JP2002098695A - Inspection method and drug for occlusion of central vein of liver after hemopoietic stem cell implantation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inspection method which can by applied for the estimation and decision of the occlusion of a central vein of the liver of a patient and for observing the recuperative progress of the patient after hemopoietic stem cell implantation, and to also provide a drug used for the occlusion of the central vein after hemopoietic stem cell implantation. SOLUTION: In the inspection method, the concentrations of vascular endothelial cell growth factors/blood vessel permeability increasing factors (VEGF/VPF) contained in the human serum are measured. The drug is composed of a medicine containing antibodies to the VEGF/VPF.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to central hepatic vein occlusion (hereinafter, referred to as SCT) after hematopoietic stem cell transplantation (hereinafter, referred to as SCT).
VOD) and a therapeutic agent for VOD after SCT, and belongs to medical and pharmaceutical technologies.

[0002]

2. Description of the Related Art SCT is used for hematopoietic diseases such as leukemia and aplastic anemia, congenital diseases such as congenital immunodeficiency disease and congenital metabolic disorder, and in recent years, the largest amount of high-dose chemotherapy and radiation therapy for solid tumors The indication has been expanding as a rescue therapy for myelosuppression, which is a side effect of the disease. SCT first attacks the bone marrow cells of the pathological host thoroughly with a large amount of anticancer drugs or whole-body irradiation (referred to as transplant pretreatment).
This is done by infusing healthy hematopoietic cells from the nor (donor). Depending on the type of donor that provides hematopoietic cells, they can be broadly classified into autologous transplantation (using their own bone marrow or peripheral blood stem cells that have been cryopreserved in advance) and allogeneic transplantation (using other human hematopoietic cells). Allogeneic transplants are further classified into two types, related and unrelated, depending on the source. Furthermore, each is distinguished between bone marrow cells themselves, peripheral blood stem cells, and cord blood.

In SCT, after pretreatment and hematopoietic cell infusion, the infused hematopoietic cells survive without being rejected and recover healthy hematopoiesis, and especially in the case of malignant disease, there is no recurrence of the original disease. Is the goal. About 1 after hematopoietic cell infusion
Within months is the time when the appearance of complications is concentrated, and whether or not the transplantation itself will succeed before saying the recurrence of the underlying disease
It is important how to overcome the early complications after transplantation. The three major complications of SCT early after transplantation are infection, acute GVHD (graft-versus-host disease, in the case of allograft), and central hepatic venous occlusion (veno-occulusive disease, VO
D). Other complications include mucosal damage to the oral cavity and gastrointestinal tract, bleeding, anemia, TMA (thrombotic mi
There are organ disorders such as croangiopathy) and renal disorder.
One month after transplantation, chronic GV
Neurological complications, such as HD and leukoencephalopathy, and recurrences are important factors that determine prognosis and quality of life (QOL).

VOD is clinically (1) unexplained weight gain (> 2%) within 30 days after transplantation (mostly concentrated during the blood recovery phase (7 to 14 days after transplantation)) and early after transplantation. Alternatively, ascites, (2) right rib costal pain or hepatomegaly, (3) jaundice (TB ≧ 2 mg / dl), two or more of the three main features are recognized, and it is clinically possible to exclude other diseases. It is recognized as a diagnostic criterion. Pathologically, it is characterized by occlusion of the central hepatic vein, necrosis of hepatocytes in the central venous area, and congestion. The above three clinical signs are based on the diagnostic criteria of congestive symptoms associated with occlusion of the central hepatic vein, portal hypertension, and hepatocellular damage resulting therefrom. Risk factors for VOD after transplantation include infection during pretreatment, viral hepatitis, long history of chemotherapy before transplantation, liver injury cases, pretreatment mainly with busulfan, liver injury during pretreatment, allogeneic transplantation (Especially transplants from unrelated persons or HLA mismatched donors) are known. Coagulation and fibrinolysis markers that fluctuate in line with the clinical course of VOD include protein C (decreased), procollagen type III (increased), factor VII (decreased), thrombomodulin (increased), vWF (increased), AT
-III (decrease), PAI-1 (increase) and the like are known. S
After CT, these coagulation abnormalities always occur even if there is no clinical VOD, and it can be said that coagulation abnormalities due to vascular endothelial cell damage are likely to occur after SCT. In addition, inflammatory cytokines such as TNF-α, IL-6, and IL-8 are known as cytokines. Many patients have clinically severe mucosal damage, fever always precedes, and CRP becomes strongly positive,
It is believed that the infection was triggered.

[0005] Because of the movement of these risk factors and markers of endothelial cell damage, VOD has been considered to be a thrombotic disease that occurs based on vascular endothelial cell damage. However, it is not uncommon for these markers to be normal before transplantation, and it has not been possible to predict the onset of VOD before transplantation. Continuous administration of heparin, administration of prostaglandin E1, administration of urso, oral use of trenthal, and regular supplementation of AT-III have been performed as preventive measures, but the results are far from satisfactory. . In recent years, clinical trials of activated protein C preparations have also started. Treatment includes symptomatic treatments such as administration of diuretics, AT-III and FFP, as well as tissue plasminogen activator (t
There is a report that fibrinolytic therapy with PA) was effective. However, since tPA treatment is early after transplantation when the platelet count is low, there are many reports of serious side effects such as intracranial hemorrhage, and the response rate is low for dangerous side effects (about 30%). It is said that the current situation is that it is impossible to take tPA treatment.

