WO2019218810A1 - Pharmaceutical composition for treating non-hodgkin's lymphoma - Google Patents

Abstract

Disclosed is a pharmaceutical composition for treating non-Hodgkin's lymphoma, comprising a protein kinase C inhibitor and rituximab. Also disclosed is a pharmaceutical composition for treating non-Hodgkin's lymphoma, comprising a first preparation formed by a protein kinase C inhibitor and a pharmaceutically acceptable carrier, and a second preparation formed by rituximab and a pharmaceutically acceptable carrier. By combining the discovery that PKC is highly phosphorylated in rituximab-resistant non-Hodgkin's lymphoma cells, and the principle of pro-apoptotic effects of a PKC inhibitor, the present invention proves, by means of researches, that the protein kinase C inhibitor can be used, alone or in combination with rituximab, for treating rituximab-tolerant relapsed/refractory non-Hodgkin's lymphoma and significantly improves the overall survival.

Description

[Name of invention established by ISA according to Rule 37.2] Pharmaceutical composition for treating non-Hodgkin's lymphoma Technical field

The present invention relates to a pharmaceutical composition for treating non-Hodgkin's lymphoma, and more particularly to a pharmaceutical composition for treating B cell non-Hodgkin's lymphoma.

Background technique

Burkitt's lymphoma (BL) is a highly invasive B-cell non-Hodgkin's lymphoma (NHL), accounting for 3-5% of lymphoma in all age groups, accounting for children. 40-50% of lymphomas are characterized by highly expressed c-MYC targets and germinal center-associated B cell genes, as well as low expressed MHC-I molecules and NF-κB target genes. Adult BL patients have a poor response to CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone)-based treatment regimens with a 2-year and 5-year overall survival (OS) of approximately 50-65% If it reaches the bone marrow or central nervous system, it will be further reduced to less than 30%. In contrast, the intensive short-term chemotherapy regimen significantly increased the survival rate of children with BL to more than 90%, and in adult BL patients, a similar treatment regimen improved survival to more than 70%. However, these treatment options inevitably encounter barriers to drug toxicity and drug resistance.

Rituximab combined with CHOP chemotherapy (R-CHOP) can increase overall survival in patients with diffuse large B-cell lymphoma (DLBCL) by at least 20%. Similarly, many single-arm clinical trials have further demonstrated the efficacy of rituximab for BL-enhanced short-term chemotherapy regimens. A recent phase III clinical trial showed that chemotherapy plus rituximab achieved a higher 3-year event-free survival rate (75% vs. 62%, P=0.024) and 3-year overall survival rate compared with chemotherapy alone ( 83% vs. 70%, P = 0.011). Therefore, the increase in rituximab is expected in the design of future BL treatment regimens, so that the production of rituximab resistance during this treatment will be the same as the treatment of DLBCL, and it is expected to occur.

Although rituximab has achieved great success in treating a wide variety of B-cell lymphomas, resistance to rituximab is a major challenge for relapsed/refractory patients and remains Approximately 50% of patients do not respond to treatment with rituximab, and patients who initially respond will eventually develop resistance to further treatment with rituximab.

Rituximab kills lymphocytes primarily by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) by binding to membrane CD20. Resistance to ADCC may be due to the intrinsic polymorphism of the Fc Fc receptor FcgammaRIIIa gene, and resistance to CDC is likely due to down-regulation of CD20 expression and upregulation of membrane complement regulatory proteins (mCRPs), particularly CD59.

In order to improve the therapeutic efficacy of rituximab, researchers in the field have made considerable efforts to improve the expression of CD20, such as by the histone deacetylase inhibitor trichostatin A or synthetic CpG oligodeoxynucleotides. , or the function of CD59 is inhibited by modified monoclonal antibody or bacterial toxin-derived ILYd4. However, only CpG oligodeoxynucleotides were further evaluated for safety in B-cell NHL patients in clinical trials (stage I) and no further clinical trials have been reported. Therefore, there is a need to find alternative treatment strategies. Therefore, on the one hand, in order to improve the antitumor effect of rituximab, on the other hand, in order to reverse the resistance to rituximab, those skilled in the art are working to develop a combination with rituximab. Pro-apoptotic agents are used in the treatment of B-cell lymphoma.

Summary of the invention

In view of the above problems in the prior art, it is an object of the present invention to provide a treatment for relapsed/refractory non-Hodgkin's lymphoma (especially non-Hodgkin's lymphoma resistant to rituximab) A pharmaceutical composition of a tumor.

To achieve the above object, the present invention first provides a pharmaceutical composition for treating non-Hodgkin's lymphoma, including a protein kinase C (PKC) inhibitor and rituximab.

Further, the pharmaceutical composition for treating non-Hodgkin's lymphoma of the present invention further comprises a pharmaceutically acceptable carrier or excipient.

Further, the protein kinase C inhibitor of the present invention includes, but is not limited to, staurosporin and its analogs, carbamazepines, biguanide maleamides, biguanide maleamide macrocycles Compounds, 2-alkylindole maleimide compounds, Balanol compounds, N-phenyl-2-pyrimidinamine derivatives, Rottlerin, H-series inhibitors, sulfonamides and sulfonylbenzoyl derivatives , a monoterpene maleimide derivative, an adenosine-5'-terminal carboxylic acid peptide derivative.

Preferably, the protein kinase C inhibitor is selected from the group consisting of militalin, a derivative thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, and a prodrug thereof.

