These are all multifactorial pathologies as they often involve simultaneously:

(1) A pathogen (ex. virus) or causative agent (ex. an allergen)

(2) Secondary bacterial infection

(3) Inflammation (inflammatory proteins)

(4) Broken cellular integrity (skin, mucus membrane, eye surface)

When symptoms appear, most of these factors are already installed and the damage is already started.

For example, in case of allergy, when an allergen comes in contact with the nasal mucosa, it is presented by antigen-presenting cell (dendetric cells) to T_H2 lymphocyte; a subset of T cells that produce a cytokine called interleukin-4 (IL-4). These T_H2 cells interact with other lymphocytes called B cells, which coupled with signals provided by IL-4, stimulates the B cell to begin production of a large amount of a particular type of antibody known as IgE. Secreted IgE circulates in the blood and binds to an IgE-specific receptor (a kind of Fc receptor called FcεRI) on the surface of other kinds of immune cells called mast cells and basophils, which are both involved in the acute inflammatory response. When these IgE-coated already sensitized cells are re-exposed to the same allergen, mast cells and basophils immediately gets activated & undergo a process called degranulation, during which they release histamine and many other inflammatory chemical mediators (cytokines, interleukins, leukotrienes, and prostaglandins) from their granules into the surrounding tissue causing several systemic effects, as vasodilation, mucous secretion, nerve stimulation, and smooth muscle contraction. Nasal mucosa inflammation kill nasal mucosa cells providing a favourable ground for microbial growth with clinical symptoms such as rhinorrhoea, itchiness, & dyspnoea.

Inflammation cause nasal mucosa damage and the formation of gaps between the cells. The broken nasal mucosa barrier allows allergens & infection to enter directly into the body leading to continuous triggering of allergen & inflammatory cascade. Chronic condition may lead to respiratory destress and the development of chronic diseases such as asthma.

The broken nasal mucosa is covered with dead cells, cellular debris, inflammatory proteins (cytokines), histamine, IgE, and bacterial proteins.

The basic physiopathology remains the same in almost all other topical diseases.

Along with the broken cellular integrity & the presence of inflammatory proteins (cytokines), disease specific proteins may vary from disease to disease. For ex:

Viral diseases: virus glycoproteins (ex. H1N1)

Wound surface: multiple MMPs (Matrix MetalloProteinases). MMPs destroy the ECM (Extracellular Matrix) onto which daughter cells attach & grow. They block healing.

Psoriasis, Eczema, Dermatitis: Growth factors (ex. EGF, FGF)

Allergy: Histamine, IgE

Asthma: TSLP, leukotrienes, histamine

An ideal treatment can only be multifactorial. It should simultaneously:

(1) Stop the entry of the causative agent,

(2) Clean the damaged surface of all the contaminants including the microbial contaminants, inflammatory proteins, virus particles, dead cells, cellular debris, allergens, histamine, IgE, pollutants, bacteria and other particles,

(3) Should stimulate cell growth to reconstitute the damaged cellular barrier and to stop chronic systemic inflammatory trigger,

(4) Non-chemical & cell friendly to allow cell growth,

(5) Fast acting,

(6) Long lasting to avoid repeated applications,

(7) Hydrating,

(8) Non-irritant, non-absorbable, topically acting, & totally safe.

Infection can only heal when the cells are healthy and cellular integrity & functions are restored.

Cells are like babies: they need support for attachment (like the ground for babies to stand or lie on), water for hydration, food for energy, oxygen to stay alive (as babies need air to breathe), total absence of chemicals, infection, and contact with “bad” proteins. If any of these conditions are not met, the cells cannot grow, cannot defend themselves, cannot heal the injury, and cannot stop the infection.

Therefore, the 1^st step towards healing should be to restore the ideal conditions for cellular repair & growth.

