WO2020067868A1 - Use of cd38 enzyme inhibitors in hiv-positive patients - Google Patents

Use of cd38 enzyme inhibitors in hiv-positive patients Download PDF

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Publication number
WO2020067868A1
WO2020067868A1 PCT/MX2019/050020 MX2019050020W WO2020067868A1 WO 2020067868 A1 WO2020067868 A1 WO 2020067868A1 MX 2019050020 W MX2019050020 W MX 2019050020W WO 2020067868 A1 WO2020067868 A1 WO 2020067868A1
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cells
hiv
nad
inhibitor
hiv infection
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PCT/MX2019/050020
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Spanish (es)
French (fr)
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Héctor Enrique ESPINOSA ARCINIEGA
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Instituto Nacional De Enfermedades Respiratorias Ismael Cosio Villegas
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • the present invention is generally related to the medical industry and to applied research in immunology, specifically in immunometabolism, specifically with the manufacture of treatments against viral infections. Particularly within the industry and manufacturing of therapies for the treatment of HIV infection.
  • a subject of the present invention is the use of CD38 enzyme inhibitors for the manufacture of targeted drugs for people with HIV infection. It includes inhibitors from traditional Mexican foods or high production fruits and / or vegetables in the country.
  • HIV infection and AIDS are a global pandemic, with around 36.7 million people living with the virus in 2016 (WHO, 2018).
  • WHO World Health Organization
  • 655 accumulated cases of HIV infection had been registered, of which 135,515 were still alive: 62,916 cases were reported with HIV infection and 72,599 with progression to AIDS (WHO, 2018) .
  • ART Antiretroviral Therapy
  • CD38 a membrane ectoenzyme, as an indicator of HIV pathogenesis
  • CD38 expression on the cell surface has been considered as a marker of progression to AIDS (Tilling et al., 2002).
  • the biological functions of CD38 have not been considered in these analyzes, nor has the link of a causal mechanism been studied.
  • CD38 has been shown to be a better predictor than viral load, various soluble mediators, and the percentage of activated T cell phenotypes (Giorgi et al., 1999; Karim et al., 2013; Liu et al., 1997).
  • CD38 is an enzyme with a biological role in the pathogenesis of HIV infection. It is important to emphasize the emphasis on the distinction between HIV infection and AIDS.
  • AIDS is the consequence of uncontrolled HIV infection, while HIV infection and "HIV disease" are terms that encompass infection without clinical manifestations and infection under treatment. antiretroviral. This discrimination was used when reviewing the scientific literature and the industrial property gazettes presented below as a direct background to the invention.
  • CD38 as an ectoenzyme
  • Savarino et al (2000) present a systematic review of the role of CD38 in HIV infection.
  • the authors describe CD38 as an ectoenzyme, an adhesion receptor, and a signaling molecule in HIV pathogenesis.
  • Savarino et al. describe CD38 as a molecule with an active role in HIV infection; Acting as a compensatory molecule that catalyzes the synthesis of nicotinamide as a mechanism for inhibiting viral replication, the work is speculative in nature, considering only in vitro infection and not chronic infection.
  • In vitro infection studies are hardly translatable to the phenomenon observed in vivo.
  • CD38 as a therapeutic target
  • CD38 has studied CD38 for its main catalytic enzymatic function, the hydrolysis of NAD +, resulting in the synthesis of second messengers cADPR, ADPR & NAADP (W02013002879 to Sinclair, David et al).
  • This document discloses the therapeutic use of small molecules as CD38 inhibitors in order to increase the concentration of NAD in cells.
  • the treatments he mentions include
  • AIDS is one of the conditions that can be treated, this statement is within a generic list of conditions. This is evident when seeing that the ailments that precede the mention of AIDS are qlaucoma and macular degeneration; and that those that follow the mention are fulminant hepatitis and diseases related to the brain (see page 64, first paragraph) (W02013002879, 2013).
  • AIDS is only one of the possible consequences of HIV infection.
  • HIV-related conditions are untreated asymptomatic infection, infection with opportunistic disease control, disease successfully treated with highly active antiretroviral therapy, as well as "Non-AIDS-Defining Illnesses" in people with HIV (even without treatment).
  • Sinclair et al disclose a wide range of CD38 inhibitors, among them are kuromanin, luteolin and luteolinidine as part of a generic listing. The present invention focuses on the prevalence of anthocyanins in fruits and vegetables of high production in Mexico or in traditional Mexican foods.
  • Sinclair et al claim as their goal the increase in NAD levels in a general way, without mentioning the relationship between overexpression of CD38, a characterizing condition of HIV, with its use. claimed. In other words, Sinclair et al do not consider CD38 as a biologically functional enzyme in HIV infection. Finally, they do not report decreased exposure of the individual's T cells to the second CD38 messengers. It is important to mention that there is no causal relationship between both events.
  • T cell memory 5 Center T cell memory 5 Center stimulated fail to express CD154, CD40 ligand, when induced to express cytokines as well as IL-2. These differences were amplified in cells from HIV patients expressing CD38. This suggests a possible CD38-mediated pathogenesis mechanism, in which the dissociation between cytokine and CD154 expression in CD4 CM T cells may impede interaction between T cells and antigen presenting cells. This in turn contributes
  • CD38 has been characterized as a biomarker of HIV progression.
  • the present invention considers CD38 as an enzyme involved in HIV pathogenesis by removal of intracellular NAD.
  • Catalytic inhibition of CD38 markedly decreases death by activation of central memory CD4 T cells in cells of an HIV positive patient.
  • Inhibition of CD38 increases the capacity of the core memory cells of an HIV patient (without treatment) to differentiate into effector memory cells, a crucial homeostatic function in the regeneration of the immune system.
  • Figure 1 shows how the core memory CD4 T cells of HIV patients overexpress both CD38 and NAMPT.
  • Figure 2 shows that the concentration of NAMPT in the blood is positively correlated with the number of CD4 T cells per microliter of blood from HIV patients.
  • Figure 3 shows how the competitive inhibitor of the CD38 enzyme can totally inhibit its catalytic activity in human lymphocytes that express CD38.
  • FIG 4 shows that inhibition of CD38 catalytic activity markedly decreases death by activation of CD4 T C M cells from an HIV patient.
  • Figure 5 shows that CD38 inhibition increases the capacity of memory cells. central (TCM) of an HIV patient (untreated) to differentiate into effector memory cells (TEM).
  • FIG. 6 shows that kuromanin (naturally occurring anthocyanin) inhibits the catalytic activity of CD38 in Peripheral Blood Mononuclear Cells (CMNSPs), proportionally to its concentration.
  • CNSPs Peripheral Blood Mononuclear Cells
  • Figure 7 shows that anthocyanin lutoelin inhibits CD38 more than ara-F-NAD at the same concentration.
  • Figure 8 shows that kuromanin can reduce the catalytic activity of CD38 also in human lymphocytes.
  • the present invention is based on a new approach towards the pathogenesis of HIV, considering CD38 as an enzyme with a biological function, and therefore, a therapeutic target for the treatment of HIV.
  • CD38 the functional enzyme
  • NAMPT overexpression indicates that the cell is seeking to reestablish homeostasis.
  • overexpression of CD38 leads to an increase in the number of CD38 molecules on the surface of CD4 + T C M cells and therefore, there are a greater number of enzymes in contact with the blood plasma.
  • HIV infection is characterized by chronic activation of CD4 + CM T cells. CD4 T cells from a subject with HIV are found in said microenvironment.
  • CD4 + T C M cells are affected by this microenvironment and the constant excretion of intracellular NAD, which causes additional stress to the mitochondria of cells. These conditions favor an adaptation of the energetic metabolism of the cells, the appearance of the Warburq effect having already been documented. This effect is characterized by the adaptation of oxidative phosphorylation to aerobic glycolysis and therefore a decrease in mitochondrial respiration. This leads to possible involvement of the mitochondrial crest and fusion between mitochondria.
  • the Warburq effect may be one of the causes that contributes to cell death.
  • CD4 + from HIV patients. Subsequently, we sought to determine the enzymatic kinetics of CD38 by using an inhibitor. As shown in Figure 3, the addition of CD38 inhibitors enables the CD38 + cell to behave similarly to a CD38- cell. Similarly, recent findings from our laboratory find that CD38- T C M CD4 cells respond to stimulation by inducing more MYC (a survival promoter) and IL-2 than CD38 +. Derived from the same, the results supported the initial hypothesis, CD38 inhibitors lead to better maintenance of the immune system (Figure 4). Importantly, we have found that CD38 inhibition in T C M CD4 cells of patients increases their survival and their differentiation to memory effector cells (Figure 5), two of their homeostatic functions.
