WO2010003308A1

WO2010003308A1 - The use of nitric oxide and signal transduction system thereof in preparing medicaments for targeted therapy of malignant tumors

Info

Publication number: WO2010003308A1
Application number: PCT/CN2009/000691
Authority: WO
Inventors: 费瑞德·穆拉德; 卞卡; 李桃·洁西卡
Original assignee: 卞化石
Priority date: 2008-07-10
Filing date: 2009-06-22
Publication date: 2010-01-14

Abstract

The use of nitric oxide and signal transduction system thereof in preparing the medicaments for targeted therapy of malignant tumors. The medicaments are selected from targeted medicaments which can alter expression or activity of inducible NOS, endothelial NOS and neural NOS and so forth.

Description

Application of Nitric Oxide and Its Information Transmission System in Preparation of Targeted Therapeutic Drugs for Malignant Tumors

Technical field

The invention relates to biotechnology, in particular to the application of nitric oxide and its information transmission system in preparing a targeted therapy for treating malignant tumors.

Background technique

Malignant tumors are the main killers of humans. A lot of research has been done to prevent and treat malignant tumors. Nitric Oxide (NO) is widely involved in information transmission activities closely related to life activities. Therefore, the association between NO and tumor growth has always attracted international attention. However, in the nearly 20 years since the Nobel Prize for Nitric Oxide was achieved, there has been no fundamental breakthrough in the relationship between NO and the diagnosis and treatment of malignant tumors. The main reason is:

1) Nitric oxide (N0) is an inorganic gas radical that has attracted attention in recent years. NO contains a pair of unpaired electrons, so it has the characteristics of free radicals. Because of its unique physical and chemical properties, NO has a biological diversity under normal conditions and affects almost all systems. N0 is highly soluble in fat and easily diffuses into adjacent tissue cells to react with target substances, producing a range of biological effects. NO is widely involved in the transmission of physiological information (for example, expanding blood vessels and acting as neurotransmitters). At the same time, excessive production of NO in pathological conditions causes extensive damage to the body.

2) The diversity of NO biological information pathways, the pathways of NO can be divided into two categories:

a) cGMP dependent pathway (NO/sGC or pGC/cGMP) and

b) cGMP independent pathway (NO/oxidative nitration).

3) Complexity of solid malignant tumors: Solid malignant tumor tissue consists of two major parts: a) tumor cells (parenchyma), and b) peripheral tissues (stroma). These two parts of nitric oxide biology are fundamentally different.

4) Information system variation of tumor cell lines: The cell lines commonly used in tumor experiments have undergone numerous generations of reproduction, and their characteristics have undergone fundamental changes. In particular, the information transfer system involving small molecules has undergone a fundamental change. 5) Distribution and expression complexity of NO-related information molecules: There are three subtypes of NO synthase. cGMP can be synthesized by at least 4 enzymes, and the decomposition system of cGMP is very complicated, and the non-physiological characteristics of malignant tumors increase the difficulty of analysis. Therefore, the existence and role of NO in malignant tumors have not been known so far.

In recent years, the correlation between inflammation and the development of cancer has been clarified to a considerable extent. The inflammatory state or the inflammatory malignant environment is a necessary condition for the growth and metastasis of malignant tumors (Fig. 1). An important marker of the inflammatory or inflammatory malignant environment is the excessive production of reactive oxygen species (ROS) / reactive nitrogen species (RNS). Excessive ROS / RS can lead to tumor biology Regulation of tumor cell growth cycle, tumor suppressor, tumor-associated genes, etc. It can be seen that nitric oxide plays an important role in all stages of inflammation and even immune development.

Although there have been some reports on the expression of nitric oxide synthase in tumors, there has been no systematic study on the correlation between the expression of inducible nitric oxide synthase (iNOS) and the degree of malignancy in the world. In addition, the correlation and systematic studies on nitric oxide and its information transmission system and malignant tumors have not been reported. Summary of the invention

The technical problem to be solved by the present invention is to study and design the application of nitric oxide and its information transmission system in the preparation of antitumor drugs.

The invention provides a nitric oxide and an information transmission system thereof for use in preparing a targeted therapy for malignant tumors.

