CN114887034B

CN114887034B - Use of LLP2A-Ale for the preparation of a medicament for the treatment and/or prevention of peripheral blood lymphopenia

Info

Publication number: CN114887034B
Application number: CN202210823845.5A
Authority: CN
Inventors: 陈继伟; 陈猛猛; 向雪香
Original assignee: Zhongshan Laibo Ruichen Biomedical Co ltd
Current assignee: Zhongshan Laibo Ruichen Biomedical Co ltd
Priority date: 2022-07-14
Filing date: 2022-07-14
Publication date: 2022-10-28
Anticipated expiration: 2042-07-14
Also published as: CN114887034A

Abstract

The present invention relates to the use of LLP2A-Ale for the preparation of a medicament for the treatment and/or prevention of peripheral blood lymphopenia. The peripheral blood lymphopenia may be due to any cause.

Description

Use of LLP2A-Ale for the production of a medicament for the treatment and/or prophylaxis of peripheral lymphopenia

Technical Field

The invention belongs to the field of medicine. In particular, the invention relates to the use of LLP2A-Ale for the preparation of a medicament for the treatment and/or prevention of peripheral blood lymphopenia.

Background

LLP2A-Alendronate (abbreviated as LLP 2A-Ale), a polypeptide compound, having the structure shown below:

。

LLP2A-Ale is a clinical candidate developed for bone diseases (osteonecrosis, osteoporosis and fracture) and is currently in the first clinical trial phase. It is a single organic molecule with two ligands covalently linked by a chemical chain, one ligand, LLP2A, is a highly derivatized synthetic tripeptide with high affinity and specificity for α 4 β 1 integrin. The other ligand is bone targeting diphosphate (Alendronate/Ale), and because alphSub>A 4 betSub>A 1 integrin is highly expressed in mesenchymal stem cells, LLP2A-Ale and the mesenchymal stem cells can be specifically combined to pull the mesenchymal stem cells to the bone surface, increase the level of vascular endothelial growth factor (VEGF-A) in vivo and improve the microcirculation of local bone tissues, promote the transformation of the mesenchymal stem cells to bone-forming cells instead of fat cells, thereby generating more bone-forming cells and stimulating new bone formation.

Currently, the common causes of peripheral blood lymphopenia are classified into the following:

1. acute exposure to myelosuppressive doses of radiation (hematopoietic syndrome of acute radiation syndrome) results in severe peripheral leukopenia (including neutropenia and lymphopenia) in patients with acute exposure to myelosuppressive doses of radiation, and for this indication, the FDA approved Neupogen/Filgrastim/G-CSF (fully humanized protein macromolecule, 175 amino acid) developed by american ann company in 2015, which promotes proliferation and maturation of bone marrow neutrophils and increases the number of neutrophils entering the blood circulation, thereby reducing the risk of severe infection. However, neupogen/Filgrastim/G-CSF has no effect on peripheral blood lymphocytes and is unable to treat the lymphopenia component of the severe pancytopenia with peripheral blood resulting from acute exposure to myelosuppressive doses of radiation. Therefore, its efficacy for this indication is limited, and there is currently no effective therapeutic drug for this severe peripheral blood lymphopenia, and this unmet medical need needs need to be filled with new drugs.

2. Peripheral blood severe lymphopenia associated with radiotherapy caused by tumor radiotherapy or radiotherapy, local radiotherapy or radiotherapy is one of the main means of treating tumor diseases, and about 70% of cancer patients need radiotherapy in the process of treating cancer. However, radiotherapy often causes many adverse events, peripheral blood Severe Lymphopenia and immune function decline are one of its serious side effects, and it is reported in literature that a high proportion (46% -71.7%) of peripheral blood circulation Acute Severe Lymphopenia (ace liver cancer, breast cancer, pancreatic cancer, lung cancer and head and neck cancer) associated with radiotherapy occurs after radiotherapy or after concurrent radiotherapy and chemotherapy, which is a serious adverse event related to radiotherapy, on the one hand, may cause tumor radiotherapy to be forced to be temporarily suspended, cannot complete the course of radiotherapy and cannot affect the treatment of tumor diseases. On the other hand, severe peripheral blood circulating lymphocyte depletion and immune function decline not only increase the risk of hospitalization for infection, but also lead to poor prognosis and shorter survival of tumor patients. At present, no effective treatment medicine exists for the severe lymphopenia of peripheral blood caused by local radiotherapy, and the huge unmet medical requirement needs to be filled by new effective medicines.

