CH718339A2 - Formulations for intravenous infusions comprising an inclusion complex of a stilbene in synergy with microamounts of melatonin. - Google Patents

Abstract

The invention relates to formulations for intravenous infusions without preservative comprising microamounts of melatonin and a stilbene such as resveratrol or pterostilbene, specifically designed and developed as a synergistic therapeutic tool, capable of drastically reducing Storm Syndrome of Cytokines (CTS) in the context of Acute Respiratory Distress Syndrome (ARDS) in patients affected by COVID-19.

Description

The invention relates to formulations for intravenous infusions without preservative comprising microquantities of melatonin and a stilbene such as resveratrol or pterostilbene, specifically designed and developed as a synergistic therapeutic tool, capable of drastically reducing the Syndrome Cytokine Storm (CTS) in Acute Respiratory Distress Syndrome (ARDS) in patients affected by COVID-19.

[0002] The invention also comprises a method making it possible to perform intravenous infusions of resveratrol in synergy with microquantities of melatonin at appropriate concentrations, in order to reach and maintain in a planned manner the plasma concentration at the state of balance of the two active substances for several consecutive days.

The synergistic action of resveratrol and microquantities of melatonin in the context of the present invention consists of a progressive and consecutive action which begins at the beginning, with the intravenous infusion of melatonin, while the plasma level of resveratrol is reached in a targeted manner by the simultaneous administration of a single oral dose of the latter.

The invention claims an inhibitory action of the formulations on the production of inflammatory cytokines, by a synergistic biochemical mechanism which leads to an immunomodulatory response.

[0005] Thanks to the endothelial protective action of melatonin and resveratrol and to that of reducing platelet reactivity, the formulations exert effective cardiovascular protection in COVID-19 patients, more particularly in those whose body mass index (BMI) is high.

[0006] In the present invention, it is also claimed that the formulations improve convalescent plasma therapy (CPT) in COVID-19 patients.

[0007] The formulations below can be safely used as adjuvant therapy in other emergency conditions caused by viral infection, such as Ebola virus or dengue hemorrhagic fever (DHF).

[0008] Inhibition of oxidative damage and toxicities of either snake venom or administered anti-venom, allowing increased efficacy of current snakebite therapy is another emergency condition in which formulations can be administered effectively and safely[70][71][72].

[0009] Continuous parenteral administration of the formulations is suitable for inhibiting the drug interaction toxicity that triggers the anaphylactic reaction following the cytokine cascade in patients in intensive care units (ICUs) as well as in hospitalized patients. .

[0010] Other emergency medical uses include, but are not limited to, severe hypoxemia or anoxia, pulmonary fibrosis, severe sepsis and septic shock of viral and bacterial origin, herpes infections, thrombosis microvascular, hypoxic-ischemic brain injury, acute infarction, hypoxic encephalopathy of the newborn, severe burns, anaphylactic shock and spinal cord injury.

The present invention relates to formulations for preservative-free intravenous infusions comprising an inclusion complex of a stilbene such as resveratrol or pterostilbene in synergy with microquantities of melatonin. These parent formulations have been specifically designed and engineered as a synergistic therapeutic tool capable of significantly reducing Cytokine Storm Syndrome (CTS) in life-threatening conditions, such as Acute Respiratory Distress Syndrome (ARDS) or acute lung injury (ALI) in patients with COVID-19.

[0012] The formulations have an inhibitory action on the production of inflammatory cytokines by a synergistic biochemical mechanism which leads to an immunomodulatory response.

The invention also includes a method of achieving and maintaining in a planned manner, by intravenous infusion, the steady-state steady-state plasma concentration of melatonin and stilbene, for as long as necessary, even several consecutive days.

The synergistic action of microquantities of melatonin and stilbene in the context of the present invention has been specifically designed and consists of a progressive and consecutive action which begins, in the initial intravenous infusion phase for melatonin, while the Plasma plateau of stilbene is reached consecutively and anticipated, thanks to the oral administration of a single dose of the latter at the start of the infusion.

[0015] In order to tackle the Cytokine Storm Syndrome (CTS) which occurs following the rapid replication of the SARS-CoV-2 virus in patients affected by COVID-19, the physicochemical characteristics of various known pharmaceutical active substances have been explored.

[0016] By implementing and deploying the most innovative technologies, two active substances have finally been identified: a biological compound belonging to the chemical family of indolamines, called melatonin, and stilbene monomers such as trans-resveratrol or pterostilbene.

Since trans-resveratrol is the best known and most studied compound of the stilbene family, trans-resveratrol is hereinafter referred to as its most important representative which, in the present invention, is associated synergistically with melatonin, whose respective abbreviations will hereinafter be „STI“ and „MLT“.

[0018] It is already known that these active substances can reverse or control a variety of human diseases. Their safety profile has also been widely proven to be favorable.

[0019] However, formulations for intravenous infusions suitable for safe and prolonged administration with one or both of these substances have never been designed and developed as a therapeutic tool before.

Furthermore, it is evident that the synergistic combination of active substances, namely the combination of STI and microamounts of MLT, capable of achieving and maintaining in a planned manner the steady state plasma concentration of two active substances, has also not been addressed.

Finally, an intravenous infusion carried out over several days with a steady-state plasma concentration of MLT and STI has not yet been identified, in order to provide maximum therapeutic benefit to patients suffering from pathologies putting their lives in danger.

[0022] MLT or N-acetyl-5-methoxytryptamine is a molecule highly conserved during evolution, belonging to the indolamine family, which is found in all microorganisms[53], plants and animals. [54][55].

[0023] Originally identified in the bovine pineal gland[56], MLT is also produced by a wide range of tissues, including the retina, thymus, spleen, heart, muscle, liver, stomach, pancreas, intestine, placenta, testes, ovaries, bone marrow, skin and hair follicle, cerebral cortex and striatum[1][50][51][52].

[0024] The MLT content of these fabrics varies. However, it gradually but constantly decreases with age, in parallel with the decrease in its production at the level of the pineal gland [57][58].

[0025] After biosynthesis, pineal MLT is immediately released into the circulatory system and reaches all biological fluids, including cerebrospinal fluid, bile, saliva, synovial fluid, sperm, ovarian follicular fluid, amniotic fluid, breast milk and tears[59][60][61].

[0026] MLT is widely known for its involvement in the human chronobiological system and there are several pharmaceutical or nutraceutical products that are currently sold in the United States as well as in many countries around the world for this specific use.

However, MLT does not only exert an action on human circadian rhythms, but it has a fundamental protective role in several physiopathological states caused by inflammation and/or oxidative stress, such as sepsis, disorders neurodegenerative diseases, diabetes, biliary and pancreatic dysfunctions, hepatotoxicity, cancer and finally as an immune system modulator.

[0028] More recently, attention has focused on the strong therapeutic potential of MLT in cardiovascular diseases (CVD).

[0029] Low endogenous levels of MLT are associated with a greater risk of myocardial infarction in women with a high body mass index [62], which confirms the crucial role of endogenous MLT in cardiovascular pathologies.

[0030] Epidemiological studies report that the nocturnal synthesis of MLT and its circulating levels are reduced in patients suffering from coronary diseases.

[0031] MLT has numerous beneficial actions in several cardiac pathologies [63][64][65][66][67].

[0032] Actions mediated by MLT receptors appear to be the cardiac protective mechanisms of MLT. Signaling pathways include membrane responses, cytoplasmic modulation of kinases, interactions with nuclear receptors, and enhancement of mitochondrial functions[68][44].

[0033] Despite all the publications on the inference and modulation of all these vital functions exerted by MLT, it is not sufficiently clear that the plasma level of MLT constantly decreases with age and that this decrease contributes to the conditions degenerative diseases of aging and related pathologies.

[0034] Reputable studies have shown that MLT has anti-inflammatory properties, while some argue that MLT is an immunostimulant.

[0035] The two points are consistent, because the MLT is in fact an immune buffer.

It acts as a stimulant in immunosuppressive conditions, or as an anti-inflammatory agent in the presence of heightened immune responses, such as the acute inflammation identified as Cytokine Storm Syndrome (CTS) which occurs in patients COVID-19. TCS occurs when large numbers of leukocytes are activated and release inflammatory cytokines, which in turn activate even more leukocytes. They include interleukin-1 (IL-1), interleukin-12 and interleukin-18, tumor necrosis factor (TNF-a), interferon gamma (IFNγ) and colony stimulating factor granulocyte-macrophage cells (GM-CSF) and play an important role in mediating the innate immune response.

[0037] The data examined in the article by Carillo-Vico et al. “support the idea of MLT as an immune buffer, acting as a stimulant under basal or immunosuppressive conditions or as an anti-inflammatory compound in the presence of heightened immune responses, such as acute inflammation.”[2].

[0038] The fact that such an important human immunomodulator is secreted so little in the elderly, leads to the consideration that this deficiency may be a cofactor which leads the elderly, infected with the SARS-CoV-2 virus, to develop the fatal acute respiratory distress syndrome (ARDS) or acute lung injury (APL).

This low level of MLT in the elderly is part of the pharmacological research carried out for this invention, with the aim of identifying an effective therapeutic tool, easily usable in intensive care units (ICUs), capable of reduce the incidence of CTS and hospitalization.

[0040] The other active substance that has been identified as capable of providing therapeutic results in COVID-19 patients is trans-resveratrol (TSI) [3,5,4'-trihydroxy-trans-stilbene] or pterostilbene [3,5-dimethoxy-4'-hydroxy-trans-stilbene] which are monomeric stilbenes widely distributed in nature.

[0041] While red grapes and red wine are the main dietary sources of STI, pterostilbene is mainly found in blueberries and grapes. These two active substances „exhibit many similarities in terms of pharmacological properties, including antioxidant, neuroprotective, anticancer, cardioprotective, analgesic, anti-atherosclerotic, anti-aging, anti-diabetic, anti-inflammatory and anti-obesity activity“[33][ 35].

[0042] STI is a naturally occurring polyphenol antioxidant found in grapes, berries, peanuts and red wine. This phytohormone is produced naturally by several plants, as a defense mechanism, under attack from pathogens such as bacteria or fungi or following oxidative stress such as exposure to UV rays.

[0043] Even though MLT and STI are both antioxidants, they have different and synergistic mechanisms of action.

[0044] STI has the greatest direct antioxidant capacity, having MLT only 5% of the antioxidant capacity of STI[4], but MLT acts indirectly as a potent free radical scavenger, through the stimulation of antioxidant enzymes and preventing the decline of glutathione peroxidase, superoxide dismutase and catalase[5][6]. „Exogenously administered MLT causes a doubling of glutathione peroxidase activity within 30 minutes in rat brain“[7].

MLT and STI both act on the upregulation of heme-oxigenase-1 and, consequently, on the reduction of the intracellular level of Reactive Oxygen Species (ROS) which are potent inducers of NLRP3 inflammasome activation[22][23]. As reported by Gozzelino et al. “The cytoprotective effect [mediated by upregulation of heme-oxygenase-1] inhibits the pathogenesis of a variety of immune-mediated inflammatory diseases.”[25].

The synergistic effect between MLT and STI is typically demonstrated by Kwon et al. observing that both compounds showed a synergistic increase in heme oxygenase-1 protein expression levels, but not in heme oxygenase-1 mRNA levels compared to MLT or STI alone. It turns out that MLT can potentiate the neuroprotective effect of STI against oxidative damage[8].

[0047] In recent years, MLT and STI have also captured the attention of scientists for their protective activity on cardiac cells.

[0048] Nduhirabandi et al. emphasize the “benefits of MLT supplementation in various cardiac pathologies and cardiometabolic disorders.” [...] „Current results from experimental studies support the potential use of MLT as an adjunctive preventive and curative therapy in heart failure“[29].

[0049] According to Sun et al. “MLT has significant effects on ischemia-reperfusion injury, chronic intermittent hypoxia injury of the myocardium, pulmonary hypertension, hypertension, heart valve disease, vascular disease, and lipid metabolism. As an inexpensive and well-tolerated drug, MLT could be a new therapeutic option for cardiovascular diseases” [30].

[0050] The STI, on the other hand, “prevents cardiac hypertrophy, contractile dysfunction and remodeling [following heart failure)“[28].

[0051] Sung et al. reported that “ITS has been shown to prevent and/or slow the progression of heart failure in animal models of heart failure induced by myocardial infarction” [29].

[0052] DiBella et al. suggested that MLT, solubilized in water with adenosine and stabilized with glycine, is able to inhibit the growth of different tumors, both in vitro and in vivo [43].

[0053] More interestingly, the combination of STI and MLT also showed a synergistic action on the in vitro maturation of porcine oocytes and the subsequent development of the embryo [10] [11].

Simple co-administration of STI and MLT to Wistar rats that underwent permanent bilateral carotid artery occlusion by surgery was beneficial in induced vascular dementia by regulating the expression of brain-derived neurotrophic factors. [13].

[0055] Igarashi et al. demonstrated that “the administration of MLT to IVF (In Vitro Fertilization) patients with poor prognosis during controlled ovarian stimulation of their cycle, could improve embryo development and clinical outcomes. Considering the beneficial effect of STI on the activation of sirtuin 1, the joint administration of STI and MLT could be more favorable“[14].

[0056] Based on the fact that STI reduces the incidence of stroke, age-related macular degeneration and Alzheimer's disease and that MLT reduces the progression of cognitive impairment in these patients , Kwon et al. demonstrated that the co-administration of STI and MLT has a synergistic antioxidant effect on the intracellular levels of Reactive Oxygen Species (ROS) caused by β-amyloid, with a beneficial pharmacological effect in the treatment of patients with Alzheimer's disease[15].

[0057] According to Abais et al. “ROS may serve as a 'kindling' or trigger to activate the NLRP3 inflammasome as well as 'incendiary' or 'effector' molecules, resulting in pathological processes” [69].

As explained in detail below, the NLRP3 inflammasome is one of the main Cytokine Storm Syndrome (CTS) complexes and both MLT and STI are able to act as sensors for ROS ( Reactive Oxygen Species).

[0059] With the outbreak of the COVID-19 pandemic in early 2020, it was learned that the SARS-Cov-2 virus induces a hyper-inflammatory state caused by upregulation of interleukin-6 (IL- 6) and immunomodulatory chemokines which can cause typical bilateral interstitial pneumonia and acute heart failure with consequent shock and rapid death.

It has been elucidated that this phenomenon is due to the activation of the NLRP3 inflammasome which is a cytosolic multiprotein complex which causes the release of biologically active interleukin-1β and interleukin 18. As reported by Vaccari et al . the NLRP3 inflammasome is the primary target of COVID-19 patients as it represents the trigger that leads to cytokine storm syndrome (CTS)[16].

[0061] MLT and STI act synergistically at different biochemical levels through various mechanisms that result in inhibition of NLRP3 inflammasome assembly and activation, leading to down-modulation production of inflammatory cytokines. More specifically, MLT and STI increase sirtuin 1 (SIRT1) by regulating enzyme expression and increasing substrate specificity, respectively. SIRT1 is an important factor in the STC scenario because it acts by down-modulating the activation of NLRP3. Moreover, a relevant signal that leads to the activation of the NLRP3 inflammasome comes from the release of ROS (Reactive Oxygen Species), calcium ions and mitochondrial DNA following mitochondrial damage [16] [ 77]. Ultimately, as both MLT and STI are potent antioxidants, they protect mitochondria by absorbing ROS and preventing mitochondrial damage with the ultimate effect of promoting mitochondrial biogenesis.

[0062] Tan et al. studied in depth the role of MLT and its implication in the protection and dynamics of mitochondria. Besides scavenging activity, they established that MLT maintains optimal mitochondrial membrane potential and preserves mitochondrial functions by inhibiting mitochondrial permeability transition pores and activating uncoupling proteins. According to these authors, MLT can accumulate in mitochondria at high concentrations compared to a concentration gradient, probably by active transport into these organelles via a mitochondrial MLT transporter [75]. STI also has an important role in preventing mitochondrial damage in synergy with MLT. By activating SIRT1, STI indirectly promotes deacetylation and activation of the peroxisome proliferator activator coactivator gamma 1 alpha (PGC-1α), which is the "master regulator" of mitochondrial biogenesis, as reported Ungvari et al.[76]. Appreciated their identification of the complex biochemical mechanism that brings about the induction of mitochondrial biogenesis by STI„by activating SIRT1 and/or AMPK, increases the regulation of Nrf2-driven antioxidant enzymes and eNOS, and decreases activated NADPH oxidases. by TNF α (which are a major extramitochondrial source of ROS). By reducing oxidative stress, STI increases the bioavailability of nitric oxide, promoting the activation of PGC1a, which confers mitochondrial protection and increases mitochondrial biogenesis.”

[0063] It is now evident that negative modulation of STC is a primary target of COVID-19 therapy. Due to this hyper-inflammatory reaction, COVID-19 patients may develop clotting problems giving rise to disseminated intravascular coagulation (DIC) which causes serious and often fatal conditions called acute respiratory distress syndrome (“ARDS”) or acute lung injury (ALI).

[0064] As soon as the COVID-19 infection escalated into a pandemic, several practitioners focused their attention on MLT and STI as a potential therapeutic tool in intensive care units (ICU) to neutralize STC.

[0065] The growing interest in MLT[17][18][19][20]and STI[21][22][23]is reflected in several recent articles published in reputable scientific media.

Nevertheless, MLT and STI, in particular the latter, are characterized by low water solubility and this property has restricted its use by intravenous infusion [78].

[0067] The current availability of pharmaceutical or nutraceutical preparations of MLT and/or STI is limited to oral use, while emergency care practitioners demand that preparations for intravenous infusions be included in the standard COVID treatment protocol. -19 and are administered at the same time.

[0068] Since the plasma concentration required to reach a steady state with therapeutic activity is quite high, a safe injectable formulation of water-soluble MLTs and STIs, as a synergistic combination, appears to be necessary to urgently in serious pathological conditions, such as the hyper-inflammatory state that causes Acute Respiratory Distress Syndrome (ARDS).

The synergistic effect exerted by MLT and STI in the progressive inhibition of the activation of the NLRP3 inflammasome has been studied.

[0070] This is the key complex, at the origin of Cytokine Storm Syndrome (CTS), which is fatal.

The combination of MLT and STI acting in synergy for certain medical indications is described in existing patents. These patents, however, do not interfere with the underlying invention and are discussed below.

[0072] Patent application HK 1258525 A1 (2019-11-15) entitled: "Composition comprising resveratrol and melatonin for reducing hair loss and/or increasing hair regrowth"[24] relates to a composition comprising 0.01% to 15% STI and 0.01% to 15% MLT. It also relates to the use of said composition for reducing hair loss and/or increasing hair regrowth in a human subject. Although this state of the art mentions a composition of MLT and STI, it does not suggest any injectable use of STI and microquantities of MLT, nor the obtaining of a steady state plasma level, of the two substances. active, for the treatment of seriously ill COVID-19 patients, which is one of the objects of the invention below.

[0073] Patent application WO2010059628A1 "Water-soluble formulations of resveratrol and their uses" describes an aqueous solution containing STI, water, and an effective amount of a solubilizer containing poloxamer 334 (Pluronic® 104), in which the solubilizer makes the STI water-soluble. The solution can be used as a medicine for the treatment of cancer. This patent application is far from describing the achievement and maintenance of steady state plasma levels of STI in synergy with microquantities of MLT which is the heart of the invention below.

[0074] Patent application CA 3097468 A1 (2019-10-24) entitled: "Mixture of resveratrol supported by a metal hydroxide and pure unsupported resveratrol for the treatment of female sterility" relates to the formulation of a food supplement for the enhancement of female fertility in humans based on the use of a defined STI stoichiometry with different pharmacokinetic profiles. The mixture is used alone or in combination with nutrients with antioxidant properties and not, used in female infertility, including MLT. This disclosure does not interfere with the formulations below designed for intravenous infusion, having a different therapeutic target and under emergency conditions.

[0075] In US Patent No. 10,342,779 B2 entitled "Anhydrous liquid melatonin composition", assigned to WORPHMED SRL, Milan (IT) the pharmaceutical composition which has been described suffers from the following limitation: it claims the use of MLT up to 10% w/v, whereas the invention below focuses on micro amounts of MLT. The synergistic action of MLT and STI has not been identified in this patent. Finally, no specific action is claimed either on the Cytokine Storm Syndrome (CTS) caused by a coronavirus, or on the cardio-protection of cardiac cells irreversibly damaged due to the hyper-inflammatory state caused. by the upregulation of interleukin 6 (IL-6) and immunomodulatory chemokines in patients.

