Application of Huanglian Shangqing tablets combined with glucocorticoids in the preparation of drugs for treating leukemia

Abstract

This invention discloses the application of Huanglian Shangqing tablets combined with glucocorticoids in the preparation of drugs for treating leukemia. The Huanglian Shangqing tablets combined with glucocorticoids can inhibit the activity of Jurkat cells in a concentration-dependent manner; inhibit the proliferation of Jurkat cells in a concentration- and time-dependent manner; and significantly induce apoptosis in the human acute lymphoblastic leukemia cell line Jurkat. In animal experiments, compared with the model group, the Huanglian Shangqing tablets combined with glucocorticoids can increase the hematocrit ratio of leukemia mice, reduce the spleen burden in mice, have no obvious toxic side effects, and improve the survival rate of leukemia mice. Therefore, the Huanglian Shangqing tablets combined with glucocorticoids have the potential value of developing drugs to reverse glucocorticoid resistance in leukemia. Furthermore, Huanglian Shangqing tablets are already used clinically, which can effectively shorten the preclinical research period and has a very good application prospect.

Description

Technical Field

[0001] This invention relates to the fields of biology and medicine, and more specifically to the application of a compound preparation, Huanglian Shangqing tablets, in combination with glucocorticoids in the preparation of drugs for treating leukemia. Background Technology

[0002] Huanglian Shangqing Tablets dispel wind and clear heat, purge fire and relieve pain. They are used for dizziness, vertigo, acute conjunctivitis, toothache, mouth ulcers, sore throat, earache and tinnitus, constipation, and scanty dark urine caused by wind-heat attacking the upper body and excessive heat in the lungs and stomach. The main ingredients include 17 medicinal herbs: Coptis chinensis, Gardenia jasminoides, Forsythia suspensa, stir-fried Vitex trifolia, Saposhnikovia divaricata, Schizonepeta tenuifolia, Angelica dahurica, Scutellaria baicalensis, Chrysanthemum morifolium, Mentha haplocalyx, Rheum palmatum, Phellodendron chinense, Platycodon grandiflorus, Ligusticum chuanxiong, Gypsum fibrosum, Inula japonica, and Glycyrrhiza uralensis. The sugar-coated tablets are yellowish-brown to brownish-yellow after removing the coating; they have a fragrant odor and a bitter taste.

[0003] Leukemia is a group of malignant clonal diseases affecting hematopoietic stem cells. It is mainly divided into acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL). Currently, the main treatment options for leukemia include chemotherapy, targeted therapy, immunotherapy, and stem cell transplantation. The primary treatment for acute lymphoblastic leukemia is chemotherapy, with the main drugs being glucocorticoids (GCs) such as prednisone (Pred) and dexamethasone (DEX). However, in patients with relapsed acute lymphoblastic leukemia, resistance to glucocorticoids is more common than with other chemotherapy drugs. Therefore, discovering drugs that reverse drug resistance and are highly effective and low in toxicity, and optimizing treatment methods are key ways to treat and improve the survival rate of leukemia patients and improve their post-recovery quality of life. Summary of the Invention

[0004] The purpose of this invention is to address the difficulty in resisting glucocorticoid resistance in leukemia by finding highly effective, low-toxicity, and reversible natural drug resources to improve the survival rate of leukemia patients and enhance their post-recovery quality of life.

[0005] To achieve the above objectives, this invention provides the application of Huanglian Shangqing tablets combined with glucocorticoids in the preparation of drugs for treating leukemia. Experiments have demonstrated that, in vitro, Huanglian Shangqing tablets combined with glucocorticoids have a significant inhibitory effect on the glucocorticoid-resistant acute lymphoblastic leukemia cell line Jurkat; in vivo, Huanglian Shangqing tablets combined with prednisone can effectively treat leukemia mice and improve their survival rate. Huanglian Shangqing tablets combined with glucocorticoids can be used to prepare drugs that reverse glucocorticoid resistance in leukemia.

[0006] Furthermore, the aforementioned Huanglian Shangqing tablets are a compound preparation made from 17 medicinal materials, including Coptis chinensis, Gardenia jasminoides, Forsythia suspensa, stir-fried Vitex trifolia, Saposhnikovia divaricata, Schizonepeta tenuifolia, Angelica dahurica, Scutellaria baicalensis, Chrysanthemum morifolium, Mentha haplocalyx, Rheum palmatum, Phellodendron chinense, Platycodon grandiflorus, Ligusticum chuanxiong, Gypsum fibrosum, Inula japonica, and Glycyrrhiza uralensis.

[0007] Furthermore, the aforementioned glucocorticoids include prednisone and dexamethasone.

[0008] Furthermore, the aforementioned leukemia is acute lymphoblastic leukemia.

[0009] Furthermore, the aforementioned drugs for treating leukemia can be any pharmaceutically permissible oral dosage form, any injectable dosage form, or a dosage form manufactured using modern technology.

[0010] This invention also provides the application of Huanglian Shangqing tablets in the preparation of drugs for reversing glucocorticoid resistance in leukemia.

[0011] Currently, the most commonly used glucocorticoids in chemotherapy regimens for lymphoma are dexamethasone (DEX) and prednisone (Pred). Pred only has biological activity after being metabolized by the liver to prednisolone. Preliminary animal experiments showed that the mortality rate of SCID mice injected with DEX intraperitoneally was significantly higher than that of other groups, even the Model group. Therefore, DEX was used for the in vitro cell experiments of this invention, while Pred was used for the in vivo animal experiments. The VP regimen, consisting of vincristine (VCR) and prednisone (Pred), is the basic framework for induction remission therapy in acute lymphoblastic leukemia. Therefore, Pred+VCR was selected as the positive control drug for animal experiments in this invention.

[0012] According to literature reports, CEM-C1 and Jurkat are both dexamethasone (DEX)-resistant acute lymphoblastic leukemia cell lines, and have been studied in multiple publications as experimental cell lines for reversing glucocorticoid resistance. In vitro, the dexamethasone-insensitive cell line Jurkat was screened using the MTT assay. In vitro testing showed that Huanglian Shangqing tablets (Y4) combined with glucocorticoids significantly inhibited the activity of Jurkat acute lymphoblastic leukemia cells. Flow cytometry analysis showed that the combined treatment significantly induced cell apoptosis, demonstrating its important potential as a drug to combat leukemia drug resistance. In vivo, results from F-MuLV-induced leukemia mouse models (Balb / c) and Jurkat cell xenograft SCID mouse models showed that the combination of Y4 and Pred increased the survival rate of mice, reduced the burden on the spleen, increased hematocrit, reduced the proportion of leukemia cells in the bone marrow, and reduced the infiltration of leukemia cells in the spleen. The therapeutic effect was significantly better than that of Pred or Y4 alone, and no obvious toxic side effects were observed.

[0013] The beneficial effects of this invention are as follows:

[0014] 1. This invention combines Huanglian Shangqing tablets with glucocorticoids to inhibit the activity of Jurkat cells in a concentration-dependent manner; it also inhibits the proliferation of Jurkat cells in a concentration- and time-dependent manner; and it significantly induces apoptosis in the human acute lymphoblastic leukemia cell line Jurkat. In animal experiments, compared with the model group, the combination of Huanglian Shangqing tablets and glucocorticoids can increase the hematocrit ratio of leukemia mice, reduce the spleen burden in mice, have no obvious toxic side effects, and improve the survival rate of leukemia mice. Furthermore, Huanglian Shangqing tablets have been used clinically, which can effectively shorten the preclinical research period and has a very good application prospect.

[0015] 2. Compared with the existing uses of Huanglian Shangqing tablets, this invention discovers a novel efficacy of Huanglian Shangqing tablets combined with glucocorticoids in treating acute lymphoblastic leukemia. It has the potential to become a drug for reversing glucocorticoid resistance in acute lymphoblastic leukemia, or to isolate compounds that can highly sensitize the affected cells. Furthermore, this drug is already on the market, which can effectively shorten the preclinical research period and has excellent application prospects. This drug has significant therapeutic effects and few toxic side effects, while also demonstrating the novel efficacy of Huanglian Shangqing tablets and the important role of repurposing an existing drug. Attached Figure Description

[0016] Figure 1 This study investigated the effect of dexamethasone (DEX) on the proliferation of CEM-C1 and Jurkat cells.

[0017] Figure 2The cell inhibition rate (a) of acute lymphoblastic leukemia cell line Jurkat after 48 hours of treatment with different concentrations of Huanglian Shangqing tablets (Y4) combined with dexamethasone (DEX) is shown in Figure 2 (b).

[0018] Figure 3 The graph shows the apoptosis (a) and apoptosis rate (b) of the acute lymphoblastic leukemia cell line Jurkat after 48 hours of treatment with different concentrations of Huanglian Shangqing tablets (Y4) combined with dexamethasone (DEX).

[0019] Figure 4 The activity of Huanglian Shangqing tablets (Y4) combined with prednisone (Pred) in F-MuLV-induced leukemia mice (Balb / c) is shown in (a) mouse appearance, (b) survival curve, (c) hematocrit, and (d) spleen weight.

[0020] Figure 5 This study compares the activity of Huanglian Shangqing tablets (Y4) in combination with prednisone (Pred) in F-MuLV-induced leukemia mice (Balb / c), where (a) represents liver weight, (b) represents heart weight, (c) represents lung weight, and (d) represents the weight of both kidneys.

[0021] Figure 6 This is a pathological section (HE staining) of the spleen of an F-MuLV-induced leukemia mouse (Balb / c).

[0022] Figure 7 This shows the expression of Hu-CD3 in peripheral blood cells of SCID mice xenografted with Jurkat cells.

[0023] Figure 8 The appearance (a), spleen weight (b), and spleen size (c) of SCID mice with Jurkat cell xenografts.

[0024] Figure 9 This is a comparison of the weight of the heart (a), liver (b), lungs (c), and kidneys (d) in different groups of SCID mice with Jurkat cell xenograft.

[0025] Figure 10 This is a pathological section (HE staining) of the spleen of an SCID mouse that underwent xenograft transplantation of Jurkat cells. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0027] Example 1

[0028] 1. Experimental Materials

[0029] RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Gibco, Inc., USA; the apoptosis kit was purchased from BD Biosciences, Inc., USA; the tetramethylazolidinyl ether (MTT) and penicillin-streptomycin mixture was purchased from Beijing Solarbio Science & Technology Co., Ltd.; Huanglian Shangqing tablets (Guizhou Bailing Pharmaceutical Co., Ltd.) were purchased from the Medical Alliance Drug Platform; dexamethasone sodium phosphate injection (DEX) was purchased from Chenxin Pharmaceutical Co., Ltd.; prednisone tablets (Pred) were purchased from Shandong Lukang Pharmaceutical Group; vincristine was purchased from Shenzhen Wanle Pharmaceutical Co., Ltd.; the acute lymphoblastic leukemia cell line Jurkat was purchased from the American Type Culture Collection (ATCC); the acute lymphoblastic leukemia cell line CEM-C1 was purchased from Shanghai Fantai Biotechnology Co., Ltd.; and NIH3T3 cells (expressing F-MuLV clone 57 vector) were kindly provided by Professor Ben-David Yaacov of the Guizhou Provincial Natural Products Research Center.

[0030] Preparation of the sample solution to be tested: Take the supernatant tablets of Coptis chinensis and prepare a stock solution of 100 mg / mL with 10% DMSO + 90% double-distilled water. Store at -20℃ and use at 4℃.

[0031] Preparation of 5% MTT solution: Weigh 0.5g of MTT powder, add 100mL of sterile PBS, dissolve at 60℃, filter sterilize through a 0.22μm filter membrane, store at -20℃, and use at 4℃ for later use.

[0032] Preparation of the triple solution: Weigh 10g of sodium dodecyl sulfate (SDS), add ddH2O to dissolve it completely, then add 5mL of isopropanol and bring the volume to 100mL with ddH2O. Finally, add 100μL of hydrochloric acid (because hydrochloric acid is volatile).

[0033] Cell lines: Jurkat and CEM-C1 were cultured in RPMI-1640 medium containing 5% FBS in a 37℃, 5% CO2 incubator.

[0034] 2. Experimental methods and results

[0035] 2.1 Screening for cell lines insensitive to dexamethasone (DEX)

[0036] To determine the DEX-resistant cell lines, CEM-C1 and Jurkat cells were treated with different concentrations of DEX. The inhibition rate of the two cell lines was detected by the MTT assay. Specifically, Jurkat and CEM-C1 cells, human acute lymphoblastic leukemia cells in the logarithmic proliferation phase, were diluted with RPMI-1640 medium containing 5% FBS at a concentration of 1×10⁻⁶. 4Cells were seeded at a density of 90 μL / well in 96-well plates, and the plates were incubated at 37°C with 5% CO2. After 4 hours, 10 μL of LDEX diluent was added to each well, with five concentrations (0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, and 100 μg / mL, diluted with RPMI-1640 serum-free medium) for each concentration, with three replicates for each concentration. A control group was also included (10 μL of RPMI-1640 serum-free medium was added to each well). The plates were then incubated at 37°C with 5% CO2. After 72 hours, 10 μL of 5% MTT solution was added to each well, and the plates were incubated at 37°C with 5% CO2. After 4 hours, 100 μL of triplet solution was added to each well, and the plates were incubated at 37°C with 5% CO2. Incubate overnight in a CO2 incubator; the next morning, measure the absorbance (OD) value of each well at a wavelength of 570 nm using a microplate reader, and calculate the inhibition rate.

[0037] Figure 1 The results showed that different concentrations of DEX (0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, and 100 μg / mL) were applied to CEM-C1 and Jurkat cells, respectively. At each concentration, Jurkat cells were less sensitive to DEX. Even after an action time of up to 72 h, the inhibition rate of DEX on Jurkat cells was still less than 20%. Therefore, Jurkat cells were selected as an in vitro cell model.

[0038] 2.2 MTT assay was used to determine the inhibitory rate of different concentrations of Huanglian Shangqing tablets (Y4) combined with dexamethasone (DEX) on leukemia cells.

[0039] Jurkat cells, a human acute lymphoblastic leukemia cell line in the logarithmic proliferative phase, were diluted with RPMI 1640 medium containing 5% FBS and injected at a concentration of 1×10⁻⁶. 4Cells were seeded at a density of 90 μL per well in 96-well plates, with 100 μL of sterile double-distilled water added to each well around the perimeter. The 96-well plates were then incubated at 37°C in a 5% CO2 incubator. After 4 hours, different concentrations of Coptis chinensis supernatant tablets (Y4) and DEX were added, 10 μL per well, with the following groups: Control, Y4-L, Y4-H, DEX, DEX+Y4-L, and DEX+Y4-H, with 5 replicates per group. The final concentrations of Huanglian supernatant tablets were: Y4-L 250 mg / L, Y4-H 500 mg / L, and DEX 200 mg / L. The tablets were incubated at 37℃ in a 5% CO2 incubator. After 24 h, 48 h, 72 h, and 96 h of incubation, 10 μL of 5% MTT solution was added to each well, and the wells were incubated at 37℃ in a 5% CO2 incubator. After 4 h, 100 μL of triple solution was added to each well, and the wells were incubated overnight at 37℃ in a 5% CO2 incubator. The next morning, the absorbance (OD) value of each well at 570 nm wavelength was measured using a microplate reader, and the growth curve was calculated.

[0040] Depend on Figure 2 As can be seen, when DEX (200 μg / mL) and different concentrations of Y4 (Y4-L: 250 μg / mL, Y4-H: 500 μg / mL) were used alone or in combination with Jurkat cells for 48 h, the combination of DEX and Y4 significantly reduced the survival rate of Jurkat cells compared with single treatment. When the Y4 concentration was 250 μg / mL and DEX was used in combination, the survival rate of Jurkat cells was 79.16%; when the Y4 concentration was 500 μg / mL and DEX was used in combination, the survival rate of Jurkat cells was 45.83%, showing a dose-dependent effect on Y4. After 24 h, 48 h, 72 h, and 96 h of treatment with DEX (200 μg / mL) and Y4 (500 μg / mL) alone and in combination, the cell viability of Jurkat cells in the DEX+Y4 combination group was significantly lower than that in the Control group, the DEX or Y4 single treatment group, and the effect was time-dependent. The results showed that the combined use of Huanglian Shangqing tablets (Y4) and DEX reduced the survival rate of Jurkat cells, with better effect than DEX or Y4 alone, and had a time- and dose-dependent effect.

