ABSTRACT

The present invention relates to hemotherapy, more particularly to a molecular iodine- enriched blood and its use in the treatment and prevention of several diseases, including cancer. The invention also relates to process for the preparation of said molecular iodine- enriched blood.

TECHNICAL BACKGROUND

Besides its effects as part of thyroid hormones, molecular iodine (I2) exerts multiple and complex actions on organs that capture it. This chemical form of iodine is internalized by a facilitated diffusion system that is evolutionary conserved, and its effects appear to be mediated by a variety of mechanisms and pathways. As an oxidized component, it directly neutralizes free radicals, induces the expression of type II antioxidant enzymes, or inactivates proinflammatory pathways. In neoplastic cells, I2 generates iodolipids that have an antineoplastic effect with mitochondrial and nuclear mechanisms that include the activation of apoptotic pathways and the inhibition of markers related to stem cell maintenance, chemoresistance, and survival.

Recently, I2 has been postulated as an immune modulator that, depending on the cellular metabolic context, can function as an inhibitor or activator of immune responses.

Iodine can be found in nature and in many states of oxidation either organic and inorganic. Molecular iodine I2, being an apolar molecule, is taken up by a facilitated diffusion system, independent of the sodium iodine symporter (NIS) or Pendrin (PEN), necessary, instead, for the absorption of iodides and all other iodine-based ionic compounds and the oxidized iodine form I2 is organified in the absence of peroxidases.

I2 diffuses through all cell membranes, including mitochondrial and nuclear membranes, where iodine, by binding to histones, is able to modulate the expression of particular genes, directly and indirectly exerting epigenetic effects.

I2 acts as a scavenger of a reactive oxygen species (ROS) like hydroxyl radicals (OH) or superoxide anions (O2) generating neutral components hypoiodous acid (HIO) or hydroiodic acid (HI). I2 in combination with arachidonic acid (AA), and generating the iodolipid 6-iodolactone (6-IL), inhibits the activity of proinflammatory enzymes like Nitric Oxide Synthase (NOS) and Cyclooxygenase type 2 (Cox2).

Studies in mammary cancer demonstrated that moderately high concentrations of molecular iodine have an antiproliferative and apoptotic effect either in vivo and in vitro. In animal and human studies, molecular iodine supplementation exerts a suppressive effect on the development and size of both benign and cancerous neoplasias. Investigations by several authors have demonstrated that these anticancer effects can be mediated by a variety of mechanisms and pathways, including direct actions, in which the oxidized iodine dissipates the mitochondrial membrane potential, thereby triggering mitochondrion-mediated apoptosis, and indirect effects through iodolipid formation and the activation of Peroxisome Proliferator-Activated Receptors Type Gamma (PPARy Peroxisome Proliferator-Activated Gamma), which, in turn, trigger apoptotic pathways, such as BAX-caspase apoptotic pathway in cancer cells or differentiation in healthy cells. I2 but not iodide (T) supplementation alleviates human mastalgia and exerts a potent antineoplasic effect on pharmaco-induced mammary tumoral progression in rats. It is known that in some pathologies, such as diabetes and cancer, blood undergoes chemi cal -physical changes, visible even under optical or electronic microscope; among the many metabolic changes are evident: oxidation of Fe, changes in hemoglobin, loss of erythrocyte membrane potential, lowering of pH, the loss of homeostasis; these factors together with many others decreases the transport of oxygen gradually and inexorably leading to a condition of persistent hypoxia and an oxidative metabolism with hyperproduction of ROS and reactive nitrogen species (RNS).

Hypoxia is a characteristic feature of locally advanced tumors resulting from an imbalance between oxygen (O2) supply and consumption. Major causative factors of tumor hypoxia are abnormal structure and function of the microvessels supplying the tumor, increased diffusion distances between the nutritive blood vessels and the tumor cells, and reduced O2 transport capacity of the blood due to the presence of disease- or treatment-related anemia. So, a common feature of most tumors is glycosylation changes and a low level of oxygen, which severity depends on tumor types, though it is generally accepted that oxygen levels in tumor tissues is poorer than in the respective normal tissues and on average it is between l%-2% O2 or even below.

Metabolic disorders, such as oxidation, dehydration and persistent hypoxia in cancer patients, determine and maintain multiple morphological and volumetric asymmetries of erythrocytes (loss of membrane potential, Fe oxidation of hemoproteins with serious consequences including Heinz Body formation).