Midostaurin is a broad-spectrum PKC inhibitor (PKC412) derived from modified staurosporins to increase selectivity for PKC and further inhibit other kinases such as FLT3, PDGFR , KIT and VEGFR2 and their metabolites. In Phase I clinical trials, chronic oral treatment with midazolam is safe and tolerable and confirmed by the FDA in clinical treatment; it has the chemical structure shown in Formula I below:

In April 2017, midazoline was approved for the treatment of acute myeloid leukemia (AML) and advanced systemic mastocytosis (SM) with FLT3 (Fms-like tyrosine kinase 3) mutations.

The present inventors have found in the study that midazolam alone or in combination with rituximab can be used for the treatment of patients with BL, especially for patients with relapsed/refractory BL, and further for other highly active Treatment of patients with non-Hodgkin's lymphoma of PKC, especially for relapsed/refractory B-cell non-Hodgkin's lymphoma with highly activated PKC.

Although the effect of rituximab on the direct induction of cytotoxicity by lymphocytes is sometimes controversial, many studies have shown that rituximab lacks pro-apoptotic ability in various NHL cells. Our series of studies have revealed that rituximab fails to induce apoptosis to any detectable extent in non-Hodgkin's lymphoma, including BL cells, and therefore apoptosis in rituximab The role played by antitumor activity is very limited. Therefore, we attempted to increase the sensitivity of rituximab therapy by combining other drugs with pro-apoptotic effects. Macromolecular reagents include humanized monoclonal mapramumab targeting TRAIL-R1, genetically engineered fusion protein scFvRit: sFasL, Apo2L/TRAIL (dulanermin), and anti-CD20-interleukin -21, and small molecule reagents including selective NEDD8 activating enzyme (NAE) inhibitor pevonedistat (MLN4924), mTOR inhibitor temsirolimus and proteasome inhibitor bortezomib, enhance the antitumor activity of rituximab on B cell NHL cells . However, their efficacy needs to be further confirmed or proven to be ineffective in clinical trials.

Finally, the inventors found in the study that midazolin strongly enhanced the cytotoxic effect of rituximab by promoting apoptosis in BL cells.

Considering the high phosphorylation of PKC in resistant BL cells, we tested the effect of midazolam on the antitumor activity of rituximab in naive and rituximab-resistant BL cells, and the experimental results confirmed its The anti-tumor activity of rituximab was strongly enhanced in vitro and in vivo (especially in rituximab-resistant BL cells), and further we found that methotrex enhances rituximab by promoting apoptosis. The cytotoxicity of monoclonal antibodies may be achieved by altering the phosphorylation of downstream signaling pathways, including Bad, Bcl-2 and NF-κB.

Based on the above findings, the present invention also provides a pharmaceutical composition for treating non-Hodgkin's lymphoma comprising rituximab and protein kinase C inhibitors of different specification unit preparations and a pharmaceutically acceptable carrier Or excipients for simultaneous, separate or sequential administration.

Further, the present invention provides a pharmaceutical composition for treating non-Hodgkin's lymphoma, comprising a first formulation of a protein kinase C inhibitor and a pharmaceutically acceptable carrier, and rituximab and a pharmaceutically acceptable A second formulation of the accepted carrier formed.

Alternatively, the dosage form of the above preparation is an injection administration preparation, a gastrointestinal administration preparation, a respiratory administration preparation, a dermal administration preparation, a mucosal administration preparation or a channel administration preparation.

Wherein, the injection administration preparation of the present invention includes, but is not limited to, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intraluminal injection, and the like; and the gastrointestinal administration preparation of the present invention refers to after oral administration. A pharmaceutical dosage form that enters the gastrointestinal tract and acts locally or absorbed to exert a systemic effect, including but not limited to powders, tablets, granules, capsules, solutions, emulsions, suspensions, and the like; Formulations include, but are not limited to, sprays, aerosols, powders, and the like; the dermal administration preparations of the present invention include, but are not limited to, external solutions, lotions, tinctures, ointments, plasters, pastes, patches The mucosal administration dosage form of the present invention includes, but is not limited to, eye drops, nasal drops, ophthalmic ointments, gargles, sublingual tablets, adhesive tablets and filming agents, etc.; Pharmaceutical preparations include, but are not limited to, suppositories, aerosols, effervescent tablets, drops, and pills.

Preferably, the protein kinase C inhibitor is selected from the group consisting of militalin, a derivative thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, and a prodrug thereof.

In a preferred embodiment of the present invention, the militalin is orally administered 1-3 times a day, each dose is 50-100 mg, and continuous administration is 10-18 days for one course of treatment; more preferably daily Oral administration twice, 50-100 mg each time, for 14 days for one course of treatment; further, the dosage of the mexetiline is 1-4 mg/Kg/day.

In another preferred embodiment of the present invention, the rituximab is intravenous, 350-400 mg/m ² body surface area, more preferably 375 mg/m ² body surface area, once a week, one for each week Treatment.

Further, the present invention also provides a protein kinase C inhibitor for use in the treatment of rituximab-resistant non-Hodgkin's lymphoma, and in the preparation of non-Hodgkin-resistant rituximab The application of the drug for the treatment of lymphoma.

Further, the present invention also provides the use of rituximab and a protein kinase C inhibitor for the treatment of non-Hodgkin's lymphoma, and for the preparation of a combination therapy for non-Hodgkin's lymphoma.

Wherein the non-Hodgkin's lymphoma is a B cell non-Hodgkin's lymphoma, or the non-Hodgkin's lymphoma is a non-Hodgkin's lymphoma resistant to rituximab, Alternatively, the non-Hodgkin's lymphoma is a non-Hodgkin's lymphoma with highly activated PKC.