Although the severity is classified into mild, moderate, and severe, most of the mild ones recover spontaneously,
The vere type has a fatality rate of 90% or more. It is difficult to predict how much the severity will progress when VOD develops, and when it becomes severe, it becomes refractory to treatment.
Although there is no effective treatment only for A treatment, considering the side effects of tPA treatment, the indication and timing are not often based on markers or the like, so it is often too late.

[0007]

SUMMARY OF THE INVENTION The object of the present invention is to provide an SC
An object of the present invention is to provide a test method which can be used for predicting the onset of VOD after T, confirming diagnosis, and monitoring the prognosis, and a therapeutic agent for VOD after SCT.

[0008]

Means for Solving the Problems The present inventors diligently studied whether or not the above problem could be solved by measuring the concentration of vascular endothelial cell growth factor / vascular permeability factor (VEGF / VPF). Thus, the present invention has been completed. That is, the present invention relates to a method for examining central hepatic vein occlusion after hematopoietic stem cell transplantation, which comprises measuring the concentration of vascular endothelial cell growth factor / vascular permeability factor (VEGF / VPF) in human serum. Vascular endothelial cell growth factor / vascular permeability factor (VE
It is a therapeutic agent for central hepatic vein occlusion after hematopoietic stem cell transplantation, which comprises a preparation containing an antibody to GF / VPF).

[0009]

DETAILED DESCRIPTION OF THE INVENTION Angiogenesis, the phenomenon of capillary endothelial cell proliferation, migration and tissue invasion, plays an important role in physiological or pathological phenomena such as fetal growth, wound healing and cancer cell proliferation. [Folkman, J. Cancer Res. 46: 467 (1986)]. Factors that induce angiogenesis include basic fibroblast growth factor (basic
fibroblast growth factor, bFGF, acidic fibroblast growth factor, aF
GF), vascular endothelial growth factor / vascular permeability factor (vascul
ar endothelial growth factor / vascular permeab
ility factor, VEGF / VPF), platelet-derived endothelial cell growth
factor, PD-ECGF), and as a substance that indirectly acts on vascular endothelial cells, transforming growth
factor-α (TGF-α), transforming growth fact
or-β (TGF-β), angiogenin, tumor necrosis fa
ctor-α (TNF-α) and the like are known [Falkman, J. & S
hing, Y. Chem., 267: 10931 (199).
2)].

Among the above-mentioned angiogenesis-inducing factors, vascular endothelial cell growth factor / vascular permeability factor (VEGF / VPF, hereinafter simply referred to as VEGF) includes normal or tumor cells of mouse, rat, guinea pig, bovine and human. It is secreted by strains and has been shown to be present in the brain, pituitary, kidney, and ovaries by tissue [Ferrara, N., et.a.
l. Endocrine Reviews 13:18 (1992)].
VEGF is also used for angiogenesis and metastasis of breast cancer [Weider, Ne
t.al. N. Engl. J. Med. 324: 1 (1991)] and angiogenesis of renal cell carcinoma [Progress of Medicine, 168: 231 (19)
94)] or angiogenesis in retinal disease [Adami
sAPet.al., Biochem.Biophys.Res.Comm., 193:
631 (1993)].

For the human VEGF gene, its cDNA
Has already been isolated, its nucleotide sequence has been determined, and its amino acid sequence has been deduced. Four types of proteins having different numbers of amino acid residues (121 amino acid residues, 165
, 189, and 206), of which 121 amino acid residues (VEGF12
1) and 165 amino acid residues (VEGF16
5) is said to be a mature protein [Ferrara N., e
t.al., Endocrine Reviews 13:18 (199
2)]. Note that VEGF121 lacks 44 amino acids at the carboxyl terminus of VEGF165, but it has not been clarified whether there is a difference between VEGF121 and VEGF165 in the action on vascular endothelial cells.

[0012] The present invention relates to VEG in human serum after SCT.
Measuring the F concentration;
VEG in human serum based on the relationship that VEGF levels in patients with VOD in patients are significantly higher compared to healthy and SCT patients but non-VOD patients
This is an inspection method of VOD after SCT by measuring F concentration. The method for measuring the concentration of VEGF in human serum is not particularly limited, but a method based on labeled immunoassay is preferred. It is also preferable to use a compound that specifically reacts with VEGF. Examples of such a compound include an antibody or receptor that binds to VEGF. As an antibody against VEGF, a monoclonal antibody, a polyclonal antibody, or a special antibody such as a humanized antibody can also be used, and these antibodies are appropriately used as a label. As the label, for example, erythrocytes, latex, radioactive elements, enzymes, luminescent substances, fluorescent substances, metal molecules, metal gels, bacteriophages, and the like can be used. On the other hand, instead of the antibody, VE
It is also possible to use known receptors for GF (Shibuya. M. et al, Oncogene 5: 519 (1990), Jaime. Ce).
tal, Science, 255: 989 (1992)).

The form of the therapeutic agent for VOD after SCT in the present invention is not particularly limited as long as it contains an antibody against VEGF, and is administered orally or parenterally.
When an injection solution or the like is used for parenteral administration, it is preferable to contain additives such as a stabilizer, a buffer, a preservative, and an isotonic agent, while tablets and capsules used for oral administration are preferable. Preferably contains a binder, an encapsulating agent, an excipient, a lubricant and the like generally used in those preparations. