very high dilutions of dexamethasone inhibit its pharmacology effect in vivo

8/12/2019 Very High Dilutions of Dexamethasone Inhibit Its Pharmacology Effect in Vivo

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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

1Faculty of Veterinary Medicine, University of Santo Amaro, Sao Paulo, Brazil; and 2Department of Pharmacology,Institute of Biomedical Sciences, University of Sao Paulo, Brazil

We evaluated the interaction of dexamethasone 10717 and 10733 M (equivalent to 7cHand 15cH) with dexamethasone in pharmacological concentrations, using as experi-

mental models: acute inflammation induced by carrageenan, Ehrlich ascitic tumour, andmigration of tumour infiltrating leukocytes (TIL). Male adult BALB=c mice (n7 pergroup) were used in all experiments. Carrageenan (1%) was injected into the footpad foroedema evaluation and into the peritoneal cavity (i.p.), for differential counting ofinflammatory 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. Thedifferential TIL was counted using smears stained by hematoxylin eosin. Treatmentswere made immediately after carrageenan inoculation or once a day, during Ehrlichtumour development, until the animals were killed. Animals were treated with thefollowing preparations: (1) phosphate buffer saline (PBS) solution; (2) dexamethasone(0.5 mg=kg for inflammation model or 4 mg=kg for tumour model) mixed with dexa-methasone 7cH or 15cH; (3) dexamethasone (same doses) mixed in PBS. Homeopathicdexamethasone partially blocked the anti-inflammatory effect of pharmacological dex-

amethasone with regard to paw oedema (two-way ANOVA, P 0.0008) and polymor-phonuclear cell migration (x2, P0.0001). No important differences were observedbetween experimental and control groups, in relation to Ehrlich tumour cells viabilityor count, or bodyweight, but potentised dexamethasone restored control levels of TILviability, compared to mice treated with pharmacological doses of dexamethasone (x2,P 0.001). The results demonstrate that a potentised substance may change its ownpharmacological effects and suggest that ultradilutions effects act mostly on hostresponse. British Homeopathic Journal(2001) 90, 198203.

Keywords:animal model; dexamethasone; ultramolecular dilutions; carrageenan;Ehrlich tumour; leukocyte migration

Introduction

In recent years, research into ultramolecular dilutionsand homeopathy has been a topic of interest in variouscountries and universities. There is a consensusamong researchers with regard to the difficulties ofestablishing a model in which it is possible to obtainreproducible results. Clinical and experimental data

obtained in studies about the effect of homeopathicpreparations in inflammatory conditions present aconsiderable degree of reproducibility.1 6

A significant number of these studies have beencarried out using the isopathy principle, meaning thata substance or an aetiological agent is prepared in apotentised form and used to treat or minimise thedisease induced by the same agent.711 The use ofisotherapy or nosodes is common in human andveterinary homeopathic clinical practice. In this con-text, some experimental studies have been made by

using nosodes from infectious agents;

1214

in vivoandin vitro experimental studies have indicated efficacyin suppressing undesirable and toxic effects of severalsubstances by their potentised forms.15 17,22,23

*Correspondence: LV Bonamin, Laboratorio de PatologiaVeterinaria, Faculdade de Medicina V eterinaria, Universidade de

Santo Amaro, Rua Prof. Eneas de Siqueira Neto, 340. 04829-300,Sao Paulo, Brasil.E-mail: [email protected] or [email protected] 18 October 2000; revised 9 January 2001;accepted 28 March 2001

British Homeopathic Journal (2001) 90, 1982032001 Nature Publishing Group All rights reserved 00070785/01 $15.00

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These results stimulated further research with theaim of a better understanding of the mechanisms andlogic of this type of therapy. The present studycontributes to this approach using dexamethasone(Decadron1) as a model of interaction betweenpotentised and pharmacological substances. Dexa-

methasone was chosen because of its well knownpharmacological effects, which facilitate its use indifferent experimental models.