3. Peripheral blood lymphopenia due to poor immune reconstitution or immune unresponsiveness in Acquired Immune Deficiency Syndrome (AIDS): the pathogenesis of AIDS is that HIV virus invades the immune system of a human body, including CD4+ T lymphocytes, mononuclear macrophages, DC and the like, and the AIDS is mainly characterized in that the number of the CD4+ T lymphocytes is continuously reduced, and finally, the cellular immune function of the human body is defective, so that various opportunistic infections and tumors are caused. The current standard treatment of AIDS (2021 guideline) is to use Antiretroviral (ART) drugs to inhibit virus replication, reestablish immunity, and prevent and reduce the incidence and mortality of AIDS-related diseases (opportunistic infection, tumor, etc.) and non-AIDS-related diseases. However, about 10% to 40% of HIV-infected individuals do not fully achieve immune reconstitution even after complete viral suppression. This phenomenon is called immune reconstitution failure and these patients are called immune reconstitution or immune non-responders (INRs). Compared with Immune Responders (IRs), the incidence and fatality rate of INRs AIDS-related diseases (infection, tumor, etc.) and non-AIDS-related diseases (cardiovascular diseases, tumor, neurocognitive disorder, osteoporosis, etc.) are remarkably increased, and the medical burden and the economic burden of families and society are increased. At present, no effective therapeutic drug is available for poor immune reconstitution or immune unresponsiveness of Acquired Immune Deficiency Syndrome (AIDS).

4. Peripheral blood lymphopenia caused by glucocorticoid use, which is caused by immunosuppression, namely, the number of circulating lymphocytes is reduced by inhibiting phagocytosis and treatment of antigen by macrophages, promoting destruction and disintegration of the lymphocytes and promoting migration of the lymphocytes out of blood vessels; the cell immunity is mainly inhibited at a small dose; in large dose, the preparation inhibits plasma cell and antibody production to inhibit humoral immunity function. The side effects caused by this glucocorticoid use are currently no effective therapeutic agents.

5. LLP2A-Ale may be an effective therapeutic drug for peripheral blood lymphopenia caused by autoimmune diseases (such as systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis).

6. The retrospective study on 52 critical patients in the quasipelas hospital is published in the lancet respiratory medicine by institutions such as the Tongji medical college of Huazhong science and technology university, the jin Yin Tan hospital in Wuhan City, the people hospital in Wuhan university and the like. Studies have shown that lymphopenia occurs in 80% of critically ill patients. Since targeted invasion of the new corona (SARS-CoV) virion destroys the cytoplasmic components of lymphocytes and causes their destruction. The authors state that the high association of blood lymphocytes with disease progression suggests that lymphocyte deficiency or incapacitation is a key cytopathology for new coronary pneumonia. Protection, maintenance or promotion of lymphocyte levels may have a favorable effect on the prevention and treatment of new coronary pneumonia. There are currently no effective therapeutic agents that increase the peripheral blood lymphocyte count.

7. Peripheral blood lymphopenia caused by blood diseases (such as Hodgkin's disease), no effective therapeutic drug for increasing peripheral blood lymphocyte count exists at present.

