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Isothermal microcalorimetric analysis of homœopathic dilutions
122
Time-serial design for clinical trials M. BRANDS Amsterdam, The Netherlands At the last GIRI conference in Paris, an urgent call for clinical trial models was made. In recent years, many clinicians have complained of not being able to carry out efficacy studies. Patient groups in clinical practice are too heterogeneous to be matched according to standards of double-blind design. Heterogeneity is caused by the complex way the human system functions: you cannot compare humans subjects on the basiS of their blood pressure only. Humans change in the form of patterns. In hom0eopathy this is common knowledge (e.g. Calcarea evolves to Sulphur). Pattern recognition is a concept used in linguistics and biology, but no adequate research methodology has so far existed for this system of pattern recognition. The 'timeserial design' has been developed in behavioural science (Hersen and Barlow, 1984) and was used recently for assessment of rehabilitation programmes after stroke (Wagenaar, 1990). It consists of alternating 'treatment' and 'no-treatment' periods. The 'no-treatment' condition is called the 'baseline'. In this period only measurements (questionnaires, oral medical history, clinical examination, biochemical parameters etc.) are taken. Subsequently treatment is given, and the measurements are repeated. The resulting trends in baseline and treatment periods are compared by linear and non-linear regression and analysis of the residue. A 'multiple baseline design' is used if there are several baseline and treatment periods, and in this case the scores on the questionnaires are based on visual analogue scales for ease of recording and data-processing. The problems with this method concern internal and external validity. As far as I know the 'time-serial design' is the only model to measure synchronous change in many parameters, i.e., changes of patterns. The model forms the basis of a current proposal for the Ministry of Health in the Netherlands to evaluate the efficacy of hom~eopathy in the treatment of AIDS.
Isothermal microcalorimetric analysis of homeopathic dilutions C. CABANER, M. BASTIDE Laboratory of Immunology, Faculty of Pharmacy, University of Montpellier, France At the 5th GIRI Meeting in Paris 1991, C.
British Homceopathic Journal
Dragan described differences between homoeopathic dilutions and solvent in a report entitled 'Composite structure of homoeopathic solutions of Belladonna as revealed by calorimetric measurements'. Using a very sophisticated instrument, namely an isothermal microcalorimetric apparatus, in the Laboratory of Pharmaceutical Physics (under the direction of Professor J. C. Sari) in Marseille, France, we tried to confirm these differences. The microcalorimetric assays were performed at 37~ using an LKB Bioactivity Monitor 2277 able to record heat flows lower than 1 mW. The heat of the reaction was measured upon mixing two solutions by constant reactant flow in the Y mixing junction. The following preparations were compared: non-succussed solvent to succussed solvent (4c) and homoeopathic dilutions to the corresponding succussed solvent. All assays were performed in water/ethanol/glycerol (WEG) in the following composition: 237 ml of sterile distilled water for injections; 73 ml of 95 % ethanol; 100 ml of glycerol. The solutions were prepared by serial one hundred-fold dilutions in W E G being agitated on an Agitelec instrument operating at 540-560 succussions/min for 5 min (vertical succussion amplitude: 25 mm). The following homoeopathic dilutions of NaC1 and BSA (Bovine Serum Albumin) were prepared: 4c, 9c, 15c,and 30c, corresponding to 10 6M, 10 16M,10 2SM and 10 58M for NaC1, and 10-9M, 10-19M, 10-3tM and 10 61M for BSA. All the tubes were stored tightly closed at +4~ until use. Preparation of the dilutions was standardized so that all the tubes remained open for same period of time to avoid variations due to evaporation of alcohol. In preliminary experiments, we verified that two measurements of the heat of reaction for the NaC1 15c/WEG 15c gave the same value. The value for non-succussed WEG/succussed (4c) WEG was +3.8 mW (endothermic reaction). All other mixtures gave an exothermic reaction. The results expressed as the mean of four or three preparations were as follows C1Na 4c/WEG 4c BSA 4c/WEG 4c C1Na 9c/WEG 9c BSA 9c/WEG 9c C1Na 15c/WEG 15c
- 1.2 mW + 14.34 ; 5.53 mW _+ 7.47 - 14.66 mW + .03 - 12.5 mW _+ 0.866 - 12.75 mW + 1.79
Volume 82, Number 2, April 1993
BSA 15c/WEG 15c - 14.00 mW + 3 BSA 30c/WEG 30c - 34.32 mW + 19.44 Given the relatively high standard error of mean for certain pairs, it is difficult to interpret the results. The technique appears to be highly sensitive to minor variations in the preparation of the individual samples. More rigorous standardization is therefore necessary. It has been found that a minor difference in the alcohol in the tube can lead to differences of several orders of magnitude of mW. It is important to note that in experiments carried out on dilutions prepared extemporaneously in W E G or different solvents (water, 20% ethanol, 60% ethanol), the heat of reaction was zero.