The present invention uses seven key biological target sequences in the nitric oxide cell information system: inducible nitric oxide synthase and other nitric oxide synthase subtypes; cellular protein oxidation and nitration; cyclic guanosine monophosphate (cGMP) Soluble ornithine cyclase (sGC); Receptors for natriuretic peptides (NPs) and other cell membranes of ornithine cyclase (pGC); phosphodiesterase subtypes; serine/threonine Specific changes in gene expression and protein function of acid-specific protein kinases [series; including protein kinase G (PKG)] in malignant tumor tissues serve as biotherapeutic targets. Based on the specificity of the seven key biological target series, the regulatory drugs can be combined according to the target detection conditions to form an anti-malignant tumor based on a nitric oxide cell information system. Tumor-targeted therapeutic drug combination. This treatment can be used for primary lesions of tumors and other organ metastasis sites. The seven key biological target series and related drug development details of the present invention include:

1) These drugs are targets for altering the expression or activity of inducible nitric oxide synthase, endothelial nitric oxide synthase, and neuronal nitric oxide synthase in malignant tumors.

2) These drugs are targets for altering protein oxidation and nitration in malignant tumors.

3) These drugs are drugs that alter the level of cyclic guanosine monophosphate in malignant tumors.

4) This class of drugs is a gene that changes the soluble guanylate cyclase subtypes of malignant tumors: α1, α2; βΚ β2, β3 subunit, or a target drug that changes its enzymatic activity.

5) These drugs are genes or protein expressions that alter the subtypes of cell membrane ornithine cyclase in malignant tumors, or target drugs that alter their enzymatic activity.

6) Such drugs are targets for altering the expression of genes or proteins of specific subtypes of diuretic diesterase in malignant tumors, or for altering the protein function of specific subtypes of phosphodiesterase in malignant tumors.

7) These drugs are genes or protein expressions that alter the serine/threonine-specific protein kinase family, or target drugs that alter the protein function of the serine/threonine-specific protein kinase family in malignant tumors.

8) Such drugs include tumor treatment drugs for ligands or activators of guanylate cyclase/sodium peptide receptors (Table 1) and analogs thereof.

9) This class of drugs includes the PDE family and its inhibitors (Table 2); other phosphodiesterase inhibitor complexes such as methylxanthine (caffeine, theophylline, theobromine, aminophylline, choline, 2 Tumor therapy for hydroxypropyl theophylline), astragalus and its analogues.

10) These drugs include nitric oxide donors, nitric oxide precursors (such as nitrite, nitrate nitrate) and their analogs.

11) Such drugs include nitric oxide tinctures, or nitric oxide scavengers and analogs thereof.

12) Such drugs include tumor therapeutic drugs for cyclic guanosine monophosphate isomers and analogs thereof.

A detailed description of the serine/threonine-specific protein kinase series involved in the present invention is as follows:

Phosphorylase kinase (PHK) is a serine/threonine-specific protein kinase that converts phosphorylase b to phosphorylase a. One of the enzymes activated by phosphorylase kinase is glycogen phosphorylase. The gene for phosphorylase kinase α is PHKA1, ΡΗΚΑ2, and the gene for phosphorylase kinase β is ΡΗΚΒ, phosphoric acid. Kinase γ gene is PHKG1, PKHG2 ₀ phosphorylase kinase by phosphorylation of reversible allosteric regulation and regulation. Protein kinase A (PKA) is a holoenzyme consisting of two regulatory subunits and two catalytic subunits. When the level of cAMP is low, the whole enzyme remains intact and is not activated by the contact reaction. When the cAMP concentration is increased (eg, the G protein coupled receptor couples Gs to activate adenylate cyclase, inhibits the phosphodiesterase that degrades cAMP), cAMP binds to two sites of the regulatory subunit, thereby Causes a conformational change that releases the catalytic subunit.

Protein kinase B (PKB) is also known as AKT kinase. The human Akt family has three genes: Aktl, Akt2 and Akt3.

Aktl participates in the cell survival pathway by inhibiting the process of apoptosis. Aktl also induces protein synthesis pathways and is therefore a key signaling protein in the cellular pathway that causes excessive proliferation of skeletal muscle and tissue growth. Because it blocks apoptosis, it promotes cell survival. Aktl is also a major cause of many types of cancer. Akt (now known as Aktl) was originally thought to be an oncogene in retroviral transformation.

Akt2 is an important signaling molecule in the insulin signaling pathway, a regulatory enzyme necessary for inducing glucose transport.

