British Homeopathic Journal (2001) 90, 198203 2001 Nature Publishing Group All rights reserved 00070785/01 $15.

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ORIGINAL PAPER

Very high dilutions of dexamethasone inhibit its pharmacological effects in vivo

LV Bonamin1*, KS Martinho1, AL Nina1, F Caviglia1 and RGW Do Rio2
Faculty of Veterinary Medicine, University of Santo Amaro, Sa o Paulo, Brazil; and 2Department of Pharmacology, Institute of Biomedical Sciences, University of Sa o Paulo, Brazil
1

We evaluated the interaction of dexamethasone 10717 and 10733 M (equivalent to 7cH and 15cH) with dexamethasone in pharmacological concentrations, using as experimental models: acute inammation induced by carrageenan, Ehrlich ascitic tumour, and migration of tumour inltrating leukocytes (TIL). Male adult BALB=c mice (n 7 per group) were used in all experiments. Carrageenan (1%) was injected into the footpad for oedema evaluation and into the peritoneal cavity (i.p.), for differential counting of inammatory cells. Ehrlich ascitic tumour cells (107 viable cells=ml) were injected i.p. and tumour cells were counted after 6 days, by the Trypan blue exclusion method. The differential TIL was counted using smears stained by hematoxylin eosin. Treatments were made immediately after carrageenan inoculation or once a day, during Ehrlich tumour development, until the animals were killed. Animals were treated with the following preparations: (1) phosphate buffer saline (PBS) solution; (2) dexamethasone (0.5 mg=kg for inammation model or 4 mg=kg for tumour model) mixed with dexamethasone 7cH or 15cH; (3) dexamethasone (same doses) mixed in PBS. Homeopathic dexamethasone partially blocked the anti-inammatory effect of pharmacological dexamethasone with regard to paw oedema (two-way ANOVA, P  0.0008) and polymorphonuclear cell migration (x2, P 0.0001). No important differences were observed between experimental and control groups, in relation to Ehrlich tumour cells viability or count, or bodyweight, but potentised dexamethasone restored control levels of TIL viability, compared to mice treated with pharmacological doses of dexamethasone (x2, P  0.001). The results demonstrate that a potentised substance may change its own pharmacological effects and suggest that ultradilutions effects act mostly on host response. British Homeopathic Journal (2001) 90, 198203. Keywords: animal model; dexamethasone; ultramolecular dilutions; carrageenan; Ehrlich tumour; leukocyte migration

Introduction
In recent years, research into ultramolecular dilutions and homeopathy has been a topic of interest in various countries and universities. There is a consensus among researchers with regard to the difculties of establishing a model in which it is possible to obtain reproducible results. Clinical and experimental data
*Correspondence: LV Bonamin, Laborato rio de Patologia Veterina ria, Faculdade de Medicina Veterina ria, Universidade de Santo Amaro, Rua Prof. Ene as de Siqueira Neto, 340. 04829-300, Sa o Paulo, Brasil. E-mail: Leoni@sti.com.br or patovet@unisa.br Received 18 October 2000; revised 9 January 2001; accepted 28 March 2001

obtained in studies about the effect of homeopathic preparations in inammatory conditions present a considerable degree of reproducibility.1 6 A signicant number of these studies have been carried out using the isopathy principle, meaning that a substance or an aetiological agent is prepared in a potentised form and used to treat or minimise the disease induced by the same agent.7 11 The use of isotherapy or nosodes is common in human and veterinary homeopathic clinical practice. In this context, some experimental studies have been made by using nosodes from infectious agents;12 14 in vivo and in vitro experimental studies have indicated efcacy in suppressing undesirable and toxic effects of several substances by their potentised forms.15 17,22,23

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These results stimulated further research with the aim of a better understanding of the mechanisms and logic of this type of therapy. The present study contributes to this approach using dexamethasone (Decadron1) as a model of interaction between potentised and pharmacological substances. Dexamethasone was chosen because of its well known pharmacological effects, which facilitate its use in different experimental models.

tion (4 C). The dexamethasone and PBS used in each experiment were always from the same batch.