Effects on highly diluted [32-adrenergic agonists on isolated guinea pig trachea E. CALLENS, H. D E B I A N E , M. C. SANTAIS, E RUFF Laboratory of Respiratory Physiopathology, Faculty of Medicine, 45 Rue des Saint-P6res, 75270 Paris, France Recent studies have described pharmacological effects of highly diluted agonists on in vitro experimental models (heart, duodenum). We have studied different bronchodilatory compounds used clinically for the treatment of asthma on an airway smooth muscle. The concentration necessary to inhibit 50% of the contraction of guinea pigs trachea induced by carbacol (ECs0) varied from 10 5M to 10-SM depending on the [32-adrenergic compounds tested. Tulobuterol and especially its metabolites (3 OH- and 4 0 H - t u l o b u t e r o l ) were found to be the most potent (Ruff E, et al. Beta-2 Adrenergic Response to Tulobuterol in Airway Smooth Muscle, Vascular Smooth Muscle and Adrenergic Nerves. J Pharmacol Exp Ther 1988; 244:173-80). Similar results have been obtained with human bronchi (ECs0 10 6M - 4x10 l~ The aim of the present study was to determine the highest dilution able to cause relaxation of guinea pig trachea basal tone for each 132-adrenergic agonist. Serial 10-fold diluted and agitated solutions (vortex) of isoproterenol and 40H-tulobuterol or solvent (isotonic saline solution or diluted Krebs-Hensleit buffer) were tested on isolated tracheal rings in Krebs-Hensleit buffer mounted on an isometric myograph (2g). The maximal relaxation obtained with 10-3theophylline was considered to be the
123 EC100. On this model, high dilutions of [3-adrenergic agonists at less than 10 2~ produced detectable relaxation of basal tone (isoproterenol: 10-25M; salbutamol: 10-25M; tulobuterol: 10 3~ and 4OH-tulobuterol 10 36M). The ECs0 were 10 19M isoproterenol, 10 24M salbutamol, 10-2SM tulobuterol and 10-26M 4 OH-tulobuterol. No relaxation was detected with diluted/agitated solvent. We observed some important variation in tracheal activity depending on the experiments. Furthermore, the pharmacological effect was not impaired by heating (90~ 20 min). We conclude that highly diluted/agitated dilutions of [32-adrenergic agonists can stimulate specific receptors. The experiments have to be repeated to confirm these results, to define their limits and the conditions which could modify the results. The mechanism of action of such high dilutions also needs to be elucidated.
Protective effect of very low concentrations of heavy metals (cadmium and cisplatin) against cytotoxic doses of these metals on renal tubular cell cultures A. DELBANCUT, 1 R DORFMAN, 2 J. CAMBAR I 1) Group of Studies of Renal Physiology and Physiopathology, University of Bordeaux, France; 2) Dolisos Laboratories, 71 Rue Beaubourg, Paris, France Cadmium, an important pollutant, and cisplatin, a widely used cytostatic drug, have strong toxic effects on renal tubules. Many agents have been proposed to reduce this renal toxicity, including chelating agents or sulfhydryl compounds. High dilutions of toxic heavy metals have been shown to reduce the lethal effect of the same metals in mice and increase metal elimination in rats. In the present study, we studied the effect of very low concentrations (or high dilutions) of cadmium or cisplatin on the cytotoxicity of these metals tested on a proximal tubular cultured cell line (LLCPK1). Cell viability was assessed by an automatic procedure using neutral red. The potential protective effect of pretreatment with succussed high dilutions ranging from 10 14 to 10 4~ of cadmium or cisplatin on the cytotoxic effect of a 24-h treatment with 2 - 5 x 10- 5 M of cadmium or I - 10 x 10-SM of cisplatin was studied. The cell culture control was pretreated with succussed dilutions of culture media followed by the same toxic treatment as the test cells.