  • NAMPT nicotinamide phosphoribosyltranseferase
  • CD38-expressing and non-expressing lymphocytes were purified by the fluorescence-activated cell sorting method (FACS) taking into account the light scattering and fluorescence properties associated with CD38 extracellular expression .
  • FACS fluorescence-activated cell sorting method
  • the cells were suspended in buffer A in the absence or presence of different concentrations of the CD38 catalytic inhibitor b-ara- 2'- deoxy-2'- fluoro-nicotinamide adenine dinucleotide (ara-F-NAD).
  • Figure 3 shows the fluorescence, proportional to the amount of product, against time, for human lymphocytes expressing CD38 without inhibitor (upper line) and with ara-F-NAD concentrations of 100 times and a thousand times the constant of inhibition (Ki).
  • the negative signal absence of catalytic activity of CD38 used the fluorescence produced
  • lOOOKi correspond to a concentration of 1.69 micromolar.
  • Core memory T cells were purified from peripheral blood mononuclear cells from HIV patients without antiretroviral treatment. These cells were subjected to two-step purification on immunomagnetic columns. The purity was greater than 90%.
  • TCR T-cell receptor
  • the core memory T cells were:
  • CD45RA and CCR7 whose expression pattern differs between CD4 T cell subpopulations with different degrees of differentiation. For example, cells
  • CMNSPs were separated as in experiments 2 and 3, and incubated with different conditions of the catalytic inhibitors. A more detailed description of each experiment as well as the
  • Figures 1-8 provide the experimental results obtained that support the role of CD38 as an active enzyme in HIV pathogenesis.
  • Figure 1 shows the difference in expression of messenger RNA of various genes, expressed as how many times more messenger RNA than in healthy control cells.
  • the central memory CD4 T cells of HIV patients overexpress both CD38 (NAD-removing enzyme) and NAMPT (nicotinamide-phosphoribosyl transferase or visphatin, synthesizing enzyme).
  • CD38 tenth gene on the X axis
  • NAMPT is the enzyme in a NAD synthesis pathway that determines the amount synthesized. This suggests that behind the negative correlation of CD38 with the preservation of CD4 T cells is a decrease in NAD and also a decrease in NAD
  • Figure 1 shows the graph of CD4 T cell counts against NAMPT concentration in plasma of HIV patients without treatment, from two cohorts with different stages of progress. This suggests that, while NAD synthesis is associated with survival of these cells, CD38, an enzyme that eliminates NAD, is negative for their survival. This supports the idea that CD38, through its catalytic activity, decreases the survival of CD4 T cells in HIV infection. In white circles, the points corresponding to a chronic cohort (Pearson regression).
  • Figure 3 shows the enzymatic kinetic determination of the minimum inhibitory concentration of the CD38 inhibitor ara-F-NAD, measured as CD38 conversion of nicotinamide 1, N6-ethenoadenine-etheno dinucleotide (ethene-NAD) in a fluorescent product.
  • the graph shows how a competitive inhibitor can inhibit the catalytic activity of CD38 at levels of lymphocytes that do not they express the enzyme.
  • the graph shows the fluorescence, proportional to the amount of product, against time, for human lymphocytes expressing CD38 without inhibitor (upper line) and with the
  • ara-F-NAD concentrations 100 Ki (Ki is the inhibition constant) and 1000 Ki, all compared to the background fluorescence, determined with non-CD38-expressing human lymphocytes (absence of CD38 activity, bottom line). Histograms representing the number of cells labeled with a viability probe, corresponding to core memory T cells that were cultured seven days after receiving polyclonal stimulation of the T cell receptor with an anti-CD38 agonist antibody, are shown in Figure 4. and an anti-CD28 agonist antibody. The peak on the right in each histogram corresponds to dead cells (which fix more marker) and the peak on the left corresponds to living cells. The numbers indicate the percentage.
  • B Cells grown with CD38 catalytic inhibitor.
  • Figure 5 shows graphs of CD45RA expression versus CCR7 expression in cultured central HIV memory T cells from a patient with 7 days in medium (A) or in medium with more CD38 inhibitor (ara-F-NAD to lOOKi). Memory cells do not express CD45RA. Central memory cells express CCR7 (right box on each graph), while effector memory T cells (obtained after 7 days of culture after stimulation) do not express it.
  • Figure 6 shows how kuromanin (naturally occurring anthocyanin) inhibits the catalytic activity of CD38, proportionally to its concentration.
  • CNSPs human peripheral blood mononuclear cells
  • ara-F-NAD upper line, black
  • Next line same cells in the presence of ara-F-NAD in 10 micromolar content. 3. Next line (gray) Same cells with a 20 micromolar concentration of
  • Kuromanina 4. Bottom line: same cells in the presence of 1.69 micromolar ara-F-NAD, an inhibitor used in our in vitro studies, capable of decreasing activity to that of the negative control (cells that do not express CD38).
  • B 1. upper line positive control of catalytic activity, consisting of peripheral blood mononuclear cells (CMNSPs) human expressing CD38, from a healthy donor, in the absence of the inhibitor (upper line, black).
  • CNSPs peripheral blood mononuclear cells
  • Next line down 1.69 micromolar ara-F-NAD CMNSPs.
  • 3 Cells with 50microM (light gray, third line) of kuromanin.
  • 4 Cells with and lOOmicromolar kuromanin (black, bottom line).
  • Figure 7 shows that anthocyanin lutoelin inhibits CD38 more than ara-F-NAD at the same concentration.
  • the upper line indicates the production of fluorescent derivative of ethene-NAD by CD38 from human peripheral blood mononuclear cells.
  • the intermediate line corresponds to the inhibited catalytic activity in the presence of ara-F-NAD.
  • the lower (dark) line corresponds to the catalytic activity inhibited by luteolin.
  • Figure 8 shows that kuromanin can reduce the catalytic activity of CD38 also in human lymphocytes.
  • the upper line black
  • the intermediate line corresponds to basal activity, corresponding to human lymphocytes that do not
  • the bottom line corresponds to the activity of human lymphocytes expressing CD38 in the presence of kuromanin at a concentration of 50 micromolar.

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Abstract

The invention relates to the use of CD38-inhibitor anthocyanins for producing a medicament for treating HIV infection. This second use is derived from the reconceptualisation of the role of CD38 as a biologically functional enzyme, supported by experimental tests that show how catalytic inhibition reduces the death of CD4 T cells and increases the capacity of same to differentiate into effector memory cells. These effects support homeostatic functions that are crucial in the regeneration of the immune system. Catalytic inhibitors come from berries, in particular, kuromanin, luteolin and luteolinidin, or traditional Mexican food such as blue corn, and can be used to inhibit the expression of CD38 even at basal levels.

Description

USO DE INHIBIDORES ENZIMÁTICOS DE CD38 EN PACIENTES  USE OF CD38 ENZYMATIC INHIBITORS IN PATIENTS
VIH POSITIVOS  HIV POSITIVES
CAMPO DE LA INVENCIÓN FIELD OF THE INVENTION
La presente invención se relaciona generalmente con la industria médica y con la investigación aplicada en inmunologia, en concreto en inmunometabolismo, en especifico con la fabricación de tratamientos contra infecciones virales. En particular dentro de la industria y fabricación de terapias para el tratamiento de la infección por VIH. The present invention is generally related to the medical industry and to applied research in immunology, specifically in immunometabolism, specifically with the manufacture of treatments against viral infections. Particularly within the industry and manufacturing of therapies for the treatment of HIV infection.
La presente invención tiene por objeto el uso de inhibidores enzimáticos de CD38 para la fabricación de medicamentos enfocados en personas con infección por VIH. Comprende inhibidores provenientes de alimentos tradicionales mexicanos o frutas y/o vegetales de alta producción en el pais. A subject of the present invention is the use of CD38 enzyme inhibitors for the manufacture of targeted drugs for people with HIV infection. It includes inhibitors from traditional Mexican foods or high production fruits and / or vegetables in the country.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
La infección por VIH y el SIDA son una pandemia global, con alrededor de 36.7 millones de personas viviendo con el virus en 2016 (WHO, 2018) . En México 5 en el mismo año, se hablan registrado 186, 655 casos acumulados de infección por VIH, de los cuáles 135,515 aún se encontraban con vida: siendo 62,916 casos reportados con infección por VIH y 72,599 con progresión hacia SIDA (WHO, 2018) . Esto representa un lOaumento respectivo de 5,145 y 4,112 casos en comparación con 2015. Se estima que en el país alrededor de 140 mil personas con VIH tienen acceso a la Terapia Antirretroviral (TARV) (CENSIDA, 2017), sin embargo, hay un segmento considerable de la población HIV infection and AIDS are a global pandemic, with around 36.7 million people living with the virus in 2016 (WHO, 2018). In Mexico 5 in the same year, 186, 655 accumulated cases of HIV infection had been registered, of which 135,515 were still alive: 62,916 cases were reported with HIV infection and 72,599 with progression to AIDS (WHO, 2018) . This represents a respective increase of 5,145 and 4,112 cases compared to 2015. It is estimated that around 140 thousand people with HIV in the country have access to Antiretroviral Therapy (ART) (CENSIDA, 2017), however, there is a considerable segment of the population
15 infectada que no ha sido diagnosticada aún. El diagnóstico y tratamiento tempranos de la infección por VIH son aspectos críticos para evitar la progresión hacia SIDA (WHO, 2018) . 15 infected that has not been diagnosed yet. Early diagnosis and treatment of HIV infection are critical aspects to prevent progression to AIDS (WHO, 2018).
Dentro del contexto mexicano y en especifico del 20 de los Institutos Nacionales de Salud, el Instituto Nacional de Enfermedades Respiratorias (INER) "Ismael COSÍO Villegas" es quién provee atención al mayor número personas con infección por VIH y que presentan SIDA (INER, 2017) . Breve descripción de CD38 Within the Mexican context and specifically from 20 of the National Institutes of Health, the National Institute of Respiratory Diseases (INER) "Ismael COSÍO Villegas" is the one who provides care to the greatest number of people with HIV infection and who have AIDS (INER, 2017 ). CD38 Brief Description
Numerosas lineas de investigación enfocadas a células T CD4 mostraron la relevancia de CD38, una ectoenzima de membrana, como un indicador de lapatogénesis de VIH (Karim et al., 2013) . CD38 ha sido utilizado como un biomarcador relevante en el avance del VIH, habiendo un amplio cuerpo que sustenta la correlación entre la expresión de CD38 y el avance de la enfermedad. En especifico, la expresión de CD38 en la superficie celular se ha considerado como un marcador de la progresión hacia SIDA (Tilling et al., 2002) . Sin embargo, no se han considerado las funciones biológicas de CD38 en dichos análisis, ni se ha estudiado el vinculo de un mecanismo causal Numerous lines of research focused on CD4 T cells showed the relevance of CD38, a membrane ectoenzyme, as an indicator of HIV pathogenesis (Karim et al., 2013). CD38 has been used as a relevant biomarker in the advancement of HIV, with a broad body supporting the correlation between CD38 expression and disease progression. Specifically, CD38 expression on the cell surface has been considered as a marker of progression to AIDS (Tilling et al., 2002). However, the biological functions of CD38 have not been considered in these analyzes, nor has the link of a causal mechanism been studied.
entre ambas variables.  between both variables.
De igual manera, se ha mostrado que CD38 es un mejor predictor que la carga viral, diversos mediadores solubles y el porcentaje de fenotipos activados de células T (Giorgi et al., 1999; Karim et al., 2013; Liu et al., 1997) . Como se mostrará en esta sección, dicho paradigma sobre CD38 difiere fundamentalmente de la presente invención, en especifico por el hecho de que consideramos a CD38 como una enzima con una función biológica en la patogénesis de la infección por VIH. Es importante recalcar el énfasis en la distinción entre la infección por VIH y el SIDA. Para un experto en el campo de la inmunologia, el SIDA es la consecuencia de una infección por VIH no controlada, mientras que la infección por VIH y la "enfermedad por VIH" son términos que abarcan la infección sin manifestaciones clinicas y la infección bajo tratamiento antirretroviral . Se utilizó esta discriminación al revisar la literatura cientifica y las gacetas de propiedad industrial que se presentan a continuación como antecedentes directos de la invención . Similarly, CD38 has been shown to be a better predictor than viral load, various soluble mediators, and the percentage of activated T cell phenotypes (Giorgi et al., 1999; Karim et al., 2013; Liu et al., 1997). As will be shown in this section, such a CD38 paradigm differs fundamentally from the present invention, specifically in that we consider CD38 as an enzyme with a biological role in the pathogenesis of HIV infection. It is important to emphasize the emphasis on the distinction between HIV infection and AIDS. For an expert in the field of immunology, AIDS is the consequence of uncontrolled HIV infection, while HIV infection and "HIV disease" are terms that encompass infection without clinical manifestations and infection under treatment. antiretroviral. This discrimination was used when reviewing the scientific literature and the industrial property gazettes presented below as a direct background to the invention.
CD38 como una ectoenzima CD38 as an ectoenzyme
Savarino et al (2000) presentan una revisión sistemática del rol de CD38 en la infección por VIH. Los autores describen a CD38 como una ectoenzima, un receptor de adhesión y una molécula de señalización en la patogénesis del VIH. A pesar de que Savarino et al. describen a CD38 como una molécula con un papel activo en la infección de VIH; actuando como una molécula compensatoria que cataliza la sintesis de nicotinamida como mecanismo de inhibición de la replicación viral, el trabajo es de carácter especulativo al considerar únicamente la infección in vitro y no la infección crónica. En materia de aplicación, los estudios de infección in vitro son difícilmente traducibles al fenómeno observado in vivo. En segunda instancia, la asociación lateral entre CD4 y CD38 que los autores reclaman como una manera de proteger a las células de la infección es insuficiente para el avance inexorable de la infección por VIH no tratada. Asimismo, sustentan un posible rol activo de CD38 mediante ensayos in vitro murinos, lo cual no es directamente aplicable al fenómeno biológico-clinico . Savarino et al (2000) present a systematic review of the role of CD38 in HIV infection. The authors describe CD38 as an ectoenzyme, an adhesion receptor, and a signaling molecule in HIV pathogenesis. Despite the fact that Savarino et al. describe CD38 as a molecule with an active role in HIV infection; Acting as a compensatory molecule that catalyzes the synthesis of nicotinamide as a mechanism for inhibiting viral replication, the work is speculative in nature, considering only in vitro infection and not chronic infection. Regarding In vitro infection studies are hardly translatable to the phenomenon observed in vivo. Second, the lateral association between CD4 and CD38 that the authors claim as a way to protect cells from infection is insufficient for the inexorable advance of untreated HIV infection. Likewise, they support a possible active role of CD38 through murine in vitro tests, which is not directly applicable to the biological-clinical phenomenon.
CD38 como un blanco terapéutico CD38 as a therapeutic target
Otros autores han estudiado a CD38 por su función enzimática catalítica principal, la hidrólisis de NAD+, dando como producto la sintesis de los segundosmensajeros cADPR, ADPR & NAADP (W02013002879 a Sinclair, David et al) . Este documento divulga el uso terapéutico de moléculas pequeñas como inhibidores de CD38 con el fin de aumentar la concentración de NAD en células. Los tratamientos que menciona incluyen Other authors have studied CD38 for its main catalytic enzymatic function, the hydrolysis of NAD +, resulting in the synthesis of second messengers cADPR, ADPR & NAADP (W02013002879 to Sinclair, David et al). This document discloses the therapeutic use of small molecules as CD38 inhibitors in order to increase the concentration of NAD in cells. The treatments he mentions include
enfermedades respiratorias, enfermedades relacionadas con la edad, enfermedades metabólicas, cáncer y colesterol. A pesar de que el documento divulga que el SIDA es una de las condiciones que pueden ser tratadas, esta afirmación se encuentra dentro de un listado genérico de padecimientos. Esto es evidente al ver que los padecimientos que preceden a la mención del SIDA son el qlaucoma y la deqeneración macular; y que aquellos que suceden a la mención son hepatitis fulminante y enfermedades relacionadas al cerebro (ver páqina 64, primer párrafo) (W02013002879, 2013) . respiratory diseases, age-related diseases, metabolic diseases, cancer and cholesterol. Although the document discloses that AIDS is one of the conditions that can be treated, this statement is within a generic list of conditions. This is evident when seeing that the ailments that precede the mention of AIDS are qlaucoma and macular degeneration; and that those that follow the mention are fulminant hepatitis and diseases related to the brain (see page 64, first paragraph) (W02013002879, 2013).