The present inventors have found in the study that protein kinase C (PKC) is highly upregulated and activated in BL cells that are resistant to rituximab, while broad-spectrum PKC inhibitors (pan-PKC inhibitors) are used alone or in combination with rituximab. The combination of monoclonal antibodies can effectively trigger apoptosis in BL cells and significantly improve overall survival, especially in BL cells that are resistant to rituximab; this result can be further extended to resistance to rituximab Non-Hodgkin's lymphoma, or non-Hodgkin's lymphoma with highly activated PKC. Thus, the above conclusions of the present invention support the use of protein kinase C alone or in combination with rituximab to treat relapsed/refractory non-Hodgkin's lymphoma, and we call for further clinical trial evaluation of its efficacy.

The concept, the specific structure and the technical effects of the present invention will be further described in conjunction with the accompanying drawings in order to fully understand the objects, features and effects of the invention.

DRAWINGS

Figure 1 is a Western blot of mCRPs expression in Ramos cells and Ramos 640 cells;

Figure 2 is a comparison of FACS analysis of mCRPs expression in Ramos cells and Ramos640 cells: compared with the original cells, the expression of CD20 is decreased, the expression of CD59 is increased, the expression of CD55 is slightly decreased, and the expression of CD46 is not different. Value ± SD, n = 3, NS = no significant difference, ** P < 0.01, *** P < 0.001, **** P < 0.0001;

Figure 3 is a Western blot of mCRPs expression in Raji cells and Raji32 cells;

Figure 4 is a comparison of FACS analysis of mCRPs expression in Raji cells and Raji32 cells: compared with the original cells, the expression of CD20 was decreased, the expression of CD59 was increased, the expression of CD55 was increased, and the expression of CD46 was decreased in Raji32 cells. SD, n=3, **P<0.01, ***P<0.001, ****P<0.0001;

Figure 5 is a Western blot of Akt phosphorylation and mCRPs expression in four groups of cells before and after PI3K inhibitor IPI-145 (1 μM): phosphorylation of Akt (S473) in Ramos640 cells and Raji32 cells compared to their original cells An increase in level, which is inhibited by treatment with the PI3K inhibitor IPI-145, resulting in decreased expression of CD20 and CD55;

Figure 6 shows the effect of IPI-145 (1 μM) on rituximab-mediated CDC in Ramos cells and Ramos 640 cells: IPI-145 alone does not increase the cell death rate of primordial and drug-resistant cells. Addition of IPI-145 to rituximab reduced rituximab-mediated CDC in primordial and drug-resistant cells, and the data were expressed as mean ± SD, n = 3, NS = no significant difference, * **P<0.001, and ****P<0.0001; RTX: rituximab (640 μg/mL); NHS: normal human serum (20% v/v);

Figure 7 is the effect of IPI-145 (1 μM) on rituximab-mediated CDC in Raji cells and Raji32 cells: addition of IPI-145 alone and addition of rituximab increased drug-resistant cells Cellular mortality, but did not increase cell death in blast cells, this combination significantly increased rituximab-mediated CDC in Raji32 cells, expressed as mean ± SD, n = 3, NS = none Significant difference, ***P<0.001, and ****P<0.0001; RTX: rituximab (32 μg/mL); NHS: normal human serum (20% v/v);

Figure 8 is a bar graph of up-regulated greater than 1.5-fold phosphorylated protein and its phosphorylation site in a phosphorylated antibody microarray;

Figure 9 is an IPA (Ingenuity Pathway Analysis) analysis result of the up-regulated protein of Figure 8, the solid line indicates direct adjustment, and the broken line indicates indirect regulation;

Figure 10 is a GSEA enrichment profile of anti-apoptotic TNFs:NF-κB:Bcl-2 pathway obtained after comparison of Ramos640 cells with Ramos cell RNA-Seq data, FDR < 0.25 is considered significant;

Figure 11 is a GSEA enrichment characteristic map of the p53 pathway obtained by comparing Ramos640 cells with Ramos cell RNA-Seq data, and FDR < 0.25 is considered significant;

Figure 12 is a Western blot of PKCα/β1/β2/γ/η expression in four BL cells and phosphorylation of PKC and its downstream anti-apoptotic proteins and the effect of PCK inhibitor methotrexate (1 μM) on them. This figure shows that up-regulation of PKC isoforms in drug-resistant cells leads to an increase in PKC phosphorylation and activates downstream anti-apoptotic proteins, while midazolin inhibits PKC and its downstream signaling;

Figure 13 is a graph showing the results of apoptosis induced by the pan-PKC inhibitor militalin in Ramos cells and Ramos 640 cells, where Control: medium control, RTX: rituximab (640 μg/mL), Mido: rice bran Tolin (1 μM); this figure demonstrates that pan-PKC inhibitors effectively induce apoptosis in na[iota]ve and resistant Ramos cells, and the addition of rituximab to midazoline significantly enhances Ramos640 cells. Pro-apoptotic effect, data are expressed as mean ± SD, n = 3, ** P < 0.01, *** P < 0.001 and **** P < 0.0001;

Figure 14 is a graph showing the results of apoptosis induced by the pan-PKC inhibitor methotrexate in Raji cells and Raji32 cells, in which Control: medium control, RTX: rituximab (32 μg/mL), Mido: rice bran Tolin (1 μM); this figure demonstrates that pan-PKC inhibitors effectively induce apoptosis in naive and drug-resistant Raji cells, and the data are expressed as mean ± SD, n = 3, **P < 0.01, ** *P<0.001 and ****P<0.0001;

Figure 15 is a Western blot of the effect of the PKC inhibitor, mitutoline (1 μM), on the expression of mCRPs in four cells, which shows that midazolin significantly reduces CD20 expression in primary and drug-resistant cells as well as in resistant cells. CD59 expression, but did not alter the expression of CD55 and CD46 in protocell or resistant cells;

Figure 16 is a graph showing the effect of the PKC inhibitor, midazoline, on rituximab-mediated CDC in Ramos cells and Ramos 640 cells, where Control: medium control, RTX: rituximab (640 μg/mL), Mido: midazolam (1 μM), NHS: normal human serum (20% v/v); this figure indicates that in the original Ramos cells, the addition of militalin did not enhance rituximab-mediated CDC, However, CDC was increased in drug-resistant cells, and the data were expressed as mean ± SD, n = 3, *** P < 0.001 and **** P < 0.0001;

Figure 17 is a graph showing the effect of the PKC inhibitor, mitolide, on rituximab-mediated CDC in Raji cells and Raji32 cells, in which Control: medium control, RTX: rituximab (32 μg/mL), Mido: midazolam (1 μM), NHS: normal human serum (20% v/v); this figure shows that in the original Raji cells, the addition of methotrex failed to enhance rituximab-mediated CDC, However, CDC was increased in drug-resistant cells, and the data were expressed as mean ± SD, n = 3, *** P < 0.001 and **** P < 0.0001;

Figure 18 is the growth of tumors on day 50 after inoculation of Raji32-Luc cells, the tumor mass is represented by the intensity of firefly luciferase activity, RTX: rituximab, Mido: methotrexate;

Figure 19 is a quantitative result of total photon flux in Figure 18, data expressed as mean ± SEM (n = 7), * P < 0.05, ** P < 0.01 and *** P < 0.001;

Figure 20 is the growth of tumors on day 70 after inoculation of Raji32-Luc cells, the tumor mass is represented by the intensity of firefly luciferase activity, "X" represents mouse death, RTX: rituximab, Mido: rice bran forest;

Figure 21 is a quantitative result of total photon flux in Figure 19, data expressed as mean ± SEM (n = 7), * P < 0.05, ** P < 0.01 and *** P < 0.001;

Figure 22 shows tumor growth on day 90 after inoculation of Raji32-Luc cells. Tumor quality is expressed by the intensity of firefly luciferase activity, "X" represents death of mice, RTX: rituximab, Mido: rice bran forest;

Figure 23 is a quantitative result of the total photon flux in Figure 21, the data is expressed as mean ± SEM (n = 7), * P < 0.05, ** P < 0.01 and *** P < 0.001;

Figure 24 is a survival curve of different groups of mice treated with drugs after inoculation of Raji32-Luc cells, and the data are expressed as mean ± SEM (n = 7). *P<0.05, **P<0.01 and ***P<0.001.

Detailed ways

Example 1 Construction of Raji Cells and Ramos Cells Against Rituximab-Mediated CDC Tolerance

Rituximab-mediated ADCC is carried out by immune cells from individual patients whose tolerance to ADCC is caused by the intrinsic characteristics of immune cells, such as the FcyRIIIa gene polymorphism. Apoptosis plays only a negligible role in the antitumor activity of rituximab. Therefore, we constructed two BL cell lines, Ramos640 and Raji32, which are resistant to rituximab-mediated complement-dependent cytotoxicity at rituximab concentrations of 640 and 32 μg/mL, respectively. The construction method is as follows:

Both BL cell lines Raji and Ramos were purchased from American type culture collection (ATCC) (Manassas, VA), and the cells were cultured in 10% (volume) fetal bovine serum (GIBCO BRL, Grand Island, NY) and 1% (by volume) penicillin/streptomycin (Ambion, Austin, TX) in RPMI 1640 medium.

As a complement resource, normal human serum (NHS) was collected from 10 healthy humans and combined to serve as a source of complement. The above serum was aliquoted and stored at -80 ° C until use. In addition, heat-killed human serum (IHS) prepared by incubating the above serum for 30 minutes in a 65 ° C water bath was used as a negative control.

Raw Raji cells and Ramos cells were separately treated with increasing concentrations of rituximab (Roche, Basel, Switzerland). In the presence of 20% (by volume) of NHS, the concentrations were increased from 4 or 40 μg/mL, respectively, to a multiple of 2 or more to 32 or 640 μg/mL, and the resulting drug-resistant cells were called Raji32 and Ramos 640, respectively. They were treated with 32 μg/mL and 640 μg/mL rituximab containing 20% (by volume) of NHS every 21 days to maintain resistance to rituximab.

Example 2 Immunoblot analysis and CDC effect assay

The present invention was subjected to immunoblot analysis according to standard methods.

The CDC effect was determined by fluorescence-activated cell sorting (FACS) analysis to detect propidium iodide (PI) staining positive cells. Specifically, after washing with PBS, the cells were incubated with fluorescein-conjugated antibody for 30 minutes, then rinsed and resuspended in PBS. Flow cytometric analysis was performed on a Cytomics FC500 MPL flow cytometer (Beckman Coulter, Brea, CA) and analyzed using FlowJo software (Ashland, OR). We used the MoFlo XDP instrument (Beckman Coulter, Brea, CA) for cell sorting based on the relevant fluorescence, using the PE annexin V apoptosis assay kit (BD Pharmingen, Inc., San Diego, CA) according to the manufacturer's instructions. Perform apoptosis analysis.

Example 3 Downregulation of CD20 and Upregulation of CD59 Lead to BL Cells to Rituximab-Mediated CDC Tolerance

Using immunoblot analysis (Figures 1 and 3) and FACS (Figures 2 and 4), we found that CD20 expression was reduced in both drug-resistant cells compared to the original cells, while CD59 expression was elevated ( Figure 1 - Figure 4). However, the expression of two other membrane complement regulatory proteins (mCRP), CD55 and CD46, was inconsistent in both resistant cells. The expression of CD55 was reduced in Ramos 640, while the expression in Raji32 was elevated; whereas the expression of CD46 in Ramos 640 was unchanged, but the expression was decreased in Raji32 (Figs. 1 - 4). These results are consistent with previous reports that reduced CD20 expression and elevated CD59 expression result in BL cells being resistant to rituximab-mediated CDC. However, since the predecessors did not obtain good results in the study of increasing CD20 expression and inhibiting CD59 function, the present invention has attempted other therapeutic strategies.

Example 4 Strong enrichment of the PI3K/Akt pathway, but its inhibition failed to reverse drug resistance

The present invention further studies the mechanism of drug resistance by protein signaling pathway analysis. This example uses a phosphorylated antibody microarray to identify functional proteins and signaling pathways that are activated in Ramos 640 cells.

We analyzed phosphorylation of the protein with a phosphorylated antibody chip (FullMoon Microsystems, Catalog #CSP100, Sunnyvale, CA) containing 269 antibodies against 131 protein phosphorylation sites and according to its established method by Wayen Biotech The company (Shanghai, China) conducted an analysis.

Fluorescence analysis showed that the phosphorylation levels of 35 proteins in Ramos640 were up-regulated (31/35) or down-regulated (4/35) by more than 1.4-fold compared to Ramos cells. Analysis of the KEGG signaling pathway of these proteins demonstrated that the PI3K/AKT signaling pathway contained the most phosphorylated protein sites in 13 and the apoptotic pathway showed the largest enrichment factor of 47.76 (as shown in Table 1).

Table 1 Enrichment pathways of Ramos 640 cells relative to Ramos cells as determined by phosphorylated antibody microarrays

Next, the PI3K inhibitor IPI-145 was used to identify whether inhibition of PI3K can increase the sensitivity of resistant BL cells to rituximab treatment. The PI3K inhibitor IPI-145 was purchased from Selleck Chemicals (Houston, TX).

Specifically, the original cells and the drug-resistant cells were seeded into 96-well plates at a density of 1×10 ⁴ cells/100 μL/well, and were divided into four groups, and the first group was cultured for 48 hours as a control, The two groups were treated with a medium containing IPI-145 at a concentration of 1 μM for 48 hours, and the third group was treated with a medium containing 20% (by volume) of NHS and rituximab at a concentration of 640 μg/mL or 32 μg/mL for 48 hours. The fourth group was treated with a medium containing 20% (by volume) of NHS and having a rituximab concentration of 640 μg/mL or 32 μg/mL and an IPI-145 concentration of 1 μM for 48 hours.

Using CytoTox-Glo ^TM cytotoxicity assay kit (Promega Corporation, Madison, WI) cytotoxicity assay performed according to its technical bulletin steps and cytotoxicity was calculated according to the following formula:

Cytotoxicity (%) = dead cell luminescence / total luminescence × 100%

The data of the present invention are expressed as mean ± standard deviation unless otherwise stated. Significant differences between the two groups were determined using a two-tailed Student's t-test of unpaired data. In all analyses, p < 0.05 was considered to be statistically significant.

The experimental results first confirmed that the PI3K/Akt signaling pathway was activated in Ramos640 and Raji32 cells compared to its original cells, and IPI-145 effectively attenuated Akt phosphorylation (S473) (Fig. 5). However, in both primary and resistant cells, treatment with IPI-145 significantly inhibited expression of CD20, while expression of the other three mCRPs including CD59, CD55 and CD46 showed little change (Fig. 5). These results subsequently led to IPI-145 having a slightly opposite effect on rituximab-mediated CDC in the original and drug-resistant cells (except for Raji32 cells) (Figures 6 and 7). In Raji32 cells, IPI-145 alone or in combination with rituximab slightly increased cell mortality compared to control or rituximab alone (Figure 7). These results suggest that PI3K/Akt may not be a valuable drug target for the promotion of rituximab treatment.

Example 5 PKC-mediated apoptotic pathway is highly activated in drug-resistant cells

Considering that the effect of rituximab in inducing apoptosis in B-NHL (B-cell non-Hodgkin's lymphoma) cells is negligible, the addition of an agent that induces apoptosis to rituximab may be a An ideal treatment strategy.

Although the apoptotic pathway was identified as the most abundant pathway in the phosphorylated antibody microarray, it contained only six proteins (Table 1), which hindered the selection of pathway modulators. Therefore, we narrowed the search by increasing the fold change of the up- and down-regulated phosphorylated protein sites in the phosphorylated antibody microarray to 1.5-fold, and the results showed that a total of 16 protein sites were up-regulated by 1.5-fold (Fig. 8).

Functional annotations with a fold change >1.5 were screened by the DAVID Bioinformatics database for functional annotation. Interestingly, all of these up-regulated proteins/sites were involved in apoptosis through the IPA pathway analysis software (QIAGEN, Duesseldorf, Germany); more importantly, IPA also It is revealed that PKC (protein kinase C) signaling regulates all of these up-regulated proteins, although the detection of PKC phosphorylation is not included in the phosphorylated antibody microarray set, the interaction network between the PKC subunit and the differential protein obtained by IPA analysis is shown in Fig. 9 is shown.

Next, we performed RNA sequencing of the original and resistant Ramos cells with RNA-seq and further performed gene enrichment analysis.