Materials and methods

Animals

Male adult BALB=c mice (25 35 g weight) bred atthe University of Sao Paulo were used, living inconventional plastic cages (maximum of 20 miceper cage), controlled temperature (22 3C) andlight cycle (12 h dark per day, lights on at 6:30 am),and receiving food and waterad libitum. All in vivoprocedures were performed in the autumn=winterperiod. Animals were randomly distributed intogroups.

Drug preparation

Dexamethasone. Decadron 4 mg=ml1 (dexametha-sone disodium phosphate) was used as a positivecontrol. The doses were adjusted for mice by dilutingthe drug in PBS (phosphate buffered saline) or inpotentised dexamethasone (see below).

PBS. The buffer was prepared in distilled waterfrom SIGMA tablets. The PBS used in differentgroups was always from the same tablet.

Potentised dexamethasone. Decadron1 (7cH) wassix-fold serially diluted (1:100) in distilled water and aseventh final dilution was made in PBS. For eachpassage, the sample was succussed. The final solutionwas 10717 M. Dexamethasone (15cH) was 14-foldserially diluted in the same manner as 7cH. The

final dilution was made in PBS. The fifteenth finalconcentration was 10733 M. The use of buffer in thelast dilution was necessary to obtain an isotonicsolution, for use by subcutaneous route.

All potentised dexamethasone was obtained byserial dilutions of 1:100 using 10ml of distilledwater or PBS as solvent and 0.1 ml of dexamethasoneas active principle. Dilutions were made in a 20 mlamber flask, whose top was smaller than the bottom.For each passage, the solution was manually agitated100 times, by hitting the flask rhythmically on anelastic surface. This procedure was described by

Samuel Hahnemann and is recommended by theBrazilian Homeopathic Pharmacopoeia.The dilutions were made on the day of administra-

tion or 1 day before and maintained under refrigera-

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

Inflammation induction

Acute inflammatory oedema was induced by admin-

istration of 0.04 ml of 1% carrageenan kappa(SIGMA), prepared in sterile distilled water, intothe left footpad of animals and the oedema wasmeasured during 5 h. The paw volume was calculatedfrom width (a) and thickness (b) measures, takenwith a micrometer 1:100 mm and calculated by theformula:18

Volume p=6 a2 b

Paw measures were taken twice, for each experimen-tal time point.

Inflammatory cell migration was induced by the

administration of 0.1 ml of 1% carrageenan kappa(SIGMA, St Louis, USA), also prepared in steriledistilled 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 awashing leukocyte suspension. Smears were preparedfrom each animal, fixed in absolute methanol(MERCK, Darmstadt, Germany) for 15 min at 4Cand stained with hematoxylin eosin for differentialcounting of leukocytes: monocytes, macrophages,polymorphonuclear cells (PMN) and lymphocytes,based on morphological parameters. Five hundredinflammatory cells per slide were counted. All experi-

ments were performed during the afternoon.

Ehrlich tumour development

The Ehrlich tumour cell line is maintained in ourlaboratory in ascitic form. For all experiments, 3 4 ml of ascitic fluid were harvested from mice inocu-lated 7 10 days before and cells were counted ina modified Neubauers chamber, using leukocytecompartments. The cell concentration was adjustedto 107 cells=ml in PBS. The Trypan blue exclusionmethod 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). Animalswere weighed daily, for 6 days, to evaluate the ascitesformation. After this period, animals were killed, 3 mlof PBS were injected into the peritoneal cavity and asample of suspended cells was harvested. Total, livingand dead tumour cells were counted, using the methodas described earlier. Smears were prepared, fixed inabsolute methanol (MERCK, Darmstadt, Germany)for 15min at 4C and stained with hematoxylin eosin for differential counting of tumour infiltrating

leukocytes (TIL), as macrophages, lymphocytes, PMNand degenerating cells of typical morphology (reduc-tion of size, irregular shaping, fragmentation ofnucleus and increased eosin-staining). Five hundred

Dexamethasone in pharmacological and ultramolecular dilutionLV Bonamin et al

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leukocytes per slide were counted. Tumour cells werenot considered.

