Application of all-trans retinoic acid combined with low-dose rituximab in the preparation of drugs for the treatment of hormone-resistant or recurrent ITP

Abstract

The present invention provides the use of all-trans retinoic acid (ATRA) combined with low-dose rituximab in the preparation of a medicament for treating steroid-resistant or recurrent ITP. The dosage of rituximab is 100 mg weekly; the dosage of ATRA is 20 mg per square meter of body surface area per day. A multicenter randomized controlled trial demonstrated that the combination of ATRA and LD-RTX demonstrated superior efficacy and safety compared to LD-RTX alone in the treatment of patients with steroid-resistant or recurrent ITP.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the use of all-trans retinoic acid combined with low-dose rituximab in the preparation of a drug for treating hormone-resistant or recurrent ITP. Background Art

[0002] Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by increased platelet destruction and insufficient platelet production. Glucocorticoids and intravenous immunoglobulin are first-line treatments. However, approximately one-third of ITP patients do not respond to first-line therapy. Furthermore, most responding patients relapse during subsequent drug reduction or discontinuation, requiring further second-line treatment. However, the optimal second-line treatment regimen remains uncertain.

[0003] Rituximab (Rituximab, also known as Rituxan, RTX) is a chimeric anti-CD20 monoclonal antibody that exerts its therapeutic effect primarily by rapidly eliminating CD20-positive B lymphocytes and regulatory T cells. It is currently widely used in the treatment of ITP. There is no gold standard for the specific dosage of Rituxan. Proposed regimens include the standard-dose regimen (SD-RTX, i.e., 375 mg per square meter of body surface area weekly for 4 weeks) and the low-dose regimen (LD-RTX, i.e., a fixed dose of 100 mg weekly for 4 weeks). Previous studies focusing on SD-RTX have demonstrated that SD-RTX can achieve a good overall response rate (OR) and sustained response rate (SR) in ITP patients. However, SD-RTX can cause infusion-related reactions in up to 20% of patients and may also increase the incidence of long-term complications, such as infections. SD-RTX also carries a higher financial burden for patients. A meta-analysis of nine studies investigating the efficacy of LD-RTX for ITP showed that LD-RTX achieved comparable ORs to SD-RTX while also reducing the incidence of infusion-related reactions and long-term complications, demonstrating its favorable safety and tolerability profile. Studies focusing on conditions other than ITP have also suggested that the risk of infection or serious infection after rituximab use is dose-related. LD-RTX also generally offers lower costs compared to SD-RTX. These advantages suggest that LD-RTX may be a promising treatment option for ITP patients. However, LD-RTX may have some limitations compared to SD-RTX: Previous studies have shown that the median time to response (TTR) for ITP patients treated with LD-RTX is 30 days, significantly longer than the median TTR for SD-RTX (10.5 days). SR is also lower in ITP patients treated with LD-RTX compared to SD-RTX.

[0004] All-trans retinoic acid (ATRA), a key metabolite of vitamin A, is involved in the proliferation and differentiation of cells. Our previous studies have confirmed the efficacy of ATRA in the treatment of ITP models, which is achieved through its induction of megakaryocyte differentiation, maturation and its immunomodulatory effects. We have also previously conducted a multicenter randomized controlled clinical trial to explore the efficacy of ATRA combined with danazol in patients with hormone-resistant or recurrent ITP. The results showed that ATRA combined with danazol can achieve an overall response rate of 82% and a 12-month sustained response rate of 62% in patients with hormone-resistant or recurrent ITP. A study by Dai et al. also evaluated the immunomodulatory therapeutic efficacy of ATRA in patients with chronic ITP, and the reported overall response rate was 54.3%.

[0005] However, there is currently no precedent for combining all-trans retinoic acid with LD-RTX to treat hormone-resistant or recurrent ITP. Summary of the Invention

[0006] The object of the present invention is to provide a pharmaceutical composition.

[0007] The pharmaceutical composition provided by the present invention comprises all-trans retinoic acid and rituximab (Rituxan, RTX), wherein the rituximab is low-dose rituximab.