[0076] Patent application No. WO2013068565A3 entitled “Melatonin-based solutions and powders for their preparation” attributed to the Italian company Eratech Srl relates to an injectable formulation based on MLT plus at least one soluble excipient and at least one surfactant, in a mixture of water and polyalkylene glycol. This disclosure does not interfere with the intravenous infusion formulations of this invention which describe microamounts of MLT in synergy with an STI inclusion complex and which do not use any surfactant and/or other substance which facilitates the solubilization of the MLT. Finally, the therapeutic use disclosed in WO2013068565A3 encompasses neonatal cerebral infarction, sleep disorders in pediatric patients and autism spectrum disorders (ASD) which have no relation to the therapeutic uses claimed by the present invention, c ie the use in pathological conditions characterized by mass inflammation caused by STC.

[0077] Patent application No. WO2015144965 entitled “Durable preparation of an injectable melatonin exhibiting long-term stability” filed by the Servicio Andaluz de Salud Sevilla (Spain) relates to an aqueous composition of MLT exhibiting surprising long-term stability and allowing high concentrations of said water-insoluble active principle. Intravenous administration of said composition is also claimed. This composition is unrelated to those of the present invention which describes a formulation having a synergistic effect between MLT and an STI, which do not contain propylene glycol or polyethylene glycol and which may contain ascorbic acid having a preservative effect. on the degradation of MLT and STI.

[0078] Patent No. WO2012156565A1 entitled „Injectable Preparation of Melatonin“ granted to the University of Granada, Seville (SP) relates to an injectable pharmaceutical composition comprising water, propylene glycol and MLT, a derivative, a salt, prodrug or solvate thereof, which does not contain any other solvent, co-solvent or dispersing agent. This prior art focuses on the intravenous injection of highly concentrated MLT dissolved in propylene glycol (PPG) between 55 and 65 mg every 24 hours. It should be emphasized, however, that it describes neither the attainment and concomitant maintenance of steady-state plasma levels of MLT and STI, nor a method for achieving this rapidly, which is the core of the study. invention below.

[0079] Several studies report that MLT, in addition to other functions, is able to activate SIRT1 and, therefore, inhibit the NLRP3 inflammasome and the production of cytokines IL1β and IL18[39][40] [41][42]. STI is able to increase the affinity of SIRT1 for their substrates. Indeed, the synergistic action of MLT and STI in this biological system is evident.

[0080] SIRT1 is a member of a family of enzymes called sirtuins. SIRT means in English “Silent Mating Type Information Regulation” and in French „regulation d'information de type d'adaptation silent“ which has its counterpart in Saccharomyces cerevisiae (gene Sir2).

SIRT1 is mainly located in the nucleus of human cells. It deacetylates transcription factors and acts as a gene regulator by silencing and suppressing the expression of certain genes. Activation of SIRT1 reduces the expression of the NLRP3 inflammasome, which has the effect of negatively modulating the entire STC[74].

[0082] The formulation below promotes the induction of SIRT1 while the body undergoes massive STC, caused by the replication of COVID-19 virus whose invasion in the human body can lead to the death of the patient.

[0083] There is also a need to develop a therapeutic method which makes it possible to rapidly reach a steady-state plasma level of two drugs administered together, independently of their pharmacokinetic properties, even if they are very different from one another. the other.

[0084] Furthermore, it is also necessary that the plasma concentration of the two active substances be constant, administered by intravenous infusion, be maintained for several consecutive days in order to provide maximum curative benefit to patients suffering from life-threatening pathologies. , such as CTS, caused by COVID-19.

[0085] With respect to the improvement of symptoms caused by TCS, mention should be made of the disclosure of patent application no. This disclosure includes methods and compositions for ameliorating symptoms or treating infectious diseases or disease states that trigger generalized cytokine release by administering one or more pharmaceutical compositions comprising a therapeutically effective amount of a curcumin extract, curcuminoids or synthetic curcumin (S-curcumin) and derivatives thereof, and lipids dissolved or dispersed in a suitable aqueous or non-aqueous medium sufficient to reduce the level of cytokines in the host. This citation discloses various cytokine release suppressing agents, including anti-inflammatory agents such as MLT (paragraph 90) and STI (paragraph 55), but does not demonstrate that these compositions are capable of simultaneously achieving and maintaining steady-state plasma levels (with respect to MLT and STI), nor a method to achieve this quickly, which is the heart of the invention below.

[0086] US5498423A entitled "Method for correcting plasma melatonin levels and pharmaceutical formulation comprising melatonin" assigned to NEURIM PHARMACEUTICALS discloses a sustained release formulation of MLT for treating deficiency of plasma levels of MLT. This state of the art does not mention a composition of MLT and STI capable of reaching and maintaining steady state plasma levels of these two substances to treat STC, nor a method to achieve this quickly, this which is the heart of the invention below.

[0087] Patent WO2016139635A1 „A pharmaceutical composition for the parenteral administration of melatonin, and a process for its preparation“ filed by Industria Farmaceutica Galenica Senese Srl on March 4, 2016 concerns pharmaceutical compositions for the parenteral administration of MLT in the form of solutions sterile aqueous solutions, exhibiting good stability even in the absence of any stabilizing excipient and having a concentration of MLT up to high amounts, sufficient for an effective medical treatment. This state of the art does not mention a composition of MLT and a stilbene capable of achieving and maintaining the steady state plasma levels of these two substances to treat the serious condition of STC, nor a method for achieving this quickly, which is the heart of the invention below.

[0088] In fact, oral preparations of MLT or STI are unable to rapidly achieve and maintain a constant plasma level necessary to neutralize/modulate STC. Furthermore, these substances are rapidly metabolized by the liver and only a small percentage of them can reach the bloodstream, following oral administration.

[0089] Zetner et al. report that a systematic search of the literature was performed and included experimental or clinical studies investigating the pharmacokinetics of alternative routes of administration of MLT in vivo.

[0090] As indicated above, in view of the synergistic antioxidant, anti-inflammatory and cardioprotective action of STI and MLT, a combination of the two active substances has been studied, but not enough to explore the therapeutic advantages of their administration by intravenous infusion in intensive care units (ICUs).

[0091] The formulations and the therapeutic methods of this invention relate to the synergistic immunomodulatory action of microquantities of MLT and of STI while the patient, infected with the SARS-CoV-2 virus undergoes, in a massive manner, the Syndrome of the Cytokine storm (CTS), caused by the replication of SARS-CoV-2 viruses, which can rapidly lead to patient death.

More particularly, it has been discovered that the combination of these two active substances has a surprisingly strong synergistic effect, in particular on the activation of SIRT1, the neutralization of ROS (Reactive Oxygen Species) and, subsequently, inhibition of the NLRP3 inflammasome and the associated production of IL1β and IL18 cytokines.

[0093] The formulation of microquantities of MLT and STI which have additional synergistic activity appears to be a remarkable advance in the fight against the COVID-19 pandemic.

[0094] It has surprisingly been found that the administration of STI and microamounts of MLT, when infused at a certain rate, accumulate in the blood to a steady state plasma level and safely at which upregulation and activation of SIRT1 is achieved with the result of inhibiting NLRP3 inflammasome activation, and hence STC, in patients with COVID-19.

As it has been widely proven that MLT and STI act simultaneously at two different biological levels, it can be assumed that they offer a higher level of protection than each active principle administered alone.

[0096] For these reasons, we have focused on the possibility of administering MLT and STI continuously by infusing the two substances simultaneously and both contained in the same infusion bag in order to accumulate them in the plasma. of the patient until a planned plasma level is reached and maintained for several consecutive days.

The co-administration of the two active substances offers an advantage due to their synergistic action relative to their effect when they are administered separately.

[0098] In addition, the infusion of the two active substances, which makes it possible to obtain a plasma level in the state of equilibrium, is much more effective than their administration by separate injection, because of their different pharmacokinetic profiles. Specifically, MLT has a rapid elimination rate compared to STI and a single injection does not bring the desired synergistic action, since the plasma level of both active substances would not occur simultaneously.

In the event that the MLT and the STI are administered separately to the same patient, in the same quantity, by two different infusion bags, this would result in a useless and above all dangerous multiplication of the liquid introduced into the body of a patient. whose kidneys, heart and lungs are already in critical condition. In this case, it seems difficult for a healthcare professional in an intensive care unit (ICUs) to synchronize the two infusions in order to obtain an identical result.

[0100] Finally and additionally, one must consider the ergonomic treatment in the intensive care unit (ICU) where other drugs are administered by intravenous infusion to a patient who suffers from life-threatening diseases and where time is of the essence. .

[0101] The possibility of administering two active substances which act in synergy on the same target by a single infusion in formulations designed and developed to act as a single drug seems more advantageous for the patient and the doctors as well as for the nursing staff.

[0102] It has surprisingly been found that the addition of ascorbic acid to the formulation subject of this invention exerts a conservative effect on the degradation of the two active substances, MLT and STI, once they are reconstituted in the final formulation at the time of intravenous infusion.

[0103] According to the present invention, it is possible to obtain a substantial improvement or even a prevention of ARDS in COVID-19 patients, because the formulation comprises active substances at micro-concentrations whose safety profile is proven.

[0104] An important aspect of the present invention relates to the method making it possible to reach, in a planned manner, the plasma level of the two active substances together thanks to a loading dose carried out simultaneously by a higher initial infusion rate and by the oral administration of an appropriate amount of one of the two drugs (the STI).

The formulation of the present invention can be used in several emergency pathological situations thanks to the antioxidant, anti-inflammatory, immunomodulatory and protective activities of the cardiovascular tissues of MLT and STI which exert a synergistic and simultaneous action.

[0106] The formulation disclosed in the present invention may also improve convalescent plasma therapy (CPT) in COVID-19 patients.

[0107] Thanks to the endothelial protective action of MLT and STI and to that of the reduction of platelet reactivity, the formulation exerts effective cardiovascular protection in COVID-19 patients, more particularly in those whose body mass index is high.

[0108] The formulation below can be safely used as adjuvant therapy in other emergency conditions caused by viral infection, such as Ebola virus or dengue hemorrhagic fever (DHF).

[0109] Inhibition of snake venom and anti-venom oxidative damage and toxicities, allowing increased effectiveness of existing snakebite therapy, is another emergency condition in which the formulation can be administered effectively and safely.

[0110] Other emergency medical uses include, but are not limited to, severe hypoxemia or anoxia, pulmonary fibrosis, severe sepsis and septic shock of viral and bacterial origin, Herpes Zoster infections, microvascular thrombosis, hypoxic-ischemic brain injury, acute infarction, hypoxic encephalopathy of the newborn, severe burns, anaphylactic shock, spinal cord injury.

The present invention provides a pharmaceutical entity consisting of three distinct elements (A, B and C) but which represent a functional and synergistic unit (FINISHED PRODUCT): A) An amber glass bottle containing microquantities of melatonin (MLT) in liquid or freeze-dried form; B) An amber glass vial containing a lyophilized powder comprising a stilbene (STI) included in liposomes or in a cyclodextrin; C) An oral dosage form based on STI.

[0112] The FINISHED PRODUCTS (A and B) are suitable for being diluted just before being used in intravenous infusion. STI can also be any of their metabolites, such as resveratrol-glucuronide or resveratrol sulfate and pterostilbene glucuronide and pterostilbene sulfate.

[0113] The FINISHED PRODUCTS (A and B) are diluted just before use in an isotonic intravenous solution (“SOLUTION FOR INFUSION”). FINISHED PRODUCT (A and B) are first diluted by adding a small amount of Water for Injection (WFI) and, after gentle mixing, the solution of each FINISHED PRODUCT is withdrawn using a sterile syringe disposable and is then injected into a sterile 1L isotonic infusion bag.

The FINISHED PRODUCT (A) contains the MLT in freeze-dried form or dissolved in a mixture of water for injection (WFI) and an organic solvent with low toxic potential.

The FINISHED PRODUCT (B) contains the STI being complexed in a cyclodextrin or in liposomes and it is in freeze-dried form.

[0116] The two FINISHED PRODUCTS (A and B) must be in such a state that the addition of the small quantity of water for injection, during dissolution, gives a perfectly clear solution, after gentle manual mixing.

[0117] In addition, the quantity of MLT and STI of the FINISHED PRODUCTS (A and B) must be such that the two drugs are soluble in the 1L isotonic SOLUTION FOR INFUSION, without any subsequent precipitation. .

In the mixture which dissolves the MLT in the FINISHED PRODUCT (A), the organic solvent with low toxic potential must preferably belong to class 3 according to the ICH Q3C directive for residual solvents.

[0119] Considering these safety parameters, the solvent has been identified as an alcohol such as ethanol, propanol and its isomers, butanol and its isomers, pentanol and its isomers or a ketone such as acetone or methyl ethyl ketone or an organosulfur compound such as dimethyl sulfoxide (DMSO). The percentage of organic solvent in the mixture can be up to 100%.

The microquantity of the MLT in the FINISHED PRODUCT (A) depends on the following parameters: 1. The solubility of the MLT in the chosen organic solvent (in the case of the variant in liquid form). 2. The infusion rate. The higher the flow rate, the lower the concentration of MLT in the SOLUTION FOR INFUSION needed to achieve the same steady state. 3. The weight of the patient. The higher the weight, the higher the concentration of MLT in the SOLUTION FOR INFUSION needed to achieve the same steady state. 4. The degree of intoxication of the patient's liver. The higher the liver intoxication, the lower the concentration of MLT in the SOLUTION FOR INFUSION needed to achieve the same steady state, since both MLT and STI are primarily metabolized by the liver. 5. The possibility of creating a predominance in the synergistic pharmacological effect between MLT and STI.

[0121] The optimal FINISHED PRODUCT (A) (in liquid form) consists of the minimum quantity of organic solvent with low toxic potential, preferably no more than 50 μl in total, just the minimum quantity to dissolve the microquantities of MLT in order to to constitute a clear and stable solution.

The quantity of organic solvent with low toxic potential must not exceed the minimum necessary to dissolve the active substance, because the FINISHED PRODUCT (A) is subsequently diluted, before being used to produce the SOLUTION FOR INFUSION.

[0123] According to the ICH Q3C guideline, “it is considered that quantities of these residual solvents [class III solvents] of 50 mg per day or less (corresponding to 5,000 ppm or 0.5% according to option 1) would be acceptable without justification. Higher quantities may also be acceptable provided they are realistic to manufacturing capacity and good manufacturing practice.”

[0124] The FINISHED PRODUCTS (A, B and C) of this invention have been developed in compliance with the strict applicable safety rules and the unwritten “patient first” rule. Patients in general, and COVID-19 patients in particular, should not be injected with unnecessary amounts of chemicals or solvents which, in cases of massive inflammation due to Cytokine Storm Syndrome (CTS), could be even more harmful.

[0125] Consequently, the safety profiles of the FINISHED PRODUCTS (A, B and C) as well as of the SOLUTION FOR INFUSION are favorable.

Since the STI is poorly soluble in water, the STI in the FINISHED PRODUCT (B) is complexed beforehand in a cyclodextrin, preferably (2-Hydroxypropyl)-β-cyclodextrin.

[0127] Alternatively, any other cyclodextrin (α-, β- or γ-cyclodextrins) provided that, based on the quantity of STI chosen, its solubility in the SOLUTION FOR INFUSION is sufficient to obtain a perfectly clear and stable solution .

[0128] It has been reported that the inclusion complex between STI and cyclodextrin can significantly increase the solubility of STI in water [46].

[0129] As reported by Nishihira et al, since the STI is sensitive to external agents such as air, light and oxidizing enzymes, the inclusion complex with cyclodextrins can overcome this limitation. This FINISHED PRODUCT (B) drug delivery system, in addition to increasing the solubility of STI in water, further protects the molecule against oxidation and photo-decomposition. It also increases its stability and bioavailability[38].

[0130] Even if the incorporation of a hydrophobic drug in a liposome is an old technique and that there are several methods making it possible to obtain a high efficiency of trapping and to produce nanoparticles having a diameter less than 220 nm, it It was surprisingly found that the incorporation of a liposome into the STI gives rise to a stable and water-soluble active substance, suitable for an intravenous infusion formulation.

[0131] Isailović et al. describe several different methods of producing liposomes incorporating STI [47].

FINISHED PRODUCT (A) should allow MLT to be conveniently delivered to a steady state plasma concentration of 2000 pg/ml, preferably 700 pg/ml.

[0133] Due to the known solubility of MLT in organic solvents, the FINISHED PRODUCT (A) (in liquid form) must contain MLT at a concentration up to 30 mg/ml in a WFI/organic solvent mixture having a quantity of organic solvent with low toxic potential up to 50 µl (provided that the solubility of the MLT in the chosen solvent is between 5 and 10 mg/ml, ie 250-500 µg in 50 µl). As shown in FIGURE 1 below, the planned plasma concentration of approximately 700 pg/ml can be maintained by infusing approximately 300 ng of MLT/min.

This objective is achieved by infusing 1 L of the SOLUTION FOR INFUSION, the final MLT concentration of which is approximately 500 μg/L.

[0135] In other words, the SOLUTION FOR INFUSION must be prepared just before use, by diluting the 50 µl of the FINISHED PRODUCT (A) (in liquid form) or by solubilising the FINISHED PRODUCT (A) (freeze-dried) with a small amount of WFI and injecting the solution into 1L of saline solution for intravenous infusion. FIGURE 1 shows the concentration of MLT in plasma after a constant infusion of approximately 320 ng/min. According to the present invention, steady-state plasma concentrations of MLT can be anticipated by initially altering the infusion rate. Even keeping the concentration of MLT constant in the SOLUTION FOR INFUSION, the desired steady state plasma level can be anticipated by increasing the infusion rate in what can be called the „loading phase“.

[0136] In a preferred version of the present invention, the steady state plasma concentration of MLT is obtained by starting with a higher infusion rate which is gradually decreased over the first 50 minutes. At this time, the equilibrium level is reached, and the infusion rate is set to a lower fixed level. This original method gives the possibility of anticipating the moment when the steady state begins, by increasing it from around 220 min to around 45 min (considering around 98% of the plasma level at steady state) . FIGURE 2 shows the difference between steady state plasma MLT concentration with and without a loading phase.

FIGURES 3 and 4 show how the initial variation in infusion rate accelerates the achievement of the targeted plasma MLT concentration in steady state.

[0138] Conversely, due to its much higher elimination half-life than that of MLT, STI would reach steady state after 2.5 days.

The pharmacokinetics of STI administered orally is described by Sergides et al. After oral administration of 500 mg to healthy male and female volunteers, it is reported that “STI is primarily metabolized in the intestines and liver, and its major metabolites are STI glucuronides and sulfates.”

[0140] [...] According to literature data, conjugated metabolites have relevant biological activities, and the efficacy of STI is also mediated by its metabolites and by accumulation in specific tissues“[34].

The bioavailability of orally administered STI is discussed by Walle et al. They compared the oral administration of 25 mg of STI to the intravenous injection of a single bolus of 0.2 mg of STI[49]. The bioavailability of STI mixed with its conjugates (glucuronide and sulfates) is about 75% but, if we consider STI alone, the bioavailability after oral administration is about 1%, since STI is rapidly metabolized in the body. intestine and in the liver after its first passage.

[0142] However, even if the STI is rapidly transformed into its conjugates, Lu et al. showed that STI sulfate is delivered to target tissues and that the parent compound, STI, is gradually regenerated, giving it beneficial in vivo effects. According to Sergides et al[34], “metabolites [of STI] have been shown to be pharmacologically active and are therefore considered to contribute to the in vivo biological effects of the parent compound.”

[0143] This is why, given that the retro-conversion of the metabolite into the parent compound in the target tissues, also described by Das et al [36], the plasma concentration of STI must be considered as a whole, with their metabolites glucuronides and sulfates, since they also exert their pharmacological activity.

[0144] In view of the above, the SOLUTION FOR INFUSION should conveniently deliver STI and its metabolite to a steady state plasma concentration of about 500 ng/ml, preferably about 120ng/ml. Therefore FINISHED PRODUCT (B) should contain up to 2 grams of inclusion complex, preferably 500 mg. As shown in FIGURE 5, the desired plasma concentration of approximately 120 ng/ml can be maintained by infusing approximately 350 µg of STI/min.

As clearly shown in FIGURE 5, STI and its metabolites accumulate very slowly in plasma and a state of equilibrium can only be reached after several days of continuous infusion. This prerogative is not compatible with the rapid intervention that is necessary in an emergency, such as ARDS in patients with COVID-19. In intensive care units (ICUs), it is vital to achieve therapeutic plasma concentration of both drugs as quickly as possible in order to take advantage of their synergistic action on STC inhibition, as well as function protection. mitochondrial.