Animal treatments

For inflammation experiments, treatment was given

immediately after carrageenan inoculation. ForEhrlich tumour experiments, treatments were madedaily, between 11:00 and 14:00 h, until the day theanimals were killed. All treatments were given sub-cutaneously, in a volume equal to 0.1 ml=10 g of weight, in a non-blinded manner. The doses were0.5 mg=kg as anti-inflammatory and 4.0 mg=kg asimmunosuppressive.

For all experiments, animals were divided into threegroups, 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 amixture of the homeopathic and pharmacologicalform of drug

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

Statistical analysis

The statistical test chosen for each parameter was

based on the Bartlett test, to check the Gaussiandistribution of samples. A parametric or non-para-metric test was applied, according to whether thedistribution was normal or not, respectively. To eval-uate the inflammatory oedema and the weight oftumour bearing mice, two-way ANOVA was used.The Kruskal Wallis test was used to evaluatetumour-cell counts. To evaluate the differential leu-kocyte counting, the w2 test was employed. For allparameters, one has fixed values ofP 0.05.

Animal welfare

All experiments were conducted according to article 3of The Declaration of Animal Rights, of 27 January1978.19

Results

The inflammatory model

The anti-oedema effect of dexamethasone (0.5 mg=kg)seen in G3 was abolished when it was prepared as amixture with potentised dexamethasone (G2), only in15cH (Figure 1A and B). The PMN cell migration wasinhibited in G3, as compared to the control group

(G1), and this inhibition was also blocked in G2, forboth 7cH and 15cH. Similarly the macrophage pre-dominance seen in G3 was not observed in G2 (Table1; 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 (Figure4A and B) were not affected by any form of dexa-methasone treatment, except in the last day before theanimals were killed (Figure 4A and B), in which micetreated with pharmacological dexamethasone (G3)showed slight loss of weight, probably related toinhibition of ascitic exudate formation by chroniccorticoid treatment. On the other hand, the presenceof potentised dexamethasone in G2 restored the con-trol levels of leukocyte degeneration, which was alsoseen in the positive control (G3) (Figure 5A and B).The effect of 15cH treatment was more evident thanthat obtained with 7cH (Table 2; Figures 4A and 5A).

Discussion

The results show that dexamethasone 7cH and 15cHmodify the inhibitory pharmacological effects on theexudative or cellular responses against carrageenanand Ehrlich tumour. The effects of 15cH were

A

0

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4

6

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10

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14

1 2 3 4 5 6

hours

paw

edem

a(mm3)

GI

GII

GIII

B

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hours

paw

edema

(mm3)

GI

GII

GIII

*

**

* *

Figure 1 Evolution of paw oedema volume (mm3) after inocu-lation with 0.04 ml of 1% kappa carrageenan. The paw volumewas measured by micrometry, during 5 h with 1 h of intervalbetween measures. G1 animals treated with PBS control;G2 animals treated with dexamethasone (0.5mg=kg) dilutedinto dexamethasone 7cH (A) or 15cH (B); G3 animals treatedwith dexamethasone (0.5 mg=kg) diluted into PBS. All treatmentswere made s.c., immediately after carrageenan inoculation intothe footpad. Results are expressed by means.d. Two-wayANOVA, *P0.0008.


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stronger than 7cH. The evaluation of ascitic formation

by bodyweight is based on the fact that Ehrlichtumour induces lipolysis,20 so the bodyweight gainin tumour bearing mice can be associated with theformation of ascites.