Inammation induction

Materials and methods

Animals

Acute inammatory oedema was induced by administration of 0.04 ml of 1% carrageenan kappa (SIGMA), prepared in sterile distilled water, into the left footpad of animals and the oedema was measured during 5 h. The paw volume was calculated from width (a) and thickness (b) measures, taken with a micrometer 1:100 mm and calculated by the formula:18 Volume p=6 a2 b Paw measures were taken twice, for each experimental time point. Inammatory cell migration was induced by the administration of 0.1 ml of 1% carrageenan kappa (SIGMA, St Louis, USA), also prepared in sterile distilled water, into the peritoneal cavity. After 4 h (corresponding to the maximum cell migration time), 3 ml of PBS were injected into the cavity to obtain a washing leukocyte suspension. Smears were prepared from each animal, xed in absolute methanol (MERCK, Darmstadt, Germany) for 15 min at 4 C and stained with hematoxylin eosin for differential counting of leukocytes: monocytes, macrophages, polymorphonuclear cells (PMN) and lymphocytes, based on morphological parameters. Five hundred inammatory cells per slide were counted. All experiments were performed during the afternoon.

Male adult BALB=c mice (25 35 g weight) bred at o Paulo were used, living in the University of Sa conventional plastic cages (maximum of 20 mice per cage), controlled temperature (22 3 C) and light cycle (12 h dark per day, lights on at 6:30 am), and receiving food and water ad libitum. All in vivo procedures were performed in the autumn=winter period. Animals were randomly distributed into groups.
Drug preparation

Dexamethasone. Decadron 4 mg=ml1 (dexamethasone disodium phosphate) was used as a positive control. The doses were adjusted for mice by diluting the drug in PBS (phosphate buffered saline) or in potentised dexamethasone (see below). PBS. The buffer was prepared in distilled water from SIGMA tablets. The PBS used in different groups was always from the same tablet. Potentised dexamethasone. Decadron1 (7cH) was six-fold serially diluted (1:100) in distilled water and a seventh nal dilution was made in PBS. For each passage, the sample was succussed. The nal solution was 10717 M. Dexamethasone (15cH) was 14-fold serially diluted in the same manner as 7cH. The nal dilution was made in PBS. The fteenth nal concentration was 10733 M. The use of buffer in the last dilution was necessary to obtain an isotonic solution, for use by subcutaneous route. All potentised dexamethasone was obtained by serial dilutions of 1:100 using 10 ml of distilled water or PBS as solvent and 0.1 ml of dexamethasone as active principle. Dilutions were made in a 20 ml amber ask, whose top was smaller than the bottom. For each passage, the solution was manually agitated 100 times, by hitting the ask rhythmically on an elastic surface. This procedure was described by Samuel Hahnemann and is recommended by the Brazilian Homeopathic Pharmacopoeia. The dilutions were made on the day of administration or 1 day before and maintained under refrigera-

Ehrlich tumour development

The Ehrlich tumour cell line is maintained in our laboratory in ascitic form. For all experiments, 3 4 ml of ascitic uid were harvested from mice inoculated 7 10 days before and cells were counted in a modied Neubauers chamber, using leukocyte compartments. The cell concentration was adjusted to 107 cells=ml in PBS. The Trypan blue exclusion method was used to determine cell viability, which was never less than 90%. The tumour was inoculated into the peritoneal cavity (0.1 ml per mouse; 106 tumour cells). Animals were weighed daily, for 6 days, to evaluate the ascites formation. After this period, animals were killed, 3 ml of PBS were injected into the peritoneal cavity and a sample of suspended cells was harvested. Total, living and dead tumour cells were counted, using the method as described earlier. Smears were prepared, xed in absolute methanol (MERCK, Darmstadt, Germany) for 15 min at 4 C and stained with hematoxylin eosin for differential counting of tumour inltrating leukocytes (TIL), as macrophages, lymphocytes, PMN and degenerating cells of typical morphology (reduction of size, irregular shaping, fragmentation of nucleus and increased eosin-staining). Five hundred
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leukocytes per slide were counted. Tumour cells were not considered.
Animal treatments

 Group III (G3): treated with dexamethasone, diluted in PBS (positive control).