En sequnda instancia y como se ha enfatizado anteriormente, el SIDA es solo una de las posibles consecuencias de la infección por VIH. Por ejemplo, dentro de los padecimientos relacionados a VIH se encuentran la infección asintomática no tratada, la infección con control de enfermedades oportunistas, la enfermedad tratada exitosamente con terapia antirretroviral altamente activa, asi como las "Non- AIDS-Defining Illnesses" en personas con VIH (incluso sin tratamiento) . Adicionalmente, Sinclair et al divulgan una amplia gama de inhibidores de CD38, entre ellos se encuentran la kuromanina, luteolina y luteolinidina como parte de un listado genérico. La presente invención se enfoca en la prevalencia de antocianinas en frutas y vegetales de alta producción en México o en alimentos tradicionales mexicanos. In the second instance, and as previously emphasized, AIDS is only one of the possible consequences of HIV infection. For example, among HIV-related conditions are untreated asymptomatic infection, infection with opportunistic disease control, disease successfully treated with highly active antiretroviral therapy, as well as "Non-AIDS-Defining Illnesses" in people with HIV (even without treatment). Additionally, Sinclair et al disclose a wide range of CD38 inhibitors, among them are kuromanin, luteolin and luteolinidine as part of a generic listing. The present invention focuses on the prevalence of anthocyanins in fruits and vegetables of high production in Mexico or in traditional Mexican foods.
Terceramente, Sinclair et al reivindican como su fin el aumento de los niveles de NAD de manera general, sin mencionar la relación entre la sobreexpresión de CD38, una condición caracterizante del VIH, con su uso reivindicado. Es decir, Sinclair et al no consideran a CD38 como una enzima biológicamente funcional en la infección por VIH. Finalmente, no divulgan la disminución de la exposición de las células T del individuo a los segundos mensajeros de CD38. Es importante mencionar que no hay relación causal alguna entre ambos sucesos. Third, Sinclair et al claim as their goal the increase in NAD levels in a general way, without mentioning the relationship between overexpression of CD38, a characterizing condition of HIV, with its use. claimed. In other words, Sinclair et al do not consider CD38 as a biologically functional enzyme in HIV infection. Finally, they do not report decreased exposure of the individual's T cells to the second CD38 messengers. It is important to mention that there is no causal relationship between both events.
Por otra parte, se ha intentado proteger a inhibidores de CD38 por la novedad en su estructura química (W02016087975 a Becherer, David et al) . Dicha invención divulga inhibidores de CD38 y métodos de tratamiento para todo tipo de padecimientos, entre los cuáles se mencionan desórdenes neurocognitivos , como ejemplo se mención a la demencia asociada a VIH-1. A pesar de que dicho padecimiento se derive de la infección por VIH, no hay materia suficiente para establecer un vinculo entre lo que revindica Becherer, tratamiento de desórdenes neurocognitivos, y el aplicar dicho tratamiento a infecciones inmunodegenerativas . Finalmente, la invención divulga novedad por que las moléculas contienen un grupo imidazol, lo cual no es el caso de los inhibidores de la presente invención. Investigación de CD38 en el INER On the other hand, attempts have been made to protect CD38 inhibitors due to the novelty in their chemical structure (W02016087975 to Becherer, David et al). Said invention discloses CD38 inhibitors and treatment methods for all kinds of conditions, among which neurocognitive disorders are mentioned, as an example mention is made of dementia associated with HIV-1. Despite the fact that said condition derives from HIV infection, there is not enough material to establish a link between what Becherer claims, treatment of neurocognitive disorders, and applying such treatment to immunodegenerative infections. Finally, the invention discloses novelty in that the molecules contain an imidazole group, which is not the case of the inhibitors of the present invention. CD38 research at INER
En publicaciones previas hemos divulgado cómo en células CD38+, y en especifico en células CD38+ de pacientes infectados con VIH, las células T de memoria 5 central (TCM) estimuladas fallan en expresar CD154, el ligando de CD40, cuando se induce a expresar citocinas, asi como IL-2. Dichas diferencias estaban amplificadas en las células provenientes de pacientes con VIH que expresaban CD38. Esto sugiere un posible lOmecanismo de patogénesis mediado por CD38, en el cual la disociación entre la expresión de citocinas y de CD154 en las células TCM CD4 puede impedir la interacción entre las células T y las células presentadoras de antigeno. A su vez, esto contribuye In previous publications we have disclosed how in CD38 + cells, and specifically in CD38 + cells of HIV - infected patients, T cell memory 5 Center (T CM) stimulated fail to express CD154, CD40 ligand, when induced to express cytokines as well as IL-2. These differences were amplified in cells from HIV patients expressing CD38. This suggests a possible CD38-mediated pathogenesis mechanism, in which the dissociation between cytokine and CD154 expression in CD4 CM T cells may impede interaction between T cells and antigen presenting cells. This in turn contributes
15 al deterioro de la inmunidad del sujeto y ayuda a explicar la relación entre la expresión de CD38 y la progresión de la enfermedad en la infección crónica por VIH. 15 to the deterioration of the subject's immunity and helps explain the relationship between CD38 expression and disease progression in chronic HIV infection.
Un segundo estudio (Olvera-Garcia, Espinosa, 20 Sieg, & Lederman, 2014) permitió esclarecer que el comportamiento de las células que expresan CD38 se observa también en respuestas de memoria especifica de antigeno de una infección crónica. El siguiente paso fue determinar si la correlación de la activación 25 de CD38 con la progresión de la enfermedad por VIH era independiente del ciclo celular de las células. Se observó como en una subpoblación de células TCM CD4 CD38 de sujetos infectados se correlaciona con un conteo menor de células T CD4+ en sangre, independientemente de la frecuencia de células en ciclo celular (y potencialmente proliferante) . Esto sugiere que la expresión de CD38 es el factor determinante de la relación entre activación crónica y avance de la infección por VIH (Würsch et al., 2016) . Posteriomente , y basado en la trayectoria de investigación, divulgamos la propuesta de un modelo de la muerte celular de células TCM CD4 basado en un análisis de transcriptoma . Este modelo nos llevó a descubrir genes sobreexpresados en células TCM CD4 de pacientes con VIH y establecer a partir de ahi un pool de genes que identificamos como una firma distintiva de la infección por VIH (Olvera-Garcia et al., 2016) . A second study (Olvera-Garcia, Espinosa, 20 Sieg, & Lederman, 2014) clarified that the behavior of cells expressing CD38 is also observed in antigen-specific memory responses of a chronic infection. The next step was to determine whether the correlation of CD38 activation with the progression of HIV disease was independent of the cell cycle of cells. It is observed as in a subpopulation of T cells CD4 CD38 C M infected subjects correlates with a lower count of CD4 + T cells in blood, regardless of the frequency of cells in the cell cycle (and potentially proliferating). This suggests that CD38 expression is the determining factor in the relationship between chronic activation and progression of HIV infection (Würsch et al., 2016). Subsequently, and based on the research trajectory, we disclosed the proposal for a model of T C M CD4 cell death based on a transcriptome analysis. This model led us to discover overexpressed genes in CD4 T C M cells from HIV patients and to establish from there a pool of genes that we identify as a distinctive signature of HIV infection (Olvera-Garcia et al., 2016).
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BREVE DESCRIPCIÓN DE LA INVENCIÓN BRIEF DESCRIPTION OF THE INVENTION
CD38 ha sido caracterizado como un biomarcador de la progresión del VIH. La presente invención considera a CD38 como una enzima envuelta en la patogénesis del VIH mediante la remoción de NAD intracelular . CD38 has been characterized as a biomarker of HIV progression. The present invention considers CD38 as an enzyme involved in HIV pathogenesis by removal of intracellular NAD.
Los inhibidores competitivos de CD38 pueden inhibir totalmente su actividad catalitica. Competitive CD38 inhibitors can totally inhibit its catalytic activity.
La inhibición catalitica de CD38 disminuye notablemente la muerte por activación de las células T CD4 de memoria centrales en células de un paciente VIH positivo. Catalytic inhibition of CD38 markedly decreases death by activation of central memory CD4 T cells in cells of an HIV positive patient.
La inhibición de CD38 aumenta la capacidad de las células de memoria centrales de un paciente con VIH (sin tratamiento) de diferenciarse a células de memoria efectora, función homeostática crucial en la regeneración del sistema inmune. Inhibition of CD38 increases the capacity of the core memory cells of an HIV patient (without treatment) to differentiate into effector memory cells, a crucial homeostatic function in the regeneration of the immune system.