The present observation contradicts the results thatwould be expected from traditional pharmacology,since animals from the G2 and G3 groups, which

received very similar doses of dexamethasone in thesame experimental conditions, presented differentbehaviours and these were statistically significant.According to the traditional point of view, the

Table 1 Differential counting (means.d.) of leukocytes in the peritoneal washing 4 h after i.p. inoculation of 1% kappa carrageenan(0.1ml)

7cH 15cH

Groups PMN MACRO LYMPH PMN MACRO LYMPH

PBS only (G1) 358.2841.31 78.2834.26 63.4221.00 407.0027.95 56.5716.88 36.4013.48Dexamethasone and potentised

dexamethasone (G2)

333.8563.90 106.8551.20 59.2827.85 419.0017.88*** 39.0018.31 41.707.18

Pharmacological dexamethasoneonly (G3)

308.2845.51* 142.1435.54** 49.5729.44 373.0014.28** 79.1443.36* 47.7012.80

Cells were counted by smears stained by hematoxylin-eosin. G1 treated with PBS (control); G2 treated with dexamethasone (0.5 mg=kg) indexamethasone 7cH (A) or 15cH (B); G3 treated with dexamethasone (0.5 mg=kg) diluted in PBS. PMNpolymorphonuclear cells;MACROmacrophages; LYMPH lymphocytes. All treatments were made s.c., immediately after carrageenan inoculation into theperitoneal cavity. *ANOVA, P0.05 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, ofleukocytes present in the peritoneal washing 4 h after i.p. inocu-lation of 1% kappa carrageenan (0.1 ml). Cells were counted bysmears stained by hematoxylin-eosin. G1 animals treated withPBS (control); G2 animals treated with dexamethasone(0.5mg=kg) diluted into dexamethasone 7cH (A) or 15cH (B);G3 animals treated with dexamethasone (0.5 mg=kg) dilutedinto PBS. LYMPH lymphocytes; MACROmacrophages;PMNpolymorphonuclear cells. All treatments were mades.c., immediately after carrageenan inoculation into the peri-toneal cavity. w2 test, *P0.0001 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 stainingmethod. G1 animals treated with PBS (control); G2 animalstreated with dexamethasone (4.0mg=kg) diluted into dexa-methasone 7cH (A) or 15cH (B); G3 animals treated withdexamethasone (4.0 mg=kg) diluted into PBS. All treatmentswere made daily, s.c. Kruskal Wallis test.


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presence of 7cH or 15cH potencies in G2 should notinterfere with the effects of dexamethasone, sincefrom the chemical view point they consisted ofPBS only, or at least of minimal concentrations ofdexamethasone.

Other experimental manipulations developed in ourlaboratory show quite different and independent beha-viours of the 7cH and 15cH homeopathic preparations

of dexamethasone used alone. In those studies, poten-

tised dexamethasone produced an increase of lympho-cyte migration into the tumour site (unpublished).

The experimental design we used is quite importantfor the comprehension of the great diversity of bio-logical effects that can be obtained from the ultra-dilutions. The use of both forms of dexamethasone(potentised and pharmacological) mixed into a singlepreparation has not been previously described in the

literature, although numerous papers have demon-

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 (4.0mg=kg) dilutedinto dexamethasone 7cH (A) or 15cH (B); G3 animals treatedwith dexamethasone (4.0 mg=kg) diluted into PBS. All treatmentswere made daily, s.c. Two-way ANOVA, *P0.04.

Figure 5 Differential counting, expressed in percentage, ofleukocytes present in the peritoneal washing 6 day after i.p.inoculation of 106 Ehrlich tumour cells. Cells were counted bysmears stained by hematoxylin eosin. G1 animals treatedwith PBS (control); G2 animals treated with dexamethasone(4.0mg=kg) diluted into dexamethasone 7cH (A) or 15cH (B);G3 animals treated with dexamethasone (4.0mg=kg) diluted

into PBS. DEGdegenerated cells; LYMPH lymphocytes;MACROmacrophages; PMNpolymorphonuclear cells. Alltreatments were made daily, s.c. w2 test, *P0.0001 in relationto G1, #P0.0012 in relation to G3.

