were S,2S)-U- 50488 HCI and (+)-trans-(R,2R)-U-50488 HC. The potencies used were potency chords consisting of 4 h 12 th and 3 0 th potencies of the respective enantiomers representing a -46- 0 concentration of U-50488 of 1.08x10' 0

M.

O Materials Methods: One study compared isopathic inhibition of(-)-U50488 toxicity injected Z i.p. at the estimated LD50 into male ICR conventional mice, with placebo. The other study compared isopathic inhibition of (-)-U-50488 toxicity with inhibition by a potency chord U-50488. The outcome was the difference in survival. Designs were prospective, blind, randomised, intention-to-treat and compared the efficacy of 2 indistinguishable arms. Protocols were otherwise identical to previous studies. Results: The isopathic study did not yield a significant result. The study comparing isopathy with enantiomer potency treatment gave a p- 00 O 10 value of 0.0047 and odds ratio of 1.97 (95% Confidence Interval: 1.23 3.14), so we reject the null hypothesis.

Conclusion: We conclude that enantiomeric potencies were superior to identically produced isopathic potencies, in terms of their ability to inhibit toxicity of (-)-U-50488 HCI injected intraperitoneally in ICR conventional mice, and provides a model for testing various assumptions of homeopathy and isopathy.

INTRODUCTION

This paper reports the results of 2 experiments. The first looks at the ability, of an isopathic preparation of(-)-U-50488, to inhibit of the toxicity of intraperitoneal injections of(-)-U-50488, and is a replication of an earlier paper, except that the (-)-isomer ofU-50488 was used to prepare potencies, instead of the (+)-isomer as in the earlier experiment.

14 U50488 has been found in various models to potently inhibit peripheral and visceral pain. This is mediated by kappa opiod receptors, but non-opioid mechanisms are also involved, such as blocking of sodium channels in neurons.1516 14. Kuzeff, Topashka-Ancheva, and Mecheva, R.P. Inhibition of(-)-trans- (1S,2S)-U50488 hydrochloride by its enantiomer in white mice a placebo-controlled, randomized study. Forsch Komplementarmed Klass Naturheilkd 11, 144-9 (2004).

Joshi, S.K. and Gebhart, G.F. Nonopioid actions ofU50,488 enantiomers contribute to their peripheral cutaneous antinociceptive effects. J Pharmacol Exp Ther 305, 919-24 (2003).

16. Su, Joshi, Kardos, and Gebhart, G.F. Sodium channel blocking actions of the kappa-opioid receptor agonist U50,488 contribute to its visceral antinociceptive effects. J Neurophysiol 87, 1271-9 (2002).

-47- 00 0 Using the resulting data from the isopathic experiment for power calculation, a second 0 CN experiment was conducted in 300 mice. Once again the same protocol was used, except that this O time potencies of the (+)-isomer and (-)-isomer of U-50488 HCI were prepared, in order to compare their ability to inhibit the toxicity ofi.p. injections of the (-)-isomer. We are therefore comparing isopathic inhibition of toxicity of an optical isomer, with enantiomeric or stereoisomeric inhibition of toxicity.

1 7 An experimental model using bioassay and other molecules such as a-methylbenzyl isocyanate, verapamil, BAY K8644 and a nicotine salt has been reported.' 8 Previous papers have described the background of using stereoisomers in homeopathy 9 20 and it is not intended to reiterate this here. This is particularly of interest 00 S 10 since a large proportion of biomolecules are stereoisomers.

Stereoisomers are molecules, which, in terms of structure, are identical to each other in all regards except for their spatial configuration. Enantiomers are molecules, which are nonsuperimposable mirror images. Living organisms are very specific in terms of their sensitivity to stereoisomers and one speaks in this regard of stereospecificity and stereoselectivity.

Enantiomers, mirror image molecules, are referred to with and prefixes. The application of chemical nomenclature in stereoisomers and some of the difficulties associated therewith are discussed elsewhere.