The erythrocytes, losing the potential, adhere to each other forming the rouleaux, thus decreasing the surface of gas exchange. Although the beneficial effects of iodine and oxygen in therapy are known, there is still a need for new therapeutic approaches which provide a better bioavailability of iodine and an improved therapeutic effect, especially, but not limited to, in cancer affected subjects.

OBJECTS OF THE INVENTION

It is a first object of the invention to provide an alternative composition to administer iodine to a subject in need thereof, especially, but not limited to, a cancer affected subject, which is highly effective.

It is another object of the invention to provide a composition, which is safe, cost effective and which can be used in the treatment and prevention of any cancer, in any subject.

It is another object of the invention to provide a process for the preparation of the composition of the invention.

It is a further object of the invention to provide the use of the composition of the invention in therapy, especially, but not limited to, in cancer therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 to 4 show peripheral blood of a cancer patient before (B) and after (A) blood purification (optical microscope).

Figures 5 to 7 show peripheral blood of a cancer patient (SEM - Scan Electron Microscopy).

Figure 8 shows peripheral blood of a cancer patient treated according to the invention.

DESCRIPTION OF THE INVENTION

According to one of its aspects, it is a subject-matter of the invention a blood enriched with molecular iodine.

Blood enriched with molecular iodine must be well oxygenated, where the expression "well-oxygenated" is defined below as a function of partial oxygen pressure. According to a preferred embodiment, is the object of the invention a purified bloodenriched with molecular iodine, characterized in that said blood is subjected to a purification treatment, before the addition of said molecular iodine.

The expression “purified blood” herein means that said blood has been subjected to a purification treatment so that it has been re-oxygenated. Such a purification treatment may be any suitable one, such as for example, GAET (Great Auto Hemotransfusion), EV- LLLT (EndoVenous -Low Level Laser Therapy), Dialysis and Plasmapheresis, which are well known in the art. The purification can be made ex vivo, i.e. samples of venous blood are purified before the treatment with iodine. The purification may be partial or total, (according to the method used).

The expressions “well-oxygenated blood enriched with molecular iodine”, “molecular iodine-enriched purified blood” are synonyms and herein indicate a blood, as herein defined, which has been added with molecular iodine.

The expression “molecular iodine” means E.

As said, the blood enriched with molecular iodine of the invention is a well oxygenated blood, where the expression "well oxygenated" indicates that a venous blood having a partial pressure of oxygen, also known as pCL, ranging from 35 to 40 mmHg, preferably about 40 mmHg. As it is known, partial pressure of oxygen is a measurement of oxygen pressure in blood. If the blood sample is not well oxygenated, that is, it does not have the partial pressure of oxygen, must then be subjected to purification for become a "purified blood" suitable for use according to the invention.

Also the expression "purified blood" therefore indicates a venous blood with a partial oxygen pressure ranging from 35 to 40 mmHg, preferably about 40 mmHg. According to a preferred embodiment, the concentration of molecular iodine in said molecular iodine-enriched purified blood ranges from 40 to 92 pg/L, more preferably 70 pg/L.

According to the present invention, if the blood sample that is to be enriched with molecular iodine has a partial oxygen pressure ranging from 35 to 40 mmHg, preferably about 40 mmHg, then it can be directly enriched by introducing molecular iodine, as will be described below.

In the case where, instead, the blood sample that must be enriched with iodine has a partial oxygen pressure of less than 35 mmHg, then said sample shall be subjected to a purification treatment as described here.

In cancer patients purification is almost always necessary before the addition of iodine. According to a preferred embodiment, in addition to the partial pressure of oxygen, as above indicated, it is important that also that the blood present the values of the analyses below in the Experimental Section are within the reference values.

Indeed, it was unexpectedly found that improving the chemi cal -physical conditions of hypoxic blood of cancer patients by a purification process, followed by the addition of molecular iodine, and subsequently administering said iodine-enriched purified blood in a cancer patient, provides an effective anti-cancer activity.

In the present invention, the purification of the blood has the aim of re-oxygenating, correcting the morphological and functional changes in erythrocytes, contrasting rouleaux formation, which are primarily caused by the oxidative and nitrosative stress of cancer cells. As said, molecular iodine (I2), at appropriate concentrations, has multiple effects, either genetic and metabolic, directly on blood and on the apoptosis of cancer cells, through an independent Caspase pathway; molecular Iodine also acts on the activation of the nuclear receptor PPARy (Peroxisome Proliferator-Activated Receptor gamma), mediated by 6-Iodiolactone, on the activation of the immune system and on histones and therefore on epigenetic control of several genes.