Table 2 Differential counting, (mean s.d.) of leukocytes present in the peritoneal washing 6 days after i.p. inoculation of 10 6 Ehrlichtumour cells

7cH 15cH

Groups PMN MACRO LYMPH DEGEN PMN MACRO LYMPH DEGEN

PBS only (G1) 3.331.63 17.834.21 10.835.49 4.833.06 5.806.90 18.4010.73 8.004.00 18.2018.80Dexamethasone and

potentiseddexamethasone (G2)

4.838.25 23.6612.22 25.165.84 29.8311.78 4.005.61 33.0015.89 24.6016.24 28.4012.74

Pharmacologicaldexamethasone only (G3)

1.710.75 26.287.08 20.288.48 50.285.28* 1.280.75 12.145.84 12.716.37 44.8520.21*

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 polymorphonuclearcells; MACROmacrophages; LYMPH lymphocytes; DEGENdegenerated cells. All treatments were made via s.c. *ANOVA, P0.05 inrelation to the other groups.


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strated the effects of isotherapy by treating animals orcells.710

Another interesting result is the absence of inter-ference of dexamethasone (in both the G2 and G3groups) upon Ehrlich tumour cell count, whereas itmodified the cellular response against the tumour. The

absence of pharmacological effects of dexamethasoneon tumour cell growth indicates specificity of theisopathic phenomenon related to the host responseagainst aggressor factors. This specificity has alreadybeen proposed by other authors21,22 and appears to belinked with organic adaptation processes, stressing thegeneral principle of homeopathy: it does not treatdiseases but improves health mechanisms in sickorganisms. In fact, as shown by Malarczyk, the useof potentised cytostatic substances induced anincrease in the immune response of tumour bearingindividuals although the toxic effect on tumour cellswas maintained.22,23

Our data, taken together, suggest a possible inter-ference (and even a predominance) of potentisedpreparations over the same drug in pharmacologicalconcentrations. Also, on account of the good reprodu-cibility of this model, the mixture of potentised andpharmacological forms of a substance could be pro-posed as a useful methodology to demonstrate thehomeopathic phenomenon.

Acknowledgements

With thanks to Dr Bernard Poitevin for the suggestionsat the beginning of our work; to Dr Madeleine Bastideand Dr. Alosio Cunha de Carvalho for their enthusiasmin discussing ideas; to Dr Josmar S Arrais de Matos, Pro-rector for Research at UNISA, and Dr Godofredo CGenofre Netto, Research Director at UNISA for thesupport. Special thanks to Fundacao de Amparo aPesquisa do Estado de Sao Paulo (FAPESP) for thefinancial support (Grant n 97=13008-5).

References

1 Davenas E, Poitevin B, Benveniste J. Effect on mouse peri-toneal macrophages of orally administered very high dilutionsof Sillicea. Eur J Pharmacol1987; 135: 313319.

2 Sainte-Laudy J, Belon P. Analysis of immunosuppressiveactivity of serial dilutions of histamine on human basophilactivation by flow cytometry. Agents Actions1993; 38(Suppl):C245 C247.

3 Bildet J, Guyot M, Bonnini F, Grignon MF, Poitevin B,Quilichini R. Mise en evidence des effets des dilutionsdApis mellifica et dApium virus vis-a-vis de lerythemeprovoque par un rayonnement UV chez le cobaye. AnnPharmacol Fr1989; 47 : 24 32.

4 Reilly DT, Taylor MA, Beattle NGM, et al. Is evidence forhomeopathy reproducible? Lancet1994; 344: 1601 1606.

5 Oberbaum M, Weisman Z, Kakinkovich A, Bentwich Z.Healing chronic wounds performed on mouse ears usingsilica (Sio2) as a homeopathic remedy. In: Bastide M (ed).Signals and Images. Dordrecht: Kluwer Academic Publishers,1997, pp. 191 199.