Studies on Akt 1 or / and Akt2 knockout mice demonstrated that Akt 1 and Akt2 have different effects. In mice with null Aktl and normal Akt2, glucose homeostasis is undisturbed, but animals are smaller in size, which is consistent with Aktl's primary role in growth. In contrast, mice with normal Aktl and knocked out Akt2 had only slight growth defects, but showed a diabetic phenotype (insulin resistance), which is consistent with the view that Akt2 is specific for the insulin receptor in the signaling pathway. . The role of Akt3 is not known, but it is clearly expressed in the brain. It has been reported that mice lacking Akt3 have smaller brains.

Protein kinase C (PKC) is actually a family of protein kinases composed of approximately 10 isozymes. Based on its second messenger needs can be divided into three subfamilies: classic, new and non-classical.

Classical PKC - includes alpha, beta, beta and gamma subunits. Its activation requires Ca2+, diglycerides (DAG) and phospholipids such as lecithin.

PKC-α (PR CA)

P C-βΙ (PRKCB1)

PKC-βΙΙ

PKC-γ (PRKCG) The novel (n) PKCs include the δ, ε, η and θ subunits, which require DAG for activation but do not require Ca2+. Thus, both classical and novel PKCs, like phospholipase C, are activated by the same signal transduction pathway.

PKC-δ (PRKCD)

PKC-ε (PR CE)

PKC-η (PRKCH)

P C-Θ (PRKCQ)

Non-classical PKC - Ca2+ and DAG are not required for activation.

PKC-ι (PRKCI)

PKC - ζ (PRKZ)

PK-N1 (PKN1)

PK-N2 (PKN2)

Ca2+/calmodulin-dependent protein kinase or CaM kinase is mainly regulated by the Ca2+/calmodulin complex. There are two types of CaM kinases:

Specificity CaM kinase: For example, myosin light chain kinase (MLCK), which phosphorylates myosin, causes muscle contraction.

The multifunctional CaM kinase: also known as CaM kinase II, plays an important role in many processes, such as neurotransmitter release, transcription factor regulation, and glucose metabolism. About 1% to 2% of the protein in the brain is CaM kinase II.

CaMK I - CAMK1, CAM 1D, CAMK1G

CaMK II - CAM 2A, CAM 2B CAMK2D, CAMK2G

CaMK IV - CAMK4

Mitogen-activated protein kinases (MAPKs) act under extracellular stimuli (mitogens) and regulate a variety of cellular activities such as gene expression, mitosis, differentiation, and cell survival/apoptosis.

MAP is associated with most non-nuclear oncogene activities and is closely related to cellular responses to growth factors such as BDNF or nerve growth factor.

Extracellular stimulation leads to AP kinase activation through a signaling cascade (MAPK cascade) consisting of MAP kinase, MAP kinase kinase (MKK or MAP2K) and AP kinase kinase kinase (MKKK or AP3K). Extracellular stimulation causes phosphorylation of the MAP2K serine and threonine residues, resulting in activation of MAP3K; MAP2K then activates AP kinase by phosphorylating its serine and tyrosine residues. From yeast to mammals, the AP kinase signaling cascade is well developed.

Currently, there are six different MAPKs in mammals:

Extracellular signal-regulated kinase (ERK1, ERK2): ERKs (aka MAP kinase) signaling pathway is first activated by growth factors and phorbol esters (a tumor growth promoter) to regulate cell proliferation and differentiation.

c-Jun N-terminal kinases (JNKs) (MAPK8, MAPK9, MAPK10) are also stress-activated protein kinases (SAPKs). P38 subunit (MAPK11, MAPK12 (= ERK6), APK13, MAPK14): JNK and p38 signaling pathways are involved in stress, such as cytokines, UV irradiation, heat shock, and osmotic shock, and are associated with cell differentiation and apoptosis.

ERK5 ERK5 (MAPK7) was recently discovered and is activated by growth factors and stress stimuli and is involved in cell proliferation.

ERK3/4 ERK3 (MAPK6) and ERK4 (MAPK4) are structurally related to atypical MAPKs, and there are SEG motifs on the active loop. The main difference is only in the C-terminal extension. ERK3 and EKR4 are mainly cytosolic proteins that bind, translocate and activate MK5 (PPAR, MAP2K5). Unlike the relative stability of ERK4, ERK3 is unstable.

ERK7/8 (MAPK15) is the newest member of the MAPKs family and functions similarly to atypical MAPKs. ERK7/8 has a C-terminus similar to ERK3/4.

