Compound of glycosaminoglycan, preparation method and application thereof
阅读说明：本技术 糖胺聚糖的化合物、其制备方法及用途 (Compound of glycosaminoglycan, preparation method and application thereof ) 是由 林华洋 于 2014-06-27 设计创作，主要内容包括：本发明涉及将药物与糖胺聚糖,例如透明质酸(HA)缀合的化合物,其中所述药物用于治疗多种疾病,例如炎症、自身免疫疾病、过敏症、感染且优选地,癌症。本发明的缀合化合物可通过用作靶药物递送载体的糖胺聚糖与CD44细胞表面受体的相互作用来增加药物在疾病的特定位点的浓度,然后增强定点递送的(site-delivered)药物的治疗功效并减少全身性副作用。(The present invention relates to compounds that conjugate drugs to glycosaminoglycans, such as Hyaluronic Acid (HA), wherein the drugs are used for the treatment of various diseases, such as inflammation, autoimmune diseases, allergy, infections and preferably cancer. The conjugate compound of the present invention can increase the concentration of a drug at a specific site of a disease by the interaction of glycosaminoglycan used as a target drug delivery vehicle with CD44 cell surface receptors, and then enhance the therapeutic efficacy of site-delivered (site-delivered) drugs and reduce systemic side effects.)
1. A conjugate consisting of a glycosaminoglycan and an active compound, wherein the active compound is directly conjugated through formation of an amide bond with the carboxyl group of the glycosaminoglycan, its derivative, or a salt thereof, and wherein the active compound is selected from the group consisting of lenalidomide, gemcitabine, nimesulide, and celecoxib.
2. The conjugate of claim 1, wherein the glycosaminoglycan is hyaluronic acid.
3. The conjugate of claim 2, wherein the hyaluronic acid has an average molecular weight of 10kDa to 2000 kDa.
4. A conjugate according to one of claims 1 to 3 for use in the treatment of cancer.
5. The conjugate of claim 4, wherein the cancer is liver cancer, hepatocellular carcinoma, cholangiocarcinoma, cholangiocellular cystadenocarcinoma, colon cancer, adenocarcinoma, lymphoma and squamous cell carcinoma, breast cancer, ductal carcinoma, lobular carcinoma, lung cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, kidney cancer, renal cell carcinoma, urothelial cell carcinoma, multiple myeloma, myelodysplastic syndrome (MDS), Hodgkin's lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia or pancreatic cancer.
6. Use of a conjugate of a glycosaminoglycan and an active compound for the preparation of a pharmaceutical composition for the treatment of cancer, wherein the conjugate is according to any one of claims 1 to 3.
7. The use of claim 6, wherein the cancer is liver cancer, hepatocellular cancer, cholangiocarcinoma, cholangiocellular cystadenocarcinoma, colon cancer, adenocarcinoma, lymphoma and squamous cell carcinoma, breast cancer, ductal carcinoma, lobular carcinoma, lung cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, kidney cancer, renal cell carcinoma, urothelial cell carcinoma, multiple myeloma, myelodysplastic syndrome (MDS), Hodgkin's lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia or pancreatic cancer.
8. A pharmaceutical composition comprising a conjugate of at least one glycosaminoglycan and an active compound in combination with at least one excipient and/or diluent, wherein the conjugate is according to any one of claims 1 to 3.
9. The pharmaceutical composition of claim 8, wherein the composition is for treating cancer.
10. The pharmaceutical composition of claim 9, wherein the cancer is liver cancer, hepatocellular cancer, cholangiocarcinoma, cholangiocellular cystadenocarcinoma, colon cancer, adenocarcinoma, lymphoma and squamous cell carcinoma, breast cancer, ductal carcinoma, lobular carcinoma, lung cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, kidney cancer, renal cell carcinoma, urothelial cell carcinoma, multiple myeloma, myelodysplastic syndrome (MDS), hodgkin's lymphoma, non-hodgkin's lymphoma, chronic lymphocytic leukemia or pancreatic cancer.
Technical Field
The present invention relates to compounds consisting of glycosaminoglycans conjugated to drugs, and also to the preparation and use of such compounds.
Background
Extracellular matrix (ECM) is a dynamic combination of interacting molecules that modulate cellular function and interaction in response to stimuli. One class of extracellular matrix macromolecules, glycosaminoglycans, are molecules known to be involved in a large number of normal and abnormal biological processes including cell migration (migration), differentiation (differentiation), proliferation (proliferation), immune response (immune response) and organization of the cytoskeleton (cytoskeletal organization).
Glycosaminoglycans (GAGs) are unbranched polymers comprising repeating disaccharide units (dissacharide units). These disaccharide units always comprise an amino sugar (N-acetylglucosamine or N-acetylgalactosamine), which in most cases is sulfated, the second sugar usually being uronic acid (glucuronic acid or iduronic acid). GAGs are highly negatively charged due to the presence of carboxyl or sulfate groups (sulfate) on most of the sugar residues of GAGs, and thus, GAGs are strongly hydrophilic. GAGs tend to adopt a highly extended conformation and form a matrix that is space-filling and resistant to compression forces. Four major groups of GAGs have been distinguished by their sugar residues, the type of bond between these residues, and the number and position of sulfate groups. They include: (1) hyaluronic Acid (HA), (2) chondroitin sulfate (chondroitin sulfate) and dermatan sulfate (dermatan sulfate), (3) heparan sulfate (heparin) and heparin (heparin), and (4) keratan sulfate (keratan sulfate).
Hyaluronic acid (Hyaluronan, also known as hyaluronic acid, hyaluronate or HA) is the simplest of GAGs. It consists of a regular repeating sequence of non-sulfated disaccharide units, in particular N-acetylglucosamine and glucuronic acid. The molecular weight can vary from 400 daltons (disaccharides) to over several million daltons. It can be present in all tissues, such as skin, cartilage and eyes, in varying amounts, and in fluid form in most adult animals. It is particularly abundant in early embryos. In articular cartilage, hyaluronic acid can form large aggregates that are important for cartilage function. In addition, cell motility and adhesion of immune cells are mediated by cell surface receptors RHAMM (receptor for hyaluronic acid mediated motility) and CD 44.
HA is directly synthesized by a single protease, Hyaluronan Synthase (HAs), which is extruded outside the cell during synthesis by the pressing of growing polymers at the inner membrane of the cell surface. In contrast, other GAGs are synthesized in the intracellular golgi apparatus and bind to some of the core proteins and are then released by exocytosis. In vivo HA degradation in vertebrate tissues is mediated by hyaluronidase and exoglycosidase (exoglycosidase) which sequentially remove sugars. Mammalian hyaluronidase has both hydrolytic and transglycosidase activities and can degrade hyaluronic acid and chondroitin. In connective tissue, hydration of hyaluronic acid in combination with water creates spaces between the tissues, creating an environment conducive to cell movement and proliferation. Hyaluronic acid plays a key role in biological phenomena related to cell motility, including rapid development, regeneration, repair, embryogenesis, embryonic development, wound healing, angiogenesis, and tumorigenesis.