RNA sequencing method:

Total RNA was extracted from Ramos and Ramos 640 cells using Trizol reagent (Invitrogen, Grand Island, NY). Total RNA from 3 different generation cells in each group was pooled separately. RNA quality was identified using Bioanalyzer 2200 (Agilent Technologies, Santa, Clara, CA) and stored at -80 °C. RNA with an RNA complete number (RIN) > 8.0 is acceptable for cDNA library construction. RNA-seq was sequenced by Shanghai Novelbio Co., Ltd., and a cDNA library was constructed for each pooled RNA sample using Ion Tatal RNA-seq kit v2.0 (Life Technologies, Gaithersburg, MD), followed by a proton sequencing process. A clean reading was obtained from the original reading by removing the linker sequence before reading the map, and then aligned with the human genome (version: GRCh37NCBI) using the MapSplice program (v2.1.6). We applied the DEseq algorithm to screen differentially expressed genes. The criteria for significance analysis and false discovery rate (FDR) analysis were as follows: (1) fold change >1.5 or <0.667; (2) FDR<0.05.

Gene set enrichment analysis method:

The present invention uses gene set enrichment analysis GSEA software (the Massachusetts Institute of Technology Broad Institute) to identify the function of differentially expressed genes found in RNA-seq. A predetermined version of the software was used to identify significantly enriched pathways, and an enrichment pathway with FDR < 0.25 was considered significant. Anti-apoptotic TNFs used in the present invention: NF-κB: Bcl-2 pathway gene set is derived from the PathCards Pathway Uniform Database (version 4.0.6.37, Weizmann Academy of Sciences) "Apoptosis and Survival Anti-apoptotic TNFs: NF-κB : Bcl-2 pathway SuperPath" consists of 42 genes. The p53 pathway gene set used in the present invention consists of 132 genes from the "p53 pathway (RnD) SuperPath" in the unified database of the PathCards pathway.

By detecting the original and resistant Ramos cells by RNA-seq and GSEA analysis, we further found that the TNFs:NF-κB:Bcl-2 pathway gene was significantly increased in Ramos640 cells compared to Ramos cells, while the p53 pathway gene was significantly increased. Significantly reduced (Figures 10 and 11). They also indicate up-regulation of anti-apoptotic genes and down-regulation of pro-apoptotic genes. Thus, these data indicate that PKC phosphorylates multiple downstream proteins, resulting in an anti-apoptotic effect in Ramos 640 cells.

Example 6 pan-PKC inhibitor Midostaurin significantly promotes apoptosis

Mitalin is a multi-kinase inhibitor originally designed to inhibit PKC and is currently approved for acute myeloid leukemia (AML) and advanced systemic mast cells with FLT3 (Fms-like tyrosine kinase 3) mutations. Hyperplasia (SM). The PKC inhibitor mitutolin used in the present invention was purchased from Selleck Chemicals (Houston, TX).

In this example, we first compared the expression levels of five PKC isoforms in naive BL cells and resistant BL cells, and found that PKCα/β2/γ/η in Ramos640 cells, PKCα/β1/β2/γ/η in Raji32 cells. , overexpression compared to its original cells, and more importantly, phosphorylation levels of all PKC isoforms in both drug-resistant cells were significantly elevated (Figure 12). In addition, we examined the effect of midazolam on the regulation of PKC phosphorylation. We observed that it strongly inhibited the phosphorylation of PKC in resistant BL cells and naive Ramos cells, but not in the original Raji cells, and it reduced PKCβ2/η in Ramos cells, PKCα/η in Ramos640 cells, and Raji32. The expression level of PKCβ1/η in the cells (Fig. 12).

Next, we examined the phosphorylation levels of several downstream PKC signaling molecules, including the Bad, Bcl-2, and NF-κB subunits p65. We first found that their phosphorylation levels were significantly elevated (Figure 12). In addition, in both drug-resistant cells, the expression level of Bad was significantly decreased, and the expression level of p65 was significantly increased (Fig. 12). All of the above changes may contribute significantly to inhibition of apoptosis, leading to the development of resistance to rituximab in Ramos640 cells and Raji32 cells. In addition, midazolin strongly inhibited the phosphorylation levels of Bad, Bcl-2 and p65 in all of the original cells and drug-resistant cells (Fig. 12). Further, midazolin significantly increased or decreased the expression of Bad or p65 in all four primordial or drug-resistant cells, and reduced Bcl-2 expression only in naive and resistant Raji cells ( Figure 12). Thus, militalin inhibits PKC and its downstream signaling, which may lead to pro-apoptotic effects in Ramos and Raji cells to varying degrees. Among them, Raji cells appear to be more resistant to midazolin-mediated PKC phosphorylation than Ramos cells because midazolin failed to inhibit PKC phosphorylation.

In addition, we tested the use of the pan-PKC inhibitor militalin (1 μM) alone or in combination with rituximab (32 μg/mL for Raji cells and 640 μg/mL for Ramos cells). Pro-apoptotic effects on naive and resistant BL cells.

Specifically, the original cells and the drug-resistant cells were seeded into 96-well plates at a density of 1×10 ⁴ cells/100 μL/well, and were divided into four groups, and the first group was cultured for 48 hours as a control, The two groups were treated with rituximab at a concentration of 640 μg/mL or 32 μg/mL for 48 hours, and the third group was treated with a medium containing 1 μM of miltazone for 48 hours. The medium was treated with a concentration of 640 μg/mL or 32 μg/mL and a midazorine concentration of 1 μM for 48 hours.