21 It could be noted here that the R and S notation of the Cahn-Ingold-Prelog convention, used in stereochemical nomenclature, was incorrectly reported in an earlier paper 1 9 and is correctly reported by Smith.

21 17. Australian patent no. AU2003208170, Indian patent application no.

2704/DELNP/2004, European patent application no. EP1487448, US patent application no. 20050119350, Canadian patent application no. CA 2505984, Hong Kong patent application no. 05105229.7, New Zealand patent application no. 535654, International Application No.: PCT/AU2003/000219 18. Kuzeff, R.M. Treatment of effect of chemicals with their ultradilute stereoisomers.

International Patent Application No.: PCT/AU2003/000219, Applicant Sempach Pty Ltd, 2003.

19. Kuzeff, Topashka-Ancheva, and Mecheva, R.P. Inhibition of (-)-propranolol hydrochloride by its enantiomer in white mice.

Forsch Komplementarmed Klass Naturheilkd 10, 309-14 (2003).

Kuzeff, Mecheva, and Topashka-Ancheva, M.N. Inhibition propranolol hydrochloride by its enantiomer in white mice--a placebo-controlled randomized study. Forsch Komplementarmed Klass Naturheilkd 11, 14-9 (2004).

21. Smith, H.J. Introduction to the Principles of Drug Design and Action, CRC Press, Boca Raton, 2006, ISBN 0-415-28877-0, Chapter 6, pages 117-183.

-48- 00 22 23 24 25 26 27 28 29 3 0 Experiments using isopathy have been previously reported. 0 Effects in rC isopathy do not appear to be of the magnitude one seems to observe in the clinic with 'correct' O homeopathic prescribing. To treat the toxicity ofintraperitoneal injections of(-)-U-50488 in an Z isopathic way, one would use potencies of(-)-U-50488. When the enantiomer is used, U-50488, we call this enantiomeric or stereoisomeric treatment. Stereospecificity and stereoselectivity dictate that the pharmacokinetics, pharmacodynamics, physiological actions and therefore symptoms of stereoisomers tend to be very different. Accordingly, it is misleading to think of enantiomeric treatment as being semi-isopathic. Also, as demonstrated in this study, the Seffects of enantiomeric treatment are different from isopathic treatment. The only reason for 00 O 10 using U50488 in this study is that it was commercially available and provided the possibility of a N1 humane method for testing the hypothesis. The method of preparation of potency used in this study was used to demonstrate aspects of examples noted elsewhere.

8 22. Cazin Cazin Gaborit Chaoui Boiron Belon Cherruault Y., Papapanayotou C. A study of the effect of decimal and centesimal dilutions of arsenic on the retention and mobilization of arsenic in the rat. Hum Toxicol 6, 315-20 (1987).

23. Fisher, House, Belon, and Turner, P. The influence of the homoeopathic remedy plumbum metallicum on the excretion kinetics of lead in rats. Hum Toxicol 6, 321-4 (1987).

24. Jonas, Lin, and Tortella, F. Neuroprotection from glutamate toxicity with ultra-low dose glutamate. Neuroreport 12, 335-9 (2001).

Jonas, W.B. Do homeopathic nosodes protect against infection? An experimental test. Altern Ther Health Med 5, 36-40 (1999).

26. Aabel, Laerum Dolvic Djupesland, P. Is homeopathic 'immunotherapy' effective? A double-blind, placebo-controlled trial with the isopathic remedy Betula for patients with birch pollen allergy. Br Homeopath J. 2000 Oct;89(4):161-8 27. Aabel, S. No beneficial effect of isopathic prophylactic treatment for birch pollen allergy during a low-pollen season: a double-blind, placebo-controlled clinical trial of homeopathic Betula 30c. Br Homeopath J. 2000 Oct;89(4):159-160.

28. Berchieri A. Jr., Turco Paiva, Oliveira Sterzo, E.V. Evaluation of isopathic treatment of Salmonella enteritidis in poultry. Homeopathy. 2006 Apr;95(2):94-97.