CD44 (also known as Pgp-1, Hermes-3, HCAM or ECMR III) is a widely expressed glycoprotein with a molecular weight of 85kDa to 90 kDa. CD44 is the major cell surface receptor for the glycosaminoglycan, Hyaluronic Acid (HA). Although CD44 specifically binds HA, certain chondroitin sulfate-containing proteoglycans are also recognized. CD44 plays a role in a variety of cellular and physiological functions, including adhesion or migration to HA, HA degradation, and tumor metastasis. CD44 has also been shown to play a role in extracellular matrix binding, cell migration, lymphocyte activation, lymphocyte homing (lymphocyte homing) and proliferation of bronchial smooth muscle cells (Gunthert et al, 1991, A new variant of glycoprotein CD44 conjugates to rat cancer cells, 5; 65(1): 13-24). The CD44 receptor exhibits a complex alternative splicing pattern in the variable region of its extracellular domain. CD44 appears to be a particularly important HA leukocyte (leukcyte) receptor and therefore may play a role in the pathogenesis of asthma. Furthermore, increased HA levels in experimental asthma in control mice were significantly reduced in antibody treated mice, confirming the role of CD44 in HA metabolism (specifically, when high molecular weight HA is broken down into proinflammatory low molecular weight forms). This is particularly important because HA-derived oligosaccharides can bind to and activate Toll receptors (Toll-like receptors). Therefore, these results are highly significant for the therapeutic effect against CD 44.
The HA-CD44 interaction may play an important role in development, inflammation, T cell recruitment and activation, lung inflammation, and tumor growth and metastasis. Altered Expression of alternatively spliced CD44 transcripts has been found in many cancers, including cancers of the stomach (F Reihani-Sabet et al, 2003, Effects of Infection and H. pyro Infection Expression of CD44 Variant Exons in Gastric Tissue, Journal of Sciences,14: 11-16).
Due to the overexpression of the CD44 receptor, malignant tumor cells (malignant tumor cells) are able to selectively take up more bioconjugates (bioconjudgements) than normal connective tissue or mesenchymal cells (mesenchyme cells). Several studies have shown that HA synthesis and uptake are associated with cancer progression and metastatic potential. Certain tumors, including many tumors found in the lung, have a condition in which a cell surface marker of CD44 is overexpressed. Breast cancer cells are known to have more HA uptake than normal cells, requiring HA for high P-glycoprotein expression, which is a major contributor to the development of multi-drug resistance (multi-drug resistance). Furthermore, invasive (invasive) breast cancer cells overexpress CD44, the major receptor for HA, and are dependent on high concentrations of HA internalized by CD44 for proliferation. Thus, nanoconjugates of chemotherapeutic drugs with HA (nanoconjugates) are effective against lymphatic metastasis (Eliaz, R.E. et al, 2004, Liposome-encapsulated doxorubicin targeted to CD44: a strain to kill CD 44-overpressing tumor cells, Cancer Res.,61(6):2592 cells 601).
Selective inhibitors of non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 (COX-2) are a group of therapeutic agents commonly used to treat pain, inflammation, and fever. More recently, more and more experiments have suggested that certain NSAIDs and selective COX-2 inhibitors may also have anti-cancer activity by participating in multiple biological events throughout the tumorigenesis process. For example, epidemiological studies have shown that the regular use of aspirin reduces the risk of developing cancer, particularly in the colon (Sandler RS. et al, 2003, A randomized tertiary of aspirin to a present chromosomal adenosine in tissues with a previous chromosomal nucleotide cancer, New England J. Med.,348: 883-. In addition, COX-2 antagonists, such as Celecoxib (Celecoxib), Rofecoxib (Rofecoxib), Nimesulide (Nimesulide), Meloxicam (Meloxicam), Etodolac (Etodolac), and the like, have also been found to have anti-cancer activity (Yamazaki R. et al, 2002, selected cyclic oncogenese-2 inhibitors show a differential activity to inhibition promotion and expression of alcohol adsorption of phenol adsorption cells, FEBS Lett.531 (2): 278-84). Further, COX-2 is chronically overexpressed in many premalignant, malignant, and metastatic human cancers, and the levels of overexpression are found to be significantly correlated with the aggressiveness, prognosis, and survival rates of some cancers (Dannenberg AJ. et al, 2003, Targeting cycloxygenase-2 in human neoplasia: ratioale and promise, Cancer Cell,4(6): 431-6). Maximum efficacy is usually limited by the dose of COX-1 associated toxicity; however, COX-2 inhibitors have been shown to have tumor-inhibiting effects in several animal models of colon, skin, lung, bladder and breast cancers (Alane T. Koki et al, 2002, Celecoxib: A Specific COX-2 Inhibitor With Anticancer Properties, Cancer Control, 9(2Suppl): 28-35).
WO94/09811 describes the use of CD44 in the treatment of inflammation or in the detection of cancer metastasis. These authors suggest that CD44 is up-regulated in inflammatory disorders and that CD44 peptide is capable of inhibiting T cell activation. However, no data or content is presented regarding the inhibition of metastasis by CD44, and no description is given of the use of CD44 for inhibiting tumor growth or angiogenesis. WO99/45942 discloses the use of HA-binding proteins and peptides, including CD44, to inhibit cancer and angiogenesis-dependent diseases. This patent application uses a 38kDa fragment of the cartilage connexin, metastatin (metastatin), and HA-binding peptide derived from this fragment to inhibit lung metastasis of B16 mouse melanoma and Lewis lung carcinoma. In the case of the HA-binding peptide, the growth of B16 melanoma on chicken CAM, and the migration of endothelial cells on HA, HAs been inhibited. In both of the above patent applications, the use of HA-binding peptides is directly related to their ability to bind hyaluronic acid.
U.S. patent No. 8,192,744 discloses that soluble recombinant CD44 hyaluronic acid binding domain (CD44 HABD) inhibits in vivo angiogenesis in chicken and mice and thereby inhibits human tumor growth from multiple sources. The invention discloses a soluble and non-glycosylated CD44 recombinant protein which is used as a novel angiogenesis inhibitor based on the targeting of a vascular cell surface receptor.
Thus, the above-mentioned prior art discloses the potential use of CD44 and suggests that any effect may depend on HA-CD44 interactions. Thus, to date, all of the utilities ascribed to HA-CD44 conjugates have been directly dependent on their ability to bind hyaluronic acid.
However, some drugs have not been successfully conjugated to hyaluronic acid and further testing should be performed to confirm the potential usefulness of HA as a site-directed delivery vehicle for active compounds. In particular, the prior art does not show the interaction of the cell surface receptors CD44 and the conjugates of HA with active compounds, which can be developed as a target transporter and used for the effective treatment and amelioration of diseases over-expressed by CD 44.
For a variety of pathologies, such as cancer, there is in fact still a need to have available therapeutic tools that balance the cytotoxic effect on tumor cells and the cytotoxic effect on normal cells with better safety characteristics.
Disclosure of Invention
The object of the present invention is to provide a novel compound based on the conjugation of HA with an active compound, which is suitable for the site-directed delivery of the active compound in diseases overexpressing the surface cell receptor CD 44.
Accordingly, the present invention provides compounds that conjugate glycosaminoglycans to a drug for the treatment of cancer diseases that are highly associated with the expression of CD 44.
In a first aspect, the present invention is directed to a compound consisting of a conjugate from a glycosaminoglycan and an active compound, wherein the active compound is conjugated to the carboxyl group of the glycosaminoglycan, its derivative, or a salt thereof via a functional group to form a covalent conjugation, and wherein the active compound is selected from the group consisting of lenalidomide, gemcitabine and a COX-2 antagonist.