[0041] 2.3 Flow cytometry detection of apoptosis

[0042] Jurkat cells, a human acute lymphoblastic leukemia cell line in logarithmic growth phase, were diluted with RPMI 1640 medium containing 5% FBS and cultured at 2.5 × 10⁻⁶ cells / mL. 5Cells were seeded at a density of 100 cells / mL in 6-well plates, 2 mL per well, and incubated at 37°C in a 5% CO2 incubator. After 4 h, different concentrations of Coptis chinensis supernatant tablets and DEX were added, with the groups being Control, Y4-L, Y4-H, DEX, DEX+Y4-L, and DEX+Y4-H, respectively. The final concentrations of Huanglian supernatant tablets were: Y4-L 250 mg / L, Y4-H 500 mg / L, and DEX 200 mg / L. Each well was supplemented with RPMI-1640 serum-free medium to ensure a consistent volume. The cells were incubated at 37°C in a 5% CO2 incubator. After 48 hours, cells were collected, and 50 μL of 1×Binding Buffer was added to each tube. The mixture was gently pipetted to mix the cells. 1.5 μL of LPI and 1.5 μL of Annexin V were added to each tube. In the Control group, one tube was left unstained (Blank group), one tube was stained with LPI, and one tube was stained with Annexin V. After staining on ice for 15 minutes in the dark, cell apoptosis was detected by flow cytometry.

[0043] Figure 3 Flow cytometry results showed that Huanglian Shangqing tablets (Y4) combined with DEX could significantly induce apoptosis in human acute lymphoblastic leukemia cell line Jurkat cells, with better effects than DEX or Y4 alone, and there was a dose-dependent effect on Y4.

[0044] 2.4F-MuLV-induced leukemia mouse model (Balb / c)

[0045] Balb / c mice were intraperitoneally injected with 100 μL of F-MuLV virus solution within 48 hours of birth. Four weeks after birth, they were randomly divided into five groups: a model group, a positive control group (prednisone + vincristine, Pred + VCR), a prednisone-only group (Pred), a Huanglian Shangqing tablet-only group (Y4), and a combination of prednisone and Huanglian Shangqing tablets (Pred + Y4). A control group (normal Balb / c mice) served as a negative control. Each group consisted of six mice, half male and half female. Drug administration began at week 6. The administration schedule was as follows: VCR was administered weekly on days 1, 8, 15, 22, and so on; Pred and Y4 were administered for 5 days, followed by a 2-day break; Pred was given at 2 / 3 of its dose in weeks 3 and 4, 1 / 3 in weeks 5, 6, 7, and 8, and maintained at 1 / 3 every other day starting in week 9; Y4 was also reduced to every other day (at the same dose) for maintenance starting in week 9. Survival curves and weight changes were recorded. At week 14 of drug administration, the remaining mice were sacrificed, and the following tissues were collected: heart, liver, spleen, lungs, and kidneys were weighed, hematocrit was measured, and spleen tissue was collected for pathological examination. Results are shown below. Figures 4-6 .

[0046] Depend on Figure 4It is evident that in the F-MuLV-induced Balb / c mouse model, the combination of Pred and Y4 increased the survival rate, increased hematocrit, and reduced the spleen burden without significant toxic side effects. Compared with the control group, the model group mice exhibited arched backs, ruffled fur, pale ears and paws, lethargy, weight loss, and limb curling (see...). Figure 4 a) Similar to Pred+VCR, Pred+Y4 treatment resulted in 100% survival in mice, while mice in the Pred-only and Y4-only groups died. The survival rate of mice in the Pred+Y4 treatment group was successively higher than that of the Pred group, Y4 group, and Model group (see...). Figure 4 b) Compared with the Model group, the Pred+Y4 treatment group significantly increased the hematocrit of mice, which was superior to the Pred or Y4 monotherapy group. The treatment effect was similar to that of Pred+VCR (see...). Figure 4 c) Compared with the Model group, the Pred+Y4 treatment group significantly reduced the spleen burden in mice, which was superior to the Pred or Y4 alone group, and the effect was similar to that of Pred+VCR (see...). Figure 4 d). By Figure 5 It is evident that F-MuLV modeling can enlarge the liver of Balb / c mice. Y4 alone does not significantly reduce the liver burden, but the combination of Pred and Y4 significantly reduces the liver burden caused by F-MuLV modeling in Balb / c mice, with a therapeutic effect similar to that of Pred+VCR (see...). Figure 5 a) Compared with the Model group or Control group, there were no significant differences in the weight of the heart, lungs, and kidneys among the groups, and no significant toxic side effects on the heart, lungs, and kidneys (see [link]). Figure 5 (b, c, d). Figure 6 It is evident that in the F-MuLV-induced Balb / c mouse model, F-MuLV modeling can disrupt the original structure of the spleen in Balb / c mice, resulting in the infiltration of a large number of leukemia cells. Pred or Y4 alone cannot improve this condition, but the combination of Pred and Y4 can significantly reduce the infiltration of leukemia cells and partially restore the spleen structure to normal.