Using CytoTox-Glo ^TM cytotoxicity assay kit (Promega Corporation, Madison, WI) cytotoxicity assay performed according to its technical bulletin steps and cytotoxicity was calculated according to the following formula:

Cytotoxicity (%) = dead cell luminescence / total luminescence × 100%

The data of the present invention are expressed as mean ± standard deviation unless otherwise stated. Significant differences between the two groups were determined using a two-tailed Student's t-test of unpaired data. In all analyses, p < 0.05 was considered to be statistically significant.

As we expected, rituximab alone treated apoptosis that was not induced in all four naive or resistant BL cells (Figures 13 and 14). However, compared with the medium control, the use of militalin alone significantly induced Ramos cells (from 4.7% to 63.3%), Ramos 640 cells (from 10.8% to 50.2%), and Raji32 cells (from 6.4% to 25.4%). Apoptosis, while Raji cells only increased slightly (from 6.6% to 10.9%), although the p value reached statistical significance (p = 0.0038) (Figure 13 and Figure 14). These results are also functionally determined. In general, Raji cells are more resistant to ampicillin-induced apoptosis than Ramos cells. In addition, the combination of midazolam and rituximab showed a slight further pro-apoptotic effect only in primordial cells but not in resistant Ramos cells and Raji cells (Figures 13 and 14), further confirming rituximab The pro-apoptotic effect of the monoclonal antibody, if any, is negligible.

Example 7 Mitalantin enhances susceptibility of resistant BL cells to rituximab-mediated CDC

In this example we used the pan-PKC inhibitor, midazolin, to identify whether inhibition of PKC can increase the sensitivity of resistant BL cells to rituximab treatment.

Specifically, the original cells and the drug-resistant cells were seeded into 96-well plates at a density of 1×10 ⁴ cells/100 μL/well, and were divided into four groups, and the first group was cultured for 48 hours as a control, The two groups were treated with a medium containing 20% (by volume) of NHS and a concentration of rituximab of 640 μg/mL or 32 μg/mL for 48 hours, and the third group was treated with a medium containing 1 μM of metoprolin. For the hour, the fourth group was treated with a medium containing 20% (by volume) of NHS and having a rituximab concentration of 640 μg/mL or 32 μg/mL and a myristicin concentration of 1 μM for 48 hours.

Using CytoTox-Glo ^TM cytotoxicity assay kit (Promega Corporation, Madison, WI) cytotoxicity assay performed according to its technical bulletin steps and cytotoxicity was calculated according to the following formula:

Cytotoxicity (%) = dead cell luminescence / total luminescence × 100%

The data of the present invention are expressed as mean ± standard deviation unless otherwise stated. Significant differences between the two groups were determined using a two-tailed Student's t-test of unpaired data. In all analyses, p < 0.05 was considered to be statistically significant.

In addition to CD20, rituximab-mediated CDC can be regulated by expression of mCRP, such as CD46, CD55, and especially CD59. We observed that midazolam (1 μM) significantly reduced the expression of CD20 in all four BL cells, whereas the decrease in CD59 expression was predominant in drug-resistant cells; it had no effect on the expression of CD55 and CD46 ( Figure 15). These results indicate that midazolin may block rituximab-mediated CDC due to decreased expression of CD20 in naive BL cells, whereas expression of both CD20 and CD59 is reduced in resistant BL cells. Therefore, its effect on resistant BL cells requires further testing. In addition, these results show the difference between the effects of IPI-145 and midazolam on the regulation of CD20 and CD59 expression, in which IPI-145 only reduces CD20 expression in resistant BL cells but does not reduce CD59 expression (Fig. 5).

Rituximab (a concentration of 32 μg/mL for Raji cells and 640 μg/mL for Ramos cells) plus NHS (20%, v/v) effectively induced CDC in naive Ramos cells and Raji cells. However, the addition of militalin (1 μM) failed to enhance the susceptibility of rituximab-mediated CDC, although it promoted apoptosis (Figures 16 and 17). This may be due to a decrease in CD20 expression and a already high cell death rate due to rituximab alone. In contrast, the addition of rituximab significantly enhanced the cytotoxic effect of militalin in the original BL cells (Figure 16 and Figure 17), indicating that the effect of rituximab-mediated CDC is greater in primary BL cells. Mitotalin induces apoptosis. Interestingly, we found that the cytotoxic effects of rituximab in combination with midazolam have synergistic effects in resistant Ramos640 cells and Raji32 cells. As shown in Figure 16, rituximab and midazoline induced 17.1% and 54.3% cell death, respectively, and their combination induced 72.8% cell death. Similarly, as shown in Figure 17, rituximab and midazoline induced 28.0% and 25.8% cell death, respectively, and their combination induced 62.9% cell death. This result may be due to the unique anti-tumor mechanism of rituximab and midazolam.

Example 8 Rituximab in combination with Mentrolidine significantly inhibited tumor growth in drug-resistant cells

Given that the apparent pro-apoptotic effects of midazolin may contribute to the improvement of the anti-tumor activity of rituximab, we inoculated a more resistant Raji32 cell transfected with a plasmid expressing luciferase. The efficacy of rituximab in combination with midazolam was tested in immunodeficient mice.

Plasmid construction and lentiviral transduction

The CDS (coding sequence) of the firefly luciferase gene was obtained from the pGL3-Basic plasmid by PCR amplification, and inserted into the pCDH cDNA clone and expression vector by EcoRI and BamHI endonuclease sites. The primers for firefly luciferase CDS amplification are as follows:

Forward primer 5'-ATGGAAGACGCCAAAAACATAAAG-3'

Reverse primer 5'-TTACACGGCGATCTTTCCGCCCTT-3'

The pCDH plasmid was co-transfected with the pMD.2G and psPAX2 plasmids in 293FT cells to generate firefly luciferase overexpressing lentivirus. The lentivirus was then added to Raji32 cell culture medium for 48 hours. All cells transfected with lentivirus in the present invention were classified using GFP and MoFlo XDP instruments (Beckman Coulter, Brea, CA) and referred to as Raji32-Luc cells.