[0008] The present invention also provides a unit-dose medicine.

[0009] The unit-dose medicine comprises several doses of all-trans retinoic acid in a unit dose of 20 mg and several doses of rituximab in a unit dose of 100 mg, and the unit doses of all-trans retinoic acid and rituximab are each packaged independently.

[0010] The ratio of the unit dose number of all-trans retinoic acid to the unit dose number of rituximab is 20-24:1, calculated based on the body surface area of 60 kg.

[0011] Furthermore, the unit dose medicine comprises 20-24 unit doses of all-trans retinoic acid and 1 unit dose of rituximab.

[0012] The use of the pharmaceutical composition composed of the above-mentioned all-trans retinoic acid and low-dose rituximab or the unit-dose medicine in the preparation of a drug for treating hormone-resistant or recurrent ITP also falls within the scope of protection of the present invention.

[0013] In the application, the dosage of rituximab is 100 mg per week; the dosage of all-trans retinoic acid is 20 mg per square meter of body surface area per day.

[0014] More specifically, rituximab was administered for 6 weeks and all-trans retinoic acid was administered for 12 weeks.

[0015] Multicenter randomized controlled trials have shown that the combination of ATRA and LD-RTX has superior efficacy and safety compared with LD-RTX alone in the treatment of patients with steroid-resistant or recurrent ITP. This may be because RTX and ATRA play different roles in the treatment of ITP. Based on the "double-hit" theory of ITP pathogenesis, the combined use of RTX and ATRA may exert a synergistic effect in the treatment of ITP by simultaneously targeting both platelet production and platelet destruction. This may overcome the two drawbacks of LD-RTX: a longer TTR and a lower SR. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Implement the test plan for the subjects.

[0017] Figure 2 Diagram of the subject screening process during the experiment.

[0018] Figure 3 The figure compares the median platelet count of subjects in the ATRA combined with LD-RTX treatment group with the median platelet count of subjects in the LD-RTX monotherapy group.

[0019] Figure 4 This is a comparison of the recurrence-free survival rate of patients in the ATRA combined with LD-RTX treatment group and the LD-RTX monotherapy group.

[0020] Figure 5 The incidence of adverse events in the ATRA combined with LD-RTX treatment group and the LD-RTX monotherapy group. DETAILED DESCRIPTION

[0021] The present invention is described below by means of specific examples, but the present invention is not limited thereto.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the reagents, biological materials, etc. used in the following examples are all commercially available unless otherwise specified.

[0023] The present invention provides a pharmaceutical composition comprising all-trans retinoic acid and rituximab (Rituxan, RTX), wherein the rituximab is low-dose rituximab.

[0024] The present invention also provides a unit-dose medicine.

[0025] The unit-dose medicine comprises several doses of all-trans retinoic acid in a unit dose of 20 mg and several doses of rituximab in a unit dose of 100 mg, and the unit doses of all-trans retinoic acid and rituximab are each packaged independently.

[0026] The use of the pharmaceutical composition composed of the above-mentioned all-trans retinoic acid and low-dose rituximab or the unit-dose medicine in the preparation of a drug for treating hormone-resistant or recurrent ITP also falls within the scope of protection of the present invention.

[0027] In the application, the dosage of rituximab is 100 mg per week; the dosage of all-trans retinoic acid is 20 mg per square meter of body surface area per day.

[0028] More specifically, rituximab was administered for 6 weeks and all-trans retinoic acid was administered for 12 weeks.

[0029] Multicenter randomized controlled trials have shown that ATRA combined with LD-RTX has better efficacy and safety than LD-RTX alone in the treatment of patients with hormone-resistant or recurrent ITP.

[0030] Example

[0031] method

[0032] Experimental design and case screening

[0033] We designed an open-label, multicenter, controlled, phase 2 clinical trial conducted in seven tertiary hospitals in China, with patient enrollment between October 2017 and January 2020. This study was reviewed by the ethics committees of each center and conformed to the Declaration of Helsinki and clinical practice guidelines.