As all the topical diseases (ex. throat infection, rhinosinusitis, allergy, psoriasis, wounds) involve cellular destruction, the presence of causative agent (viruses, bacteria, allergens) & multiple disease related proteins (ex. histamine, inflammatory cytokines, MMPs, leukotrienes, TSLP proteins). The aim of VITROBIO research was to find a topically applicable treatment capable to clean any live biological surface of all these contaminants rapidly without being absorbed in the body.

Such a treatment must be cell friendly & non-cytotoxic to allow cell growth and lesion repair within a shortest period.

It is known that the cells are like babies and for growth, they require a clean, chemical free, oxygenated, hydrated & contaminant free environment. If these basic conditions are not met, cells cannot grow & disease cannot cure. A clean tissue starts healing immediately.

An intact and healthy skin or mucus membrane should not allow entry of any foreign material (ex. allergens, bacteria) into the body which intern should stop further inflammatory &/or allergen cascade and disease progression.

Such a treatment can also be designed as a long-lasting, hydrating, protective & absorbent liquid barrier to stop further infection and to trap undesired protein molecules present on the infected / damaged tissue.

Introducing adjustable osmotic properties in the barrier should allow hypotonic liquid exudation from inside towards outside the tissue. The liquid flow should detach & drain all the contaminants from the surface including newly liberated virus particles, microorganisms, protein molecules, & all other free-floating particles, thereby replacing the use of chemical drugs: antiseptics, antibiotics, antihistamine, anti-inflammatory, & hydrating medicines.

It is also possible to incorporate specific protein binding polymeric substances in the film capable to mechanically trap the targeted protein(s) on the diseased surface.

Based on this hypothesis, a 20-year R&D program was launched in 1994 with the following results:

(1) Conceiving a topically applicable protective film: Multiple natural, non-irritant, food grade ingredients were tested in different concentrations to conceive a thin or thick, osmotically adjustable liquid film, depending on the mode of application envisaged. We conceived & patented a glycerol-based solution termed VB-Gy (Int. PCT/FR99/01340), 18 times more osmotic than sea water (3.2% NaCl). The physical properties of the protective film & its osmotic activity was adjusted as per the therapeutic aim. For example, higher osmotic activity is desired to detach bacteria from a highly infected surface such as the throat mucosa or to break open sinus-blocking biofilms but a thick & allergen absorbent film is needed to stop allergen contact with the nasal mucosa.

(2) Rendering the film flexible & resistant to mechanical pressures: To improve the duration of retention of such films on live biological membranes & to minimize the frequency of applications, we used the polymer – macromolecule binding technology as some specific polymeric structures may bind with certain macromolecules (technique used to introduce flexibility in plastic films) and/or proteins. We selected > 182 synthetic or natural polymeric molecules (ex. plant tannins) using different extraction techniques and evaluated their binding properties with the molecules of the protective film. 32 inert, non-cytotoxic polymers were finally retained. Only a few individual or associations of polymers rendered the film flexible & resistant to mechanical pressure while conserving its non-irritant, cell-friendly, & topically applicable properties. The film is formed instantly, can remain on the infected surface for 4-6h, can conserve its osmotic properties, and can protect the lesion like a bandage during this period. The filmogen bandage was patented worldwide in 2013 (PCT/EP2013/061835).

Applying such a film on any live biological membrane instantly generates a strong osmotic flow from inside towards outside the live biological tissue onto which the film is applied thereby detaching & draining all the free floating molecules (including bacteria, viruses, & proteins) from the surface. A clean surface starts healing immediately.

(3) Identifying undesired proteins implicated in each pathology: This work was conducted by establishing specific in vitro models for each pathology. For ex., to identify the role of 27 known wound matrix-destroying MMP proteins, an ECM specific to each cell type was prepared & cells were grown on the surface of ECM in the presence or absence of different concentrations of each MMP. ECM-destroying MMPs (8/27) were then incubated with individual polymers to select best polymeric association capable of mechanically trapping the selected molecule without any interaction with the underlying live tissue.