29. Velkers, te Loo, Madin, F.,van Eck, J.H. Isopathic and pluralist homeopathic treatment of commercial broilers with experimentally induced colibacillosis. Res Vet Sci, 2005, Feb;78(1):77-83.

de Almeida, Campos, Herrera, Bonamin, L.V.,da Fonseca, A.H.

Effects of homeopathy in mice experimentally infected with Trypanosoma cruzi.

Homeopathy. 2008 Apr;97(2):65-9.

-49- 00 O MATERIALS AND METHODS 04 oal icewe 0 The protocol is the same as reported elsewhere.

1 4 19 20 Male ICR conventional mice were Z bred in Slivnitza animal house, and transferred to the laboratory animal house (temperature 20-22 0 C, photoperiod 7am to 7pm), 1 week before the experiment to allow acclimatization.

Experiments were conducted in accordance with laws prevailing in Bulgaria. During breeding mice were fed their standard diet, available at the Institute of Zoology. The design is prospective, blind, randomized, placebo-controlled, and intention-to-treat. The studies were both blinded and randomized, using indistinguishable treatment and placebo arms in 00 O 10 the case of the isopathic study, and 2 indistinguishable treatment arms in the study comparing isopathic with enantiomeric treatment (isopathy/enantiomer study). The design is simple and the only thing adding a patina of complexity to the description, relates to the fact that people who were working on a given arm of the experiment for a given batch of mice, did not touch, handle or contaminate the other arms. The and isomer potencies and placebo arms were in this sense physically separated, although all work except potency and placebo preparation, and cleaning of glassware, was conducted in one room.

Sample size of the isopathic study, N=158, was the same as in a previous study. 14 The isopathic results reported herein were then used to calculate sample size of the subsequent study comparing isopathic with enantiomeric treatment. It was calculated that between 150- 450 mice would be required in each arm to achieve statistical significance; a total of between 300-900 male mice were needed, for power of 0.975 at the 5% level.31 Since the vivarium at the Institute of Zoology can only house about 300 mice, it was decided prospectively to stop the experiment if it achieved significance after the first batch of 300 mice was admitted to the study. If significance were not achieved, the experiment would have been stopped at either the 600 mouse stage or, if necessary, after 900 mice, to assess the difference, if any, between the activity of isopathic treatment compared with enantiomeric treatment. It transpired that significance was achieved with the first batch of 300 mice.

31. Desu M.M. Raghavarao D. Nonparametric Statistical Methods For Complete and Censored Data. Chapman Hall/CRC, Boca Raton, Florida (2004).

o Mice were allowed food and water ad libitum at all stages of the experiment up until just before CI the injection of (-)-U-50488 HCI, with the exception of the brief period of abstinence O minutes) on any occasions after giving oral liquids by capillary tube.

All glassware such as test tubes used more than once during the experiment were washed with tap water, copiously rinsed with distilled water, hot-air dried and finally heated for 2 hours at 200 0 C before being considered clean and reusable. The same glassware was used for preparation and storage of and (+)-U-50488 HCI potencies used in the 2 treatment arms of the Sexperiment. In addition, as in previous experiments' 4 1920 cages and water bottles were washed 00 0 10 after each experiment with "Pur+Aloe Vera" (pH neutral, Henkel, Austria, 245605), mixed C 50:50 with Belina ACE (Greece). Bottles were then copiously rinsed with distilled water and treated according to the protocol mentioned above for glassware in general. Both these products are commercially available in Sofia.

Preparation of (+)-U-50488 HCL potency for Isopathic vs. Enantiomeric Experiment One person was responsible for manufacture of potencies. Isomers of U-50488 were manufactured by Sigma. Molecular weight 405.79. The and (+)-U-50488 potencies were manufactured on different days, to avoid the theoretical risk of contaminating the potencies with the (+)-potencies and vice versa.