Time-serial design for clinical trials M. BRANDS Amsterdam, The Netherlands At the last GIRI conference in Paris, an urgent call for clinical trial models was made. In recent years, many clinicians have complained of not being able to carry out efficacy studies. Patient groups in clinical practice are too heterogeneous to be matched according to standards of double-blind design. Heterogeneity is caused by the complex way the human system functions: you cannot compare humans subjects on the basiS of their blood pressure only. Humans change in the form of patterns. In hom0eopathy this is common knowledge (e.g. Calcarea evolves to Sulphur). Pattern recognition is a concept used in linguistics and biology, but no adequate research methodology has so far existed for this system of pattern recognition. The 'timeserial design' has been developed in behavioural science (Hersen and Barlow, 1984) and was used recently for assessment of rehabilitation programmes after stroke (Wagenaar, 1990). It consists of alternating 'treatment' and 'no-treatment' periods. The 'no-treatment' condition is called the 'baseline'. In this period only measurements (questionnaires, oral medical history, clinical examination, biochemical parameters etc.) are taken. Subsequently treatment is given, and the measurements are repeated. The resulting trends in baseline and treatment periods are compared by linear and non-linear regression and analysis of the residue. A 'multiple baseline design' is used if there are several baseline and treatment periods, and in this case the scores on the questionnaires are based on visual analogue scales for ease of recording and data-processing. The problems with this method concern internal and external validity. As far as I know the 'time-serial design' is the only model to measure synchronous change in many parameters, i.e., changes of patterns. The model forms the basis of a current proposal for the Ministry of Health in the Netherlands to evaluate the efficacy of hom~eopathy in the treatment of AIDS.
Isothermal microcalorimetric analysis of homeopathic dilutions C. CABANER, M. BASTIDE Laboratory of Immunology, Faculty of Pharmacy, University of Montpellier, France At the 5th GIRI Meeting in Paris 1991, C.
British Homceopathic Journal
Dragan described differences between homoeopathic dilutions and solvent in a report entitled 'Composite structure of homoeopathic solutions of Belladonna as revealed by calorimetric measurements'. Using a very sophisticated instrument, namely an isothermal microcalorimetric apparatus, in the Laboratory of Pharmaceutical Physics (under the direction of Professor J. C. Sari) in Marseille, France, we tried to confirm these differences. The microcalorimetric assays were performed at 37~ using an LKB Bioactivity Monitor 2277 able to record heat flows lower than 1 mW. The heat of the reaction was measured upon mixing two solutions by constant reactant flow in the Y mixing junction. The following preparations were compared: non-succussed solvent to succussed solvent (4c) and homoeopathic dilutions to the corresponding succussed solvent. All assays were performed in water/ethanol/glycerol (WEG) in the following composition: 237 ml of sterile distilled water for injections; 73 ml of 95 % ethanol; 100 ml of glycerol. The solutions were prepared by serial one hundred-fold dilutions in W E G being agitated on an Agitelec instrument operating at 540-560 succussions/min for 5 min (vertical succussion amplitude: 25 mm). The following homoeopathic dilutions of NaC1 and BSA (Bovine Serum Albumin) were prepared: 4c, 9c, 15c,and 30c, corresponding to 10 6M, 10 16M,10 2SM and 10 58M for NaC1, and 10-9M, 10-19M, 10-3tM and 10 61M for BSA. All the tubes were stored tightly closed at +4~ until use. Preparation of the dilutions was standardized so that all the tubes remained open for same period of time to avoid variations due to evaporation of alcohol. In preliminary experiments, we verified that two measurements of the heat of reaction for the NaC1 15c/WEG 15c gave the same value. The value for non-succussed WEG/succussed (4c) WEG was +3.8 mW (endothermic reaction). All other mixtures gave an exothermic reaction. The results expressed as the mean of four or three preparations were as follows C1Na 4c/WEG 4c BSA 4c/WEG 4c C1Na 9c/WEG 9c BSA 9c/WEG 9c C1Na 15c/WEG 15c
- 1.2 mW + 14.34 ; 5.53 mW _+ 7.47 - 14.66 mW + .03 - 12.5 mW _+ 0.866 - 12.75 mW + 1.79
Volume 82, Number 2, April 1993
BSA 15c/WEG 15c - 14.00 mW + 3 BSA 30c/WEG 30c - 34.32 mW + 19.44 Given the relatively high standard error of mean for certain pairs, it is difficult to interpret the results. The technique appears to be highly sensitive to minor variations in the preparation of the individual samples. More rigorous standardization is therefore necessary. It has been found that a minor difference in the alcohol in the tube can lead to differences of several orders of magnitude of mW. It is important to note that in experiments carried out on dilutions prepared extemporaneously in W E G or different solvents (water, 20% ethanol, 60% ethanol), the heat of reaction was zero.