The glycosaminoglycan of the conjugate according to the invention is preferably hyaluronic acid.
Furthermore, the glycosaminoglycan conjugate according to the present invention is preferably used for the treatment of cancer diseases.
Thus, in a second aspect, another object of the present invention is the use of a compound consisting of a conjugate from a glycosaminoglycan and an active compound, wherein the active compound is conjugated via a functional group to the carboxyl group of the glycosaminoglycan, its derivative, or a salt thereof to form a covalent conjugation, and wherein the active compound is selected from the group consisting of lenalidomide, gemcitabine and COX-2 antagonists, for the treatment of cancer and for the preparation of a pharmaceutical composition for said therapeutic treatment.
In yet a further aspect, the object of the present invention is a process for the preparation of a compound consisting of a conjugate from a glycosaminoglycan and an active compound, wherein the active compound is conjugated to the carboxyl group of the glycosaminoglycan, its derivative or a salt thereof via a functional group to form a covalent conjugation, and wherein the active compound is selected from the group consisting of nardoxylamine, gemcitabine and a COX-2 antagonist.
Drawings
For a fuller description of the invention, reference is made to the embodiments thereof which are illustrated in the accompanying drawings. The drawings attached hereto form a part of the specification. The accompanying drawings, however, should not be taken to limit the scope of the invention.
Figure 1 shows the affinity of HA in normal and damaged colon tissue by fluorescence index.
FIG. 2 shows the fluorescence results of HA-dye compounds acting on HCT15 cell line and HT29 cell line with different time courses, wherein FIG. 2A represents HCT15 cell line for 6 hours; FIG. 2B shows the HCT15 cell line at 12 hours; FIG. 2C shows HT29 cell line at 6 hours; FIG. 2D shows the HT29 cell line at 12 hours.
Figure 3 shows the structure of the HA-lenalidomide conjugate.
Figure 4A shows the cytotoxic effect of free lenalidomide, HA and HA-lenalidomide conjugate on HT29 cell line.
Figure 4B shows the cytotoxic effect of free lenalidomide, HA and HA-lenalidomide conjugate on HCT15 cell line.
FIG. 5A shows authentic nimesulide (containing NO)2Basic group, NiNO2) And hydrogenation modified product (containing NH)2Radical, NiNH2) The structure of (1).
FIG. 5B shows HA-NiNH2Structure of the conjugate.
FIG. 6A shows NiNO2And NiNH2Cytotoxic effects in HT29 or HCT 15.
FIG. 6B shows NiNO2、NiNH2HA, and HA-NiNH2Cytotoxic effect of the conjugate in HT 29.
Figure 7A shows the total body weight of mice in each of the three groups over 24 days.
FIG. 7B shows control, NiNO2Group and HA-NiNH2Tumor suppression in groups.
Figure 8 shows the synthesis and structure of HA-celecoxib.
Figure 9A shows the cytotoxic effect of HA, celecoxib, and HA-celecoxib conjugates on the HT29 cell line.
Figure 9B shows the cytotoxic effect of HA, celecoxib, and HA-celecoxib conjugates on the GBM401 cell line.
Figure 10 shows the synthesis and structure of HA-gemcitabine conjugates.
Figure 11 shows the cytotoxic effect of HA-gemcitabine conjugate on a549 cell line.
Figure 12 shows the cytotoxic effect of HA-gemcitabine conjugate on GBM401 cell line.
Detailed Description
The objects, advantages and novel features of the invention will become apparent from the following detailed description of the invention when considered in conjunction with the drawings.
Generally, a drug taken orally or injected into the circulatory system must reach its targeted treatment area directly, and since the drug concentration and specificity is not so high at the targeted site, the effect of the drug on targeting diseases and normal organs is very similar. Thus, in many cases, the administration of an effective amount of an active compound is impaired and limited by the safety profile of the active compound.
To improve therapeutic efficacy, combining therapeutic efficacy with good safety profiles, one strategy is to modify the drug by covalently binding it to a carrier to make it more target selective for the disease region. As previously predicted, this is particularly desirable in the field of anti-tumor therapy.
With this aim, the inventors conceived a strategy to explore the interaction between Hyaluronic Acid (HA) and its receptor CD44 for targeted delivery of active substances.
After long-term studies and experiments with HA, the inventors have established a strategy to maintain relatively high concentrations of drug at the target site versus normal tissues or organs.
The results from which the present invention originates are fully described in the examples and are briefly summarized below.
In fact, the present invention is supported by experimental results showing that hyaluronic acid with different average molecular weights HAs a higher adhesion index in wounded tissues compared to normal tissues, and HA with low average molecular weight performs better than HA with high average molecular weight. In particular, as shown in fig. 1, comparing the difference between three different average molecular weight HA adhered to injured colon tissue, the fluorescence index of the adhesion of injured colon tissue to 350kDa HA is higher than that of the other two average molecular weight HA (average molecular weight 2000kDa ═ 2MDa and 1000kDa ═ 1 MDa). In addition, even normal colon tissue or injured colon tissue HAs a fluorescence index for adhesion to 1MDa HA higher than 2MDa HA. This result demonstrates that HA can adhere more specifically to sites of inflammation, which prompted the inventors to further create the invention and to verify whether the unique feature of tissue adhesion of hyaluronic acid (suggested to be caused by the interaction of HA with its surface cell receptor CD 44) is preserved when the glycosaminoglycan is conjugated with other compounds.
Thus, the inventors further conjugated the drug to HA to verify whether HA can be used as a targeted delivery tool for delivering the drug to sites rich in CD 44. As previously described, when CD44 is overexpressed during the course of inflammation, infection, or cancer, the relevant drug can easily reach and remain and maintain relatively high concentrations at the targeted site, as ligand HA can adhere to receptor CD 44. With the adhesion of HA to sites of inflammation or sites rich in CD44, the drug should specifically aggregate at the site due to the relatively high concentration of the conjugated drug at said site and thus correspondingly reduce the amount of drug used, with better safety profile.
To confirm that the drug or dye HAs been successfully conjugated to HA and further confirm HA adhesion, the inventors of the present invention performed an experiment involving conjugation of the dye to HA (HA-dye) and treatment of cell lines and mice, respectively. Fig. 2A and 2B show experiments on cell line HCT15 (colorectal adenocarcinoma with less CD 44) at different working times, while fig. 2C and 2D show experiments on cell line HT29 (colorectal adenocarcinoma with abundant CD 44) at different working times. The results of HT29 (fig. 2C and 2D) show that the HA-dye HAs been successfully conjugated and adhered to the CD 44-rich region of HT29 (fig. 2C) and even into HT29 cells (fig. 2D). That means that the strategy of the present invention is suitable and effective and also means that the drug or dye can be conjugated to HA and that HA retains its ability to bind to CD 44.