Statistical analysis

For inammation experiments, treatment was given immediately after carrageenan inoculation. For Ehrlich tumour experiments, treatments were made daily, between 11:00 and 14:00 h, until the day the animals were killed. All treatments were given subcutaneously, in a volume equal to 0.1 ml=10 g of weight, in a non-blinded manner. The doses were 0.5 mg=kg as anti-inammatory and 4.0 mg=kg as immunosuppressive. For all experiments, animals were divided into three groups, containing seven or eight mice each:  Group I (G1): treated with PBS (negative control)  Group II (G2): treated with dexamethasone, diluted in potentised dexamethasone (7cH or 15cH, depending on the experiment), which resulted in a mixture of the homeopathic and pharmacological form of drug

The statistical test chosen for each parameter was based on the Bartlett test, to check the Gaussian distribution of samples. A parametric or non-parametric test was applied, according to whether the distribution was normal or not, respectively. To evaluate the inammatory oedema and the weight of tumour bearing mice, two-way ANOVA was used. The Kruskal Wallis test was used to evaluate tumour-cell counts. To evaluate the differential leukocyte counting, the w2 test was employed. For all parameters, one has xed values of P  0.05.
Animal welfare

All experiments were conducted according to article 3 of The Declaration of Animal Rights, of 27 January 1978.19

Results
14 12 paw edema (mm3) 10 8 6 4 2 0 1 2 3 hours B 18 16 paw edema (mm3) 14 12 10 8 6 4 2 0 1 2 3 4 5 6 hours * * * * * GI GII GIII 4 5 6 A GI GII GIII

The inammatory model

The anti-oedema effect of dexamethasone (0.5 mg=kg) seen in G3 was abolished when it was prepared as a mixture with potentised dexamethasone (G2), only in 15cH (Figure 1A and B). The PMN cell migration was inhibited in G3, as compared to the control group (G1), and this inhibition was also blocked in G2, for both 7cH and 15cH. Similarly the macrophage predominance seen in G3 was not observed in G2 (Table 1; Figure 2A and B).
The Ehrlich tumour model

Tumour cell growth and viability (Figure 3A and B), and the body weight of tumour bearing mice (Figure 4A and B) were not affected by any form of dexamethasone treatment, except in the last day before the animals were killed (Figure 4A and B), in which mice treated with pharmacological dexamethasone (G3) showed slight loss of weight, probably related to inhibition of ascitic exudate formation by chronic corticoid treatment. On the other hand, the presence of potentised dexamethasone in G2 restored the control levels of leukocyte degeneration, which was also seen in the positive control (G3) (Figure 5A and B). The effect of 15cH treatment was more evident than that obtained with 7cH (Table 2; Figures 4A and 5A).

Figure 1 Evolution of paw oedema volume (mm3) after inoculation with 0.04 ml of 1% kappa carrageenan. The paw volume was measured by micrometry, during 5 h with 1 h of interval between measures. G1 animals treated with PBS control; G2 animals treated with dexamethasone (0.5 mg=kg) diluted into dexamethasone 7cH (A) or 15cH (B); G3 animals treated with dexamethasone (0.5 mg=kg) diluted into PBS. All treatments were made s.c., immediately after carrageenan inoculation into the footpad. Results are expressed by mean s.d. Two-way ANOVA, *P 0.0008. British Homeopathic Journal

Discussion
The results show that dexamethasone 7cH and 15cH modify the inhibitory pharmacological effects on the exudative or cellular responses against carrageenan and Ehrlich tumour. The effects of 15cH were