6 Belon P, Cumps J, Ennis M, et al. Inhibition of humanbasophil degranulation by successive histamine dilutions:results of a European multi-centre trial. Inflamm Res 1999;48(Suppl 1): S17 S18.

7 Tisseyre H. Analyse et evaluation de la recherche fondamen-tale en homeopathie: proposition de criteres methodologiques.These Doctorat Pharmacie, UniversiteMontpellier I, 1996.

8 Cambar J, Desmouliere A, Cal JC,et al. Influence de ladmin-istration de dilutions infinitesimales de Mercurius Corrosivussur la mortalite induite par le chlorure mercurique chez lasouris. Bull Soc Pharmacol Bordeaux 1983; 122: 30 38.

9 Fisher P, House I, Belon P, Turner P. The influence of thehomeopathic remedy. Plumbum Metallicum on the excretionkinetics of lead ints. Human Tox 1987; 6: 321 324.

10 Larue F, Cal JC, Guilleman J, Cambar J. Influence du facteurdilution sur leffet protecteur de Mercurius Corrosivus vis-a-vis de la toxicite induite par le chorure mercurique chez lasouris. Homeopathie Francaise 1985; 73: 375 380.

11 Vischniac I. Influence des doses infinitesimales de plomb surlevolution de lintoxication au plomb chez lanimal. Homeo-pathie Francaise 1965; 53: 21 33.

12 Stopes C, Woodward L. The use and efficacy of a homeopathicnosode in the prevention of mastitis in dairy herds: a farm surveyof practicing users.Bull Organ Agr1990; 10: 610.

13 Day CEI. Isopathic prevention of kennel cough is vaccina-tion justified?J Inter Assoc Vet Homeopathy 1987; 2: 4551.

14 Saxton J. The use of canine distemper nosode in diseasecontrol. Int J Vet Homeopathy 1991; 5: 8 12.

15 Cazin JC, Cazin M, Gaborit JLL,et al. A study of the effect ofdecimal and centesimal dilution ofArsenicumon the retention

and mobilisation of arsenic in the rat. Human Tox 1987; 6:315 320.16 Blostin R.Arsenicum albumand neurotoxic poisoning in dogs

and cats. In: Bastide M (ed). Signals and Images. Dordrecht:Kluwer Academic Publishers, 1997, pp 237 243.

17 Oberbaum M, Cambar J. Hormesis: dose dependent reverseeffects of low and very low doses. In: Endler PC and Shulte J(eds). Ultra High Dilution Physiology and Physics. Dor-drecht: Kluwer Academic Publishers, 1994, pp 5 18.

18 Giraldi T, Perissin L, Zorzet S, Rapozzi V. Stress, melatoninand tumour progression in mice. Ann NY Acad Sci USA 1994;719: 526 535.

19 Chapouthier G and Nouit JC (eds).The Universal Declarationof Animal Rights, Comments and Intentions. Paris: LFDA,1998.

20 Balint Z. Lipolytic activity in adipose tissue of mice bearingEhrlich ascites carcinoma.Cancer Biochem Biophys. 1991;12:4552.

21 Delbancut A. Contribution a letude des effets de hautesdilutions de metaux vis-a-vis de la cytotoxicite du Cadmiumsur des cultures de cellules tubulaires renales. These DoctoratCellular Biology, UniversiteBordeaux II, 1994.

22 Malarczyk E. Potentiated Partusisten inverts the effect ofprenatal usage of this drug. Annals of the GIRI meeting.Monaco, 28 November 1999.

23 MalarczykE. Hormetic inversionof thedrugoverdosageeffects.Annals of the GIRI meeting. Monaco, 28 November 1999.


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very high dilutions of dexamethasone inhibit its pharmacology effect in vivo

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