Adhesion conditions of free dye as well as HA-dye were performed on mouse HT29 and HCT15 cell lines for 4 weeks. Free dye was injected into the tail vein of mice. The results show that there is no difference in adhesion results between two cancer cells with different CD44 expression. The proportion of the adhering area of HT29 was 50.15%, while that of HCT15 was 49.86%. However, when HA-conjugated dyes were injected into the tail vein of mice, CD44 expressed more cancer cells HT29 showed a tremendous concentration of HA-conjugated dyes, while CD44 expressed less HCT15 showed very limited results. The ratio of the adhesion area of HT29 was 74.15%, while that of HCT15 was 25.85%. The results may indicate that when the dye is conjugated with hyaluronic acid, the concentration of the dye is increased due to the adhesion of HA to the CD44 rich site.
Diseases highly associated with CD44 include cancer, infection, and inflammation. In preferred embodiments of the invention, for example, the cancer includes colon cancer, fibrosarcoma, breast cancer, adenocarcinoma, and brain glioblastoma.
In the present description and in the claims, the term "drug" or "active compound" or "agent" encompasses anti-cancer drugs. Most anticancer drugs can be classified into alkylating agents (alkylating agents), antimetabolites (anti-metabolites), anthracyclines (anthracyclines), plant alkaloids (plant alkaloids), topoisomerase inhibitors (topoisomerases), and other anticancer drugs (other anticancer-drugs).
In a preferred embodiment, conjugation of the anti-cancer drug includes, but is not limited to, lenalidomide, gemcitabine, celecoxib, and nimesulide.
Nimesulide is a widely used selective COX-2 antagonist with better gastrointestinal safety than other NSAIDs. Recently, Nimesulide has been speculated to act as an anticancer drug by inducing the expression of p21, a tumor suppressor gene, and to inhibit the mammalian target of rapamycin-related pathway (mTOR), which is an essential pathway for cell growth, cell proliferation, cell motility, cell survival, and protein synthesis of cancer cells (Zhang YJ. et al, 2011, mTOR signaling is involved in cell death and Nimesulide administration of tumor cell growth and vitro survival via COX-2 independent pathway.
Lenalidomide, a 4-amino-glutamyl analog of thalidomide, is a synthetic compound obtained by modifying the chemical structure of thalidomide to increase its potency and reduce its teratogenic and neurological side effects (v. Kotla et al, 2009, Mechanism of action of Lenalidomide in pharmaceutical compositions, Journal of Hematology and Oncology,2: 36). Lenalidomide has been demonstrated to have anti-angiogenic, anti-tumorigenic, and immunomodulatory activity by virtue of its peculiar (anecdotes) immunomodulatory activity in Erythema Nodosum (ENL) (j. sheskin,1980, The Treatment of lepra interaction in leukemia, International Journal of Dermatology,6:318-322) and autoimmune disorders (e.atra and e.i. sato,1993, Treatment of The clinical trial of systemic lupus erythematous with clinical and Experimental rhematology, 11(5): 487-93). Lenalidomide has been found to have anti-angiogenic properties and has become a drug with activity against a variety of hematological and solid malignancies such as myelodysplasia, multiple myeloma, chronic lymphocytic leukemia, primary systemic amyloidosis, non-hodgkin's lymphoma, myelofibrosis with myelogenous metaplasia (myelofibrosis with myelogenous metaplasia) and Waldenstrom's macroglobulinemia (Venumadhav koa et al, 2009, Mechanism of action of Lenalidomide in hematological malignans, Journal of biology & Oncology,2: 36). The clinical evidence of the therapeutic potential of lenalidomide in various malignant conditions is consistent with the degree of pharmacodynamic effects that have been demonstrated by a variety of mechanisms in different hematological malignancies in vitro and in animal models. Lenalidomide can up-regulate the tumor suppressor gene p 21; and thereby induce apoptosis of cancer cells (Verhelle D. et al, 2007, Lenalidomide and CC-4047inhibit the promotion of malignant B cells in which cancer cell is expanded normal CD34+ promoter cells. cancer Res.,67(2): 746-55). Lenalidomide has also been shown to greatly reduce the expression of the angiogenic factors VEGF and interleukin-6 (IL-6) in multiple myeloma; further reducing angiogenesis and thus contributing to the clinical therapeutic activity in multiple myeloma (Gupta D. et al, 2001, Adherence of multiple myelomas cells to bone marrow linear cells increase vascular endothelial growth factor section: thermal applications. Leukemia,15(12): 1950-61).
The object of the present invention is to bind or conjugate HA with the aforementioned drugs via the carboxyl, hydroxyl or amino group of HA with or without a linker or spacer to achieve working effects at specific positions and at specific times. Thus, HA as a targeted delivery vehicle carrying the drug to specific sites with abundant CD44 may result in better therapeutic effect and safety.
As used herein, generally, the term "linker" or "spacer" refers to an organic moiety that connects two moieties of a compound. The linker typically comprises a direct bond or atom such as oxygen or sulfur, e.g., SS, NH, C (O) NH, SO2、SO2Units of NH, or a chain of atoms, e.g. substituted or unsubstituted alkyl, in which one or more methylene groups may be replaced by O, S, SO2、NH、NH2Or C (O) interval or end. The term "linker" or "spacer" of the invention may be absent and denotes any compound present between the drug and HA that can be removed by chemical means, enzymatic means or spontaneous decomposition; it also comprises at least one other group useful for attaching the drug, such as an amino group, a thiol group, an additional carboxyl group, and the like. The linker or spacer may be a polypeptide, peptide or lipid.
Suitable linkers or spacers are, for example, straight-chain or branched aliphatic, aromatic or araliphatic (araliphatic) C2-C20Dicarboxylic acids, amino acids, peptides.
When present, the linker serves to build an arm (arm) or spacer between the hyaluronic acid and the drug. The linker is attached to HA via an amide, carboxyl, hydroxyl or amino linkage on one side and to the drug via any possible covalent type bond on the other side.
When the linker or spacer is a dicarboxylic acid, it may be the hydroxy group of the compound that the drug forms an ester bond with the carboxy group. When the linker or spacer is a dihydrazide, it may be the free carboxyl group of HA that forms an amide bond with the amino group. Preferred linkers or spacers are succinic acid for drugs, adipic hydrazide for HA.
In a preferred embodiment, the present invention provides a compound consisting of a conjugate from a glycosaminoglycan, preferably hyaluronic acid, and an active compound, wherein the active compound is conjugated to the carboxyl group of the glycosaminoglycan, derivative thereof, or salt thereof via a functional group to form a covalent conjugation, and wherein the active compound is selected from the group consisting of lenalidomide, gemcitabine and a COX-2 antagonist.
The active compound lenalidomide, gemcitabine or the preferred COX-2 antagonist nimesulide or celecoxib may preferably be bound directly via the functional carboxy group of HA and the-NH 2 group of the active compound.
In a preferred embodiment of the invention, the direct covalent conjugation between one functional carboxyl group of HA and the active compound or the indirect covalent conjugation via a linker may be an amide or ester bond.
In the case of indirect conjugation via a linker, the linker is selected from the group consisting of a polypeptide, a peptide, a lipid, an amino acid, or a linear or branched aliphatic, aromatic or araliphatic C2-C20A dicarboxylic acid.
Preferred HA for conjugation HAs an average molecular weight comprised in the range of 10kDa to 2000kDa and the conjugation involves at least 40% of the carboxyl groups of HA.