8. Peripheral lymphopenia due to genetic diseases (ataxia-telangiectasia, diGeorge analysis, lung combined immunological syndrome, wiskott-Aldrich syndrome) is due to ineffective lymphopoiesis due to abnormalities in the quality and quantity of stem cells. Other causes, such as Wiskott-Aldrich syndrome, may be caused by accelerated destruction of T cells. An effective treatment modality is stem cell transplantation, such as bone marrow transplantation or umbilical cord blood transplantation.

9. Peripheral blood lymphopenia caused by protein loss enteropathy (including Amyloidosis, celiac disease, crohn's disease, allergic colitis, regional entirities, zinc specificity), no effective therapeutic agent for increasing peripheral blood lymphocyte count is currently available.

10. In some cases, such as peripheral lymphopenia due to strenuous physical exercise (athlete training), severe stress, malnutrition (e.g., cancer or end-stage cancer patients), eating disorders (anorexia nervosa), etc., there is currently no effective therapeutic agent for increasing the peripheral blood lymphocyte count.

Disclosure of Invention

Because alpha 4 beta 1 integrin is also expressed in lymphocytes and LLP2A-Ale is also specifically bound with the lymphocytes, the research of the invention finds that the number of peripheral blood lymphocytes is obviously increased after the experimental animals receive the LLP2A-Ale treatment, and suggests that LLP2A-Ale can reduce the attachment of lymphocytes on the blood vessel wall by interfering the binding of the lymphocytes and the vascular endothelial cells after being specifically bound with the lymphocytes, thereby causing the increase of the number of the lymphocytes in the peripheral blood circulation.

The present investigators also found through rat experiments that after inducing antibodies IgM and IgG using KLH (keyhole Limpet Hemocyanin), the positive control drug cyclophosphamide (cyclophosphamide) significantly reduced the serum IgM and IgG antibody levels in vivo as expected, while the treatment with intravenous administration of LLP2A-Ale did not increase nor reduce the levels of antibodies IgM and IgG compared to the negative control group (Vehicle group), demonstrating that LLP2A-Ale did not affect its immune function after specifically binding to rat lymphocytes.

By utilizing the action mechanism of the medicament, the invention further researches the application of the LLP2A-Ale and discovers that the LLP2A-Ale can be used for treating the peripheral blood lymphopenia caused by any reason.

The invention provides the use of the compound LLP2A-Ale for the preparation of a medicament for the treatment and/or prevention of peripheral blood lymphopenia.

The peripheral blood lymphopenia includes peripheral blood lymphopenia due to any reason, and specifically includes but is not limited to: hematopoietic syndrome of acute radiation syndrome presents with severe peripheral blood pancytopenia; peripheral blood severe lymphopenia and total blood leukopenia caused by tumor local radiotherapy or radiotherapy and chemotherapy; peripheral blood lymphopenia due to poor immune reconstitution or immune unresponsiveness in Acquired Immune Deficiency Syndrome (AIDS); peripheral blood lymphopenia with glucocorticoid use; peripheral blood lymphopenia due to autoimmune disease; peripheral blood lymphopenia due to infectious diseases; peripheral blood lymphopenia due to hematological disorders; peripheral blood lymphopenia due to genetic diseases; protein loss enteropathy-induced peripheral blood lymphopenia; peripheral blood lymphopenia due to physiological causes.

Specifically, the hematopoietic syndrome of acute radiation syndrome refers to severe peripheral blood leukopenia in patients who are acutely exposed to myelosuppressive dose radiation (hematopoietic syndrome of acute radiation syndrome), and can be treated with LLP2A-Ale alone or in combination with other drugs (such as Neupogen/Filgrastim/G-CSF, etc.).

The autoimmune diseases comprise systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis and the like.

The infectious diseases include tuberculosis, hepatitis B, AIDS, neocoronary disease, etc.

Such as Hodgkin's disease, etc.

The genetic diseases comprise ataxia telangiectasia (ataxia-telangiectasia), diGeorge abnormality (DiGeorge abnormality), severe combined immunodeficiency syndrome (anomaly, segment combined immunodeficiency syndrome), wiskott-Aldrich syndrome (Wiskott-Aldrich syndrome), and the like.