Effects on highly diluted [32-adrenergic agonists on isolated guinea pig trachea E. CALLENS, H. D E B I A N E , M. C. SANTAIS, E RUFF Laboratory of Respiratory Physiopathology, Faculty of Medicine, 45 Rue des Saint-P6res, 75270 Paris, France Recent studies have described pharmacological effects of highly diluted agonists on in vitro experimental models (heart, duodenum). We have studied different bronchodilatory compounds used clinically for the treatment of asthma on an airway smooth muscle. The concentration necessary to inhibit 50% of the contraction of guinea pigs trachea induced by carbacol (ECs0) varied from 10 5M to 10-SM depending on the [32-adrenergic compounds tested. Tulobuterol and especially its metabolites (3 OH- and 4 0 H - t u l o b u t e r o l ) were found to be the most potent (Ruff E, et al. Beta-2 Adrenergic Response to Tulobuterol in Airway Smooth Muscle, Vascular Smooth Muscle and Adrenergic Nerves. J Pharmacol Exp Ther 1988; 244:173-80). Similar results have been obtained with human bronchi (ECs0 10 6M - 4x10 l~ The aim of the present study was to determine the highest dilution able to cause relaxation of guinea pig trachea basal tone for each 132-adrenergic agonist. Serial 10-fold diluted and agitated solutions (vortex) of isoproterenol and 40H-tulobuterol or solvent (isotonic saline solution or diluted Krebs-Hensleit buffer) were tested on isolated tracheal rings in Krebs-Hensleit buffer mounted on an isometric myograph (2g). The maximal relaxation obtained with 10-3theophylline was considered to be the
123 EC100. On this model, high dilutions of [3-adrenergic agonists at less than 10 2~ produced detectable relaxation of basal tone (isoproterenol: 10-25M; salbutamol: 10-25M; tulobuterol: 10 3~ and 4OH-tulobuterol 10 36M). The ECs0 were 10 19M isoproterenol, 10 24M salbutamol, 10-2SM tulobuterol and 10-26M 4 OH-tulobuterol. No relaxation was detected with diluted/agitated solvent. We observed some important variation in tracheal activity depending on the experiments. Furthermore, the pharmacological effect was not impaired by heating (90~ 20 min). We conclude that highly diluted/agitated dilutions of [32-adrenergic agonists can stimulate specific receptors. The experiments have to be repeated to confirm these results, to define their limits and the conditions which could modify the results. The mechanism of action of such high dilutions also needs to be elucidated.
Protective effect of very low concentrations of heavy metals (cadmium and cisplatin) against cytotoxic doses of these metals on renal tubular cell cultures A. DELBANCUT, 1 R DORFMAN, 2 J. CAMBAR I 1) Group of Studies of Renal Physiology and Physiopathology, University of Bordeaux, France; 2) Dolisos Laboratories, 71 Rue Beaubourg, Paris, France Cadmium, an important pollutant, and cisplatin, a widely used cytostatic drug, have strong toxic effects on renal tubules. Many agents have been proposed to reduce this renal toxicity, including chelating agents or sulfhydryl compounds. High dilutions of toxic heavy metals have been shown to reduce the lethal effect of the same metals in mice and increase metal elimination in rats. In the present study, we studied the effect of very low concentrations (or high dilutions) of cadmium or cisplatin on the cytotoxicity of these metals tested on a proximal tubular cultured cell line (LLCPK1). Cell viability was assessed by an automatic procedure using neutral red. The potential protective effect of pretreatment with succussed high dilutions ranging from 10 14 to 10 4~ of cadmium or cisplatin on the cytotoxic effect of a 24-h treatment with 2 - 5 x 10- 5 M of cadmium or I - 10 x 10-SM of cisplatin was studied. The cell culture control was pretreated with succussed dilutions of culture media followed by the same toxic treatment as the test cells.