The glycosaminoglycan conjugates according to the invention are preferably used for the treatment of cancer diseases and preferably in the most preferred embodiment of the invention the cancer disease is selected from the group consisting of liver cancer, hepatocellular carcinoma, cholangiocarcinoma, cholangiocellular cystadenocarcinoma (cholangiocellular cystadenocarinoma), colon cancer, adenocarcinoma, lymphoma and squamous cell carcinoma, breast cancer, ductal carcinoma, lobular carcinoma, lung cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, kidney cancer, renal cell carcinoma, urothelial cell carcinoma, multiple myeloma, myelodysplastic syndrome (MDS), hodgkin lymphoma, non-hodgkin lymphoma, chronic lymphocytic leukemia or pancreatic cancer.
Accordingly, the present invention provides anti-cancer drug conjugates of HA and lenalidomide, HA and gemcitabine, HA and nimesulide, and HA and celecoxib, wherein the lenalidomide, gemcitabine, and celecoxib are each attached to the skin via the-NH at lenalidomide2Amide bonds are formed between the groups and-COOH groups of HA to be conjugated with HA; furthermore, for nimesulide, -NO2The radical being modified to-NH2Such that nimesulide can be covalently bound to the-COOH group of HA via an amide bond to form HA-NiNH2A conjugate. The structure of the HA-lenalidomide conjugate is shown in figure 3. True nimesulide (NiNO)2) With hydrogenation products NiNH2Is shown in FIG. 5A, while HA-NiNH2The structure of the conjugate is shown in fig. 5B. The structure of the HA-celecoxib conjugate is shown in figure 10. In one embodiment, the results of the present invention show that HA-lenalidomide conjugates show a great cytotoxic effect compared to lenalidomide or HA, respectively, in a CD 44-enriched cell line (HT29) (fig. 4A); however, the trend of this synergistic effect was less pronounced in the cell line HCT15 (fig. 4B). This result of the invention shows that under HA-lenalidomide treatment, the tendency of cell viability in HCT15 with less abundant CD44 is higher than HT29 with abundant CD44, meaning that with CD 44-rich cell lines, HT29 is more sensitive to HA-lenalidomide treatment; however, the effect of lenalidomide on cell viability was almost the same in both cell lines. The results indicate that lenalidomide does not interact with CD44, and that HA, when conjugated with lenalidomide, does enhance the therapeutic efficacy of lenalidomide compared to the same drug amount.
In another embodiment, the results of the present invention show that, in both HT29 or HCT15, true nimesulide (with nitro functionality,-NO2) The cytotoxic effect of (A) is generally superior to that of the hydrogenated modification product (having an amino function, -NH)2) In particular at higher doses (fig. 6A). However, when NiNH2When conjugated to HA, HA-NiNH2Significantly more cytotoxic than NiNH alone2Or NiNO2Cytotoxicity (fig. 6B). The results are the same for the embodiment where HA is conjugated to lenalidomide. Thus, the cytotoxic effect of anticancer drugs can be enhanced by conjugation with HA.
Furthermore, in the animal experimental embodiment, the results of the present invention show that HA-NiNH2Has a tumor-inhibiting effect in comparison with NiNO2Or the tumor suppression effect of the control group was more effective (fig. 7B). The results indicate that the average body weight of each mouse in the three groups was nearly the same over 24 days (fig. 7A), showing that there were no significant side effects such as weight loss; however, when NiNO was compared in the control group2Or HA-NiNH2The tumor volumes showed distinct differences, HA-NiNH2The group had better tumor suppression than either nimesulide or the control (fig. 7B). The results indicate that the HA-NiNH of the present invention2Conjugate than NiNO alone2Has better treatment effect.
The results show that the cytotoxic effect of HA-celecoxib tends to be better than HA and celecoxib in HT29 cells and GBM8401 cells (fig. 9A and 9B). And the results indicate a tendency for HA-celecoxib cytotoxic effects to increase in a549 cells (fig. 11) and GBM8401 cells (fig. 12).
In summary, the anti-therapeutic efficacy of lenalidomide and nimesulide conjugated to HA was compared to that of lenalidomide and nimesulide alone, and significantly improved cytotoxic effects and tumor suppression efficacy, respectively. The above-mentioned results show that the present invention makes a great contribution to enhancing the therapeutic effect of cancer drugs including lenalidomide and nimesulide.
For the treatment of diseases, preferred embodiments of the formulations or dosage forms of the invention include excipients to formulate administration dosage forms for ocular, otic, oral, nasal, respiratory, gastrointestinal, circulatory administration or topical use. More preferred embodiments of oral dosage forms are selected from the group consisting of solid dosage forms, solutions (including but not limited to suspensions), tablets (including but not limited to controlled release tablets), and capsules (including but not limited to enteric coated capsules). More preferred embodiments of the gastrointestinal administration form are selected from the group consisting of solid dosage forms, perfusates (perfusion), enemas (enemas), suppositories (suppositoriy), and solutions (including but not limited to suspensions). More preferred embodiments of the circulatory system or systemic administration form are selected from the group consisting of Intravenous (IV), Intramuscular (IM) and Subcutaneous (SC) injection. More preferred embodiments of the topical administration form are selected from the group consisting of perfusates, enemas, suppositories, sprays, inhalants and drops.
The process for the preparation of a compound consisting of a conjugate from a glycosaminoglycan and an active pharmaceutical compound, object of the present invention, comprises the following steps:
preparing an aqueous solution of a glycosaminoglycan, preferably hyaluronic acid;
preparing an aqueous solution of lenalidomide, gemcitabine or a COX-2 antagonist with N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide;
mixing and stirring the two solutions at room temperature for at least 10 hours to obtain a mixed solution; and dialyzing the mixed solution for several days. The following examples are provided to illustrate various embodiments of the present invention and are not intended to limit the invention in any way.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
Example 1: adhesion of hyaluronic acid to colonic tissue (IVIS image System-vision 3)
The method comprises the following steps:
1. 0.25 g of high molecular weight sodium hyaluronate powder (HHA; Mw: 2 MDa; Freda) and 0.25 g of low molecular weight sodium hyaluronate powder (LHA; Mw:0.35 MDa; Freda) were added to 50ml of PBS buffer (phosphate buffered saline), respectively, to form a 0.5% solution, followed by stirring for 6 hours until the powders were completely dissolved. 0.25 g of medium molecular weight sodium hyaluronate powder (MHA; Mw:1 MDa; Freda) was added to 50ml of PBS buffer, followed by stirring for 6 hours until the powder was completely dissolved and ready for use in the following steps.
