In the search for new therapies novel drugs and medications are being discovered, developed and tested in laboratories. Highly diluted substances are intended to enhance immune system responses resulting in reduced frequency of various diseases, and often present no risk of serious side-effects due to its low toxicity. It was developed and tested several highly diluted tinctures and it was described the biological activity of M1, M2, and M8. Cytotoxicity, cytokines released and NF- B activation were determined after in vitro treatment. Cell viability, oxidative response, lipid peroxidation, bone marrow, and lymph node cells immunophenotyping were accessed after in vivo treatment. None of the tested highly diluted tinctures were cytotoxic. All compounds enhanced the innate immunity, but M1 also augmented acquired immunity and M2 diminished B lymphocytes, responsible for acquired immunity.

Biological assays are intended to understand better what happens within a cell when a molecule interacts with its target. In order to determine the efficacy and safety of new drugs, there is a clear move within the pharmaceutical industry towards an increased emphasis on cell-based assays. Well-structured methodological studies are useful to understand how highly diluted tinctures work.

It was certified high quality, physicochemical composition, and endotoxin-free conditions of its matrices. Dilution procedures have followed the standard methodology described at the Homeopathic Pharmacopoeia. In brief, starting from a whole extract (MT) or initial decimal dilutions (dH or x) of several tinctures, the highly diluted tinctures were obtained by serial decimal dilutions in distilled water (1 part of tincture to 9 parts of water – v/v). In between each dilution, the solution was vigorously manual shaken (by hand) with 100 strong vertical strokes against a soft pad. The resulting aqueous solution is colorless and odorless and contains less 0.1% alcohol. These were stored in the dark at room temperature.

These highly diluted tinctures were shown to modulate immune responses. After using different products and analyzing the different results carefully, we arrived at three complexes produced with homeopathic techniques, coded as M1, M2, and M8. The results obtained using these products were surprising and some were even published in scientific journals with good impact. These combinations of highly diluted tinctures could be used as therapeutic interventions in disorders where the immune system is compromised.

It was used as the most actual and diverse methodology, with several controls: Confocal, Histochemistry, Electron Microscopes, Flow Cytometer, Slide Scanner, Matrigel matrix invasion, ImageJ software, etc. It was demonstrated that these medicines prevent lung and subcutaneous melanoma growth, decreased angiogenesis inside tumor (p<0.01);  decreased some extracellular matrix molecules, as Perlecan and Hyaluronic Acid, decreasing metastasis (in Transwell plates, p<0.01); activated macrophages, that can activate others in immune system and other structures as endothelial cells and fibroblasts; modulated cytokines like TNFα, IFNγ, IL10; increased not only the differentiation to NK cells (inflow cytometer p<0.001) but also the cytotoxicity of NK cells (p<0.05);  reactive oxygen species (ROS) and nitric oxide (NO) production was modulated. 

Therefore, M1, M2, and M8 are homeopathic complex medicines (HCM) with immunomodulatory properties, without toxicity nor mutagenic effects. This homeopathic immunotherapy can kindly restore the immune system to recognize tumor cells and these highly diluted tinctures could be used as therapeutic interventions in disorders where the immune system is compromised.