[0146] In another preferred variant of the present invention, in order to drastically anticipate the permanent regime of the STI and to synchronize it with the constant plasma concentration of the MLT, an oral quantity of STI, represented by the FINAL PRODUCT ( C) is administered just before starting the intravenous infusion of the two substances. Depending on the pharmacokinetic properties of STI, the amount of oral dose may be different, but in the case of STI it is preferably about 85% of the total dose infused over one day. If the infusion is chosen at a dose of 500 mg/day, the oral dose would be approximately 425 mg. Single oral administration and simultaneous infusion of STI achieve steady-state plasma concentrations after approximately 4 hours instead of 2.5 days. FIGURE 6 shows the difference between plasma STI concentration at steady state with and without a loading phase. With the oral loading dose (FINISHED PRODUCT (C)), it is possible to reach steady state 15 times faster than without a loading dose. FIGURES 7 and 8 show plasma concentrations of MLT and STI with and without a loading phase, respectively.

In another embodiment of the present invention, an appropriate amount of ascorbic acid is added as a preservative to inhibit the degradation of MLT and STI once the FINISHED PRODUCTS (A and B) are reconstituted in the SOLUTION FOR INFUSION. The amount of ascorbic acid mineral salt replaces the sodium salt of an injectable saline solution in order to maintain the iso-osmolality of the SOLUTION FOR INFUSION.

According to this last embodiment of the present invention, it provides a pharmaceutical entity consisting of four distinct elements (A, B, C and D) but which still represent a functional and synergistic unit (FINISHED PRODUCT): A) A amber glass bottle containing microquantities of MLT in liquid or freeze-dried form; B) An amber glass vial containing a lyophilized powder comprising the STI embedded in liposomes or in a cyclodextrin; C) An oral dosage form based on STI. D) A glass vial containing 10 ml of a concentrated solution of the mineral salt of ascorbic acid, ready to be withdrawn using a sterile disposable syringe and then injected into the 1L infusion bag so as to adjust the osmolality of the SOLUTION FOR INFUSION and serve as a preservative to inhibit the degradation of MLT and STI.

EXAMPLES

Example 1

Preparation of the MLT FINISHED PRODUCT

[0149] Materials and equipment: Melatonin (purity >=98%) - Sigma Aldrich - M5250 2-Propanol (purity >=99.8%) - Sigma Aldrich - 59300 250ml amber glass beakers Syringe Filters - Thomas Scientific - 1211K47 HSW Norm-Ject® Luer Lock Syringes - Thomas Scientific - 1224M80 Laboratory magnetic stirrer 2mL Amber Sample Vials Autoclaved - Thomas Scientific - 1230K78 Adjustable volume pipette 10-100 µl Sterile disposable pipettes - Thomas Scientific - 7733W58

The whole procedure is carried out in a class A clean room and in a dark environment in order to protect the MLT from photo-degradation by UV light.

94.4 mg of MLT, weighed accurately using an analytical balance, are placed in a 250 ml amber glass beaker and 78.6 g of 2-propanol are poured into it. After mixing for approximately 1-2 minutes, the clear MLT solution is filtered through a 0.22 µm cut-off filter directly into a sterile 250 ml amber glass beaker. The filtered and sterilized solution is then aliquoted into sterile 2 ml amber glass vials.

Example 2

Preparation of FINISHED PRODUCT containing liposomes incorporating STI.

The preparation is carried out according to one of the methods described by Bonechi et al. 48. The liposomal formulation was made with phospholipids and cholesterol in a molar ratio of 75:25. The whole procedure is performed at room temperature and in a dark environment to avoid photo-degradation of STI.

[0153] Materials and equipment: Trans-Resveratrol, purity >= 99% (3,5,4'-trihydroxy-trans-stilbene) US Pharmacopeia - Sigma Aldrich - 1602105 - Mw: 228.24 5α-Cholestan-3β-ol, Dihydrocholesterol, purity >= 97.5% - Sigma Aldrich - 8415130005 - Mw: 388.68 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, >=99% - Sigma Aldrich - P4329 - Mw: 734.04 Methylene chloride, purity >= 99.8% - Sigma Aldrich - 270997 Ethanol, purity 96% - European Pharmacopoeia - Sigma Aldrich - E1860100 Water for Injection (WFI) 250ml amber glass beakers Laboratory magnetic stirrer Rotary evaporator Bandelin SONOPULS HD 2070 ultrasonic homogenizer water bath Liquid Nitrogen Dewar LiposoFast device freeze dryer Syringe Filters, Millex 33mm, Durapore PVDF 0.22µm, Sterile - Thomas Scientific - 1211K47 HSW Norm-Ject® Luer Lock Syringes - Thomas Scientific - 1224M80 2mL Amber Sample Vials Autoclaved - Thomas Scientific - 1230K78 Adjustable volume pipette 10-100 µl Sterile disposable pipettes - Thomas Scientific - 7733W58

50 ml of 0.1 M STI in 96% ethanol are mixed with 50 ml of 1.2 M 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (75%) and 5α -Cholestan-3β-ol (25%).

The solution is dried under vacuum overnight. Then the dried lipid film is rehydrated by adding 100 ml of WFI and mixed. The lamellar dispersion was homogenized by eight freeze-thaw cycles in liquid nitrogen and a water bath at 50°C. Then, the solution is sonicated by five cycles of 20 s at 70% power level of the sonicator and, finally, extruded through 100 nm polycarbonate membranes (27 passes) with a LiposoFast apparatus.

The final liposomal nano-suspension is then sterilized by filtration through a 0.22 μm sterilizing filter. The sterile solution is aliquoted into the final sterile amber glass vials and lyophilized.

Example 3

Preparation of the FINISHED PRODUCT containing the STI inclusion complex in cyclodextrin.

The preparation is carried out according to one of the methods described by Kolling et al.[45]. The whole procedure is performed at room temperature and in a dark environment to avoid photo-degradation of STI.

[0158] Materials and equipment: Trans-Resveratrol, purity >= 99% (3,5,4'-trihydroxy-trans-stilbene) US Pharmacopeia - Sigma Aldrich - 1602105 - Mw: 228.24 (2-Hydroxypropyl)-p-cyclodextrin pharma grade - Cyclolab - 27 - Mw: 1135.0 + n*(58.1) ULTRA-TURRAX High Speed Homogenizer - IKA Ethanol, purity 96% - European Pharmacopoeia - Sigma Aldrich - E1860100 Water for Injection (WFI) 250ml amber glass beakers Laboratory magnetic stirrer Rotary evaporator freeze dryer Syringe filters, - Thomas Scientific - 1211K47 HSW Norm-Ject® Luer Lock Syringes - Thomas Scientific - 1224M80 2mL Amber Sample Vials Autoclaved - Thomas Scientific - 1230K78 Adjustable volume pipette 10-100 µl Sterile disposable pipettes - Thomas Scientific - 7733W58;[0159] 80 ml of 0.1 mM (2-hydroxypropyl)-β-cyclodextrin (10.38 mg or 8 µmol) are prepared in WFI by vigorous homogenization with a homogenizer at high speed ULTRA-TURRAX at 3,200 rpm while maintaining the temperature at 25°C. 20 ml containing 8 μmol of STI (1.83 mg) dissolved in 20 ml of ethanol are added with stirring and give a complete homogeneous suspension. The ethanol is evaporated under vacuum and the solution is then sterilized by filtration through a 0.22 µm sterilizing filter. The sterile solution is aliquoted into the final sterile amber glass vials and lyophilized. ;Example 4;Alternative method for the preparation of the FINAL PRODUCT containing the STI inclusion complex in cyclodextrin.;[0161] The preparation is carried out according to Hielscher ultrasonic technology. The whole procedure is performed at room temperature and in a dark environment to avoid photo-degradation of STI. ;[0162] Materials and equipment: Trans-Resveratrol, purity >= 99% (3,5,4'-trihydroxy-trans-stilbene) US Pharmacopeia - Sigma Aldrich - 1602105 - Mw: 228.24 (2-Hydroxypropyl)-β-cyclodextrin pharma grade - Cyclolab - 27 - Mw: 1135.0 + n*(58.1) UP200St - Powerful Hielscher Ultrasonic Laboratory Homogenizer Ethanol, purity 96% - European Pharmacopoeia - Sigma Aldrich - E1860100 Water for Injection (WFI) 250ml amber glass beakers freeze dryer Syringe Filters, Millex 33mm, Durapore PVDF 0.22µm, Sterile - Thomas Scientific - 1211K47 HSW Norm-Ject® Luer Lock Syringes - Thomas Scientific - 1224M80 2mL Amber Sample Vials Autoclaved - Thomas Scientific - 1230K78 Adjustable volume pipette 10-100 µl Sterile disposable pipettes - Thomas Scientific - 7733W58

4.38 mmol of (2-hydroxypropyl)-β-cyclodextrin are mixed with 4.38 mmol of STI with the minimum quantity of a mixture of solvents (96% ethanol: WFI 1:9). The mixture is sonicated for 15 minutes at 180W. The solution is sterilized by filtration through a 0.22 µm sterilizing filter, aliquoted into sterile amber glass vials and lyophilized.

Example 5

Preparing the SOLUTION FOR INFUSION

According to one embodiment of the present invention, the pharmaceutical packaging (FINISHED PRODUCT) designed in the context of this invention contains: A) An amber glass bottle containing the microquantities of MLT in liquid or freeze-dried form; B) An amber glass vial containing a lyophilized powder comprising the STI embedded in liposomes or in a cyclodextrin; C) An oral dosage form based on STI. D) A glass vial containing 10 ml of a concentrated solution of the mineral salt of ascorbic acid, ready to be withdrawn using a sterile disposable syringe and then injected into the 1L infusion bag so as to adjust the osmolality of the SOLUTION FOR INFUSION and serve as a preservative to inhibit the degradation of MLT and STI. 1. Solution (D) is injected into a 1L WFI amber sachet suitable for intravenous infusion (E). 2. 1.5 ml of WFI is injected into vial (A). The FINISHED PRODUCT (A), containing the MLT, is rehydrated or diluted and gently mixed. Then, the contents of the vial are injected into the sachet of WFI (E) 3. 1.5 ml of WFI are injected into the vial (B). The FINISHED PRODUCT (B) STI powder is rehydrated, and the solution is gently mixed. Next, the contents of the vial are injected into the bag of WFI (E) 4. Before starting the infusion, the appropriate amount of STI is administered to the patient orally (FINISHED PRODUCT (C). According to the present invention, this procedure makes it possible to drastically anticipate the attainment of steady-state plasma levels of stilbene and its derivatives.