Molecular iodine (and not iodide or other iodine ionic derivatives) crossing all membranes by facilitated diffusion, enters cells, organelles, mainly mitochondria, and nucleus. Iodine plays an essential role in the mitochondria in which it activates apoptosis of cancer cells, activates the immune system, regulates nuclear receptors and the direct and indirect expression of several genes.

Applicant has now unexpectedly found that the administration of well oxygenated blood (optionally through purification) added with molecular iodine restores metabolic balance, pushing stem cells towards proper differentiation, inducing apoptosis only in cancer cells, stimulating the immune system and reactivating aerobic respiration. These and other factors contribute to reversing cancer directing by apoptosis and indirectly by depriving it of its hypoxic micro-environment and re-establishing a metabolic aerobic environment suitable for differentiation, as it happens during the implantation of embryonic stem cells in the uterus.

As molecular iodine and oxygen are two oxidizing elements in equilibrium with each other, the infusion of iodine in a non-purified (not well oxygenated) blood would lead to further oxidative damage or it would not be bioavailable to the cells, as it would immediately form chemical bonds with other amphiphilic or apolar compounds.

So, the combination of molecular iodine and blood, having the partial pressure of oxygen in the indicated range, offers many advantages in cancer therapy:

1) purification (oxygenation) of blood allows blood to re-oxygenate and re-oxygenate tissues, reducing hypoxia, which, as is known, is a essential factor to maintain an environment conducive to cancer development of cancer and other numerous degenerative and non-degenerative pathologies; the purification also restores the membrane potential of red blood cells, reduce carbohydrates, especially L-Fucose that leads mitochondria to anaerobic respiration, rebalances receptors including EGFR (Epidermal Growth Factor Receptor) and NOTCH that activate mitosis, all contributing to the goal of detoxifying the body from the tumor siege.

2) Molecular iodine treatment reactivates apoptosis of cancer cells, regulates the immune system, reactivates aerobic respiration together with oxygen, reactivates cell differentiation, reactivates receptors, including the PPARy nuclear receptor and epigenetically regulates the expression of numerous genes; also, molecular iodine does not have the same effects as iodide on heart and thyroid, is not absorbed differently as a function of the expression of NIS and Pendrin Receptors, but absorbed by diffusion facilitated by all biological membranes: cellular, mitochondrial and nuclear; finally molecular iodine crosses the blood-brain barrier and the action of molecular iodine does not depend on enzymatic bioavailability.

All these advantages make the use of molecular iodine-enriched purified blood an effective cancer hemotherapy to restore homeostasis and thus prevent the restoration of metabolic conditions predisposing to the process of metastasis formation and/or cancer recurrence; the treatment is easier, safe and cost effective with respect of conventional cancer therapies, it can also be used in association with them.

In addition to the presence of correct amounts of oxygen, the blood purification of patients cancer also serves to eliminate substances interfering with iodine. For example, L-Fucose, a methyl pentose that is present in higher concentrations in the blood of cancer patients, is able to react with iodine and it is therefore useful to reduce concentrations in the blood through purification, to promote the bioavailability of the iodine. The purification then is useful to decrease the concentration so that the iodine infusion reaches the tissues through the blood.

According to a preferred embodiment, the blood enriched with molecular iodine according to the invention has a partial oxygen pressure ranging from 30 to 40 mmHg and is free or substantially free of compounds that interact with iodine, such as L-Fucose. Hemotherapy could be used to achieve and/or maintain response to conventional treatments.

Besides, the treatment is economical and painless and does not affect the quality of life of the cancer patient, which already compromised by the disease and by any cancer treatments previously administered.

With reference to the Figures, it can be the stacking of red blood cells to "rouleaux" and the alterations of their morphology in cancer patient’s blood to aggravate hypoxia; indeed, the morphological, functional and metabolic changes of the red blood cells compromise their potential for the extraction and transport of oxygen in the blood.

It is known that persistent hypoxia promotes anaerobic glycolysis, the undifferentiated state of cells, the microenvironment favorable to cancer cells.