Los inhibidores competitivos kuromanina, luteolina y luteolinidina pueden tener un segundo uso para la elaboración de medicamentos para una el tratamiento de una población VIH positiva. BREVE DESCRIPCIÓN DE LOS FIGURAS Competitive inhibitors kuromanin, luteolin and luteolinidine may have a second use for the manufacture of drugs for the treatment of an HIV positive population. BRIEF DESCRIPTION OF THE FIGURES
Un mejor entendimiento será posible a través de las siguientes descripciones detalladas de las figuras, donde: La figura 1 muestra cómo las células T CD4 de memoria centrales de pacientes con VIH sobreexpresan tanto CD38 como NAMPT . A better understanding will be possible through the following detailed descriptions of the figures, where: Figure 1 shows how the core memory CD4 T cells of HIV patients overexpress both CD38 and NAMPT.
La figura 2 muestra que la concentración de NAMPT en la sangre se correlaciona positivamente con el número de células T CD4 por microlitro de sangre de pacientes con VIH. Figure 2 shows that the concentration of NAMPT in the blood is positively correlated with the number of CD4 T cells per microliter of blood from HIV patients.
La figura 3 muestra como el inhibidor competitivo de la enzima CD38 puede inhibir totalmente su actividad catalitica en linfocitos humanos que expresan CD38. Figure 3 shows how the competitive inhibitor of the CD38 enzyme can totally inhibit its catalytic activity in human lymphocytes that express CD38.
La figura 4 muestra que la inhibición de la actividad catalitica de CD38 disminuye notablemente la muerte por activación de las células TCM CD4 de un paciente con VIH. La figura 5 muestra que la inhibición de CD38 aumenta la capacidad de las células de memoria centrales (TCM) de un paciente con VIH (sin tratamiento) de diferenciarse a células de memoria efectora (TEM) . Figure 4 shows that inhibition of CD38 catalytic activity markedly decreases death by activation of CD4 T C M cells from an HIV patient. Figure 5 shows that CD38 inhibition increases the capacity of memory cells. central (TCM) of an HIV patient (untreated) to differentiate into effector memory cells (TEM).
La figura 6 muestra que la kuromanina (antocianina de origen natural) inhibe la actividad catalítica de CD38 en Células Mononucleares de Sangre Periférica (CMNSPs), en forma proporcional a su concentración. Figure 6 shows that kuromanin (naturally occurring anthocyanin) inhibits the catalytic activity of CD38 in Peripheral Blood Mononuclear Cells (CMNSPs), proportionally to its concentration.
La figura 7 muestra que la antocianina lutoelina inhibe CD38 más que ara-F-NAD a la misma concentración. Figure 7 shows that anthocyanin lutoelin inhibits CD38 more than ara-F-NAD at the same concentration.
La figura 8 muestra que la kuromanina puede reducir la actividad catalítica de CD38 también en linfocitos humanos. Figure 8 shows that kuromanin can reduce the catalytic activity of CD38 also in human lymphocytes.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN DETAILED DESCRIPTION OF THE INVENTION
La presente invención se sustenta en un nuevo enfoque hacia la patogénesis de VIH, considerando a CD38 como una enzima con una función biológica, y por ende, un blanco terapéutico para el tratamiento del VIH. The present invention is based on a new approach towards the pathogenesis of HIV, considering CD38 as an enzyme with a biological function, and therefore, a therapeutic target for the treatment of HIV.
CD38: la enzima funcional CD38: the functional enzyme
Al considerar a CD38 como una enzima con una función biológica, debiamos ver resultados que soportaran dicha hipótesis. El primero de estos se derivó de los resultados publicados en 2016. Dentro de los genes identificados en células TCM CD4+ como caracterizantes de la infección por VIH (Olvera-Garcia et al., 2016), uno que resaltó fue NAMPT, que codifica para citocina visfatina. Este gen fue de gran interés dado que la visfatina es la enzima determinante de la tasa de sintesis de NAD de una de las rutas de sintesis de NAD. Como se puede ver en la figura 1, a mayor concentración de NAMPT en plasma, mayor cantidad de células T CD4+. When considering CD38 as an enzyme with a biological function, we should see results that support this hypothesis. The first of these was derived from the results published in 2016. Among the genes identified in CD4 + T C M cells as characteristic of HIV infection (Olvera-Garcia et al., 2016), one that stood out was NAMPT, which encodes for visfatin cytokine. This gene was of great interest since visphatin is the NAD synthesis rate determining enzyme of one of the NAD synthesis pathways. As can be seen in Figure 1, the higher the plasma NAMPT concentration, the greater the quantity of CD4 + T cells.
Estos resultados preliminares sirvieron como sustento para seguir indagando en el rol de CD38, en especifico en considerarlo como una enzima con una función biolóqica. Si NAMPT limita la síntesis de NAD, y CD38 está activamente removiendo NAD intracelular, entonces la célula posiblemente está sobreexpresando NAMPT como una manera de producir más NAD y compensar el efecto de CD38. Aunado a ello, la sobreexpresión de NAMPT mostró una correlación benéfica para los pacientes con proqresión de la infección por VIH. These preliminary results served as support to continue investigating the role of CD38, specifically in considering it as an enzyme with a biological function. If NAMPT limits the synthesis of NAD, and CD38 is actively removing intracellular NAD, then the cell is possibly overexpressing NAMPT as a way to produce more NAD and compensate for the effect of CD38. In addition, NAMPT overexpression showed a beneficial correlation for patients with HIV infection.
Activación inmune y el efecto Warburq Immune activation and the Warburq effect
Es importante mencionar que las células no importan NAD del medio, sino más bien sus precursores y NAMPT cataliza la sintesis intracelular de NAD. Por lo tanto, la sobreexpresión de NAMPT indica que la célula está buscando reestablecer homeostasis. Por otra parte, la sobreexpresión de CD38 lleva a un aumento de la cantidad de moléculas de CD38 en la superficie de células TCM CD4+ y por lo tanto, hay un mayor número de enzimas en contacto con el plasma de la sanqre. A su vez, la infección por VIH está caracterizada por una activación crónica de lascélulas TCM CD4+. Las células T CD4 de un sujeto con VIH se encuentran en dicho microambiente . It is important to mention that cells do not import NAD from the medium, but rather their precursors and NAMPT catalyzes the intracellular synthesis of NAD. Therefore, NAMPT overexpression indicates that the cell is seeking to reestablish homeostasis. On the other hand, overexpression of CD38 leads to an increase in the number of CD38 molecules on the surface of CD4 + T C M cells and therefore, there are a greater number of enzymes in contact with the blood plasma. In turn, HIV infection is characterized by chronic activation of CD4 + CM T cells. CD4 T cells from a subject with HIV are found in said microenvironment.
Las células TCM CD4+ se ven afectadas por dicho microambiente y la constante excreción de NAD intracelular, lo cual qenera estrés adicional a las mitocondrias de las células. Estas condiciones propician una adaptación del metabolismo enerqético de las células, habiéndose documentado ya la aparición del efecto Warburq. Dicho efecto se caracteriza por la adaptación de la fosforilación oxidativa a la qlicólisis aeróbica y por ende una disminución de la respiración mitocondrial . Esto desemboca en una posible afectación a la cresta mitocondrial y a la fusión entre mitocondrias. El efecto Warburq puede ser una de las causas que contribuye a la muerte celular. CD4 + T C M cells are affected by this microenvironment and the constant excretion of intracellular NAD, which causes additional stress to the mitochondria of cells. These conditions favor an adaptation of the energetic metabolism of the cells, the appearance of the Warburq effect having already been documented. This effect is characterized by the adaptation of oxidative phosphorylation to aerobic glycolysis and therefore a decrease in mitochondrial respiration. This leads to possible involvement of the mitochondrial crest and fusion between mitochondria. The Warburq effect may be one of the causes that contributes to cell death.
Inhibición de CD38 CD38 inhibition
Habiendo identificado el medio en el que se encuentran y el cambio metabólico que presentan las células TCM CD4+, buscamos analizar si al activar células CD38+ y CD38-, se podra modificar la función de la célula mediante la adición de inhibidores de CD38. Como se puede ver en la fiqura 2, en células sin activar, la inhibición de CD38 lleva a una mayor expresión del mismo. Por otra parte, en células activadas, la inhibición de CD38 abóle por completo su expresión. Estos resultados experimentales siquieron sustentando el nuevo paradiqma de CD38, en especifico porque representan condiciones más cercanas a en las que se encuentran las células TCM Having identified the medium in which they are and metabolic change have T CM CD4 + cells, we sought to analyze whether the enabled cells CD38 + and CD38, may modify cell function by adding inhibitors of CD38. As can be seen in Figure 2, in unactivated cells, inhibition of CD38 leads to increased expression of CD38. On the other hand, in activated cells, CD38 inhibition completely abolishes its expression. These experimental results continued to support the new CD38 paradiqma, specifically because they represent conditions closer to those found in T C M cells.