(4) Introducing specific anti-protein properties in the film: Polymeric extracts of film-forming, natural or synthetic sources were prepared in diverse ways and their specific binding properties for the group of proteins involved in a pathology were evaluated. The protein – polymer binding depends on: (1) the affinity of the polymer for the protein; (2) the structures of proteins & tannins; (3) the length & molecular weight of polymers; (4) the isoelectric point of the protein to be neutralized; (5) the pH of the product; and (6) the tannin – protein molecular ratio.

The osmotic activity, polymeric binding, & absorbent properties of the film were adjusted according to the type of surface to be treated to maximise efficacy & safety.

Mode of action & advantages compared to existing drugs: (1) A new generation of 1^st multitarget medical devices or drugs as a filmogen bandage for topical pathologies; (2) Possibilities to incorporate simultaneous protective, cleaning, hydrating, and protein removing properties in a single product (3) Filmogen bandage protecting the injured surface from infection, allergens & external aggressions; (4) Osmotic activity attracting hypotonic liquid from the damaged surface, creating a strong hypotonic liquid outflow from inside the lesion, detaching & draining all surface contaminants instantly (within 5-10 min); (5) Liquid flow equally keeping the lesion hydrated over many hours; (6)  Removing undesired protein molecules; (7) No systemicinteractions (8) Clinical results proving instant and high effectiveness without any side effects; (9) Possibilities to register specific treatments as medical devices due to exclusively topical & mechanical mode of action.

Future competition: Current R&D is focused on patentable, chemical or biological molecules. Multi-target drug research is still in infancy.

1^st oral TRPA1 channel-blocking nutritional supplement for the prophylactic treatment of Migraine & recurrent headache

Incidence of Migraine: Nearly 12-15% of world population (>600 million persons) suffers from migraine, and many more from recurrent headache. WHO ranked migraine as the most disabling neurological disorder, costing the US > $19 billion and the EU > €27 billion annually. Over 3 million persons are diagnosed as migraineurs in France.

Migraine physiopathology:

Current hypothesis: Migraine & headaches are considered to be neurovascular pathologies triggered due to hypersensitivity of brain vessels and meninges to some chemo-sensors. The cause of the hypersensitivity of some individuals to the stimuli which triggers migraine is still not known. The stimuli cause an electrical storm in the brain, followed by release of a shower of hormones & neuromediators leading to: serotonin (5-hydroxy tryptamine or 5-HT) dysregulation, activation of trigeminovascular system, mitochondrial disfunction, vasodilation, and symptoms of headache & migraine. What triggers migraine still remains obscure, but serotonin level perturbation is considered the key factor. Therefore, up till now, all the key treatments were directed to modulate serotonin levels with an effort to reduce the pain sensation and inflammation. There are many serotonin (5-HT) receptors in the brain and in different organs of the body, interacting with each other. In the absence of exact knowledge about the role of these 5-HT receptors & their sub-types, different drugs target different sites such as:

5-HT_1B – Ergotamine, Sumatriptan, Zolmitriptan, Methiothepin (antipsychotic), Pindolol (antidepressant).

5-HT_1D – Tryptamin, Sumatriptan, Ergotamine, Vortixetin (antidepressant)

5-HT_2A – Tryptamine, Trazodone (antidepressant), Ketanserine (antihypertensive).

5-HT_2C – Funfluramine (anti-aneroxigen), Prozac (antidepressant) Ergotamine, Sumatriptan, Zolmitriptan, Methiothepin (antipsychotic), Pindolol (antidepressant).

 5-HT_1B – Ergotamine, Sumatriptan, Zolmitriptan, Methiothepin (antipsychotic), Pindolol and many other antidepressants, Methylsergide.

Triptans are 5-HT_1B/D receptor agonists but 5-HT_1F receptors are now considered as the major targets.