The protein-loss bowel disease includes Amyloidosis (Amyloidosis), celiac disease (celiac disease), crohn's disease, ulcerative colitis (ulcerative colitis), regional enteritis (regional enteritis), zinc deficiency (Zinc deficiency), and the like.

Such physiological causes include certain conditions such as intense physical exercise (athlete training), severe stress, malnutrition (e.g., cancer or end-stage cancer patients), eating disorders (anorexia nervosa), and the like.

Drawings

FIG. 1 is a graph showing the effect of Warran ray on the body weight of mice in example 1;

FIG. 2 is a graph showing the effect of Walian radiation on mouse lymphocytes in example 1 (unit: 10) ⁹ a/L) plot;

FIG. 3 is a bar graph of the effect of warrior radiation on mouse bone marrow cells;

FIG. 4 is a graph showing the effect of Rab001-US (0.4 mg/kg) and Rab001-CN (0.1 mg/kg,0.4mg/kg,1.6 mg/kg) on the number of circulating lymphocytes in mice in the treatment protocol of example 2-1;

FIG. 5 is a graph showing the effect of Rab001-US (0.4 mg/kg) and Rab001-CN (0.1 mg/kg,0.4mg/kg,1.6 mg/kg) on the number of mouse leukocytes in the treatment protocol of example 2-1; note: p <0.05 by Tukey (one-way anova); * P <0.01; * P <0.001 VS, 6D or 13D; a is P <0.05; b is P <0.01; p <0.001 VS, baseline;

FIG. 6 is a bar graph showing the effect of Rab001-US (0.4 mg/kg) and Rab001-CN (0.1 mg/kg,0.4mg/kg,1.6 mg/kg) on the number of mouse bone marrow cells in the treatment protocol of example 2-1; note: p <0.05 by Tukey (one-way anova); * P <0.01; * P <0.001 vehicle group VS non-irradiated group;

FIG. 7 is a graph showing the effect of Rab001-US (0.4 mg/kg) and Rab001-CN (0.1 mg/kg,0.4mg/kg,1.6 mg/kg) on the number of circulating lymphocytes in mice in the prevention protocol of example 2-2;

FIG. 8 is a graph showing the effect of Rab001-US (0.4 mg/kg) and Rab001-CN (0.1 mg/kg,0.4mg/kg,1.6 mg/kg) on the number of circulating lymphocytes in mice in the prevention protocol of example 2-2; note: p <0.05 by Tukey (one-way anova); * P <0.01; * P <0.001 VS, 6D or 13D; a is P <0.05; b is P <0.01; p <0.001 VS, baseline;

FIG. 9 is a bar graph showing the effect of Rab001-US (0.4 mg/kg) and Rab001-CN (0.1 mg/kg,0.4mg/kg,1.6 mg/kg) on the number of mouse bone marrow cells in the prevention protocol of example 2-2; note: p <0.05 by Tukey (one-way anova); * P <0.01; * P <0.001 vehicle group VS non-irradiated group.

Detailed Description

The invention is further described in the following examples, which are not intended to limit the scope of the invention.

Example 1 modeling of systemic radiation-induced lymphopenia in mice

Effect of warian UNIQUE-6MV radiation on blood and bone marrow cell counts in C57 mice.

1. Main experimental materials and instruments

Materials: 12C 57BL/6 mice were purchased from Shanghai Spiker laboratory animals, inc. Reagent: 2.5ul, 10ul, 200ul, 1000ul pipettors (eppendorf, germany); trypan blue. The instrument comprises the following steps: super clean bench (Suzhou purification SW-CJ-1FD, china); biological inverted microscope (japanese OLYMPUS IX 51); microplate reader (BioTek ELx800, usa); an oscillator (Shanghai province of China WH-2 analytical instruments).

2. The experimental method comprises the following steps:

male C57BL/6 mice were 12, acclimatized for 1 week, randomized into groups of 3 mice each for 4 groups. 4 groups are respectively irradiated with warian UNIQUE-6MV rays with different doses once (0 Gy, 3Gy, 6Gy and 12 Gy), 100 mu L of whole blood is respectively taken for routine blood detection at 0h, 1h, 6h, 24h, 3d, 7d and 10d, the animals are killed after 10 days of molding, and bone marrow is collected for bone marrow cell counting.

Cell counts were performed using trypan blue method: (1) cell collection: taking mouse bone marrow cells, centrifuging for 5 minutes at 1000 rpm, discarding supernatant, and resuspending the cells with 1mL of PBS; (2) trypan blue staining: sucking 10ul cell suspension, placing in a plastic centrifuge tube, adding 10ul trypan blue staining solution, mixing gently, and staining for 1min; (3) cell counting: and sucking a proper amount of dyed cell suspension, coating the cell suspension in a cell counting plate, and observing under an inverted optical microscope, wherein the blue dyed cells are dead cells, and the non-dyed cells are active cells.

3. As a result:

(1) Effect of warian radiation on mouse weight

The results are shown in Table 1-1 and FIG. 1: the weight gain of mice decreased with one irradiation in the dose level range of 3-12Gy, and was correlated with the irradiation dose.

The results show that a single 3-12 warrior irradiation reduces body weight by about 5% on day 10 post irradiation.

TABLE 1-1 Effect of warian radiation on mouse body weight (g)

(2) Effect of warian radiation on mouse lymphocytes

The results are shown in tables 1-2 and FIG. 2.

(1) Within the dose range of 3-12Gy, 1 irradiation induced significant lymphopenia (P <0.05, even P < 0.001), which was correlated to the irradiation dose, and which occurred 1h after irradiation.

(2) In the 3Gy and 6Gy dose groups, the lymphocyte count tendency returned to the baseline value (3 Gy =32.18%,6Gy = 45.29%) in 10 days of single irradiation, and no tendency was observed for the lymphocyte count to return to the baseline value after 10 days of 12Gy dose irradiation.

(3) In the 6Gy irradiation group, the baseline value of the 1 st mouse is obviously lower than that of lymphocytes of other groups (1.84Vs4.16 to 10.09), and the baseline value of the mouse is obviously abnormal and can be judged as a discrete value; the lymphocyte count of the 2 nd mouse at the 24h time point is restored to the baseline value, and the lymphocyte count at other time points is obviously lower than the baseline value, and the value is obviously abnormal and can be judged as a discrete value.

The results show that irradiation with a single 3-12 watt-ampere ray inhibits lymphocyte viability for up to 10 days.

TABLE 1-2 Effect of warian radiation on mouse lymphocyte counts (Unit: 10) ⁹ /L）

Note: * P <0.05, P <0.01, P < 0.001.

(3) Effect of warian radiation on mouse bone marrow cells

The results are shown in tables 1-3 and FIG. 3, showing: within the irradiation dose range of 3-12Gy, after 10 days of 1 irradiation, the total cell number and the viable cell number of bone marrow were significantly less than those of the non-irradiated group, and significant myelosuppression was exhibited, and the irradiation-induced myelosuppression was correlated with the irradiation dose (P <0.05 or P < 0.01).

The results show that irradiation with a single 3-12 watt-lian ray inhibited the number of bone marrow viable cells for up to 10 days.

TABLE 1-3 Effect of Validan radiation on the number of bone marrow cells in mice after 10 days of irradiation (unit: 10) ⁶ One)

Note: * P is less than 0.05; * P < 0.01.

Example 2

LLP2A-Ale was evaluated for therapeutic/prophylactic effects in a mouse model of systemic radiation-induced lymphopenia.