So, the good oxygenation of blood (optionally obtained through its purification) and iodine addition aim to:

- reduce oxidative stress,

- restoring the membrane potential of blood cells,

- improve the oxygenation of hemoglobin and the release of oxygen to tissues.

The actual therapeutic effects of iodine occur when molecular iodine reaches cancer cells: -inducing apoptosis in cancer cells; -activating: a proper differentiation, the immune system and several receptors including PPARy.

PPAR receptors play a pivotal role in restoring metabolic balance in healthy tissues, activating differentiation, actively counteracting inflammation and invasion of cancer cells in the body.

Mitochondria also play a fundamental role in the induction of apoptosis and the activation of aerobic respiration against anaerobic glycolysis typical of cancer cells.

According to another of its aspects, it is a subject-matter of the invention a process for the preparation of molecular iodine-enriched blood, which comprises the following steps: a. optionally subjecting a sample of blood to a purification treatment, to obtain a reoxygenated blood, wherein purified blood is as above defined; b. adding a water solution of molecular iodine to the purified blood sample of step a., preferably to a concentration of 40 to 92 pg/L.

As above defined, said purification treatment is preferably selected from: GAET, EV- LLLT, Dialysis and Plasmapheresis, although any suitable purification treatment, more suitable for the patient’s clinical condition, can be used.

Preferably, the water solution of molecular iodine of step b. has a concentration of molecular iodine from 0.1 mM to 1 mM, more preferably 0.5 to 1 mM, most preferably about 1 mM.

In practice, a water solution having the desired concentration of molecular iodine is prepared and added to the purified blood of step a..

According to a preferred embodiment, a ImM h water solution may be prepared according to the following redox

2KI + H2O2 + 2HC1 = I ₂ + 2KC1 + 2H ₂ O The water solution must be sterile, free of stabilising substances, and preferably stored at 0°- 4°C for 48 hours maximum, preferably in glass bottles.

As an alternative, stabilized solutions of molecular iodine can be prepared and used.

As a standard practice, 0,2ml of a ImM I2 water solution (50 pg I2) are added into a 200 ml blood bag/bottle, said blood having been previously purified according to the invention.

The molecular iodine-enriched blood of the invention is to be administered by infusion in a subject in need thereof, according to the known methods of hemotherapy.

It is advisable that the infused amount is initially lower than the RDA (Recommended Dietary Allowance - RDA- for Iodine related to age - ICCIDD) and subsequently increased up to the RDA.

RDA for iodine are the following:

* International Council for Control of Iodine Deficiency Disorders

It is evident for the skilled in the art, that for the purpose of determining the amount of molecular iodine to be administered, also the weight of the patient is to be considered; the higher the body weight, the higher the amount. For instance, if the body weight exceeds 80 kg, it would be advisable to increase the molecular iodine administered by 20% of the patient’s weight, preferably maintaining the same concentration of iodine in the blood bag.

The blood may be either a compatible donor blood, or a previously drawn blood of the same cancer patient.

In this second case, purification in vivo and/or ex vivo is necessary.

In any case it is useful to carry out all the blood controls, partial oxygen pressure and analyses indicated below, and it is advisable that the administration is carried out immediately after the preparation of molecular iodine-enriched blood of the invention in the subject in need thereof.

According to another of its aspects, the molecular iodine-enriched blood of the invention for its use in therapy, especially for use in the treatment and/or prevention of cancer, and/or in the treatment and/or prevention of metastases represents another subject-matter of the invention.

The blood of the invention is useful for the treatment of any cancer or metastasis, either solid or liquid.

As said, it has surprisingly been found that, when the blood sample does not have the partial oxygen pressure shown here, purifying the blood prior to the addition of molecular iodine allows its improved absorption and bioavailability, through facilitated diffusion and also prevents cross-reactions. Indeed, blood purification allows red blood cells to transport oxygen to cells and mitochondria to reactivate respiration and ATP production, gradually induces apoptosis of cancer cells.

The molecular iodine-enriched blood of the invention represents an effective and safe treatment that does not interfere with the heart and/or thyroid gland and is a cost-effective clinical treatment. Preferably, said treatment is repeated, for instance 1 to 6 months apart, preferably 2-5 months apart, more preferably 2 months apart, after the suitable clinical controls, such as routine post diagnosis checks, as TAC PET, RMN, blood test and others.