[0047] 2.5 SCID mouse model of Jurkat cell xenograft

[0048] Thirty-five SCID mice (6-7 weeks old, weighing 18-20g) were selected, and Jurkat cells in the logarithmic growth phase were used. 1×10⁻⁶ cells were used. 7The model was established by intravenous injection of 200 μL of Hu-CD3 cells per cell. Four weeks later, flow cytometry was used to detect Hu-CD3 expression in peripheral blood to determine model success. Successfully modeled mice were randomly divided into four groups: Model, Pred+VCR, Pred, Y4, and Pred+Y4. Normal SCID mice (Control group) served as a negative control. Each group consisted of six mice, half male and half female. Drug administration began the following day and continued for two weeks. Mice were sacrificed after two weeks. Hematocrit was measured, and the weights of the heart, liver, spleen, lungs, and kidneys were weighed. Spleen tissue was collected for pathological examination. Results are shown below. Figures 7-10 .

[0049] Depend on Figure 7 As can be seen, Hu-CD3 expression was detected in mouse peripheral blood by flow cytometry, and the Hu-CD3 expression level exceeded that of the control group (peripheral blood of normal SCID mice) by more than 5%. Figure 8 It is evident that in the SCID mouse model of Jurkat cell xenograft, the combination of Pred+Y4 reduced the spleen burden in mice without significant toxic side effects; compared with normal SCID mice (Control), the Model group mice had reduced body weight, rough fur, and curled limbs (see...). Figure 8 a) The spleen weight and volume of mice in the Model group were significantly larger than those in the Control group. Pred+Y4 combined treatment reduced spleen weight and volume, bringing them closer to normal levels, demonstrating a better therapeutic effect than Pred or Y4 alone (see...). Figure 8 (b, c). Figure 9 As can be seen, there were no significant differences in the weight of the heart, liver, lungs, and kidneys among the groups compared to the Model or Control groups, and no significant toxic side effects were observed on the heart, liver, lungs, and kidneys. Figure 10 It is evident that in the SCID mouse model induced by Jurkat cell xenograft, the combined use of Pred and Y4 can reduce the infiltration of leukemia cells in the spleen of mice. In the SCID mouse model induced by Jurkat cell xenograft, the original structure of the spleen in SCID mice is disrupted, resulting in a large number of leukemia cells infiltrating. Pred or Y4 alone cannot improve this, but the combined use of Pred and Y4 significantly reduces leukemia cell infiltration and partially restores the spleen structure to normal.

[0050] The results above indicate that Huanglian Shangqing tablets combined with glucocorticoids have the potential value of being developed into a drug to reverse glucocorticoid resistance in leukemia.

Claims

1. Huanglian Shangqing tablets combined with glucocorticoids for treating leukemia, wherein the glucocorticoids are prednisone or dexamethasone, and the leukemia is acute lymphoblastic leukemia. 2.The use of Huanglian Shangqing tablets combined with glucocorticoids in the preparation of a drug for treating leukemia according to claim 1, characterized in that: The drug can adopt any oral or injection dosage form allowed in pharmacy.

3. Huanglian Shangqing tablets for preparing a drug for reversing glucocorticoid resistance of leukemia, wherein the glucocorticoids are prednisone or dexamethasone, and the leukemia is acute lymphoblastic leukemia.

Images