2. Fluorescent HA (HA-f) was prepared by the following steps: (1) 0.39 g of MES free acid (2- (N-morpholino) ethanesulfonic acid, Calbiochem) was dissolved in 100ml dd water. (2) Solution A: 65mg of fluorescamine powder (isomer I, Fluka) are dissolved in 9ml of 95% ethanol (EtOH) solution and stirred for 10 minutes in the dark. (3) Solution B: 359mg of EDC powder (N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride, Sigma) were dissolved in 9ml of MES buffer and stirred for 10 minutes. (4) solution C: 216mg of NHS powder (N-hydroxysuccinimide, Sigma) was dissolved in 9ml of MES buffer and then stirred for 10 minutes. (5) 3mL of solution A was slowly dropped into 50mL of 0.5% HA solution, followed by stirring for 10 minutes while keeping out of light. (6) 3ml of the solution B and 5ml of the solution C were respectively dropped into the solution of the step (5), followed by stirring for 10 minutes while keeping out of the light. (7) 0.02M MES buffer was slowly dropped into the solution of step (6) until the volume reached 100ml, followed by stirring at room temperature for 24 hours under protection from light. (8) The reacted product was poured into 5L of deionized water as a dialysate in a dialysis tube (MW: 12000 to 14000), and then stirred in the dark at 4 ℃ for 5 days with the dialysate replaced every 12 hours until the dialysate did not fluoresce. (9) The dialyzed liquid was dispensed into 50c.c. plastic centrifuge tubes, then stored overnight in a-20 ℃ freezer, and then dried in the dark in a lyophilizer. (10) The dried HA-f powder was stored in a freezer at-20 ℃. (11) 50mg of HA-f powder was slowly added to 10ml of PBS buffer, followed by stirring for 6 hours until the powder was completely dissolved.
3. Colonic tissues of 7-8 week-old SD rats (Sprague-Dawley rats) were dissected off by a dissecting knife, then washed with PBS buffer, then cut into 3-4cm lengths, and finally soaked in PBS buffer.
4. Injured colon tissue was prepared by brushing the brush longitudinally 20 times and then soaked in PBS buffer.
5. Normal and injured colon tissue was placed in 12-well plates, and then 1ml of 0.5% HA-f solution was added to each well, and shaken at room temperature for 2 hours. After 2 hours excess HA-f solution was aspirated by a pipette, then soaked in PBS buffer for 10 minutes, after which the PBS buffer was removed, and repeated three times.
6. Clean colon tissue was placed in a 12-well plate with lining tissue facing up and then placed on a platform (dock) of IVIS (in vivo imaging system). Default parameters were set to GFP (green fluorescent protein) with excitation at 465nm and emission at 500nm, and images were recorded by software.
7. All values were calculated as the mean of the observed values. Histological indices were analyzed using student's t-test.
As a result: the fluorescence indices were quantified and aligned as shown in FIG. 1. The fluorescence index of normal colon tissue is defined as 1. The test values of other colon tissues are corrected by the defined values. The results show that HA of the same average Mw adheres to injured colon tissue with a fluorescence index significantly higher than normal colon tissue (P < 0.01). Comparing the difference between three different average molecular weight HA's adhered to the injured colon tissue, the fluorescence index of the adhesion of the injured colon tissue to 350kDa HA was higher than that of the other two average molecular weight HA's (2MDa and 1 MDa). Further, even normal colon tissue or injured colon tissue HAs a fluorescence index for adhesion to 1MDa HA higher than 2MDa HA.
Example 2: HA-dye conjugation procedure and HA-dye in vitro imaging
Method
All of the following processes of HA-dye conjugation must be kept in the dark.
Synthesis of HA-ADH
1 HA (0.34MDa,50mg) was dissolved in water to give a concentration of 4 mg/ml.
A25-fold excess (114.8mg) of ADH (adipoyl trap) was added to the solution.
The pH of the reaction mixture was adjusted to 4.75 with 30.1N HCl.
4. Next, 1 equivalent (25.1mg) of EDC was added as a solid. The pH of the reaction mixture was maintained at 4.75 by the addition of 0.1N HCl.
5. After 15 minutes of reaction, the reaction was quenched by adding 0.1N NaOH to adjust the pH of the reaction mixture to 7.0.
6. The reaction mixture was transferred to a pre-treated dialysis tube (cut-off Mw: 3500) and thoroughly dialyzed against 100mM NaCl, then 4 cycles of 25% EtOH/water and finally water. The solution was then filtered through a 0.2 μm cellulose acetate membrane, snap frozen and lyophilized.
The degree of substitution of ADH is measured by 1H NMR. Synthesis of HA-ADH-FITC:
1. 88mg of HA-ADH (DS ═ 36%) were dissolved in 35ml of water.
2. 9.5mg FITC was dissolved in 10ml DMSO.
3. The HA-ADH solution was mixed with the FITC solution.
4. After stirring at room temperature for 48 hours, the solution was dialyzed alternately with 0.3M sodium chloride and pure water for 3 days using MWCO 12000-14000 dialysis bag.
5. The solution was then freeze dried for 2 days.
6. Finally, the degree of substitution was determined by UV spectroscopy.
In vitro imaging of HA-dyes
(1) Mixing 1X 105HT29 cells and HCT15 cells (human colon carcinoma, CD44 positive cells) were seeded on microscope slides in 3.5cm dishes.
(2) 1 μ M of HA-dye (HA: 0.34MDa) was added to the cells separately for the indicated time with the indicated dye concentration.
(3) After incubation, cells were washed in PBS and then fixed in 3.7% formaldehyde.
(4) Observation of the interaction between HA-dye and cells was performed by confocal microscopy.
As a result: the fluorescence view can show the adhesion sites and amounts of the dyes on HCT15 (fig. 2A and 2B) and HT29 (fig. 2C and 2D). The results show that the dye HAs been successfully conjugated to HA and HA increases HA-dye concentration at the CD44 abundant site on HT29, whereas HT29 HAs stronger fluorescence consistent with more abundant CD44 than HCT 15. It was even demonstrated that the HA-dye could enter the cells (FIG. 2D). In HT29 (more CD 44), HA-dye accumulated after 6 hours of treatment and was internalized after 12 hours of treatment. In HCT15 (less CD 44), this was not observed after 6 or 12 hours of HA-dye treatment.
Example 3: cell lines and xenograft tumor models
Method
1. Cell culture conditions and passages
(1) Cell culture conditions:
HT 29: high glucose DMEM, 10% FBS, 1% sodium pyruvate, 1% penicillin, streptomycin, and neomycin.
HCT 15: DMEM/F12, 10% FBS, 1% sodium pyruvate, 1% penicillin, streptomycin and neomycin.
(2) Passage:
(I) the medium was removed and discarded.
(II) the cell layer was briefly washed with 1 XPBS to remove all traces of serum containing trypsin inhibitors.
(III) to the culture flask add 1ml of 0.25% trypsin-EDTA solution, and under the microscope to observe the cells until the cell layer is dispersed (usually in 5 to 15 minutes). Then, 9mL of complete growth medium was added and the cells were gently pipetted out.
(IV) Add an appropriate aliquot of the cell suspension to a fresh dish (seed transfer rate 1:3 to 1: 8).
(V) incubate cultures in a 37 ℃ incubator (5% CO 2).
2. Xenograft tumor model
(1) HT29 and HCT15 cells (2X 10)7Cells/mouse) were subcutaneously injected into the right and left buttocks (upper sides of right and left hind limbs) of 8-week-old male nude mice, respectively.
(2) When the size of the xenograft tumor is 400-500 mm after 3-4 weeks3IVIS experiments can be started.
IVIS experiment
(1) After anesthesia by isoflurane, the ratio of parameter f/stop: 8, exposure time is 3 seconds, excitation wavelength is 633nm or 635nm, and an image of the xenografted nude mice is obtained as a blank at measured emission wavelength of 668 nm. The instrument used was Xenogen IVIS 200.
(2) Mu.l of 12.5. mu.M free dye or 200. mu.l of HA-dye solution (HA-dye containing 12.5. mu.M dye and 0.1mg HA (HA: 1.12 MDa)) were injected intravenously via the tail vein, respectively.