Xenograft model

Eight-week-old female SCID mice were purchased from SLAC (Shanghai Laboratory Animal Center, Shanghai Experimental Animal Center). Raji32-Luc cells were resuspended in PBS, and then each mouse was intraperitoneally injected with 1.5 x 10 ⁷ cells. Mice were divided into 4 groups (7 mice per group) based on the difference in drug administration, namely saline, rituximab, midazoline and rituximab plus militalin. On the 8th, 12th and 16th day after tumor inoculation, 118.4 mg/kg rituximab was intraperitoneally injected, and 8, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18 after tumor inoculation. On days 19, 20 and 21, the rats were intragastrically administered with miltazone 20 mg/kg. The amount of saline administered was the same as that of rituximab. Tumor growth was monitored by bioluminescence on days 50, 70, and 90 after tumor inoculation, and D-luciferin (Promega, Madison, WI) was intraperitoneally injected into mice (150 mg/kg), and ten minutes later, intraperitoneal injection was performed. Mice were anesthetized with pentobarbital (50 mg/kg) and bioluminescence detection was then performed using an In-Vivo MS FX PRO system (Bruker, Billerica, MA). Luminescence images were captured with an exposure time of 30 seconds and the signal intensity of the tumors was measured by Bruker MI software. The survival time of each mouse was recorded up to 120 days. All animal experiments were carried out in strict accordance with the experimental protocol approved by the Animal Ethics Committee of Shanghai Medical College of Fudan University.

The data of the present invention are expressed as mean ± standard deviation unless otherwise stated. For the total photon flux of the animal model, significance was determined by a one-tailed Mann Whitney test. We used the Mantel-Cox test to compare the survival rates of the two groups of xenograft models. In all analyses, p < 0.05 was considered to be statistically significant.

On the 50th day after tumor inoculation, we examined the growth of the tumor. The results are shown in Figure 18 and Figure 19. The total photon flux is used to express the growth of the tumor according to the saline control group, rituximab group, rice bran. The order of the tolin group, the rituximab + miltazone group, in which all adjacent groups showed statistically significant differences in tumor mass.

The results on the 70th day after tumor inoculation are shown in Fig. 20 and Fig. 21, and 3 of the 7 mice in the saline group died, and 1 of the 7 mice in the rituximab group died. All 7 mice in the rituximab + miltazone group survived (Figure 20). Tumor blocks in surviving mice showed similar results, ie the tumor growth rate of the saline group or the rituximab group was much higher than that of the midazolin or combination treatment group (Fig. 21).

The results on the 90th day after tumor inoculation are shown in Figure 22 and Figure 23. In the saline group, the rituximab group, the metformin group, and the combination treatment group, there were 1, 1 and 4, respectively. Six mice survived (Figure 22). Tumor blocks in surviving mice treated with saline were larger than tumor blocks in rituximab-treated surviving mice (Fig. 23).

In addition, the survival curve analysis of Figure 24 showed that the use of midazolin alone or in combination with rituximab significantly prolonged survival compared to rituximab alone, indicating induction by midazolam. Pro-apoptotic effects may be necessary and beneficial for the treatment of BL, and militalin may be used as a supplemental therapeutic, especially in the treatment of rituximab-resistant BL.

The above has described in detail the preferred embodiments of the invention. It should be understood that many modifications and variations can be made in the present invention without departing from the scope of the invention. Therefore, any technical solution that can be obtained by a person skilled in the art based on the prior art based on the prior art by logic analysis, reasoning or limited experimentation should be within the scope of protection determined by the claims.

Claims (10)

1. A pharmaceutical composition for treating non-Hodgkin's lymphoma, comprising a protein kinase C inhibitor and rituximab.
2. The pharmaceutical composition of claim 1 further comprising a pharmaceutically acceptable carrier or excipient.
3. The pharmaceutical composition according to claim 1 or 2, wherein the protein kinase C inhibitor is selected from the group consisting of militalin, a derivative thereof, a pharmaceutically acceptable salt thereof, a solvate thereof and a former thereof One or more of the medicines.
4. A pharmaceutical composition for treating non-Hodgkin's lymphoma, comprising a first formulation comprising a protein kinase C inhibitor and a pharmaceutically acceptable carrier, and rituximab and a pharmaceutically acceptable A second formulation of the carrier.
5. The pharmaceutical composition according to claim 4, wherein the preparation is in the form of an injection administration preparation, a gastrointestinal administration preparation, a respiratory administration preparation, a dermal administration preparation, a mucosal administration preparation or a cavity. Dosage preparation.
6. The pharmaceutical composition according to claim 4, wherein the protein kinase C inhibitor is selected from the group consisting of militalin, a derivative thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, and a prodrug thereof.
7. The use of rituximab and protein kinase C inhibitors in the preparation of a combination therapy for non-Hodgkin's lymphoma.
8. The use according to claim 7, wherein the non-Hodgkin's lymphoma is a B cell non-Hodgkin's lymphoma.
9. The use according to claim 7, wherein said non-Hodgkin's lymphoma is a non-Hodgkin's lymphoma resistant to rituximab.
10. The use according to claim 7, wherein the non-Hodgkin's lymphoma is a non-Hodgkin's lymphoma with highly activated PKC.

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