Dexamethasone in pharmacological and ultramolecular dilution LV Bonamin et al Table 1 (0.1 ml) Differential counting (mean s.d.) of leukocytes in the peritoneal washing 4 h after i.p. inoculation of 1% kappa carrageenan 7cH Groups PBS only (G1) Dexamethasone and potentised dexamethasone (G2) Pharmacological dexamethasone only (G3) PMN 358.28 41.31 333.85 63.90 308.28 45.51* MACRO 78.28 34.26 106.85 51.20 142.14 35.54** LYMPH 63.42 21.00 59.28 27.85 49.57 29.44 PMN 407.00 27.95 419.00 17.88*** 373.00 14.28** 15cH MACRO 56.57 16.88 39.00 18.31 79.14 43.36* LYMPH 36.40 13.48 41.70 7.18 47.70 12.80

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Cells were counted by smears stained by hematoxylin-eosin. G1 treated with PBS (control); G2 treated with dexamethasone (0.5 mg=kg) in dexamethasone 7cH (A) or 15cH (B); G3 treated with dexamethasone (0.5 mg=kg) diluted in PBS. PMN polymorphonuclear cells; MACRO macrophages; LYMPH lymphocytes. All treatments were made s.c., immediately after carrageenan inoculation into the peritoneal cavity. *ANOVA, P  0.05 in relation to the other groups; **ANOVA, P  0.02 in relation to the other groups; ***ANOVA, P  0.02 in relation to G3.

Figure 2 Differential counting, expressed in percentage, of leukocytes present in the peritoneal washing 4 h after i.p. inoculation of 1% kappa carrageenan (0.1 ml). Cells were counted by smears stained by hematoxylin-eosin. G1 animals treated with PBS (control); G2 animals treated with dexamethasone (0.5 mg=kg) diluted into dexamethasone 7cH (A) or 15cH (B); G3 animals treated with dexamethasone (0.5 mg=kg) diluted into PBS. LYMPH lymphocytes; MACRO macrophages; PMN polymorphonuclear cells. All treatments were made s.c., immediately after carrageenan inoculation into the peritoneal cavity. w2 test, *P 0.0001 in relation to G3.

Figure 3 Live and dead Ehrlich tumour cells counting (cell=ml106), made at day 6 after i.p. inoculation of 106 tumour cells. Cells were counted by using Trypans blue staining method. G1 animals treated with PBS (control); G2 animals treated with dexamethasone (4.0 mg=kg) diluted into dexamethasone 7cH (A) or 15cH (B); G3 animals treated with dexamethasone (4.0 mg=kg) diluted into PBS. All treatments were made daily, s.c. Kruskal Wallis test.

stronger than 7cH. The evaluation of ascitic formation by bodyweight is based on the fact that Ehrlich tumour induces lipolysis,20 so the bodyweight gain in tumour bearing mice can be associated with the formation of ascites.

The present observation contradicts the results that would be expected from traditional pharmacology, since animals from the G2 and G3 groups, which received very similar doses of dexamethasone in the same experimental conditions, presented different behaviours and these were statistically signicant. According to the traditional point of view, the
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Figure 4 Bodyweight evolution (g) after i.p. inoculation of 106 Ehrlich tumour cells. G1 animals treated with PBS (control); G2 animals treated with dexamethasone (4.0 mg=kg) diluted into dexamethasone 7cH (A) or 15cH (B); G3 animals treated with dexamethasone (4.0 mg=kg) diluted into PBS. All treatments were made daily, s.c. Two-way ANOVA, *P  0.04.