The most commonly used symptomatic treatments are analgesics & anti-inflammatory drugs, but a few migraine patients also showed excellent improvement with Vit.B2, B12, and tryptophan, when administered regularly in the right quantities. Some plants also show good results in some patients, particularly to prevent migraine attacks.

The diversity of drugs used to treat migraine proves that migraine & headache physiopathology is very complex, difficult to understand, and nobody knows where to act once the migraine attack is triggered. In the absence of exact knowledge, 5-HT modulating agents (agonists & antagonists) still constitute the 1^st line of treatment (70%) to treat severe cases of migraine only, while ergotamines occupy the 2^nd position (about 15%). These drugs also act on other 5-HT receptors present elsewhere in the body, and therefore produce multiple side effects. Moreover, 30-40% patients do not respond to triptans, these drugs have a slow onset, and they cause progressive hepatotoxicity. Their use is therefore limited to treat severe cases of migraine only, while most of the patients take analgesics &/or anti-inflammatory drugs to get symptomatic relief. In the absence of any effective & safe treatment, nearly 70% patients with occasional or recurrent headache / migraine opt for self-treatment.

Current R&D pipeline:

Since the discovery of triptans in 1972, migraine treatment research is still concentrated on symptomatic treatments. Multiple chemical & biological drugs are under development, such as CGRP antagonists, 5-HT1F ‘antagonists’ (but most already failed due to severe toxicity), NO inhibitors, neuroinflammatory & glutamatergic targets, oxerin 1 & 2 receptor-antagonists, neuromodulation nerve stimulators, pituitary adenyl cyclase activity-inhibitors, phosphodiesterase-inhibitors etc.

Why? Because the market potential is > US$ 10 billion /year!

What is wrong with these R&D strategies?

5-HT is designated as the key target, therefore all the pharma industries follow the same target. They have industrial logic because the time (>12 years) & cost (>US$ 1 billion) required to develop a new drug for a new or a known target is so high that everybody follows a “me too” strategy instead of taking risks to focus on an unknown target. They need to work on a single chemical or biological molecule which can be patented, and on an acceptable target to minimize the risks.

VITROBIO / Naturveda Research hypothesis

Since 2001, VITROBIO scientists have been convinced that due to the highly complex physiopathology of headache & migraine, and the involvement of multiple neuromediators, only a multiple-target approach may ease migraine symptoms. Targeting multiple targets requires associating different target chemicals or biologicals in a single drug which is extremely difficult as it is nearly impossible to obtain the drug registration if 2 or more molecules are associated:

The best migraine therapy should:

1. Avoid the triggering cause of migraine (impossible as cause(s) are not known)
2. Block the origin of migraine

The best solution therefore resides in blocking the mechanism involved in migraine triggering. Stopping the migraine trigger mechanism should stop the migraine cascade, and this approach would require only 1 molecule. Such an approach may also be employed as a preventive treatment.

In 2002, we found that certain parts of the Salix sp. plant (from which salicylic acid is derived) are used since ancient ayurvedic period (2000 BC) to treat pain and headache, with notable but limited efficacy.

One other plant, Tanacetum parthenium, is also known to prevent migraine since ancient periods, but its efficacy is not always proven.

We studied the 5-HT receptor profile of the extracts of various parts of these two plants, prepared using different techniques, and found that they modulate 5-HT receptors differently as shown below:

As the association of the 2 plant extracts had a different 5-HT receptor modulation profile compared to triptans, we decided to conduct a clinical trial.

The study was performed after obtaining government authorization, at the Central Government Hospital of Clermont-Ferrand in France, in the Dept. of EEG & Neurology, under the responsibility of Prof. Pechadre.

14 severe migraine patients received the association of 2 plants at a dose of 300 mg/day, twice daily, for 84 days.

Mig-RL treatment significantly reduced the frequency, the intensity as well as the minimum, maximum & average duration of migraine attacks.

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