Mos/Raf kinase is part of the APKK kinase family and is activated by growth factors. The function of this enzyme is to stimulate cell growth. The current inhibition of Raf has become a target for novel anti-tumor metastatic drugs that can reduce cell proliferation by inhibiting APK cascades.

Pelle is a serine/threonine kinase that phosphorylates itself, as does Tube and Toll.

Other members include: Rhol, Racl, VE-cadherin, Tiaml, Epacl, Rapl, pll5-RhoGEF, phospho-Vav2, and pan~Vav2, di-phospho-MLC, phospho-PKA substrate, phospho-PAKl, and PAK1 The malignant tumor includes - Brain malignant tumors: astrocytoma, oligodendroglioma, ependymoma, brain (ridge) membrane, and embryo (tissue) tumor.

Lung cancer: is the primary cause of human death from cancer s

Thyroid cancer: There are four types of thyroid cancer, one of which is follicular carcinoma. Hurthle cell thyroid cancer, unlike follicular thyroid cancer, has an incidence of about 4% in thyroid cancer.

Bladder cancer is the most common malignant tumor of the urinary tract.

Breast cancer

Cervical cancer

Ovarian cancer

Uterine cancer

Hodgkin's disease is a malignant tumor derived from lymph nodes. It occurs at any age, but is more common in children and adolescents.

non-Hodgkin's lymphoma

Bone cancer: Most bone tumors are secondary tumors that develop from the spread of primary malignant tumor cells in other parts of the body to the bone.

Bone marrow cancer: The vast majority of myeloma is a secondary tumor that is caused by the spread and metastasis that occurs in other parts of the body. '

Melanoma

Gallbladder cancer: According to the American Cancer Society, gallbladder cancer is the fourth most common malignant tumor in the gastrointestinal tract, accounting for about 2 to 3% of all cancers in the United States.

Colon and rectal cancer is the third most common malignant tumor in men and the second most common malignant tumor in women. Esophageal cancer: It usually occurs between 50 and 70 years old.

Gastrointestinal tumors: The gastrointestinal tract, or the digestive system, digests the food we eat, absorbs nutrients, and excretes the residue as a form of feces.

Kidney cancer: "Kidney" means a part related to the kidney; "Cancer" refers to a type of malignant tumor that occurs in glandular, dermal, and mucosal epithelial cells.

Leukemia is a malignant tumor of hematopoietic tissue. Hematopoietic tissues include bone marrow, lymph nodes, and spleen. Although leukemia is the leading cause of death in children aged 3 to 14 years, leukemia is also common in adults.

Liver cancer occurs mostly after 45 years of age, and males are more likely to be female. Lymphoma is a malignant tumor of the lymphatic system. The lymphatic system includes lymph nodes, lymph glands, and spleen, which are spread throughout the body and are connected to each other by capillary lymphatic vessels.

Oral, pharyngeal and laryngeal cancers include leukoplakia or erythema, mucosal ulceration of the mouth and throat.

Pancreatic cancer is a gland located behind the stomach. Pancreatic juice secreted by the pancreas includes enzymes that can digest food, as well as insulin secretion from islet cells, which helps regulate blood sugar levels.

Rectal Cancer: The American Cancer Society believes that the United States is one of the countries with the highest incidence of rectal cancer in the world, second only to prostate cancer, lung cancer and skin cancer.

Prostate cancer

Testicular cancer

Sarcoma: A malignant tumor that occurs in muscle, bone, cartilage, and fibrous tissue. The sarcoma is named after the type of tissue it was originally grown on.

skin cancer

Gastric cancer is common worldwide, and in the past 40 years, the incidence of gastric cancer in the United States has decreased significantly.

Pharyngeal cancer: The most common throat tumor is laryngeal cancer.

Ureteral Tumor: The ureter is a conduit that connects the kidneys to the bladder and delivers urine to the bladder through muscle contraction. Eye cancer is relatively rare. It affects many areas of the eye: the orbit and its protective tissues, the eyeballs, the eye mask and the eyelids. The malignant tumor targeted therapeutic agent of the present invention comprises oral, parenteral, transdermal, topical, rectal, subcutaneous, intramuscular, intravenous, vaginal, intranasal. , bronchial administration, direct infiltration, peritoneal administration or local perfusion of drugs.

The ligands or activators of the guanylate cyclase/sodium peptide receptors of the present invention and their mainly distributed cells and tissues are summarized in Table 1:

Table 1. Ligand or activator of guanylate cyclase/sodium peptide receptor and its major distribution of cells and tissues

GC/NP (guanylate ring

Ligand distribution organization

Chemase/Natriuretic Peptide Receptor

ANP (cardiac natriuretic peptide) / BNP (brain natriuretic peptide) GC-A NPRA dirty, adrenal gland, heart, pulmonary vascular bed, ovary, GC/ P (guanylate ring

Ligand distribution organization

Chemase/Natriuretic Peptide Receptor

(Type A natriuretic peptide receptor) testis, Leydig tumor (MA-10) cells, neuroblastoma cells, brain, intestine, olfactory nerve and other tissues

GC-B/NPRB vascular bed, fibroblasts, heart, lung, adrenal gland,

CNP (C-type natriuretic peptide)

(B-type natriuretic peptide receptor) brain, ovary, cartilage and other tissues

ANP, BNP, CNP GC-C / NPRC kidney, adrenal gland, lung, blood vessel, heart, intestine, brain

(Natriuretic peptide, brain natriuretic peptide, C-type natriuretic peptide) (C-type natriuretic peptide receptor) and other tissues

Guanylyn/uroguanylyn GC-C /NPRC

Large intestine, intestine, kidney

(guanosine / guanosine) (C-type natriuretic peptide receptor)

Orphan (according to the United States the Orphan Drug

GC-D

Act of January 1983 ("ODA" development of the olfactory nerve epithelium

(D-type natriuretic peptide receptor)

Drug)

Orphan (according to the United States the Orphan Drug

GC-E

Act of January 1983 ("ODA" development of the retina, pineal gland

(E-type natriuretic peptide receptor)

Drug)

Orphan (according to the United States the Orphan Drug

GC-F

Act of January 1983 ("ODA" development of the retina

(F-type natriuretic peptide receptor)

Drug)

Orphan (according to the United States the Orphan Drug

GC-G

Act of January 1983 ("ODA" development of skeletal muscle, lung, intestine, kidney and other tissues

(G-type natriuretic peptide receptor)

Drug)

ROS-GC (no middle)

Calmodulin-binding protein

Text)

Orphan (according to the United States the Orphan Drug

Act of January 1983 ("ODA" development of GC-Y-X1 (no Chinese) C. elegans sensory nerve

Drug)

NO (-nitrogen oxide), CO carbon monoxide) soluble cyclase smooth muscle, platelets, kidney, lung and other tissues

The PDE family and its inhibitors involved in this issue are summarized in Table 2:

Table 2. PDE family and its inhibitors

The invention clarifies the substantial relationship between the NO information system and the malignant tumor in the international arena, and develops the core technology for the treatment of malignant tumors related to the NO Nobel Prize theory. This core therapeutic technique is closely related to the diagnostic techniques described in the inventor's other patent. After the diagnosis and distribution of NO information system-related molecules in malignant tumors are clearly diagnosed, combined with the cell division cycle and differentiation characteristics of malignant tumors, the concept of comprehensive treatment and system biology of Chinese medicine is absorbed, and the malignant tumor target based on the biological regulation of nitric oxide is performed. The directional treatment drugs comprehensively reveal that nitric oxide has the targeting, wideness, effectiveness, low toxicity or no toxicity, and has great clinical value.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 Schematic diagram of the relationship between inflammation, iNOS, protein tyrosine nitration and malignant tumors Figure 2 Non-reactive sGC stimulation and reduction of cGMP levels in malignant tumors

Figure 3 and Figure 4 Specificity of sGC mRNA expression and sGC protein expression in malignant tumors (M = malignant) Figure 5 Good telomerase reverse transcriptase expression in malignant tumor samples

Figure 6 Comparison of colorimetric analysis of benign meningioma (M71), malignant meningioma (M6) and glioma (D54) after treatment with 8Br-cGMP

Figure 7 NPR-A (GC-A), NPR-B (GC-B) and GC-C mRNA expression in benign and malignant tumor samples (BM = benign meningioma; MM = malignant meningioma; G = glioma) )

Figure 8 Schematic diagram of the natriuretic peptide family and receptors

Figure 9 and Figure 10 ANP, BNP plus phosphodiesterase inhibitors IBMX treated different concentrations of glioma cells and detected their proliferation on days 4 and 6, respectively. CON is a blank control group.

detailed description:

Example 1 Inducible expression of nitric oxide synthase in malignant tumors

The present inventors performed a sample of human benign and malignant tumors approved by a tumor pathologist in the United States, and used only primary tumor cells or tumor tissues for biological analysis of nitric oxide. Revealed the inducibility of variation in malignant tumors. Nitric oxide synthase expression lays the foundation for the use of iNOS detection in the treatment of malignant tumors. Experimental results show that there is no or only a very small amount of inducible nitric oxide synthase (iNOS) expression in benign tumors. However, as the degree of malignancy of the tumor increased, the expression of inducible nitric oxide synthase (iNOS) was also correlated (Fig. 1). The expression of iNOS in the environment of protein tyrosine nitration-induced inflammatory malignant tumors leads to excessive production of reactive oxygen species (ROS) / reactive nitrogen species (RNS), which causes cell damage mainly by 0N00- guide. NO combines with 0 ₂ - to form 0N00_, while 0N00 - is highly oxidizing and can react with proteins, lipids, carbohydrates and nucleic acids in shock, inflammation, ischemia-reperfusion injury, respiratory diseases and neurodegenerative diseases It plays an important role. 0N00 can also react with active molecules in the body, such as endogenous antioxidants (ascorbic acid, vitamin E), sulfonium groups and aromatic compounds. 0Ν0 (causing oxidation of proteins, oxidizing, hydroxylating and nitrating the sulfur-containing groups, also nitrating protein tyrosine residues (Figure 1), which alters intracellular signaling pathways (eg, tyrosine phosphorylation) Influence). Undoubtedly, this study shows It alters protein oxidation and nitration in malignant tumors and is associated with tumor therapy. Example 2 Variation test of cyclic guanosine monophosphate (cGMP) and soluble ornithine cyclase (sGC) in malignant tumors Cyclic guanosine monophosphate (cGMP) can be soluble in ornithine cyclase (sGC) or membranous ornithine cyclase (pGC) synthesized. Soluble ornithine cyclase synergistically produces enzyme activity from two subunits, α and β. Cyclic guanosine monophosphate (cGMP), once synthesized, can pass cGMP-dependent protein kinases (PKGs), Cyclic mediated ion channels (CNGs), cGMP-regulated phosphodiesterase (PDEs), and other pathways play a role.

Our experimental results show that the expression of inducible nitric oxide synthase (iNOS) is also correlated with the increase of tumor malignancy. In malignant tumors, how does the large amount of nitric oxide production (NO) synthesized by highly expressed iNOS contribute to its downstream information system? In order to understand the mechanism, we used NONOate (-nitrogen oxide supply) and Bay412272 (soluble ornithine cyclase specific stimulator) in the experiment, as shown in (Figure 2), unlike benign tumors, malignant tumors. The soluble ornithine cyclase does not respond to the stimulation of nitric oxide and soluble ornithine cyclase-specific stimulators. The level of cyclic guanosine monophosphate (cGMP) is very low. To further decipher the cyclic guanosine monophosphate (cGMP) in malignant tumors. ) the reason for the low level, used

SDSPAGE protein electrophoresis and specific antibodies were used to detect the protein expression of soluble ornithine cyclase alpha and beta subunits in benign and malignant tumors. The results showed that soluble ornithine cyclase α and β were found in malignant tumors. The protein expression of the subunits disappeared or was extremely weak (Fig. 3 and Fig. 4).

For more stringent molecular biology analysis, real-time RT-PCR was performed on the gene (mRNA) expression of soluble ornithine cyclase alpha and beta subunits in benign and malignant tumors (7700 Prizm Sequence Detector System) As shown in Fig. 3, the gene expression of soluble ornithine cyclase alpha and beta subunits in malignant tumors disappeared or was extremely weak. The results revealed mutations in cyclic guanosine monophosphate (cGMP) and soluble ornithine cyclase (sGC) in malignant tumors (Fig. 2, 3), using primary tumor cultured cells or tumor tissues for biological analysis of nitric oxide. Variations in cyclic guanosine monophosphate (cGMP) and soluble ornithine cyclase (sGC) in malignant tumors (Fig. 2, 3, 4), not only for the detection of cGMP and sGC for the diagnosis of malignant tumors, but also for the identification of tumor diffusion tracking The basis is also to provide a patent basis for the gene and protein expression of soluble guanylate cyclase subtypes or the activity of soluble guanylate cyclase in therapeutically altered malignancies.

Human telomerase reverse transcriptase The present inventors also used benign and malignant tumor samples for human telomerase Reverse transcriptase (hTERT) RT-PCR (7700 Prizm Sequence Detector System) detection. As shown in Figure 5, human telomerase reverse transcriptase gene expression is very high in malignant tumors, while benign tumor samples are extremely weak. The reliability of human benign and malignant tumor samples approved by the inventors of the United States for examination by a tumor pathologist is further demonstrated.

Example 3 Treatment of malignant tumors by regulating intracellular cyclic guanosine monophosphate (cGMP) levels

In order to develop a novel treatment strategy for malignant tumors from the experimental results of low levels of cyclic guanosine monophosphate (cGMP) in malignant tumors, the present invention firstly carried out experiments on the supplementation of cGMP with malignant tumors (Fig. 6). 8-bromoguanosine 3',5-cyclic monophosphate (8Br-cGMP) is a cyclic guanosine preparation that can pass through the cell membrane, and is treated with 8Br-cGMP for benign (M71) and malignant tumors (M6 and D54). The results showed that the growth of malignant tumors (M6 and D54) was significantly inhibited (statistics P < 0.01), while the growth of benign (M71) tumors had little effect. This experiment not only demonstrated the regulation of intracellular loops. Phosphate guanosine (cGMP) levels can be used in the preparation of anti-malignant tumor therapeutics and show little effect on the growth of benign tumors with low or no side effects.

Example 4 Natriuretic peptide family receptor and its membrane membranous ornithine cyclase

Natriuretic peptides (NPs) mainly include atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), c-type natriuretic peptide, CNP And synthetic natriuretic peptide (VAP), a group of peptides that are important in maintaining the body's water and salt balance, blood pressure stability, cardiovascular and kidney functions. The natriuretic peptide family acts on it. The two receptors are cell membrane ornithine cyclase (pGC) g|3 _: NPR-A/GC-A and NPR-B/GC-B. (Fig. 8)

In order to more closely analyze the molecular biology of the cyclic guanosine monophosphate (cGMP), the present inventors conducted a comprehensive analysis of the expression of the receptor gene (mRNA) of the natriuretic peptide family in benign and malignant tumors. As shown in Figure 7, real-time RT-PCR (7700 Prizm Sequence Detector System) results showed no correlation between the expression of the natriuretic peptide family receptor and the malignancy of the tumor. The experimental data used the natriuretic peptide family receptor. Ligand-mediated upregulation of cyclic guanosine monophosphate (cGMP) provides a basis for the relationship between the natriuretic peptide family receptor and its membrane membranous ornithine cyclase and malignant tumors, suggesting that the expression of soluble ornithine cyclase in malignant tumors Mutation, and thus, whether the tumor expresses the natriuretic peptide family receptor or which type of natriuretic peptide family receptor is expressed, is not only directly related to the level of production of the cyclic guanosine monophosphate (cGMP) of the tumor tissue, but also the natriuretic peptide family. Receptor ligands are used to provide a basis for the detection of malignant drug preparation.

Example 5 Regulation of intracellular cyclic guanosine monophosphate (cGMP) levels and malignant tumor treatment trials by phosphodiesterase information system

Phosphodiesterase activity is directly related to the intracellular second messenger, cAMP and cGMP concentrations. There are 11 phosphodiesterase subtypes that have been identified. The use of phosphodiesterase inhibitors opens up new hopes for the treatment of a variety of diseases, depending on the cell and tissue distribution of each phosphodiesterase subtype, and the altered expression under various pathological conditions. Summarize the clinical application in recent years and analyze its mechanism of action to provide a basis for further development applications.

Although atrial natriuretic peptide (ANP) or brain natriuretic peptide (BNP) acts on its receptors: NPR-A/GC-A and NPR-B/GC-B can cause intracellular cyclic guanosine monophosphate (cGMP) levels Increase (Fig. 9, 10), but when the inventors used atrial natriuretic peptide (ANP) or brain natriuretic peptide (BNP) alone, the inhibitory effect on the growth of malignant tumors was not significant. However, we treated malignant cells with atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) plus the phosphodiesterase inhibitor IBMX, as shown in Figure 7, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were concentration-dependent inhibition of the growth of malignant tumor cells (5X10- ⁵ M and 5X10- ⁶ M, statistically P <0. 01).