(3) Photographs of IVIS images were taken after 5 minutes, 10 minutes, 30 minutes, and 1 hour, 2 hours of the scheduled time. The observation parameters and instruments are as briefly described in step 1. Mice were sacrificed and then dissected 2h after injection to analyze the fluorescence distribution in the gut.
As a result: fluorescence images showed that the free dye was almost uniformly distributed in HT29 (left) and HCT15 (right). The proportion of the adhering area of HT29 was 50.15%, while that of HCT15 was 49.86%. However, HA-dyes may especially adhere more to the CD44 rich site of HT29 than to HCT15 which HAs less CD44 than HT 29. The proportion of the adhering area of HT29 was 74.15%, while that of HCT15 was 25.85%. This result demonstrates that HA can promote dye accumulation at sites that are rich in CD 44.
Example 4: synthesis of HA-lenalidomide conjugates
Method
1. 50mg HA (10K-700KDa) was dissolved in 25ml DD water.
2. 25.1mg EDC and 15.1mg NHS were mixed in 2ml DD water and stirred for 5 min at room temperature.
3. The HA solution was neutralized by adding 1.31ml NaOH.
4. 3.4mg lenalidomide was dissolved in 2ml Dimethylsulfoxide (DMSO) solution.
5. The mixture (HA, EDC, NHS and lenalidomide) was stirred at room temperature for 12 hours.
6. The mixture was dialyzed against excess DD water using a dialysis bag (MWCO: 3500) for 2-3 days.
7. The HA-lenalidomide powder was obtained by dehydration from the HA-lenalidomide solution by a freeze dryer.
As a result: figure 3 shows the structure of the HA-lenalidomide conjugate.
Example 5: in vitro cytotoxicity of lenalidomide
The method comprises the following steps:
1. HT29 cells were plated at 1X 10 per well4The cells were seeded at low density in 96-well plates in a medium containing high glucose DMEM, 10% FBS, 1% sodium pyruvate, 1% penicillin, streptomycin and neomycinAnd (4) element.
2. HCT15 cells were plated at 1X 10 per well4Cells were seeded at low density in 96-well plates in media containing DMEM/F12, 10% FBS, 1% sodium pyruvate, 1% penicillin, streptomycin, and neomycin.
3.1 day (24 hours) after inoculation, cells were each cultured in a culture medium containing the following drugs at the indicated doses for 24 hours: lenalidomide: 400. mu.M, 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M and 0. mu.M; HA: 4mg/mL, 2mg/mL, 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.0625mg/mL, 0.3125mg/mL, and 0 mg/mL; HA-lenalidomide: 400. mu.M, 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M and 0. mu.M.
4. The effect of the drug on cell viability was evaluated using an assay that cleaves the yellow dye 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2H-tetrazolium bromide (MTT) to purple formazan crystals based on dehydrogenase activity in mitochondria.
5. After 24h of drug treatment, the medium was removed and the cell layer was washed with medium, followed by MTT (0.5mg/mL) diluted in medium and incubated for 4h at 37 ℃ in an incubator (5% CO 2).
6. Then 100. mu.L/well DMSO was added to the cells and the optical density of the cell homogenate was measured at 570nm using an ELISA plate reader.
7. The fraction of viable cells was calculated by dividing the average optical density obtained from the treated cells by the average optical density from the untreated control cells.
As a result: the results show that HA-lenalidomide conjugate HAs a cell killing effect in CD 44-rich cell lines (HT29) compared to lenalidomide or HA alone (fig. 4A). Similarly, this trend of synergy was also found in the cell line HCT15 (fig. 4B).
Example 6: synthesis of HA-NiNH2 conjugate
Hydrogenation of NiNO2
1. 500mg of nimesulide (NiNO)2) Completely dissolved in 20ml of ethyl acetate, and then 200mg of 5% Pd/C (palladium on carbon) was added to the solution as a catalyst. The air in the bottle is evacuated under continuous stirring, andthe air was replaced with hydrogen gas up to 1atm, followed by stirring for 24 hours.
2. The purity of the hydrogenation product was identified by thin layer chromatography (TLC silica gel slide 60F254) at a wavelength of 254nm, with the mobile phase being hexane: ethyl acetate ═ 2: 1.
3. after product identification, the Pd/C was removed by filtration, followed by rotary evaporation to remove residual solvent.
4. The hydrogenation product was dissolved in hexane: ethyl acetate ═ 1: 1 for further purification.
5. Purify using a silica gel column and elute with an elution solution (hexane: ethyl acetate ═ 1: 1).
6. The colored fractions (fractions) were collected and the structural concentration was determined by UV and NMR, respectively, to confirm the yield of the hydrogenation product NiNH 2.
7. The NiNH2 powder was obtained by freeze-drying.
HA-NiNH2Synthesis of conjugates
1. 50mg HA (10-700 kDa) was dissolved in 25ml DD water.
2. 25.1mg EDC and 15.1mg NHS were mixed in 1ml DD water and stirred for 5 min at room temperature.
3. 3.65mg of NiNO2Dissolved in 1ml DMSO and then slowly added dropwise over 3 minutes to the HA/EDC/NHS solution using a syringe.
4. The mixture (HA, EDC, NHS and NiNH)2) Stir at room temperature for 12 hours in the dark.
5. The mixture was dialyzed against excess DD water using a dialysis bag (MWCO: 3500) for 2-3 days.
6. From HA-NiNH by freeze-dryer2Dewatering the solution to obtain HA-NiNH2And (3) powder.
As a result: FIG. 5A shows authentic nimesulide (NiNO)2) And the product NiNH2The structure of (1). FIG. 5B shows HA-NiNH2Structure of the conjugate.
Example 7: NiNO2In vitro cytotoxicity of
The method comprises the following steps:
1. HT29 cells were plated at 1X 10 per well4Cells were seeded at low density in 96-well plates in media containing high glucose DMEM, 10% FBS, 1% sodium pyruvate, 1% penicillin, streptomycin, and neomycin.
2. HCT15 cells were plated at 1X 10 per well4Cells were seeded at low density in 96-well plates in media containing DMEM/F12, 10% FBS, 1% sodium pyruvate, 1% penicillin, streptomycin, and neomycin.
3.1 day (24 hours) after inoculation, cells were cultured in culture medium containing the indicated doses of the following drugs for 24 hours: NiNO2(represents having NO)2True nimesulide of the group): 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M and 0. mu.M; NiNH2(represents having NH)2Nimesulide of group): 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M and 0. mu.M; HA: 4mg/mL, 2mg/mL, 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.0625mg/mL, 0.3125mg/mL, and 0 mg/mL; HA-NiNH 2: 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M and 0. mu.M.
4. The effect of the drug on cell viability was evaluated using an assay that cleaves the yellow dye 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2H-tetrazolium bromide (MTT) to purple formazan crystals based on dehydrogenase activity in mitochondria.
5. After 24hr drug treatment, the media was removed and the cell layer was washed with media, followed by MTT (0.5mg/mL) diluted in media and incubated for 4 hours in an incubator (5% CO2) at 37 ℃.
6. Then 100. mu.L/well DMSO was added to the cells and the optical density of the cell homogenate was measured at 570nm using an ELISA plate reader.