To make the (+)-potencies, 10mg (+)-trans-(1R,2R)-U-50488 HCI ((+)-trans-(lR,2R)-3,4- Dichloro-N-methyl-N-[2-(1 -pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride), or U50488, was added to one ml distilled water in a 10 ml Hamilton Laboratory Glass test tube with a glass stopper. Twenty forceful downward succussions were given to the fluid in the test tube by hand, in a vertical line at a rate of between 0.75-1 Hertz or about 45 succussions per seconds.

A non-heparinized disposable hematocrit capillary tube (75mm/75microlitre, Hirschmann Laborgerate, product code no. 910 01 75, 40 drops Iml, 1 drop 0.025 ml) was used to add 3 drops of this solution to another test tube containing 7 ml 40% ethanol B.P. ten ml Hamilton Laboratory Glass test tubes were used again.

-51- 00 Note that the test tube should not be more than two-thirds full, there must be sufficient I room in the test tube for the fluid to collide violently with the test tube walls when it is o succussed. This second test tube was succussed 20 times at 0.75-1 Hertz. Three drops of Z this potency were added to a third 10ml Hamilton test tube containing once again 7ml ethanol B.P. The same process was repeated until the 2 9 th dilution or potency was reached.

The 30 th potency was prepared from the 2 9 th in the same manner. It should be noted that ethanol produces froth when succussed in this way. The rate of succussion was performed sufficiently slowly to enable this froth to settle down substantially between each individual succussion. 0.75-1 Hz should be sufficiently slow for this purpose.

00oO Potencies were stored in the dark away from direct sun and avoiding sources of electromagnetic energy such as power cables, computers, television, metal surfaces, refrigerators and other electrical equipment. The potencies were not stored near substances with strong smells. Having prepared in the above manner the 4 t h 12 th and 3 0 th potencies, these 3 potencies were then mixed in the following manner: ml of liquid was removed from the tube with the 4 th potency and added to a 10 ml Hamilton Laboratory test tube. Likewise 1.5 ml was removed from the 12h" potency and added to the same tube. Then 1.5 ml was removed from the 3 0 th potency and added to the tube. Therefore a homeopathic complex, containing more than one potency was prepared. The contents were given 20 forceful downward succussions at .0.75-1 Hertz, and this mixture or complex was called The mixture of the 4 t h 12 th and 3 0 th potencies was done quickly with minimal delay, within 30 seconds, so that the contents were quickly mixed and then succussed. This H1 complex was then used for the rest of the experiment. Is was stored carefully in the dark wrapped with white paper and was minimally handled.

Subsequently, on each day of the experiment, 3 drops of H 1 were added to a test tube containing 7 ml 40% ethanol. This was immediately given 20 succussions at 0.75-1 Hz to produce 'H2'.

The test tube containing H2 was then wrapped with white paper. It was this final potency, H2 that was administered to mice. Using molecular weight of 405.79 available from www.sigmaaldrich.com this represents a 1.08 xl 010 M solution. Subsequently, the H2 potency was subjected to randomisation as described below, and as a result was then designated as 'solution A' or 'solution if the potency was designated 'solution then the -52- 00 O potency became 'solution B' according to randomization.

O Preparation of (-)-U-50488 HCL potency for Isopathic Experiment and Isopathic vs.

Z Enantiomeric Experiment (-)-trans-(1S,2S)-U-50488 HC1 and (+)-trans-(lR,2R)-U-50488 HCI, molecular weight 405.79, are used for potency preparation. The preparation of the initial complex of(-)-U50488 HCL, or (-)-trans-(1 S,2S)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide Shydrochloride, potency corresponding to H1 described already above, is produced exactly as per 00 the description for (+)-U-50488 potency, but was done on a different day. e.g. if the (+)-U50488 potency was made on a Friday morning, then the (-)-U-50488 HCL potency can be made at the same time of day on the following Monday. The only difference is that instead of starting with (+)-U-50488 one should start with 10mg (-)-U-50488. Therefore, in all our experiments we took care to separate manufacture, use, and storage of the and (-)-isomer potencies.