CD4+ de pacientes con VIH. Posteriormente, buscamos determinar la cinética enzimática de CD38 mediante el uso de un inhibidor. Como se muestra en la figura 3, la adición de inhibidores de CD38 permite a la célula CD38+ tener un comportamiento similar a una célula CD38-. De igual manera, hallazgos recientes de nuestro laboratorio encuentran que las células TCM CD4 CD38-, responden a la estimulación induciendo más MYC (un promotor de sobrevida) e IL-2que las CD38+. Derivado de lo mismo, los resultados sustentaban la hipótesis inicial, los inhibidores de CD38 llevan a un mejor mantenimiento del sistema inmune (Figura 4) . De importancia, hemos encontrado que la inhibición de CD38 en células TCM CD4 de pacientes aumenta su sobrevida y su diferenciación a células de memoria efectoras (Figura 5), dos de sus funciones homeostáticas . CD4 + from HIV patients. Subsequently, we sought to determine the enzymatic kinetics of CD38 by using an inhibitor. As shown in Figure 3, the addition of CD38 inhibitors enables the CD38 + cell to behave similarly to a CD38- cell. Similarly, recent findings from our laboratory find that CD38- T C M CD4 cells respond to stimulation by inducing more MYC (a survival promoter) and IL-2 than CD38 +. Derived from the same, the results supported the initial hypothesis, CD38 inhibitors lead to better maintenance of the immune system (Figure 4). Importantly, we have found that CD38 inhibition in T C M CD4 cells of patients increases their survival and their differentiation to memory effector cells (Figure 5), two of their homeostatic functions.
Finalmente, y al observar un efecto inhibitorio inducido por ara-F-NAD, el último paso fue analizar si la kuromanina, la leutolinidina y/o la leutolinapodian asemejar la inhibición catalítica de ara-F- NAD. Finally, and observing an inhibitory effect induced by ara-F-NAD, the last step was to analyze whether kuromanin, leutolinidine and / or leutolinpodian resemble the catalytic inhibition of ara-F-NAD.
En primera instancia comparamos la actividad de la kuromanina con ara-F-NAD para inhibir a CD38 humano (Figura 6) . Asimismo analizamos la inhibición catalítica de CD38 por luteolina en células mononucleare s CD38+ de sangre periférica (Figura 7) . En última instancia, realizamos una verificación de la inhibición por kuromanina por la población linfocitica de las células mononucleares de sangre periférica (Figura 8) . Firstly, we compared the activity of kuromanin with ara-F-NAD to inhibit human CD38 (Figure 6). We also analyzed the catalytic inhibition of CD38 by luteolin in CD38 + mononucleare s of peripheral blood (Figure 7). Ultimately, we perform a verification of kuromanin inhibition by the lymphocytic population of peripheral blood mononuclear cells (Figure 8).
Por lo tanto, tanto la investigación en la 5 literatura como experimental sustentan un nuevo paradigma funcional de CD38, y por lo tanto, un segundo uso en VIH de inhibidores de CD38 como un tratamiento adicional a la TARV, en pacientes que aún no lo han comenzado. Como se puede ver en la figura 101, los pacientes que ingresan al INER presentan una mayor progresión de la infección por VIH, vista en el conteo de células T CD4, y por lo tanto se podrían beneficiar de la presente invención. Therefore, both research in the literature and experimental support a new functional CD38 paradigm, and therefore, a second use of CD38 inhibitors in HIV as an additional treatment to ART, in patients who have not yet done so. started. As can be seen in figure 101, patients entering the INER show a greater progression of HIV infection, seen in CD4 T cell count, and therefore could benefit from the present invention.
Aplicación industrial Industrial application
15 En los últimos años, la producción de Berries ha tenido un gran aumento, incluso desplazando al Tequila como el producto con más exportaciones de Jalisco (Romo, 2018) . México produce alrededor de 390.24 Mt de berries, de los cuáles el consumo nacional abarca 20231.09 Mt, es decir alrededor del 60% de la producción nacional se consume en el país (Dirección de Investigación y Evaluación Económica y Sectorial, 2016; SAGRPA, 2017) . Por otra parte, el maiz azul representa uno de los alimentos tradicionales mexicanos, y junto con el maiz blanco, representa una fuente importante de alimentación en la población mexicana. El denominador común de ambos alimentos es que tanto las berries ( Paredes-López , Cervantes-Ce a, Vigna-Pérez, & Hernández-Pérez , 2010) como el maiz azul (Jansom, Bhamarapravati , & Itharat, 2014) presentan una alta concentración de antocianinas , entre las que se encuentran diversos inhibidores de la actividad catalítica (de enzima) de CD38, y que ambos alimentos conllevan una amplia tradición de preparaciones. De manera más especifica, ambos productos no son solo de gran producción nacional, sino que también son relevantes en la investigación y desarrollo de alimentos (Paredes-López et al., 2010) 15 In recent years, the production of Berries has had a great increase, even displacing Tequila as the product with the most exports from Jalisco (Romo, 2018). Mexico produces around 390.24 Mt of berries, of which national consumption covers 20,231.09 Mt, that is, around 60% of national production is consumed in the country (Directorate for Research and Economic and Sector Evaluation, 2016; SAGRPA, 2017) . On the other hand, blue corn represents one of the traditional Mexican foods, and together with white corn, it represents an important source of food in the Mexican population. The common denominator of both foods is that both berries (Paredes-López, Cervantes-Ce a, Vigna-Pérez, & Hernández-Pérez, 2010) and blue corn (Jansom, Bhamarapravati, & Itharat, 2014) have a high concentration anthocyanins, among which are various inhibitors of the catalytic (enzyme) activity of CD38, and that both foods carry a long tradition of preparations. More specifically, both products are not only of great national production, but are also relevant in food research and development (Paredes-López et al., 2010)
EVIDENCIA EVIDENCE
Experimento 1 Experiment 1
La determinación de nicotinamida- fosforribosiltranseferasa (NAMPT) se hizo por el método de ELISA en plasma total. La determinación de cuentas de células T CD4+ por microlitro de sangre total (de los mismos pacientes) se hizo por citometria de flujo. Los resultados se muestran en la figura 2. The determination of nicotinamide phosphoribosyltranseferase (NAMPT) was made by the ELISA method in total plasma. Determination of CD4 + T cell counts per microliter of whole blood (from the same patients) was done by flow cytometry. The results are shown in figure 2.
Experimento 2 Experiment 2
Se partió de células mononucleares de sangre periférica obtenidas de paquete globulares, por separación en gradiente de densidad en ficoll. De ellas, los linfocitos que expresan de CD38 y los que no lo expresan, se purificaron por el método de clasificación de células activada por fluorescencia (FACS) tomando en cuenta las propiedades de dispersión de la luz y fluorescencia asociada a la expresión extracelular de CD38. Las células fueron suspendidas en solución amortiguadora A en ausencia o presencia de concentraciones diferentes del inhibidor catalitico de CD38 b- ara- 2'- desoxi- 2'- fluoro- nicotinamida adenin dinucleótido (ara-F-NAD) . La actividad catalítica de CD38 en las distintas condiciones se midió como conversión del análogo de NAD (sustrato de la enzima) nicotinamida 1 , N6-etenoadenin- eteno dinucleótido (eteno-NAD) en un producto It was started from peripheral blood mononuclear cells obtained from globular bundles, by density gradient separation in ficoll. Of these, CD38-expressing and non-expressing lymphocytes were purified by the fluorescence-activated cell sorting method (FACS) taking into account the light scattering and fluorescence properties associated with CD38 extracellular expression . The cells were suspended in buffer A in the absence or presence of different concentrations of the CD38 catalytic inhibitor b-ara- 2'- deoxy-2'- fluoro-nicotinamide adenine dinucleotide (ara-F-NAD). Activity Catalytic CD38 under the various conditions was measured as conversion of the NAD analog (enzyme substrate) nicotinamide 1, N6-ethenoadenine-ethene dinucleotide (ethene-NAD) into a product
fluorescente . La cantidad de este producto en el medio se midió como emisión de fluorescencia a 410nm (excitando con fuente de 300nm) . En la figura 3 se ve la fluorescencia, proporcional a la cantidad de producto, contra tiempo, para linfocitos humanos que expresan CD38 sin inhibidor (linea superior) y con las concentraciones de ara-F-NAD de 100 veces y mil veces la constante de inhibición (Ki) . Como señal negativa (ausencia de actividad catalítica de CD38) se utilizó la fluorescencia producida fluorescent. The amount of this product in the medium was measured as fluorescence emission at 410nm (exciting with 300nm source). Figure 3 shows the fluorescence, proportional to the amount of product, against time, for human lymphocytes expressing CD38 without inhibitor (upper line) and with ara-F-NAD concentrations of 100 times and a thousand times the constant of inhibition (Ki). The negative signal (absence of catalytic activity of CD38) used the fluorescence produced
por linfocitos humanos que no expresan CD38 (linea inferior) . lOOOKi corresponden a una concentración 1.69 micromolar. by human lymphocytes that do not express CD38 (bottom line). lOOOKi correspond to a concentration of 1.69 micromolar.