REFERENCES

[0165] [1] „Significance of High Levels of Endogenous Melatonin in MammalianCerebrospinal Fluid and in the Central Nervous System“ - Curr Neuropharmacol. 2010 Sep; 8(3): 162-167 - Dun-Xian Tan, Lucien C Manchester, Emilio Sanchez-Barcelo, Maria D Mediavilla, and Russel J Reiter

[0166] [2] „Melatonin: Buffering the Immune System“ - Int J Mol Sci. 2013 Apr; 14(4): 8638-8683 - Antonio Carrillo-Vico, Patricia J. Lardone, Nuria Álvarez-Sánchez, Ana Rodriguez-Rodriguez, and Juan M. Guerrero

[0167] [3] „Bioactive compounds in wine:Resveratrol,hydroxytyrosol and melatonin:Areview“ - February 2012, Food Chemistry 130(4):797-813 - M.I.Fernandez-Mar, R. Matées, M.C. Garcia-Parrilla, B Puertas, E. Cantos-Villar

[0168] [4] „Contribution to determining the antioxidant capacity of melatonin inorodispersible tablets-comparison with reference antioxidants“ - Arch Med Sci 2020 Apr 5;16(4):871-877 - Herminia Muñoz, Sergio García, Adolfina Ruiz

[0169] [5] „Melatonin stimulates antioxidant enzymes and reduces oxidative stress inexperimental traumatic brain injury: the Nrf2-ARE signaling pathway as apotential mechanism“ - Free Radic Biol Med 2014 Aug;73:1-11 - Ke Ding, Handong Wang, Jianguo Xu, Tao Li, Li Zhang, Yu Ding, Lin Zhu, Jin He, Mengliang Zhou

[0170] [6] „Regulation of antioxidant enzymes:A significant role for melatonin“ - February 2004 Journal of Pineal Research 36(1): 1-9 - Carmen Rodriguez, Juan Carlos Mayo, Rosa M Sainz, Isaac Antolin

[0171] [7] „Melatonin stimulates brain glutathione peroxidase activity“ - Neurochem Int 1995 May;26(5):497-502 - L R Barlow-Walden, R J Reiter, M Abe, M Pablos, A Menendez-Pelaez, L D Chen , B Poeggeler

[0172] [8] „Melatonin synergistically increases resveratrol-induced heme oxygenase-1expression through the inhibition of ubiquitin-dependent proteasome pathway:apossible role in neuroprotection“ - J Pineal Res 2011 Mar; 50(2): 110-23 - Kyoung Ja Kwon, Jung Nam Kim, Min Kyeong Kim, Jongmin Lee, Louis J Ignarro, Hee-Jin Kim, Chan Young Shin, Seol-Heui Han

[0173] [9] „A combination of resveratrol and melatonin exerts chemopreventive effectsin N-methyl-N-nitrosourea-induced rat mammary carcinogenesis“ - Eur J Cancer Prev 2012 Mar;21(2):163-70 - Terézia Kiskova, Cem Ekmekcioglu, Miroslava Garajová, Peter Orendáš Bianka Bojková, Nikita Bobrov, Walter Jäger, Monika Kassayová, Theresia Thalhammer

[0174] [10] „Synergistic effects of resveratrol and melatonin on in vitro maturation of porcine oocytes and subsequent embryo development“ - Theriogenology 2018 Jul 1;114:191-198 - Sanghoon Lee, Jun-Xue Jin, Anukul Taweechaipaisankul, Geon A Kim , Byeong Chun Lee

[0175] [11] „Resveratrol compares with melatonin in improving in vitro porcine oocytematuration under heat stress“ - J Anim Sci Biotechnol 2016 Jun 3;7:33 - Yu Li, Jing Wang, Zhenzhen Zhang, Jinyun Yi, Changjiu He, Feng Wang, Xiuzhi Tian, Minghui Yang, Yukun Song, Pingli He, Guoshi Liu

[0176] [12] „Anti-aromatase effect of resveratrol and melatonin on hormonal positivebreast cancer cells co-cultured with breast adipose fibroblasts“ - Toxicology in Vitro Volume 28, Issue 7, October 2014, Pages 1215-1221 - Suthat Chottanapund, Panida Navasumrit, Potchanee Hunsonti, Supatchaya Timtavorn, Mathuros Ruchirawat, Martin Van den Berg

[0177] [13] „Effect of Melatonin and Resveratrol against Memory Impairment and Hippocampal Damage in a Rat Model of Vascular Dementia“ - Neuroimmunomodulation 2016;23(5-6):318-331 - Dongfang Shen, Xiaoyan Tian, Wenxu Sang, Rongrong song

[0178] [14] „Effects of melatonin and resveratrol on embryo development and clinical outcomes in poor-prognosis IVF patients“ - Fertility and Sterility Poster session | Volume 110, ISSUE 4, SUPPLEMENT, e195-e196, September 01, 2018 - H. Igarashi, N. Aono, Y. Nakajo, H. Hattori, M. Takahashi, M. Koizumi, T. Hashimoto, K. Kyono

[0179] [15] „Melatonin Potentiates the Neuroprotective Properties of ResveratrolAgainst Beta-Amyloid-Induced Neurodegeneration by Modulating AMP-ActivatedProtein Kinase Pathways“ - J Clin Neurol 2010 Sep;6(3): 127-37 - Kyoung Ja Kwon, Hee- Jin Kim, Chan Young Shin, Seol-Heui Han

[0180][16] “The Inflammasome in Times of COVID-19“ - Front. Immunol., 08 October 2020 - Juan Carlos de Rivero Vaccari, W. Dalton Dietrich, Robert W. Keane and Juan Pablo de Rivero Vaccari

[0181][17] “Targeting the NLRP3 Inflammasome in Severe Covid-19“, Freeman TL et al. School of Medicine at Mount Sinai New York - MINI REVIEW ARTICLE - Frontiers in Immunology, June 23, 2020

[0182][18] “Melatonin as a Potential Adjuvant Treatment of COVID-19?” Rev Med Virol. 2020 Apr 21 Anderson GI, Reiter Russel - Clinical Research Communication (CRC) Scotland & London - University of Texas Health Sciences at San Antonio, Texas, USA. - Rev. Med Virol. 2020 April 21

[0183] [19] „COVID-19:Melatonin as a potential adjuvant treatment“ - March 2020, Life Sciences 250: 117583 - Rui Zhang, Xuebin Wang, Leng Ni, Xiao Di, Baitao Ma, Shuai Niu, Changwei Liu, Russel J Reiter - Life Sci, 2020 Jun 1; 250; 117583

[0184] [20] „Clinical Trials for Use of Melatonin to Fight against COVID-19 Are UrgentlyNeeded“ Nutrients 2020 Aug 24; 12(9):2561 - Konrad Kleszczynski, Andrzej T Slominski, Kerstin Steinbrink, Russel J Reiter, Nutrients. 2020 Sep; 12(9): 2561

[0185] [21] „Potential therapeutic effects of Resveratrol against SARS-CoV-2“ - Acta Virol 2020;64(3):276-280 - L H Ramdani, K Bachari

[0186] [22] „Resveratrol and Copper for treatment of severe COVID-19:an observationalstudy“ - Indraneel Mittra, Rosemarie de Souza, Rakesh Bhadade, Tushar Madke, P.D. Shankpal, Mohan Joshi, Burhanuddin Qayyumi, Atanu Bhattacharya, Vikram Gota, Sudeep Gupta, Pankaj Chaturvedi, Rajendra Badwe

[0187] [23] „Caloric restriction, resveratrol and melatonin: Role of SIRT1 and implications for aging and related-diseases“ - Mech Aging Dev 2015 Mar;146-148:28-41 - Margarita R Ramis, Susana Esteban, Antonio Miralles, Dun-Xian Tan, Russel J Reiter

[0188] [24] „Composition comprising resveratrol and melatonin for reducing hair loss and/or increasing hair regrowth“ - Canada patent CA2958794A1 - Patent application HK 1258525 A1 (2019-11-15)

[0189] [25] „Melatonin enhances endogenous heme oxygenase-1 and represses immuneresponses to ameliorate experimental murine membranous nephropathy“ - J Pineal Res 2012 May;52(4):460-9 - Chia-Chao Wu, Kuo-Cheng Lu, Gu-Jiun Lin, Hsin-Yi Hsieh, Pauling Chu, Shih-Hua Lin, Huey-Kang Sytwu

[0190] [26] „Heme-oxygenase-1 upregulated by melatonin:potential protection againstburn-induced oxidative gastric mucosal injury“ - June 2015 Journal of IMAB - Annual Proceeding (Scientific Papers) 21(2):779-783 - Minka Alexandrova Hristova, Ganka Bekyarova, Maria Tzaneva

[0191] [27] „Resveratrol upregulates heme oxygenase-1 expression via activation ofNF-E2-related factor 2 in PC12 cells“ - Biochem Biophys Res Commun 2005 Jun 17;331(4):993-1000 - Chu-Yue Chen, Jung-Hee Jang, Mei-Hua Li, Young-Joon Surh

[0192][28] „Mechanisms of Cell Protection by Heme-Oxygenase-1“ - Annu. Rev. Pharmacol. Toxicol. 2010.50:323-354 - Raffaella Gozzelino, Viktoria Jeney, Miguel P. Soares

[0193][29] „Melatonin in Heart Failure:A Promising Therapeutic Strategy?“ - Molecules. 2018 Jul; 23(7): 1819 - Frederic Nduhirabandi and Gerald J. Maarman

[0194] [30] „Effects of melatonin on cardiovascular diseases: progress in the past year“- Curr Opin Lipidol. 2016 Aug; 27(4): 408-413 - Hang Sun, Aaron M. Gusdon and Shen Qu

[0195][31] “Cardioprotective Effect of Resveratrol in a Postinfarction Heart Failure Model“ – Oxid Med Cell Longev 2017; 2017:6819281 - Adam Riba 1 2, Laszlo Deres 1 2 , Balazs Sumegi 2 3 4, Kalman Toth 1 2 4, Eszter Szabados 1 2, Robert Halmosi

[0196] [32] „Therapeutic potential of resveratrol in heart failure“ - Ann N Y Acad Sci 2015 Aug;1348(1):32-45 - Miranda M Sung, Jason R B Dyck

[0197] [33] „Resveratrol and pterostilbene: A comparative overview of their chemistry, biosynthesis, plant-source and pharmacological properties“ - Journal of Applied Pharmaceutical Science Vol. 9(07), pp 124-129, July 2019 - Eric Wei Chiang Chan, Chen Wai Wong, Yong Hui Tan, Jenny Pei Yan Foo, Siu Kuin Wong, Hung Tuck Chan

[0198] [34] „Bioavailability and safety study of resveratrol 500 mg tablets in healthymale and female volunteers“ - Exp Ther Med. 2016 Jan; 11(1): 164-170 - CHRISTAKIS SERGIDES, MARINELA CHIRIL, LUIGI SILVESTRO, DAPHNE PITTA, and ANDREAS PITTAS