Figure 5 Differential counting, expressed in percentage, of leukocytes present in the peritoneal washing 6 day after i.p. inoculation of 106 Ehrlich tumour cells. Cells were counted by smears stained by hematoxylin eosin. G1 animals treated with PBS (control); G2 animals treated with dexamethasone (4.0 mg=kg) diluted into dexamethasone 7cH (A) or 15cH (B); G3 animals treated with dexamethasone (4.0 mg=kg) diluted into PBS. DEG degenerated cells; LYMPH lymphocytes; MACRO macrophages; PMN polymorphonuclear cells. All treatments were made daily, s.c. w2 test, *P 0.0001 in relation to G1, #P  0.0012 in relation to G3.

presence of 7cH or 15cH potencies in G2 should not interfere with the effects of dexamethasone, since from the chemical view point they consisted of PBS only, or at least of minimal concentrations of dexamethasone. Other experimental manipulations developed in our laboratory show quite different and independent behaviours of the 7cH and 15cH homeopathic preparations of dexamethasone used alone. In those studies, poten-

tised dexamethasone produced an increase of lymphocyte migration into the tumour site (unpublished). The experimental design we used is quite important for the comprehension of the great diversity of biological effects that can be obtained from the ultradilutions. The use of both forms of dexamethasone (potentised and pharmacological) mixed into a single preparation has not been previously described in the literature, although numerous papers have demon-

Table 2 Differential counting, (mean s.d.) of leukocytes present in the peritoneal washing 6 days after i.p. inoculation of 106 Ehrlich tumour cells 7cH Groups PMN MACRO LYMPH DEGEN PMN MACRO 15cH LYMPH DEGEN

PBS only (G1) 3.33 1.63 17.83 4.21 10.83 5.49 4.83 3.06 5.80 6.90 18.40 10.73 8.00 4.00 18.20 18.80 Dexamethasone and 4.83 8.25 23.66 12.22 25.16 5.84 29.83 11.78 4.00 5.61 33.00 15.89 24.60 16.24 28.40 12.74 potentised dexamethasone (G2) Pharmacological 1.71 0.75 26.28 7.08 20.28 8.48 50.28 5.28* 1.28 0.75 12.14 5.84 12.71 6.37 44.85 20.21* dexamethasone only (G3) Cells were counted by smears stained by hematoxylin-eosin. G2 treated with PBS (control); G2 treated with dexamethasone (4.0 mg=kg) diluted into dexamethasone 7cH (A) or 15cH (B); G3 treated with dexamethasone (4.0 mg=kg) diluted into PBS. PMN polymorphonuclear cells; MACRO macrophages; LYMPH lymphocytes; DEGEN degenerated cells. All treatments were made via s.c. *ANOVA, P  0.05 in relation to the other groups. British Homeopathic Journal

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strated the effects of isotherapy by treating animals or cells.7 10 Another interesting result is the absence of interference of dexamethasone (in both the G2 and G3 groups) upon Ehrlich tumour cell count, whereas it modied the cellular response against the tumour. The absence of pharmacological effects of dexamethasone on tumour cell growth indicates specicity of the isopathic phenomenon related to the host response against aggressor factors. This specicity has already been proposed by other authors21,22 and appears to be linked with organic adaptation processes, stressing the general principle of homeopathy: it does not treat diseases but improves health mechanisms in sick organisms. In fact, as shown by Malarczyk, the use of potentised cytostatic substances induced an increase in the immune response of tumour bearing individuals although the toxic effect on tumour cells was maintained.22,23 Our data, taken together, suggest a possible interference (and even a predominance) of potentised preparations over the same drug in pharmacological concentrations. Also, on account of the good reproducibility of this model, the mixture of potentised and pharmacological forms of a substance could be proposed as a useful methodology to demonstrate the homeopathic phenomenon.

Acknowledgements
With thanks to Dr Bernard Poitevin for the suggestions at the beginning of our work; to Dr Madeleine Bastide sio Cunha de Carvalho for their enthusiasm and Dr. Alo in discussing ideas; to Dr Josmar S Arrais de Matos, Prorector for Research at UNISA, and Dr Godofredo C Genofre Netto, Research Director at UNISA for the ` support. Special thanks to Fundac a o de Amparo a Pesquisa do Estado de Sa o Paulo (FAPESP) for the nancial support (Grant n 97=13008-5).

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