[0034] ITP was diagnosed according to standard procedures, including age greater than 18 years and platelet count less than 30 × 10 9 / L or patients with hormone-resistant or recurrent ITP with bleeding symptoms are enrolled. Hormone resistance or recurrence is defined as patients who have previously received the maximum dose of glucocorticoids for at least 4 weeks, but the patient has failed to achieve a response state, or the patient relapses during the process of hormone reduction. If the patient is also using other concomitant treatments (including glucocorticoids, danazol, cyclosporine, etc.), and the dose of the concomitant treatment has been stable in the past 4 weeks, and the dose of the concomitant treatment can continue to be stable within 4 weeks after the start of the study (until the initial response is evaluated), the patient can also be included in the clinical trial. Considering the safety of the experiment, patients with life-threatening bleeding symptoms (such as intracranial hemorrhage) were not included.

[0035] Our eligibility criteria also excluded patients who had received RTX prior to enrollment. Patients with malignant tumors, pregnancy or lactation, osteoporosis, HBV / HCV infection, HIV infection, current infection, acute coronary syndrome, heart failure, severe hepatic or renal insufficiency, or significant adverse reactions to previous hormonal therapy were excluded. Pregnant or lactating patients were also excluded. Patients were recruited during routine outpatient visits, and all patients provided written informed consent prior to participation in the clinical trial.

[0036] Randomization and blinding

[0037] After confirming eligibility, we randomly assigned patients in a 2:1 ratio to either ATRA plus LD-RTX or LD-RTX alone using precoded envelopes at Peking University People's Hospital, ensuring concealed group assignment. Due to the open nature of this trial, the experimental group assignments were not concealed from the investigators or participants.

[0038] Experimental Procedure

[0039] All patients received a fixed dose of RTX 100 mg weekly for 6 weeks; patients in the combination therapy group received ATRA 20 mg per square meter of body surface area orally daily for 12 weeks. Follow-up was weekly for the first 4 weeks, then every 2 weeks until week 24, and then every 4 weeks until week 52. Figure 1 At each follow-up visit, we will conduct a clinical assessment to test the patient's complete blood count, urine routine, liver and kidney function, fasting and postprandial blood glucose levels, and record the use of rescue treatment, bleeding scores and side effects. If a patient experiences a serious adverse event during the study or the patient requests to withdraw from the trial, the patient can withdraw from the clinical trial. In addition, if the platelet count measured twice in a row (at least 2 weeks apart) is greater than 300×10 9 If the platelet count is less than 10 × 10 / L, stop all treatment for the patient. Salvage therapy is defined as any new treatment to prevent bleeding or increase platelet count, including platelet transfusion, IVIg, and splenectomy, or an increase in the treatment dose. 9 / L, the patient needs rescue treatment. If the patient's platelet count is between 10-30×10 9 If the patient's blood pressure is within the range of 1 / 2L, whether the patient should start rescue therapy depends on the physician's assessment of the bleeding tendency, the patient's preference, the patient's tolerance for drug side effects, daily lifestyle and activities, and the patient's expectations. Patients who receive rescue therapy or withdraw for any reason are defined as non-responders (NR). In addition, patients who discontinue treatment for any reason are also considered NRs.

[0040] ending

[0041] The primary endpoints included overall response rate (OR) and sustained response rate (SR). OR was defined as platelet count reaching 30×10-10 on at least two occasions (at least 7 days apart) without other ITP-specific treatment. 9 / L and the platelet count doubled compared with the baseline platelet count at enrollment, and the patient had no clinical manifestations of bleeding; SR was defined as a patient with a platelet count greater than 30×10 9 / L or 2 times or more of the baseline value, without bleeding symptoms and without the need for rescue treatment for at least 6 months from the time the response is achieved.

[0042] Secondary endpoints of the study included complete response rate (CR), initial response rate, time to response (TTR), duration of response, NR, bleeding events, and safety. CR was defined as platelet counts ≥100×10-10 on at least two occasions at least 7 days apart. 9 / L and no clinical manifestations of bleeding. Initial response was defined as achieving a response within 4 weeks after enrollment and initiation of treatment. NR was defined as a platelet count less than 30×10 9 The patients were defined as those with platelet counts exceeding 300 / L or who failed to achieve a doubling of the baseline platelet count or who had bleeding symptoms. TTR was defined as the time from the start of treatment to response. Duration of response was defined as the time from the start of response to the end of response or the end of follow-up.

[0043] Bleeding was scored according to the WHO criteria: 0, no bleeding; 1, spotty bleeding; 2, mild bleeding; 3, massive bleeding; 4, life-threatening bleeding. Adverse events (AEs) were defined according to the fifth edition of the Common Terminology Criteria for Adverse Reactions.

[0044] Data Analysis

[0045] On the basis of our preliminary studies and the literature, we hypothesized that the addition of ATRA to LD-RTX would increase the proportion of patients with an OR from 60% to 85% and the proportion of patients with a SR from 35% to 60%. We used a two-sided Pearson chi-squared test at the 0.05 level to assess the superiority of the combination therapy, allowing for an 18% dropout rate. To achieve 80% power, we required 111 patients in the ATRA plus LD-RTX group and 56 patients in the LD-RTX monotherapy group.

[0046] Efficacy end points and baseline characteristics were analyzed using the full data set of all randomized participants. Patient data were summarized as categorical variables (frequency and percentage) and continuous variables (median and interquartile range (IQR)). Fisher's exact test was used to assess differences in primary and secondary end points and adverse events. We compared differences between the two groups using the Pearson chi-square test for categorical variables and the Student t test or Mann-Whitney U test for continuous variables. Duration of response and time to relapse were calculated using the Kaplan-Meier method, and the log-rank test was used to assess potential differences between the groups. We used logistic regression to assess the association of risk factors with 6-month sustained response (SR).

[0047] All statistical tests were two-sided, and P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS Statistics 24 (IBM SPSS Statistics, Armonk, NY, USA). This trial is registered with ClinicalTrials.gov, number NCT03304288.

[0048] result

[0049] Case enrollment and follow-up

[0050] From October 2017 to January 2020, we screened 181 eligible cases, of whom 13 were excluded. A total of 168 patients were included in the ITT analysis: 112 patients received ATRA combined with LD-RTX, and 56 patients received LD-RTX monotherapy. Among the 132 randomly assigned patients, 3 patients did not receive the intervention due to withdrawal of informed consent and were not included in the safety analysis. 14 patients (12.5%) in the ATRA combined with LD-RTX treatment group (1 due to adverse reactions, 6 due to the need for other interventions, 6 due to loss of follow-up, and 1 due to death) and 8 patients (14.3%) in the LD-RTX monotherapy group (1 due to adverse reactions, 4 due to the need for other interventions, and 3 due to loss of follow-up) discontinued treatment midway. Figure 2 ).

[0051] Baseline characteristics were well balanced between the ATRA plus LD-RTX group and the LD-RTX monotherapy group (Table 1). The median age of the subjects in the ATRA plus LD-RTX group was 46.0 years, compared with 44.5 years in the LD-RTX monotherapy group. Fifty-nine (52.7%) subjects in the ATRA plus LD-RTX group were male, compared with 24 (42.9%) subjects in the LD-RTX monotherapy group. The median duration of ITP was 20.0 months in the ATRA plus LD-RTX group and 20.5 months in the LD-RTX monotherapy group. All enrolled patients had failed first-line glucocorticoid therapy. In the ATRA plus LD-RTX combination group, the median number of previous treatments was 2.0. The most commonly used treatments other than glucocorticoids were recombinant human thrombopoietin and platelet receptor agonists (n=21, 18.8%) and danazol (n=21, 18.8%). In the LD-RTX monotherapy group, the median number of previous treatments was also 2.0. Danazol (n=15, 26.8%) and intravenous immunoglobulin (n=14, 25.0%) were the two most common treatments other than glucocorticoids. The median platelet count at enrollment in the ATRA plus LD-RTX group was 17.0 × 10 9 / L, while the median platelet count of subjects in the LD-RTX monotherapy group was 19.0×10 9 / L. Furthermore, all patients were assessed for bleeding at enrollment, and the majority of patients in both groups had a bleeding score of 0: 53 (47.3%) in the ATRA plus LD-RTX group and 31 (55.4%) in the LD-RTX monotherapy group. Forty (35.7%), 11 (9.8%), and five (4.5%) patients in the ATRA plus LD-RTX group, respectively, had bleeding scores of 1, 2, or 3, while 17 (30.4%), five (8.9%), and two (3.6%) patients in the LD-RTX monotherapy group had bleeding scores of 1, 2, or 3, respectively. The median follow-up was 360 days in the ATRA plus LD-RTX group and 358 days in the LD-RTX monotherapy group. All patients were followed for more than 11 months.

[0052] Efficacy evaluation

[0053] As shown in Table 2, during follow-up, 90 patients (80.4%) in the ATRA combined with LD-RTX group achieved an OR, compared with 33 patients (58.9%) in the LD-RTX monotherapy group (P = 0.003), indicating that the combination of ATRA and LD-RTX treatment improved the overall response rate. A total of 45 patients (40.2%) in the ATRA combined with LD-RTX group achieved a CR, compared with 14 patients (25.0%) in the LD-RTX monotherapy group (P = 0.052). Age (P = 0.611), sex (P = 0.668), and the number of previous treatments (P = 0.589) were not associated with the OR. An initial response was achieved in 58 patients (51.8%) in the ATRA combined with LD-RTX group, compared with 24 patients (42.9%) in the LD-RTX monotherapy group (P = 0.275). The median platelet count of subjects in the ATRA combined with LD-RTX treatment group was higher than that in the LD-RTX monotherapy group from the 10th week of follow-up ( Figure 3 ).

[0054] For patients who achieved an OR, the median time to response (TTR) was 28.0 (IQR 21.0-42.0) days in the ATRA plus LD-RTX group and 28.0 (IQR 28.0-49.0) days in the LD-RTX monotherapy group (TTR: P = .050). Therefore, the combination of ATRA and LD-RTX did not significantly shorten the TTR. During follow-up, 68 patients (60.7%) in the ATRA plus LD-RTX group achieved a SR, compared with 23 patients (41.1%) in the LD-RTX monotherapy group. Furthermore, 56 patients (50.0%) in the ATRA plus LD-RTX group and 19 patients (33.9%) in the LD-RTX monotherapy group remained in an ongoing response at 12 months (P = .048). During the follow-up period, 43 of the 90 patients (47.8%) who achieved a response in the ATRA combined with LD-RTX group relapsed, while 26 of the 33 patients (78.8%) who achieved a response in the LD-RTX monotherapy group relapsed. The median duration of response was 11.0 months in the ATRA combined with LD-RTX group and 10.0 months in the LD-RTX monotherapy group (P = 0.024). We continued to perform logistic regression analysis to explore possible predictive factors for achieving SR in the two groups of patients. Initial response and CR were potential variables associated with SR. The relapse-free survival rate of patients in the ATRA combined with LD-RTX group was significantly higher than that in the LD-RTX monotherapy group (P = 0.007, Figure 4 ).

[0055] The highest bleeding score was recorded for all patients during follow-up. There was no significant difference in the highest bleeding score between the ATRA combined with LD-RTX group and the LD-RTX monotherapy group (P = 0.618). With the increase in platelet count, the patients' bleeding symptoms improved.

[0056] Salvage therapy was administered to 21 patients (18.8%) in the ATRA plus LD-RTX group, a significantly lower proportion than in the LD-RTX monotherapy group (n = 19, 33.9%, P = 0.029). Specifically, 13 patients (11.6%) in the ATRA plus LD-RTX group received intravenous glucocorticoids, 3 patients (2.7%) received intravenous immunoglobulin, 3 patients (2.7%) received platelet transfusions, and 2 patients (1.8%) had their concomitant therapy dose increased. In the LD-RTX monotherapy group, 10 patients (17.9%) received intravenous glucocorticoids, 7 patients (12.5%) received intravenous immunoglobulin, 6 patients (10.7%) received platelet transfusions, and 3 patients (5.4%) had their concomitant therapy dose increased. No patients underwent splenectomy during follow-up.

[0057] Security

[0058] Figure 5 The incidence of adverse events in the two groups was generally mild. The adverse events recorded during the study were mainly graded 1-2, and no grade 4 or more severe adverse events occurred. The two most common adverse events in the ATRA combined with LD-RTX treatment group were dry skin (n=45, 40.2%) and dizziness and headache (n=21, 18.8%), which were considered to be a manifestation of ATRA treatment. In the LD-RTX monotherapy group, a total of 12 (21.4%) and 8 (14.3%) patients developed fever and upper respiratory tract infection, respectively. Other recorded adverse events included urinary tract infection, gastrointestinal infection, and muscle and joint pain. Rash, abnormal mood, insomnia, palpitations, stomach pain, liver damage, etc. were also recorded adverse events, but the incidence was relatively low. In addition, one patient in the ATRA combined with LD-RTX treatment group died of intracranial hemorrhage in the fourth week after enrollment.

[0059] Table 1. Baseline characteristics of enrolled patients

[0060]

[0061]

[0062] Data are expressed as percentage (%) or median (IQR, interquartile range). ATRA, all-trans retinoic acid; LD-RTX, low-dose rituximab; TPO, thrombopoietin.

[0063] Table 2. Comparison of response and prognosis between the ATRA combined with LD-RTX treatment group and the LD-RTX monotherapy group

[0064]

[0065] The data are expressed as percentage (%) or median (IQR, interquartile range).

[0066] ATRA, all-trans retinoic acid; LD-RTX, low-dose rituximab.

Claims

1. Use of a pharmaceutical composition in the preparation of a drug for treating hormone-resistant ITP, the pharmaceutical composition comprising all-trans retinoic acid and rituximab, wherein: Rituximab is low-dose rituximab.

2. Use of a unit-dose drug in the preparation of a drug for treating hormone-resistant ITP, wherein the unit-dose drug comprises several doses of all-trans retinoic acid in a unit dose of 20 mg and several doses of rituximab in a unit dose of 100 mg, wherein the all-trans retinoic acid unit dose and the rituximab unit dose are each independently packaged.

3. The use according to claim 2, characterized in that In the unit-dose medicine, the ratio of the unit dose number of all-trans retinoic acid to the unit dose number of rituximab is 20-24:

1.

4. The use according to claim 3, characterized in that The unit dose medicine contains 20-24 all-trans retinoic acid unit doses and 1 rituximab unit dose.

5. The use according to claim 1 or 2, characterized in that: The dosage of rituximab is 100 mg per week; the dosage of all-trans retinoic acid is 20 mg per square meter of body surface area per day.

6. The use according to claim 1 or 2, characterized in that: Rituximab was continued for 6 weeks, and all-trans retinoic acid was continued for 12 weeks.

7. Use of a pharmaceutical composition in the preparation of a drug for treating recurrent ITP, the pharmaceutical composition comprising all-trans retinoic acid and rituximab, wherein: Rituximab is low-dose rituximab.

8. Use of a unit-dose drug in the preparation of a medicament for treating recurrent ITP, the unit-dose drug comprising several doses of all-trans retinoic acid in a unit dose of 20 mg and several doses of rituximab in a unit dose of 100 mg, the unit doses of all-trans retinoic acid and rituximab being independently packaged.

9. The use according to claim 8, characterized in that In the unit-dose medicine, the ratio of the unit dose number of all-trans retinoic acid to the unit dose number of rituximab is 20-24:

1.

10. The use according to claim 9, characterized in that The unit dose medicine contains 20-24 all-trans retinoic acid unit doses and 1 rituximab unit dose.

11. The use according to claim 7 or 8, characterized in that: The dosage of rituximab is 100 mg per week; the dosage of all-trans retinoic acid is 20 mg per square meter of body surface area per day.

12. The use according to claim 7 or 8, characterized in that: Rituximab was continued for 6 weeks, and all-trans retinoic acid was continued for 12 weeks.

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