[0199] [35] „Pharmacokinetics,oral bioavailability,and metabolic profile of resveratroland its dimethylether analog,pterostilbene,in rats“- Cancer Chemother Pharmacol. 2011 Sep; 68(3): 593-601 - Izet M. Kapetanovic, Miguel Muzzio, Zhihua Huang, Thomas N. Thompson, and David L. McCormick

[0200] [36] „The impact of aqueous solubility and dose on the pharmacokinetic profiles of resveratrol“ - Pharm Res 2008 Nov;25(11):2593-600. - Surajit Das, Hai-Shu Lin, Paul C Ho, Ka-Yun Ng

[0201] [37] „Inclusion of trans-resveratrol in methylated cyclodextrins: synthesis and solid-state structures“ - Beilstein J Org Chem. 2014; 10: 3136-3151 - Lee Trollope, Dyanne L Cruickshank, Terence Noonan, Susan A Bourne, Milena Sorrenti, Laura Catenacci, and Mino R Caira

[0202] [38] „Resveratrol inclusion complex with β-cyclodextrin (RCD):characterization and evaluation of toxicity in wistar rats.“ - Int J Pharm Pharm Sci 9(1): 27-33. - NISHIHIRA VSK et al. 2017

[0203] [39] „Melatonin and sirtuins:A „not-so unexpected“ relationship“ - J Pineal Res 2017 Mar;62(2). - Juan C Mayo, Rosa M Sainz, Pedro Gonzalez Menéndez, Vanesa Cepas, Dun-Xian Tan, Russel J Reiter

[0204][40] „Melatonin Stimulates the SIRT1/Nrf2 Signaling Pathway CounteractingLipopolysaccharide (LPS)-Induced Oxidative Stress to Rescue Postnatal Rat Brain“- CNS Neurosci Ther 2017 Jan;23(1):33-44 - Shahid Ali Shah, Mehtab Khan, Myeung-Hoon Jo, Min Gi Jo, Faiz Ui Amin, Myeong Ok Kim

[0205] [41] „Sirtuin1 Role in the Melatonin Protective Effects Against Obesity-RelatedHeart Injury“ - Front Physiol. 2020; 11:103. - Gaia Favero, Caterina Franco, Alessandra Stacchiotti, Luigi Fabrizio Rodella, and Rita Rezzani

[0206] [42] „Induction of SIRT1 by melatonin improves alcohol-mediated oxidative liverinjury by disrupting the CRBN-YY1-CYP2E1 signaling pathway“ - J Pineal Res 2020 Apr;68(3): e12638. Sung-Eun Lee, Hong Koh, Dong Jin Joo, Balachandar Nedumaran, Hwang-Ju Jeon, Chul-Seung Park, Robert A Harris, Yong Deuk Kim

[0207] [43] „Melatonin with adenosine solubilized in water and stabilized with glycinefor oncological treatment-technical preparation, effectivity and clinical findings“- Neuro Endocrinol Lett 2017 Dec;38(7):465-474 - Giuseppe Di Bella, Luciano Gualano , Luigi DiBella

[0208] [44] „Melatonin:The Hormone of Darkness and its Therapeutic Potential andPerspectives“ - IntechOpen Book Series, 2020 - Marilena Vlachou

[0209] [45] „Resveratrol and resveratrol-hydroxypropyl-β-cyclodextrin complexrecovered the changes of creatine kinase and Na+,K+-ATPase activities found in the spleen from streptozotocin-induced diabetic rats“ - Biomedical Sciences An. Acad. Arms. Cienc. vol.91 no.3 Rio de Janeiro 2019 -Epub Sep 09, 2019 - JENIFER KOLLING et al.

[0210] [46] „Enhancing the delivery of resveratrol in humans:if low availability is theproblem,what is the solution?“ - Molecules 2014, 19, 17154-17172 - James M. Smoliga, Otis Blanchard

[0211] [47] „Resveratrol loaded liposomes produced by different techniques“ - Innovative Food Science & Emerging Technologies Volume 19, July 2013, Pages 181-189 - Bojana D.Isailović, Ivana T.Kostic, AlenkaZvonar, Verica B.Ðorđević, Mirjana Gasperlin, Viktor A. Nedović, Branko M. Bugarski

[0212][48] “Using Liposomes as Carriers for Polyphenolic Compounds:The Case of Trans-Resveratrol“ - PLoS One. 2012; 7(8): e41438. Published online 2012 Aug 22. - Claudia Bonechi, Silvia Martini, Laura Ciani, Stefania Lamponi, Herbert Rebmann, Claudio Rossi, and Sandra Ristori

[0213] [49] „High absorption but very low bioavailability of oral resveratrol in humans“- Drug Metab Dispos 2004 Dec;32(12):1377-82 - Thomas Walle, Faye Hsieh, Mark H DeLegge, John E Oatis Jr, U Kristina Walle

[0214] [50] „Gene expression of the key enzymes of melatonin synthesis in extrapinealtissues of the rat“- J Pineal Res 2001 May;30(4):243-7 - J Stefulj, M Hôrtner, M Ghosh, K Schauenstein, I Rinner, A Wölfler, J Semmler, P M Liebmann

[0215] [51] „Extrapineal melatonin:analysis of its subcellular distribution and dailyfluctuations“ - J Pineal Res 2012 Mar;52(2):217-27 - Carmen Venegas, José A Garcia, Germaine Escames, Francisco Ortiz, Ana López, Carolina Doerrier, Laura Garcia-Corzo, Luis C López, Russel J Reiter, Darío Acuña-Castroviejo

[0216] [52] „Extrapineal melatonin:sources,regulation,and potential functions“ - Cell Mol Life Sci 2014 Aug;71(16):2997-3025 - Dario Acuña-Castroviejo, Germaine Escames, Carmen Venegas, Maria E Diaz- Casado, Elena Lima-Cabello, Luis C López, Sergio Rosales-Corral, Dun-Xian Tan, Russel J Reiter

[0217][53] “Ubiquitous melatonin-presence and effects in unicells, plants and animals“- Trends Comp. Biochem. Physiol. 2, 25-45 - Hardeland R., Fuhrberg B.

[0218][54] „Melatonin in plants“ - Nutr. Rev. 2001 59, 286-290. 10.1111/j.1753-4887.2001 - Reiter R.J., Tan D.X., Burkhardt S., Manchester L.C.

[0219] [55] „Functional roles of melatonin in plants, and perspectives in nutritional and agricultural science“ - J. Exp. Bot. 2012 63, 577-597. 10.1093/jxb/err256 - Tan D.X., Hardeland R., Manchester L.C., Korkmaz A., Ma S., Rosales-Corral S., et al.

[0220] [56] „Isolation of melatonin, the pineal gland factor that lightens melanocyte S1“- J. Am. Chem. Soc. 1958, 80, 2587-2587. 10.1021/ja01543a060 - Lerner A.B., Case J.D., Takahashi Y., Lee T.H., Mori W.

[0221] [57] „Decreased MT1 and MT2 melatonin receptor expression in extrapinealtissues of the rat during physiological aging.“ - J. Pineal Res. 2009, 46, 29-35. 10.1111/j.1600-079X.2008.00604.x - Sanchez-Hidalgo M., Guerrero Montavez J. M., Carrascosa-Salmoral Mdel P., Naranjo Gutierrez Mdel C., Lardone P. J., de la Lastra Romero C. A

[0222] [58] „Physiological melatonin levels in healthy older people: a systematic review. “- J. Psychosom. Res. 2016, 86, 20-27. 10.1016/j.jpsychores. 2016.05.005 - Scholtens R. M., van Munster B. C., van Kempen M. F., de Rooij S. E.

[0223] [59] „Melatonin rhythm in human milk“ - J. Clin. Endocrinol. Metab. 1993, 77, 838-841 - Illnerova H., Buresova M., Presl J.

[0224] [60] „Extrapineal melatonin: sources, regulation, and potential functions“ - Cell Mol. Life Sci. 2014, 71, 2997-3025. 10.1007/s00018-014-1579-2 - Acuna-Castroviejo D., Escames G., Venegas C., Diaz-Casado M.E., Lima-Cabello E., Lopez L.C., et al.

[0225][61] „Presence of melatonin in human tears“ - J. Optom. 2017, 10, 3-4. 10.1016/j.optom.2016.03.002 - Carracedo G., Carpena C., Concepción P., Díaz V., García-García M., Jemni N., et al.

[0226] [62] „A nested case-control study of the association between melatonin secretion and incident myocardial infarction“ - Heart 2017, 103, 694-701. 10.1136/heartjnl-2016-310098 - McMullan C.J., Rimm E.B., Schernhammer E.S., Forman J.P.

[0227] [63] „Beneficial effects of melatonin in cardiovascular disease“ - Ann. Med. 2010, 42, 276-285 - Reiter R.J., Tan D.X., Paredes S.D., Fuentes-Broto L.

[0228] [64] „Cardioprotective effect of melatonin against ischaemia/reperfusion damage“ - Front. Biosci. (Elite Ed) 2013, 5, 305-315. 10.2741/E617 - Lochner A., Huisamen B., Nduhirabandi F.

[0229] [65] „A review of melatonin as a suitable antioxidant against myocardialischemia-reperfusion injury and clinical heart diseases“ - J. Pineal Res. 2014, 57, 357-366. 10.1111/jpi.12175 - Yang Y., Sun Y., Yi W., Li Y., Fan C., Xin Z., et al.

[0230] [66] „Effects of melatonin on cardiovascular diseases: progress in the past year“- Curr. Opinion. Lipidol. 2016, 27, 408-413. 10.1097 - Sun H., Gusdon A.M., Qu S.

[0231] [67] „Melatonin:protection against age-related cardiac pathology“ - Aging Res. Rev. 2017, 35, 336-349. 10.1016 - Favero G., Franceschetti L., Buffoli B., Moghadasian M. H., Reiter R. J., Rodella L. F., et al.