Experimento 3 Experiment 3
Las células T de memoria centrales se purificarona partir de células mononucleares de sangre periférica de pacientes con VIH sin tratamiento antirretroviral . Estas células se sometieron a purificación en dos pasos en columnas inmunomagnética . La pureza fue mayor al 90%.Core memory T cells were purified from peripheral blood mononuclear cells from HIV patients without antiretroviral treatment. These cells were subjected to two-step purification on immunomagnetic columns. The purity was greater than 90%.
Una vez obtenidas, se estimuló su receptor de células T (TCR) durante dos horas usando anticuerpos monoclonales agonistas dirigidos a las moléculas de superficie CD38 y CD28, en presencia de la citocina IL-2. Después se cultivaron 7 dias a 37°C en atmósfera con 5% de bióxido de carbono y humedad del 100%. La determinación de viabilidad se hizo con una sonda molecular fluorescente que se une a aminasde superficie celular, más abundantes en células muertas (pico derecho en cada gráfica, A y B, de la figura 4) . Once obtained, their T-cell receptor (TCR) was stimulated for two hours using agonist monoclonal antibodies directed at surface molecules CD38 and CD28, in the presence of IL-2 cytokine. They were then cultivated 7 days at 37 ° C in an atmosphere with 5% carbon dioxide and 100% humidity. Viability determination was made with a fluorescent molecular probe that binds to cell surface amines, most abundant in dead cells (right peak in each graph, A and B, of figure 4).
Las células T de memoria centrales fueron The core memory T cells were
obtenidas y cultivadas con la misma metodología que fue mostrada anteriormente y se marcaron con  obtained and cultivated with the same methodology that was previously shown and marked with
anticuerpos dirigidos a moléculas de superficie  antibodies directed at surface molecules
CD45RA y CCR7 cuyo patrón de expresión difiere entre subpoblaciones de células T CD4 con distintos grados de diferenciación. Por ejemplo, las células  CD45RA and CCR7 whose expression pattern differs between CD4 T cell subpopulations with different degrees of differentiation. For example, cells
diferenciadas (de memoria efectoras) no expresan ni CD45RA ni CCR7. Los resultados se pueden ver en la figura 5.  differentiated (from memory effectors) do not express CD45RA or CCR7. The results can be seen in figure 5.
Experimento 4 Experiment 4
Se separaron las CMNSPs como en los experimentos 2 y 3, y se incubaron con diferentes condiciones de los inhibidores catalíticos. Una descripción más detallada de cada experimento asi como los  CMNSPs were separated as in experiments 2 and 3, and incubated with different conditions of the catalytic inhibitors. A more detailed description of each experiment as well as the
resultados se encuentran en la descripción detallada de las figuras. Descripción detallada de las figuras Results are found in the detailed description of the figures. Detailed description of the figures
En las figuras 1-8 se proporcionan los resultados experimentales obtenidos que sustentan el rol de CD38 como una enzima activa en la patogénesis del VIH. En la figura 1 se muestra la diferencia en expresión de RNA mensajero de diversos genes, expresada como cuántas veces más RNA mensajero que en células de controles sanos. Las células T CD4 de memoria centrales de pacientes con VIH sobreexpresan tanto CD38 (enzima removedora de NAD) , como NAMPT (nicotinamida-fosforribosil transferasa o visfatina, enzima sintetizadora ) . CD38 (décimo gen en el eje X) es la enzima principal removedora de NAD, mientras que NAMPT es la enzima de una ruta de sintesis de NAD que determina la cantidad sintetizada. Esto sugiere que detrás de la correlación negativa de CD38 con la preservación de células T CD4 se encuentra una disminución de NAD y además la disminución de NADFigures 1-8 provide the experimental results obtained that support the role of CD38 as an active enzyme in HIV pathogenesis. Figure 1 shows the difference in expression of messenger RNA of various genes, expressed as how many times more messenger RNA than in healthy control cells. The central memory CD4 T cells of HIV patients overexpress both CD38 (NAD-removing enzyme) and NAMPT (nicotinamide-phosphoribosyl transferase or visphatin, synthesizing enzyme). CD38 (tenth gene on the X axis) is the major NAD-removing enzyme, while NAMPT is the enzyme in a NAD synthesis pathway that determines the amount synthesized. This suggests that behind the negative correlation of CD38 with the preservation of CD4 T cells is a decrease in NAD and also a decrease in NAD
(esperable en las células T CD4 de pacientes con VIH) disminuye su capacidad de sobrevida. La figura 1 se ha divulgado previamente en Olvera-Garcia et al (2016) y se agregó para mostrar dónde termina la materia que ya es del dominio público y dónde comienza la investigación nueva pertinente a la presente invención. En la figura 2 se muestra la gráfica de cuentas de células T CD4 contra concentración de NAMPT en plasma de pacientes con VIH sin tratamiento, provenientes de dos cohortes con estados de avance diferentes. Esto sugiere que, mientras la sintesis de NAD se asocia con sobrevida de estas células, CD38, enzima que elimina NAD, es negativa para su sobrevida. Esto apoya la idea de que CD38, a través de su actividad catalítica, disminuye la sobrevida de las células T CD4 en la infección por VIH. En circuios blancos, los puntos correspondientes a una cohorte crónica (regresión de Pearson) . En puntos negros, pacientes de una cohorte extremadamente avanzada (Rho de Spearman) . En ambos casos la correlación es estadísticamente significativa (p<0.05). Esto apoya la idea de que CD38, a través de su actividad catalítica, disminuye la sobrevida de las células T CD4 en la infección por VIH. (expected in CD4 T cells of HIV patients) decreases their survival capacity. Figure 1 has been previously disclosed in Olvera-Garcia et al (2016) and was added to show where the matter that is already in the public domain ends and where the new research relevant to the present invention begins. Figure 2 shows the graph of CD4 T cell counts against NAMPT concentration in plasma of HIV patients without treatment, from two cohorts with different stages of progress. This suggests that, while NAD synthesis is associated with survival of these cells, CD38, an enzyme that eliminates NAD, is negative for their survival. This supports the idea that CD38, through its catalytic activity, decreases the survival of CD4 T cells in HIV infection. In white circles, the points corresponding to a chronic cohort (Pearson regression). In blackheads, patients from an extremely advanced cohort (Spearman's rho). In both cases the correlation is statistically significant (p <0.05). This supports the idea that CD38, through its catalytic activity, decreases the survival of CD4 T cells in HIV infection.