Furthermore in these experiments and all previous experiments using stereosiomeric treatment, it was preferred to manufacture potencies in the morning, before 9.30 whenever experimental design permitted.

Preparation of Indistinguishable Placebo for Isopathic Experiment The same person who made potencies throughout the experiments, also made placebo. In the isopathic experiment, placebo was always manufactured before potency. Placebo was made by adding 3 drops of 40% ethanol from a hematocrit capillary tube to a 10ml test tube containing 7 ml 40% ethanol BP made indistinguishable placebo. This was given 20 succussions as per the treatment preparation. Fresh placebo was prepared on each day of the experiment immediately before preparation of the potency, H2. Therefore, in all our experiments we took care to separate manufacture, use and storage of potency from placebo. The same ethanol stock bottle was used for placebo and potency preparation in this study.

-53

OO

0 Randomization and blinding of potencies of placebo and (-)-U50488 for the Isopathic CN Experiment, and and (-)-U50488 potencies for the Isopathic vs. Enantiomeric O Experiment For the isopathy/enantiomer study, the H2 potencies for the randomized batches of mice were prepared each morning before 9.30 am, as described above. Mice were subsequently selected and randomized about 4.30 pm on the same day. New H2 potencies were prepared daily for each new batch of mice in advance. In order to make the H2 potencies the person Swho made potencies throughout the experiments took the HI preparations of the and 00 0 10 isomers by himself into a closed room. After making the respective H2 potencies, which C involves just one step of dilution and succussion as described above, the tubes were wrapped in such a way that paper extended partially up the side of each test tube, half way. At this stage and subsequently, each tube was only touched or handled by the portion that was wrapped with paper, with the exception of handling of the ground glass stopper. Then one sticky label was marked with the letter and another sticky label was marked with the letter A coin was tossed to determine if A or B was to correspond to the (-)-isomer potency or the (+)-isomer potency. This randomisation was done each day. The appropriate label was then affixed to each test tube according to the randomisation. Each day solution A or B was dated, and this date was written on the paper that surrounded the test tube, and also the particular batch of mice for which they were to be used was noted.

In the case of the isopathy study, the person responsible throughout the experiment for making potency and placebo, entered a closed room and performed randomization according to coin toss. After coin toss, this person made placebo first and then wrapped the test tube with paper as already described above. The appropriate randomization code, A or B, was affixed. Then the H2 potency of(-)-isomer was prepared and the appropriate code was attached, as described for the isopathy/enantiomer study.

The remaining description in this paper refers to and potencies and refers to the procedurefor the Isopathic vs. Enantiomeric Experiment. However, to determine the processfor the isopathic experiment, simply replace references to the '(+)-potencies' with 'placebo'. '(+)-potencies' are those preparations made from (+)-U-50488 HC.

-54o00 After the initial randomization, these tubes were given to a second person (person 2) who I was not present during the first randomisation and also did not manufacture the potencies.

rPerson 2 performed the final randomisation and blinding of solutions of and Z potencies just before the experiment, and did not handle the mice during the experiment and was absent from the vivarium during the experiment. Person 2 covered the previous A and B stickers affixed to the paper wrapper covering the 2 test tubes, by wrapping another layer of paper secured with tape around the tubes, thereby completely obscuring the A and B codes underneath. Person 2 then repeated the coin toss and allocated new A and B stickers to the test tubes. This was to prevent possible contamination of the (+)-isomer potency with the 00 isomer potency or vice versa. The procedure described in this section was done in order to N, minimize risk of cross contaminating solutions used for the different arms. It is perhaps not necessary, however, we wanted to allow for this possibility.

The 2 blind randomisation codes produced in this way were kept in the personal possession of the respective randomisers, and were not revealed until data had been collected. In this way nobody involved in the experiment knew the order of randomisation and the experiment was blinded.