[0232] [68] „Melatonin for a Healthy Heart Rhythm“ - IntechOpen Book Series, 2020, DOI: 10.5772/intechopen.91447 - Natalia Jorgelina Prado, Margarita Segovia-Roldan, Emiliano Raúl Diez and Esther Pueyo

[0233] 69] „Redox Regulation of NLRP3 Inflammasomes:ROS as Trigger or Effector?“ - Antioxid Redox Signal. 2015 May 1; 22(13): 1111-1129 - Justine M. Abais, Min Xia, Yang Zhang, Krishna M. Boini, and Pin-Lan Li

[0234] [70] „Melatonin inhibits snake venom and antivenom induced oxidative stress and augments treatment efficacy“ - Acta Trop 2017 May;169:14-25 - Rachana D Sharma, Gajanan D Katkar, Mahalingam S Sundaram, Basavarajaiah Swethakumar, Kesturu S Girish , Kempaiah Kemparaju

[0235] [71] „Protective effects of melatonin against oxidative damage induced byEgyptian cobra (Naja haje) crude venom in rats“ - Acta Trop 2015 Mar;143:58-65 - Ahmed E Abdel Moneim, Francisco Ortiz, Roberto C Leonardo- Mendonça, Roberto Vergano-Villodres, Jose Antonio Guerrero-Martínez, Luis C López, Darío Acuña-Castroviejo, Germaine Escames

[0236] [72] „The possible effects of melatonin in Cerastes cerastes gasperettii venom-mediated toxicity and oxidative damage in mice“ - Current Science Vol. 110, No. 8 (25 April 2016), p. 1505-1512 - Mohamed K. Al-Sadoon, Marwa M. S. Diab, Amira A. Bauomy, Ahmed E. Abdel Moneim and Bilal A. Paray

[0237] [73] „Influence of glucuronidation and reduction modifications of resveratrol on its biological activities“ - Chembiochem. 2013;14:1094-1104 - Lu DL, Ding DJ, Yan WJ, Li RR, Dai F, Wang Q, Yu SS, Li Y, Jin XL, Zhou

[0238] [74] „Negative regulation of NLRP3 inflammasome by SIRT1 in vascularendothelial cells“ - Immunobiology 2017 Mar;222(3):552-561 - Yanxiang Li, Xiaofeng Yang, Yanhao He, Weirong Wang, Jiye Zhang, Wei Zhang, Ting Jing, Bo Wang, Rong Lin

[0239][75] „Melatonin:A Mitochondrial Targeting Molecule Involving MitochondrialProtection and Dynamics“ - Int J Mol Sci. 2016 Dec; 17(12): 2124 - Dun-Xian Tan, Lucien C. Manchester, Lilan Qin, and Russel J. Reiter

[0240][76] „Mitochondrial Protection by Resveratrol“ - Exerc Sport Sci Rev. 2011 Jul; 39(3): 128-132. - Zoltan Ungvari,1 William E. Sonntag,1 Rafael de Cabo,2 Joseph A. Baur,3 and Anna Csiszar

[0241][77] „The role of mitochondria in NLRP3 inflammasome activation“ - Mol Immunol 2018 Nov;103:115-124 - Qiuyun Liu, Danyan Zhang, Diyu Hu, Xiangmei Zhou, Yang Zhou

[0242] [78] „Administration of resveratrol:What formulation solutions to bioavailabilitylimitations?“ - J Control Release 2012 Mar 10;158(2):182-93 - A Amri, J C Chaumeil, S Sfar, C Charrueau

Claims (29)

1. Pharmaceutical formulation comprising a stilbene inclusion complex (STI) in synergy with microquantities of melatonin (MLT). 2. The pharmaceutical formulation of claim 1 is further characterized in that MLT and STI have a purity content of >98% and that STI is preferably trans-resveratrol (3,5,4'-trihydroxy-trans- stilbene), not limited to pterostilbene (3,5-dimethoxy-4'-hydroxy-trans-stilbene). 3. The pharmaceutical formulation of claim 1 best identified as a finished product (FINISHED PRODUCT), containing the two ingredients, MLT and STI, in either concentrated or dry form and in separate vials, each of which may be resuspended or dissolved before use, before being further diluted and finally in a final liquid (DILUENT INJECTABLE) to constitute a single solution for intravenous infusion (SOLUTION FOR INFUSION), to be administered to a human being. 4. The FINISHED PRODUCT according to claim 3 is further characterized in that STI is in the form of a freeze-dried powder and that the quantity of STI is 2 grams at the most, preferably 500 mg. 5. The SOLUTION FOR INFUSION according to claim 4 is further characterized in that STI is a stable inclusion complex, made with cyclodextrin which can be any cyclodextrin (α-, β- or γ-cyclodextrins) provided that, based on the quantity and type of STI chosen, its solubility in the SOLUTION FOR INFUSION is sufficient to obtain a completely clear and stable solution. 6. The inclusion complex according to claim 4 is further characterized in that STI is a stable inclusion complex in liposomes, which are composed of a mixture of phospholipids and cholesterol derivatives. 7. The pharmaceutical formulation containing the MLT according to claim 2 is further identified as being a FINISHED PRODUCT in the form of a lyophilized powder or in a liquid form. 8. The FINISHED PRODUCT containing MLT in liquid form according to claim 7 is characterized in that the solvent is a mixture of water for injection (WFI) and an organic solvent with low toxic potential. 9. The FINISHED PRODUCT containing the MLT in liquid form according to claim 8 is further characterized in that the organic solvent with low toxic potential can be an alcohol such as ethanol, propanol and its isomers, butanol and its isomers , pentanol and its isomers or a ketone such as acetone or methyl ethyl ketone or an organosulfur compound such as dimethyl sulfoxide (DMSO) or any other organic solvent with low toxic potential belonging to class 3 of the ICH Q3C directive for residual solvents. 10. The FINISHED PRODUCT containing the MLT in liquid form according to claim 9 is further characterized in that the content of the organic solvent with low toxic potential is in a percentage ranging up to 100%. 11. The FINISHED PRODUCT containing the MLT in liquid form according to claim 10 is typically characterized in that the content of organic solvent with low toxic potential of the SOLUTION FOR INFUSION is such that the quantity injected never exceeds 5,000 ppm or 0.5%, preferably not more than 50 mg/day. 12. The FINISHED PRODUCT containing MLT in liquid form according to claim 7 is further specified that the concentration of MLT is up to 30 mg/ml, depending on the degree of solubility of MLT in the chosen solvent, preferably between 5 and 10mg/ml. 13. The FINISHED PRODUCT containing the MLT in freeze-dried form according to claim 3 is further characterized in that the amount of MLT is up to 1500 µg, preferably between 250 and 500 µg. 14. The SOLUTION FOR INFUSION of claim 3 is designed to be an administrable parenteral formulation, preferably an intravenous infusion formulation suitable for safe administration over several consecutive days. 15. The active pharmaceutical ingredients of claim 2 comply with the endotoxin requirements of max. 300 I.U./gram and to the microbiological count text for a total aerobic microbial count („TAMC“) of maximum 10-2 CFU/gr and a total combined yeast and mold count („TYMC“) of maximum 10 CFU/g . 16. The INJECTABLE DILUENT of claim 3 may be a one liter saline infusion bag (0.9% isotonic saline solution) or, alternatively, a one liter WFI infusion bag in which a concentrated solution sterile ascorbate salt (ASC) was injected to obtain an isotonic solution. While in the first variant the SOLUTION FOR INFUSION consists of MLT, STI and salt (0.9%), in the second variant the SOLUTION FOR INFUSION consists of MLT, STI and ASC. 17. The ASC of claim 16 is used as a preservative to prevent degradation of MLT and STI, once reconstituted in SOLUTION FOR INFUSION, prior to infusion into the patient. The amount of ASC is such as to ensure the isotonicity of the SOLUTION FOR INFUSION, but it should not exceed 3 grams. 18. The SOLUTION FOR INFUSION according to claims 3 and 16 for administration by infusion should allow the convenient administration of STI and its metabolites to a steady state plasma concentration of up to 500 ng/ml. , preferably up to about 120 ng/ml. 19. The SOLUTION FOR INFUSION according to claims 3 and 16 should allow easy intravenous administration of an amount of MLT to achieve a patient's steady-state plasma concentration of up to 2000 pg/ ml, preferably up to about 700 pg/ml. 20. A method of administering the SOLUTION FOR INFUSION containing the pharmaceutical formulation of claim 1, in order to achieve and maintain the patient's plasma concentration of MLT and STI in a targeted steady state in a planned manner. 21. The method of claim 20 is characterized in that the patient's steady-state plasma concentration of MLT is anticipated by starting with a higher infusion rate (up to about 5 times higher, from preferably up to about 3 times higher) which is gradually decreased until the targeted steady state is achieved, with the infusion rate set to a lower fixed level. 22. The method of claim 21 is characterized by the fact that the steady state plasma concentration of STI is anticipated by oral administration of STI just before starting the infusion of MLT and STI. The amount of this oral dose of STI being between 50% and 150%, preferably 85% of the total dose of STI infused over a day. 23. The INFUSION SOLUTION of claims 3 and 16 for administration of intravenous infusion in humans, suitable for drastically reducing Cytokine Storm Syndrome (CTS) in life-threatening conditions, such as Acute Respiratory Distress Syndrome (ARDS) in COVID-19 patients. 24. The INFUSION SOLUTION of claims 3 and 16 for the administration of intravenous infusion in humans, suitable for reducing platelet reactivity, providing effective cardiovascular protection in COVID-19 patients, in particular in patients having a high body mass index. 25. The SOLUTION FOR INFUSION of claims 3 and 16 for administration of intravenous infusion in humans, suitable for enhancing convalescent plasma therapy (CPT) in COVID-19 patients which comprises the administrable solution for intravenous infusion of any of the claims. 26. The SOLUTION FOR INFUSION of claims 3 and 16 for the administration of the intravenous infusion in humans, capable of preventing hemorrhages in critical health conditions due to Ebola hemorrhagic fever (EHF), Dengue hemorrhagic fever (FHD) and neonatal hydrocephalus due to Zika. 27. The INFUSION SOLUTION of claims 3 and 16 for administration of intravenous infusion in humans, suitable for inhibiting snake venom and anti-venom oxidative damage and toxicities, providing increased efficacy of the existing therapy for poisonous snakebites. 28. The SOLUTION FOR INFUSION of claims 3 and 16 for the administration of the intravenous infusion in humans, capable of inhibiting the toxicity of the drug interaction which triggers the anaphylactic reaction following the cascade of cytokines in patients in intensive care units (ICUs) and in hospitalized patients. 29. Other emergency medical uses of the FINISHED PRODUCT according to claims 3 and 16 include, but are not limited to, severe hypoxemia or anoxia, pulmonary fibrosis, severe viral and bacterial sepsis and septic shock, herpes zoster infections, microvascular thrombosis, hypoxic-ischemic brain injury, infarction, hypoxic encephalopathy of the newborn, severe burns, anaphylactic shock, spinal cord injury.