1. Main experimental materials and instruments

Materials: 72C 57BL/6 mice (purchased from Shanghai Spiker laboratory animals Co., ltd.). Reagent: 0.9% physiological saline, rab001-CN (i.e., compound LLP2A-Ale, manufactured by Ducheng Nuo Biotech, inc., of this company), rab001-US (i.e., compound LLP2A-Ale, synthesized by Bachem, USA, inc., of this company).

2. Experimental methods

Group-1 baseline control: no radiation is performed and no drug is injected.

Group-2 age-matched non-irradiated group: without irradiation, 0.9% normal saline was injected.

Group-3 vehicle group: irradiated and injected with 0.9% normal saline.

Group-6, group-5, group-4: irradiating, weighing 1.6mg of Rab001-CN drug, adding 10ml of normal saline to dissolve to obtain working solution with concentration of 0.16mg/ml, and subpackaging each animal with 100 mu L/10g of body weight for injection; 0.4mg/kg dosage, taking 1ml of high-concentration working solution, adding 3ml of normal saline, and diluting by 4 times; the 0.1mg/kg administration dose can be diluted by the above working solution physiological saline.

Group-7: irradiating, weighing Rab001-US0.4mg, adding 10ml normal saline, dissolving to obtain working solution with concentration of 0.04mg/ml, and subpackaging each animal 100 μ L/10g weight for injection.

3. The experimental scheme is as follows:

example 2-1 therapeutic Effect of LLP2A-Ale on a mouse model of systemic radiation-induced lymphopenia.

The research aims are as follows: the therapeutic effect of LLP2A-Ale on a mouse model of systemic radiation-induced lymphopenia was evaluated.

Experiment number: rab001_ mouse _3 Gy-dose _202112.

Research and design: c57BL/6 male mice (n =42, 6/group), age 6-8 weeks.

The test steps are as follows: peripheral blood severe pancytopenia and lymphopenia were induced upon irradiation (3 Gy, irradiation 1 time at the start of the test on day one), rab001-CN (4 different doses, 0, 0.1, 0.4, 1.6mg/kg or Rab001-us0.4 mg/kg) was administered intravenously after days 7, 14 and 21, respectively, once a week for a total of 3 doses; at 0.5h post-dose on day 21 of the experiment, blood samples of 0.2 ml were taken from all dosing groups (n = 3) for analysis of drug concentration in plasma. On day 28 of the experiment, all mice received euthanasia, and two femurs were harvested and the total and viable cell numbers in the bone marrow were measured. The efficacy of LLP2A-Ale treatment after intravenous administration (increasing the number of peripheral blood lymphocytes) was tested in a model of pancytopenia, which caused severe peripheral blood after systemic irradiation of mice.

The experimental protocol is shown in the following table:

example 2-preventive Effect of LLP2A-Ale on systemic radiation-induced lymphopenia mouse model.

The research aims are as follows: the preventive effect of LLP2A-Ale on a mouse model of systemic radiation-induced lymphopenia was evaluated.

Experiment number: rab001_ mouse _3 Gy-dose _202110.

Research and design: c57BL/6 male mice (n = 42), age 6-8 weeks.

The experimental steps are as follows: rab001-CN (4 different doses: 0, 0.1, 0.4, 1.6mg/kg or Rab001-US0.4 mg/kg) was administered intravenously after the first day of 3Gy dose irradiation, at 4 different doses: 0, 0.1, 0.4, 1.6mg/kg or 0.4mg/kg, respectively, once a week for a total of 3 doses. At 0.25h post-dose on day 14 of the experiment, blood samples of 0.2 ml were taken from all dosing groups (n = 3) for analysis of drug concentration in plasma. On day 28 of the experiment, all mice received euthanasia, and two femurs were harvested and the total and viable cell numbers in the bone marrow were measured. The efficacy of LLP2A-Ale after intravenous administration for the prophylactic period (increasing the number of peripheral blood lymphocytes) was tested in a model of pancytopenia, which causes severe peripheral blood after systemic irradiation of mice.