En la figura 3 se muestra la determinación por cinética enzimática de la concentración mínima inhibitoria del inhibidor de CD38 ara-F-NAD, medida como conversión por CD38 de nicotinamida 1,N6- etenoadenin- eteno dinucleótido (eteno-NAD) en un producto fluorescente . En la gráfica se ve cómo un inhibidor competitivo puede inhibir la actividad catalítica de CD38 a niveles de linfocitos que no expresan la enzima. En la gráfica se muestra la fluorescencia, proporcional a la cantidad de producto, contra tiempo, para linfocitos humanos que expresan CD38 sin inhibidor (linea superior) y con las Figure 3 shows the enzymatic kinetic determination of the minimum inhibitory concentration of the CD38 inhibitor ara-F-NAD, measured as CD38 conversion of nicotinamide 1, N6-ethenoadenine-etheno dinucleotide (ethene-NAD) in a fluorescent product. . The graph shows how a competitive inhibitor can inhibit the catalytic activity of CD38 at levels of lymphocytes that do not they express the enzyme. The graph shows the fluorescence, proportional to the amount of product, against time, for human lymphocytes expressing CD38 without inhibitor (upper line) and with the
concentraciones de ara-F-NAD de 100 Ki (Ki es la constante de inhibición) y 1000 Ki, todas comparadas con la fluorescencia de fondo, determinada con linfocitos humanos que no expresan CD38 (ausencia de actividad de CD38, linea inferior) . En la figura 4 se muestran histogramas que representan la cantidad de células marcadas con una sonda de viabilidad, correspondiente a células T de memoria centrales que fueron cultivadas siete dias después de recibir una estimulación policlonal del receptor de células T con un anticuerpo agonista anti- CD38 y un anticuerpo agonista anti-CD28. El pico de la derecha en cada histograma corresponde a las células muertas (que fijan más marcador) y el pico de la izquierda corresponde a las células vivas. Los números indican el porcentaje. A. Células cultivadas sin el inhibidor de CD38 (ara-F-NAD), B. Células cultivadas con el inhibidor catalítico de CD38. ara-F-NAD concentrations of 100 Ki (Ki is the inhibition constant) and 1000 Ki, all compared to the background fluorescence, determined with non-CD38-expressing human lymphocytes (absence of CD38 activity, bottom line). Histograms representing the number of cells labeled with a viability probe, corresponding to core memory T cells that were cultured seven days after receiving polyclonal stimulation of the T cell receptor with an anti-CD38 agonist antibody, are shown in Figure 4. and an anti-CD28 agonist antibody. The peak on the right in each histogram corresponds to dead cells (which fix more marker) and the peak on the left corresponds to living cells. The numbers indicate the percentage. A. Cells grown without CD38 inhibitor (ara-F-NAD), B. Cells grown with CD38 catalytic inhibitor.
En la figura 5 se muestran gráficos de expresión de CD45RA contra expresión de CCR7 en células T de memoria centrales de un paciente con VIH cultivadas 7 dias en medio (A) o en medio más inhibidor de CD38 (ara-F-NAD a lOOKi) . Las células de memoria no expresan CD45RA. Las células de memoria centrales expresan CCR7 (cuadro derecho en cada gráfica) , mientras que las células T de memoria efectoras (obtenidas después de 7 dias de cultivo después de estimulación) no la expresan. Figure 5 shows graphs of CD45RA expression versus CCR7 expression in cultured central HIV memory T cells from a patient with 7 days in medium (A) or in medium with more CD38 inhibitor (ara-F-NAD to lOOKi). Memory cells do not express CD45RA. Central memory cells express CCR7 (right box on each graph), while effector memory T cells (obtained after 7 days of culture after stimulation) do not express it.
En la figura 6 se muestra como la kuromanina (antocianina de origen natural) inhibe la actividad catalítica de CD38, en forma proporcional a su concentración. A: 1.- Linea superior: actividad catalítica de células mononucleares de sangre periférica (CMNSPs) humanas que expresan CD38, provenientes de un donador sano, en ausencia del inhibidor de la actividad catalítica de CD38 ara-F- NAD (linea superior, negra) . 2.- Linea siguiente: mismas células en presencia de ara-F-NAD en contentración 10 micromolar. 3. Linea siguiente (gris) Mismas células con una concentración 20 micromolar de Figure 6 shows how kuromanin (naturally occurring anthocyanin) inhibits the catalytic activity of CD38, proportionally to its concentration. A: 1.- Upper line: catalytic activity of human peripheral blood mononuclear cells (CMNSPs) expressing CD38, from a healthy donor, in the absence of the inhibitor of the catalytic activity of CD38 ara-F-NAD (upper line, black ). 2.- Next line: same cells in the presence of ara-F-NAD in 10 micromolar content. 3. Next line (gray) Same cells with a 20 micromolar concentration of
kuromanina. 4. Linea inferior: mismas células en presencia de 1.69 micromolar de ara-F-NAD, inhibidor utilizado en nuestros estudios in vitro, capaz de disminuir la actividad hasta la del control negativo (células que no expresan CD38) . B: 1. linea superior control positivo de actividad catalítica, consistente en células mononucleares de sangre periférica (CMNSPs) humanas que expresan CD38, provenientes de un donador sano, en ausencia del inhibidor (linea superior, negra) . 2. Linea siguiente hacia abajo: CMNSPs con 1.69 micromolar de ara-F-NAD. 3: Células con 50microM (gris claro, tercera linea) de kuromanina. 4: Células con y lOOmicromolar de kuromanina (negro, linea inferior) . Kuromanina. 4. Bottom line: same cells in the presence of 1.69 micromolar ara-F-NAD, an inhibitor used in our in vitro studies, capable of decreasing activity to that of the negative control (cells that do not express CD38). B: 1. upper line positive control of catalytic activity, consisting of peripheral blood mononuclear cells (CMNSPs) human expressing CD38, from a healthy donor, in the absence of the inhibitor (upper line, black). 2. Next line down: 1.69 micromolar ara-F-NAD CMNSPs. 3: Cells with 50microM (light gray, third line) of kuromanin. 4: Cells with and lOOmicromolar kuromanin (black, bottom line).
En la figura 7 se muestra que la antocianina lutoelina inhibe CD38 más que ara-F-NAD a la misma concentración . La linea superior indica laproducción de derivado fluorescente del eteno-NAD por CD38 de células mononucleares de sangre periférica humanas. La linea intermedia (gris) corresponde a la actividad catalitica inhibida en presencia de ara-F-NAD. La linea inferior (oscura) corresponde a la actividad catalitica inhibida por la luteolina. Figure 7 shows that anthocyanin lutoelin inhibits CD38 more than ara-F-NAD at the same concentration. The upper line indicates the production of fluorescent derivative of ethene-NAD by CD38 from human peripheral blood mononuclear cells. The intermediate line (gray) corresponds to the inhibited catalytic activity in the presence of ara-F-NAD. The lower (dark) line corresponds to the catalytic activity inhibited by luteolin.
En la figura 8 se muestra que la kuromanina puede reducir la actividad catalitica de CD38 también en linfocitos humanos. La linea superior (negra) corresponde a la fluorescencia producto de la actividad catalitica de CD38 presente en linfocitos humanos que expresan CD38. La linea intermedia (gris claro) corresponde a la actividad basal, correspondiente a los linfocitos humanos que no Figure 8 shows that kuromanin can reduce the catalytic activity of CD38 also in human lymphocytes. The upper line (black) corresponds to the fluorescence product of the catalytic activity of CD38 present in human CD38-expressing lymphocytes. The intermediate line (light gray) corresponds to basal activity, corresponding to human lymphocytes that do not
expresan CD38. La linea inferior (gris oscuro) corresponde a la actividad de los linfocitos humanos que expresan CD38 en presencia de kuromanina a una concentración 50 micromolar. express CD38. The bottom line (dark gray) corresponds to the activity of human lymphocytes expressing CD38 in the presence of kuromanin at a concentration of 50 micromolar.

Claims

REIVINDICACIONES Habiéndose descrito la invención como antecede, se reclama como propiedad lo contenido en las siguientes reivindicaciones: CLAIMS Having described the invention as above, the content of the following claims is claimed as property:
1. El uso de antocianinas inhibidoras de CD38 para la fabricación de un médicamente para el tratamiento de la infección por VIH. 1. The use of CD38 inhibitor anthocyanins for the manufacture of a medically for the treatment of HIV infection.
2. El uso según la reivindicación 1 caracterizado porque las antocianinas inhibidoras de CD38 comprenden frutas y vegetales de alta producción en México.  2. The use according to claim 1 characterized in that the CD38 inhibitor anthocyanins comprise fruits and vegetables of high production in Mexico.
3. El uso según la reivindicación 2 caracterizado porque las frutas y vegetales de alta producción en México comprenden berries.  3. The use according to claim 2 characterized in that the fruits and vegetables of high production in Mexico comprise berries.
4. El uso según la reivindicación 3 caracterizado porque las berries comprenden al arándano azul ( Vaccinium spp . ) , la frambuesa {Rubus idaeus) y la zarzamora ( Rubus ulmifolius) .  4. The use according to claim 3, characterized in that the berries comprise the blueberry (Vaccinium spp.), The raspberry {Rubus idaeus) and the blackberry (Rubus ulmifolius).
5. El uso según la reivindicación 1 caracterizado porque las antocianinas inhibidoras de CD38 provienen de alimentos tradicionales mexicanos .  5. The use according to claim 1 characterized in that the CD38 inhibitor anthocyanins come from traditional Mexican foods.
6. El uso según la reivindicación 5 caracterizado porque los alimentos tradicionales mexicanos comprenden al maiz azul {Zea mays L.).  6. The use according to claim 5 characterized in that traditional Mexican foods include blue corn (Zea mays L.).
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Citations (2)

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