Procedure on the evening before intraDeritoneal injections Mice were given oral and (-)-isomer potencies blind and according to randomisation for the first time shortly after their selection at 4.30 pm on the day prior to intraperitoneal U50488 injection, and also at 9.30 am the following day immediately after measurement of body weight.

Eighteen mice were selected each afternoon about 4.30 pm, on the day prior to their receiving intraperitoneal injections. All 18 selected mice were initially placed in one cage.

All cages used in the experiment contained a constant amount of unautoclaved nonirradiated softwood (pine) bedding just covering the cage floor. One mouse at a time was removed from this cage, and a coin was tossed to decide if it was to be allocated to group or group As soon as 9 mice were in either group or group then the remaining mice of the 18 selected, were allocated to the group that did not yet contain 9 mice. Group A and B mice were then administered their respective A or B solutions, as per randomization.

00 r To give oral fluids on the two.occasions prior to i.p. injection, at 4.30 pm the day before o i.p. injections, and also at 9.30am the following day (on the morning of i.p. injection), a Z mouse was taken from its cage by an experienced mouse handler and held in the supine position. This person removed approximately 0.075ml of the respective blinded and randomised homeopathy (or placebo) from the storage test tubes (containing solutions A or B which corresponded to either (-)-isomer potency or (+)-isomer potency as determined by earlier randomisation). The filled hematocrit tube was introduced just behind the incisors of the mouse such that it was impelled to drink quickly. The tube was removed as soon as 00 possible and the mouse was held for another 20 seconds in the supine position. Each mouse 7 was then returned to its communal cage (size II indicated above). The drinking water bottle and food were not placed in the cage until 10 minutes later.

Group and group mice were housed in 3 differently sizes of cages depending on the stage of the experiment, with stainless steel mesh roof, cradle for inserting a water bottle, and grill onto which could be placed dry food. The cage sizes are (width x length x height): I. 30x33x 15 cm for adaptation of the mice prior to the onset of the experiment 11. 18x33x15 cm for selection of the mice prior to injection, and III 15x24x15 cm into which mice were individually placed after injection of i.p. 50488 Two investigators and four laboratory assistants were involved in the performance of the study.

A mouse handler was a person who either picked up mice from their cages, and held them still while they receive i.p. injections of (-)-U-50488, or administered the or potencies by capillary tube. One of the investigators administered i.p. injections for every experiment. One laboratory assistant performed randomisation and blinding of solutions only and was referred to above as 'person and the remaining 3 laboratory assistants were mouse handlers. One of the investigators also acted as a mouse handler, to make a total of 4 mouse handlers, and a fifth person who administered i.p. injections. The person giving i.p.

injections, did not otherwise handle the mice after injections.

-56- O Two mouse handlers were needed to pick up mice and hold them while another 2 handlers C1 administered oral solutions, or alternatively handlers were used to hold mice in a supine O position while they received i.p. injections. These four mouse handlers who picked up Z animals from cages, and held them while the mice received i.p. injections or oral solutions via capillary tubes, had been divided into two groups of two handlers for the experiment on each day. This was done by placing the numbers 1-4 on each of 4 identical cards, which were placed in a box, which was shaken. Each handler then withdrew the cards blind.

Handlers who in this way selected the numbers one or two were paired for the experiment and handled the 'group A' mice on a given day. Those who extracted numbers 3 and 4 00 O 10 handled the "group B" mice on a given day of the experiment. Therefore, the various arms

C

of the blinded and randomized experiments for a given batch of mice were kept separate.

This was done to avoid contamination of the and (-)-isomer potencies with each other.

Procedure on the morning of the intraperitoneal injections At 9.30 am each day mice were placed in a clean strong plastic bag one at a time and suspended from a Pesola 30g or 60g scale (Pesola, Switzerland, www.pesola.ch), to measure weight in grams. Identical separate plastic bags were used for separate arms of the experiment. This weight was used to calculate dosage of intraperitoneal (-)-U-50488. After weight measurement each mouse was given its blinded and randomized potency or placebo.