The experimental protocol is shown in the following table:

4. the experimental results are as follows:

4.1 Example 1 mice were systemically subjected to the radiation-induced lymphopenia model building results (see figure 1).

The increase in body weight of the mice was reduced by about 5% on day 10 after irradiation with a dose level in the range of 3-12Gy for one irradiation; meanwhile, lymphopenia occurs after 1h of irradiation, the radiation of single 3-12Gy A ray inhibits the activity of the lymphocytes for 10 days, and the number of the radiation-inhibited bone marrow living cells also lasts for 10 days.

4.2 Example 2-1 therapeutic Effect of LLP2A-Ale on a mouse model of systemic radiation-induced lymphopenia.

1. Lymphocyte and whole blood leukocyte counts (see fig. 4, fig. 5).

(1) Non-irradiated group: the white blood cell and lymphocyte counts of the whole group of mice were maintained at baseline (normal) levels.

(2) Irradiation + vehicle group: at 6h after 3Gy disposable irradiation, the white blood cell count and the lymphocyte count of the mice in the whole blood of the mice are obviously reduced by over 80 percent (p is less than 0.001 ) compared with the baseline respectively, and the low-level state maintenance time of the white blood cell count and the lymphocyte count is 2 weeks; these cell counts were restored and maintained at normal levels for the remainder of the experiment until the end of the experiment.

(3) Irradiation + Rab001-US group: at 1h after 7d and 14d dosing, a significant increase in whole blood leukocytes and lymphocytes was observed with Rab001-US at the 0.4mg/kg dose level (14 d: p < 0.001), but at 6h after dosing (at 7d and 14 d) the leukocyte and lymphocyte counts fell back to the pre-dose levels compared to pre-dose. No significant difference in the total blood leukocyte and lymphocyte counts from those before dosing was observed at 6h after 21d dosing.

(4) Irradiation + Rab001-CN group: a significant increase in the leukocyte and lymphocyte counts was observed 1h after 14d administration compared to before administration (1.6 mg/kg group: p < 0.001), and the increase was correlated with the dose. Leukocytes and lymphocytes fell back to pre-dose levels 6h post-dose (at 7d and 14 d). Since blood was collected 1h after the 21d administration to evaluate the blood concentration, the efficacy could not be evaluated 1h after the 21d administration. No significant difference in total blood leukocyte and lymphocyte counts from those before dosing was observed as well at 6h after 21d dosing.

2. Bone marrow viable cells and total cell counts (see figure 6).

(1) The total cell number and the viable cell number were not significantly different in the non-irradiated group compared to the baseline control group, and the total cell number was not much changed but the viable cell number was significantly reduced by 29.43% in the irradiated + vehicle group compared to the non-irradiated group (P < 0.001).

(2) Irradiation + Rab001-US group: the total cell number was not significantly changed, but the number of viable cells increased by 11% compared to the irradiation + vehicle group.

(3) Irradiation + Rab001-CN group Total cell number had no effect but viable cell number increased by 13.37%, 10.20%, 22.82%, respectively, which increase was correlated with the dose administered. But the increase was still below the non-irradiated group level, showing that the test drug did not stimulate the bone marrow to produce new cells, but partially prevented and alleviated the damage of the bone marrow cells by irradiation.

1. Lymphocyte and whole blood leukocyte counts (see fig. 7, fig. 8).

(2) Irradiation + vehicle group: compared with the baseline, the white blood cell count and the lymphocyte count of the mice are respectively obviously reduced by more than 80 percent (p is less than 0.001 ) 1h after receiving 3Gy single-time irradiation, and the low-level state maintaining time of the white blood cell count and the lymphocyte count is 14d; these cell counts recovered and were maintained at normal levels for the remainder of the experiment until the end of the experiment.