7. The fraction of viable cells was calculated by dividing the average optical density obtained from the treated cells by the average optical density from the untreated control cells.
As a result: the results show that true nimesulide (with NO) is present in either HT29 or HCT152Group) is generally superior to the modified product (with NH)2Group) was used (fig. 6A). However, when NiNO2When conjugated to HA, HA-NiNH2Significantly more cytotoxic than NiNH alone2Or NiNO2(FIG. 6B).
Example 8: tumor growth inhibition in xenograft nude mouse model
The method comprises the following steps:
1. mixing HT29 (2X 10)7Cell/mouse) was injected subcutaneously into the right hip (upper right leg) of an 8-week-old female BALB/c athymic (nu +/nu +) mouse.
2. When the size of the xenograft tumor is less than 100mm3Tumor growth inhibition experiments can be initiated and designated as day 0.
3. During the experiment, tumor size and body weight were measured every 3 or 5 days.
4. Tumor volume was calculated as: 1/2(4 π/3) (L/2) (W/2) H; where L is the length of the tumor, W is the width of the tumor, and H is the height of the tumor.
5. Mice were divided into PBS-control, NiNO2Or HA-NiNH2Different groups of treatments.
6. Injecting NiNO through tail vein at intervals of 48 or 72 hours2(1.5mg/kg)、 HA-NiNH2(equivalent to 1.5mg/kg NiNO)2) Or a dose of PBS to the mice.
7. Tumor size and weight changes were recorded for each mouse.
As a result: the results showed that the average body weight of each mouse was almost the same (fig. 7A); however, when comparing control, NiNO2And HA-NiNH2The tumor volume showed significant difference for each group of (1), among which HA-NiNH2Group comparisons to NiNO2The group and the control group had better tumor suppression effect (fig. 7B). The results indicate that the HA-NiNH of the present invention2The conjugate has more NiNO than NiNO alone2Better treatment effect.
Example 9: synthesis of HA-celecoxib conjugates
Method
1. 100mg HA (10K-700KDa) was dissolved in 25ml DD water.
2. 0.8 equivalents of tetrabutylammonium hydroxide (TBA-OH) was added to the HA solution and stirred for 16 hours.
3. The solution was dried and HA-TBA was obtained as a white solid.
4. 40mg of HA-TBA was dissolved in 1ml of DD water, and then 30mg of EDC and 18 mg of NHS powder were added to the solution, and stirred at room temperature for 5 minutes.
5. Celecoxib, 4mg, was dissolved in 2ml dimethyl sulfoxide (DMSO) solution.
6. The mixture (HA-TBA, EDC, NHS and celecoxib) was stirred at room temperature for 72 hours.
7. The ratio of the mixture to DMSO and DD water was made 2: 1 for 1 day and the solution was changed 3 times.
8. The mixture was dialyzed against 0.3M NaCl using dialysis bags (MWCO: 1200-1400) for 2 days and the solution was changed 2 times a day.
9. The HA-celecoxib powder is obtained by dehydration from the HA-celecoxib solution by a freeze dryer.
As a result: figure 8 shows the synthesis and structure of HA-celecoxib conjugates.
Example 10: in vitro cytotoxicity of celecoxib
Method
1. HT29 cells were plated at 1X 10 per well4Cells were seeded at low density in 96-well plates in media containing high glucose DMEM, 10% FBS, 1% sodium pyruvate, 1% penicillin, streptomycin, and neomycin.
2. GBM8401 cells were plated at 1X 10 per well4Cells were seeded at low density in 96-well plates in media containing DMEM, 10% FBS, 1% sodium pyruvate, 1% penicillin, streptomycin, and neomycin.
3.1 day (24 hours) after inoculation, cells were cultured in medium containing the indicated doses of the following drugs for 24 hours: HA-celecoxib: 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M and 0. mu.M.
4. The effect of the drug on cell viability was evaluated using an assay that cleaves the yellow dye 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2H-tetrazolium bromide (MTT) to purple formazan crystals based on dehydrogenase activity in mitochondria.
5. After 24hr drug treatment, the media was removed and the cell layer was washed with media, followed by MTT (0.5mg/mL) diluted in media and incubated for 4 hours in an incubator (5% CO2) at 37 ℃.
6.100 μ L/well of DMSO, and the optical density of the cell homogenate was measured at 570nm using an ELISA plate reader.
7. The fraction of viable cells was calculated by dividing the average optical density obtained from the treated cells by the average optical density from the untreated control cells.
And (4) fruit: the results show that the cytotoxic effect of HA-celecoxib in HT29 cells as well as GBM8401 cells HAs a trend superior to HA and celecoxib (fig. 9A and 9B).
Example 11: synthesis of HA-gemcitabine conjugates
The method comprises the following steps:
1. 50mg HA (10-700 kDa) was dissolved in 25ml DD water.
2. 25.1mg EDC and 15.1mg NHS were mixed in 2ml DD water and stirred for 5 min at room temperature.
3. The HA solution was neutralized by adding 1.44ml NaOH.
4. 3.9mg gemcitabine was dissolved in 1ml DD water and 1ml DMSO solution and then slowly added dropwise to the HA/EDC/NHS solution over 3 minutes using a syringe.
5. The mixture (HA, EDC, NHS and gemcitabine) was stirred in the dark at room temperature for 12 hours.
6. The mixture was dialyzed against excess DD water for 2-3 days using a dialysis bag (MWCO: 12000-14000).
7. The HA-gemcitabine powder was obtained by dehydration from the HA-gemcitabine solution by a freeze dryer.
As a result: figure 10 shows the synthesis method and structure of HA-gemcitabine conjugates.
Example 12: in vitro cytotoxicity of gemcitabine
Method
1. A549 cells at 1X 10 per well4Low cell density seeded in 96-well plates containing high glucose DMEM, 10% FBS, 1% sodium pyruvate, 1% penicillinStreptomycin and neomycin.
2. GBM8401 cells were plated at 1X 10 per well4Cells were seeded at low density in 96-well plates in media containing DMEM, 10% FBS, 1% sodium pyruvate, 1% penicillin, streptomycin, and neomycin.
3.1 day (24 hours) after inoculation, cells were cultured in medium containing the indicated doses of the following drugs for 48 hours: HA-gem: 400. mu.M, 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M and 0. mu.M;
4.1 day (24 hours) after inoculation, cells were cultured in medium containing the indicated doses of the following drugs for 24 hours: HA-celecoxib: 400. mu.M, 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M and 0. mu.M.
5. The effect of the drug on cell viability was evaluated using an assay that cleaves the yellow dye 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2H-tetrazolium bromide (MTT) to purple formazan crystals based on dehydrogenase activity in mitochondria.
6. After 24hr drug treatment, the media was removed and the cell layer was washed with media, followed by MTT (0.5mg/mL) diluted in media and incubated for 4 hours in an incubator (5% CO2) at 37 ℃.
7. Then 100. mu.L/well DMSO was added to the cells and the optical density of the cell homogenate was measured at 570nm using an ELISA plate reader.
8. The fraction of viable cells was calculated by dividing the average optical density obtained from the treated cells by the average optical density from the untreated control cells.
As a result: the results indicate a trend of increased cytotoxicity of HA-gemcitabine in a549 cells (fig. 11) and 20 GBM8401 cells (fig. 12).