Homeopathy and placebo fluids were given to mice using disposable Hirschmann hematocrit capillary tubes mentioned above.

It should be noted that the manner of administration and (-)-isomer potencies after i.p. injection was different from that described above for mice prior to injection, due to the unconscious state of the mice after i.p. injection. After injection each mouse was given only 1 drop (0.025ml) of or (-)-potency or placebo, according to randomisation and blinding, by hematocrit capillary tube, instead of almost the full contents of the capillary tube (0.075ml) at times of administration prior to i.p. injection. This was done in order too avoid respiratory obstruction, which would be more common in sleeping mice administered larger amounts of oral fluids.

-57- 0 Intraperitoneal injections of(-)-U-50488 HCI at an estimated LD50 dose of 25mg/kg were C given in the isopathic study. This estimated LD50 was in accordance with a previous Sstudy. 14 In the subsequent isopathy vs. enantiomer study an estimated LD50 of Z was used, since it was deemed to more accurately reflect LD50. The weighed mice were sequentially removed from their individual size II cages between 10-11 am and held gently but firmly in the supine position. They were each injected i.p. with a solution (-)-U50488 HCI, diluted in Normal Saline for injection B.P. The volume of this solution administered to mice intraperitoneally was 0.lml/lOgm body weight, such that each mouse received an estimated LD50 of (-)-U-50488 HCI i.p. Injections were given using a 0.5ml insulin syringe 00 0 10 with 27-gauge needle, intraperitoneally into the left lower quadrant of the abdomen. After "1 injection the mice were held in the supine position for approximately 2-5 minutes until asleep or heavily sedated.

and (+)-Isomer potencies were subsequently also administered at 5, 10, 15, and 20, 50, 80 and 110 minutes after U50488 injection. Commencement of the U50488 injections started between 10-11 am each day. On these occasions each mouse was given and (-)-isomer potencies in the same sequence in which they had received their injections.

After receiving i.p. injections mice were sequentially placed into individual size III cages with softwood bedding but no lid. They remained there until the process of administration of oral potency or placebo was finished. The number of mice alive at 9 am the next morning was the end point of the experiment.

-58- Results Isopathic Study The results of the study are shown in table 1 below: Table 1: Cross tabulation of'arm' vs 'alive/dead' for isopathy study Treatment by Result Treatment(Treatment) Result(Result) Frequency Row Pct ALIVE DEAD Total medicine 47 32 79 59.49 40.51 placebo 41 38 79 51.90 48.10 Total 88 70 158 Under the null hypothesis that: the proportions of alive and dead are the same for medicine and placebo the Chi-Square 0.9215 with 1 degree of freedom, which has a probability of 0.3371. So we accept the null hypothesis at the 0.05 significance level.

Results Isopathy vs. Enantiomer study The data was analysed according to the prospectively planned Chi-Square test and logistic regression analysis as performed in previous experiments.

The results of the Isopathy vs Enantiomer study are given in table 2: -59- Table 2 cross tabulation of 'arm' vs alive/dead for Isopathy/enantiomer study Table of Outcome by Treatment Outcome alive/dead Treatment Frequency Row Pct (+)-isomer (-)-isomer Total Alive -number 99 74 173 Percentage 57.23 42.77 Dead number 51 76 127 Percentage _40.16 59.84 Total number 150 150 300 0 p-1 00 O 0 Under the null hypothesis of no difference in survival proportions between treatments this table has a chi-square statistic with a value of 8.534 with 1 degree of freedom. The chance of this value occurring given the null hypothesis is 0.0035. Therefore we reject the null hypothesis at the 0.05 significance level. This table also gives an estimate of the odds ratio vs of 1.9936 with a 95% confidence interval of 1.2518 to 3.1751.

In previous papers we analyzed by chi-square as well as logistic regression, and found as one might expect in large, blinded and randomized samples, that the results agreed with each other well, using either method.