(3) Irradiation + Rab001-US group: at a dose of 0.4mg/kg of LLP2A-Ale, mice were dosed immediately after irradiation, and the lymphocyte count and whole blood leukocyte count were measured after 1h, and the results were increased or maintained at baseline levels compared to those before 0h dosing. Whereas after administration at 7d and 14d, the positive drug resulted in a significant increase in whole blood leukocytes and lymphocytes (p <0.001, p-restricted 0.001) compared to pre-administration, but the leukocyte and lymphocyte counts fell back to pre-administration levels 24h post-administration.

(4) Irradiation + Rab001-CN group: administration immediately after irradiation, the 0.1mg/kg dose group prevented lymphocyte reduction and increased leukocyte numbers 1h after administration compared to baseline, and no effect was observed for the remaining two groups (0.4, 1.6mg/kg dose) at this time point. At the time points 7d and 14d of administration, the test drug group significantly increased the lymphocyte count and the whole blood leukocyte count (p <0.05, p < -0.05) at 1h after administration as compared to pre-administration (4 d, 11 d), and a correlation of drug effects with the administered dose was observed. But the leukocyte and lymphocyte counts fell back to pre-dose levels 24h after dosing.

2. Bone marrow viable cells and total cell counts (see figure 9).

(1) The non-irradiated group showed a smaller change in total bone marrow cell and viable cell numbers than the baseline group.

(2) Irradiation had little effect on total bone marrow cell number but significantly reduced 22.8% bone marrow viable cell number (p < 0.001) in the irradiated + vehicle group compared to the non-irradiated group.

(3) There was no significant difference in total and viable bone marrow cell counts compared to the irradiation + vehicle group, regardless of Rab001-US or Rab 001-CN.

And (4) conclusion:

example 1 mice receive a model of radiation-induced lymphopenia systemically.

The data established for the model of systemic irradiation-induced lymphopenia in mice indicate that systemic irradiation of mice 1 time results in total leukocyte and lymphopenia in the peripheral circulation and a decrease in the viable cell count in bone marrow, similar to the clinical situation of acute exposure to sub-lethal doses of systemic irradiation (the most severe form of lymphopenia).

Treatment experimental data show that 3Gy dose radiation irradiation once can cause obvious reduction of white blood cell, lymphocyte and bone marrow living cell counts in whole blood of mice; LLP2A-Ale (either Rab001-US or Rab 001-CN), intravenous injection of experimental drug can cause transient leucocyte, significant increase of lymphocyte count and partial prevention of bone marrow suppression induced by irradiation, the drug effects are related to the administration dose, and the results show that the experimental drug has no influence on the proliferation of the bone marrow cells, and are consistent with the previously observed LLP2A-Ale drug action mechanism, namely, the increase of the number of peripheral blood lymphocytes does not cause more lymphocytes to enter the peripheral blood circulation by stimulating the proliferation of the bone marrow cells, but influences the migration of the lymphocytes and the distribution of the lymphocytes in vivo.

The prevention experiment data shows that the 3Gy dose of radiation irradiation can cause obvious reduction of the counts of white blood cells, lymphocytes and bone marrow living cells in the whole blood of the mouse; LLP2A-Ale (either Rab001-US or Rab 001-CN) can cause transient white blood cell and lymphocyte counts to be obviously increased by intravenous injection of experimental drugs, and the drug effects are related to the administration dose. However, no significant effect was observed on radiation-induced myelosuppression, either Rab001-US or Rab 001-CN. Consistent with the previously observed mechanism of action of LLP2A-Ale drugs, increasing the number of peripheral blood lymphocytes does not result in more lymphocytes entering the peripheral blood circulation by stimulating the proliferation of bone marrow cells, but by affecting the migration of lymphocytes and their distribution in the body.

The above results indicate that LLP2A-Ale can be used for the prevention and treatment of lymphopenia caused by systemic irradiation.

Claims

1. Use of the compound LLP2A-Ale for the manufacture of a medicament for the treatment of radiation-induced peripheral blood lymphopenia; wherein the structure of the compound LLPA-Ale is as follows:

。