The results indicate that by determining the expression variation of phosphodiesterase subtypes in malignant tumors, it provides a basis for targeting the increase of the level of cGMP production in tumor tissues, and the detection of phosphodiesterase subtypes as Targeted inhibitors provide a basis for drugs for detecting malignant tumors.

Claims

Rights request

1. A nitric oxide and an information delivery system thereof for use in the preparation of a targeted therapy for malignant tumors.

2. The use of a nitric oxide and an information delivery system thereof for the preparation of a targeted therapy for malignant tumors according to claim 1, wherein the drug is an inducible nitric oxide synthase in a malignant tumor , endothelial nitric oxide synthase, and a target drug for the expression or activity of neuronal nitric oxide synthase.

3. The use of a nitric oxide and an information delivery system thereof according to claim 1, in the preparation of a targeted therapy for malignant tumors, characterized in that the drugs are used to alter protein oxidation and nitration in malignant tumors. Target drug.

4. The use of a nitric oxide and an information delivery system according to claim 1 for the preparation of a targeted therapy for malignant tumors, characterized in that the drug is a drug for altering the level of cyclic guanosine monophosphate in a malignant tumor.

5. The use of a nitric oxide and an information delivery system thereof according to claim 1, in the preparation of a targeted therapy for malignant tumors, characterized in that the drug is a soluble guanylate cyclase in a malignant tumor. Each subtype: α1, α2; β β2, subunit gene or protein expression, or a target drug that changes its enzymatic activity.

6. The use of a nitric oxide and an information delivery system thereof according to claim 1, in the preparation of a targeted therapy for malignant tumors, characterized in that the drug is a cell membrane guanosine cyclase in a malignant tumor. Gene or protein expression of each subtype (including GCA, GCB, GCC), or a target drug that alters its enzymatic activity.

7. The use of a nitric oxide and an information delivery system thereof according to claim 1, in the preparation of a targeted therapy for malignant tumors, characterized in that the drug is a specific subtype of phosphodiesterase in malignant tumors. A gene or protein expression, or a target drug that alters the protein function of a specific subtype of phosphodiesterase in a malignant tumor.

8. The use of a nitric oxide and an information delivery system thereof according to claim 1, in the preparation of a targeted therapy for malignant tumors, characterized in that the drug is a serine/threonine-specific protein kinase. Family gene or protein expression, or a target drug that alters the protein function of the serine/threonine-specific protein kinase family in malignant tumors.

9. The use of a nitric oxide and an information delivery system thereof according to claim 1, in the preparation of a targeted therapy for malignant tumors, characterized in that the drug comprises guanylate cyclase/natriuretic peptide. Tumor therapeutic agents for ligands or activators and their analogs.

10. A nitric oxide and an information delivery system thereof according to claim 1 for preparing a malignant tumor targeting The use in therapeutic drugs is characterized in that the drug comprises a tumor therapeutic agent of the PDE family and its inhibitors or phosphodiesterase inhibitor complexes and analogs thereof.

11. The use of a nitric oxide and an information delivery system thereof according to claim 1, in the preparation of a targeted therapy for malignant tumors, characterized in that the drug comprises a nitric oxide donor, a nitric oxide precursor material Tumor treatment drugs (such as nitrite nitrite, nitrate nitrate) and their analogs.

12. The use of a nitric oxide and an information delivery system thereof for the preparation of a targeted therapy for malignant tumors according to claim 1, characterized in that the drug comprises a nitric oxide bismuth agent or a nitric oxide scavenger And its analogs for the treatment of tumors.

13. The use of a nitric oxide and an information delivery system thereof according to claim 1, in the preparation of a targeted therapy for malignant tumors, characterized in that the drug comprises a cyclic guanosine monophosphate isomer and the like. Tumor treatment drugs.

14. The use of a nitric oxide and an information transmission system thereof according to any one of claims 1 to 10 for the preparation of a targeted therapy for malignant tumors, characterized in that the targeted therapy for malignant tumors comprises oral administration. Parenteral administration for transdermal administration, external application, rectal administration, subcutaneous administration, intramuscular administration, intravenous administration, vaginal administration, intranasal administration, bronchial administration, direct infiltration, peritoneal administration or partial perfusion Drug.