The molecular iodine-enriched blood of the invention is therefore suitable to be used as an effective solid and liquid cancer therapy. Therapy can be used without age or gender limits.

The molecular iodine-enriched blood may be also used in combination with chemotherapies, or other cancer therapies, such as radiotherapy.

So, according to another of its aspects, the invention relates to the molecular iodine- enriched purified blood of the invention in combination with further drugs, chemotherapies and/or other cancer therapies, for use in the treatment and/or prevention of cancer and metastasis.

According to another of its aspects, the invention relates to a method for the treatment and/or prevention of cancer and metastasis which comprises administering to a subject in need thereof the molecular iodine-enriched blood of the invention.

According to another of its aspects, the invention relates to a method for the treatment and/or prevention of cancer and metastasis, which comprises administering to a subject in need thereof a molecular iodine-enriched blood of the invention in combination with other drugs and/or cancer therapies.

According to another of its aspects, the invention relates to the molecular iodine-enriched blood of the invention, for use in therapy, especially in the treatment and/or prevention immunological diseases, inflammatory diseases, diabetes, viral and degenerative diseases, thyroiditis and optionally preeclampsia. According to another of its aspects, the invention relates to a method for the treatment and/or prevention of immunological diseases, inflammatory diseases, diabetes and viral and degenerative diseases, thyroiditis and optionally preeclampsia which comprises administering to a subject in need thereof a molecular iodine-enriched blood of the invention.

According to another of its aspects, the invention relates to a method for the treatment and/or prevention of immunological diseases, inflammatory diseases, diabetes and viral and degenerative diseases, thyroiditis and optionally preeclampsia which comprises administering to a subject in need thereof a molecular iodine-enriched blood of the invention, after its purification.

Experimental Section

Example 1

Preparation of a molecular iodine-enriched blood bag

(i) A ImM E water solution is prepared according to the following redox

2KI + H2O2 + 2HC1 = I ₂ + 2KC1 + 2H ₂ O and the water solution must be sterile, free of stabilising substances, stored at 0°- 4°C for 48 hours maximum, in glass bottles;

(ii) 0,2ml of a I2 water solution ImM (50 pg I2) are added into a 200 ml blood container for infusion, after purification of said blood;

(iii) shaking slowly the bag or the glass bottle.

Example 2

Tests to be performed on blood

In order to check the effectiveness of the hemotherapy of the invention and monitor the patient’s response, blood tests must be carried out at all stages; in particular, it is necessary to check the functionality of erythrocytes for oxygen transport. The shape and integrity of the membranes will be checked with the optical microscope and the experimental phase also with the SEM. Blood tests of both a donor and a patient, before purification, may be performed in vivo or ex vivo.

The study involves several blood tests before and after each treatment:

1. In vivo or ex vivo before and after optional purification

2. Ex vivo before and after iodine infusion.

1. In vivo or ex vivo before and after optional purification:

• Blood Observation under the Optical Microscope

• ScvO2

• SvO ₂

• Hemogasanalysis

• Hemochrome

• Glycemia

• L-Fucose

• LDH

• Viscosity

• VES

• Glutathione

• Sideremia

• Transferrin

Red blood cell enzymes:

• ATPases, • Acetylcholinesterase.

• Adenylate Cyclase,

• Anion Transport Protein,

• Glyceraldehyde 3 -Phosphate Dehydrogenase,

• Glucose-6-Phosphate Dehydrogenase (G6PD)

• Pyruvate Kinase

• Protein Kinases, (Spectrin)

• Transport of Cations (Calcium, Sodium, Potassium).

2. Ex vivo before and after iodine infusion:

• Blood Observation under the Optical Microscope

• Venous Gas Analysis

• Hemochromie

• Glycemia

• L-Fucose

• LDH

• Viscosity

• VES

• Glutathione

• Sideremia

• Transferrin

Red blood cell enzymes:

• ATPases,

• Acetylcholinesterase. • Adenylate Cyclase,

• Anion Transport Protein,

• Glyceraldehyde 3 -phosphate dehydrogenase,

• Glucose-6-phosphate dehydrogenase (G6PD) • Pyruvate kinase

• Protein kinases, (Spectrin)

• Transport of cations (calcium, sodium, potassium).

For the evaluation of the clinical reports of each examination, reference is made to the official international ranges. The same tests should be performed in vivo in the patient after treatment, according to the invention, in addition to routine analysis.