1 4 1 9 20 Also as one would expect in a large randomized sample, the weights and treatment arms were similar: (+)-mice N=150, mean weight 19.67 gm, median weight 19.5gm, sd=2.74 gm. (-)-mice N=150, mean weight 19.4gm, median weight 19.5gm, sd=2.91. This is not significant with p=0.41. However, use of logistic regression provides an extra layer of confidence that there are unlikely to be any random arm interactions, which might cause a spurious result. Although animals were administered i.p. U-50488 according to body weight, drug elimination is not necessarily linearly related to weight, since smaller animals have a higher metabolic rate.

32 Therefore we have adjusted the results for weight with logistic regression. As in previous studies, this was part of the prospective design.

32. Whittow GC Comparative Physiology ofThernoregulation Mammals 2, Minimal Metabolism and Body Size. New York, Academic Press, 1971, pp 30-36.

00 o Logistic Regression is a statistical method for analysing dichotomous response data (in this case 'outcome' alive/dead) while accommodating adjustments for one or more numeric covariates O (treatment and weight). A model with a treatment, weight and treatment by weight interaction was fitted. The interaction was not significant so a model with weight and treatment was fitted and is shown in table 3: Table 3 Parameter Estimates from Logistic Regression (+)-isomer treatment versus(-)-isomer treatment 00

O

(N

Standard Parameter arameter Estimate Error Pr ChiSq Intercept -1.7654 0.8552 0.0390 Treatment 0.6755 0.2392 0.0047 Weight 0.0896 0.0433 0.0383 Under the null hypothesis of no difference in survival proportions between treatments, the 'treatment factor in the above table has a p-value of 0.0047. Therefore we reject the null hypothesis at the 0.05 significance level. This gave an odds ratio (OR) of 1.97 Confidence Interval: 1.23 3.14).

Discussion There was a non-statistically significant increase of 7.6% survival in the treatment group compared with the placebo group in the isopathic study presented in this paper. This compares with a statistically significant 19.5% increase in survival in an identical previous study 1 4 where the only difference was that an identically manufactured complex of 50488 potency was used instead of using (-)-U-50488 HC1. The study comparing enantiomeric treatment showed a clear superiority of enantiomeric over isopathic treatment in terms of mouse survival.

Results were obtained with minimal pre-treatment and multiple tests were not performed to determine optimal potencies or dilutions. That it was possible to obtain evidence of effects -61 00 under such conditions supports the robust nature of the hypothesis. Molecules were selected r purely on the basis of stereochemistry and commercial availability.

Z Post hoc a logistic model was fitted to 1000 bootstrap samples, where a large number of datasets, which are the same size as the original data set, are randomly drawn from the original dataset with replacement, producing 1000 estimates of the treatment OR, and thereby statistically replicating the study. The average of this distribution of estimates was 2.03 and 95% of this distribution occurred within a range of 1.21 3.25, the median of the estimates was 1.96. Bootstrap logistic regression post hoc confirmed that the power of the 00oO isopathy/enantiomer study is in excess of 98%.

The experimental model has been robust with a clearly defined unequivocal end-point. Mice were not moribund at the end of the experiment and thus did not complicate interpretation of the outcome. There are no inherent problems with poor remedy selection, which can plague clinical studies and effectively render them under powered, since all subjects receive the same remedy preparation. It is possible to quickly achieve data on relatively large sample sizes. Our experiment on 300 mice takes 17 days.

It should be noted that mice were administered potencies and placebo in 40% ethanol. Mice accepted this very well. Since mice in all arms were given solutions in ethanol, this has no relevant bearing on the results. Furthermore, we doubt that potencies in water are stable, and wonder if they lose biological activity within hours if not minutes.

If one were to generalize the application of optical isomers as used in the present study, then one could design experiments using any optical isomer where it was sought to modulate that isomer's toxicity. Any substance can be toxic depending on the dose and context. Therefore, possible experiments investigating the present effect could involve many types of compounds including, many pharmaceuticals, drugs of addiction, cytokines, neurotransmitters, enzymes, hormones, proteins, amino acids, etc.

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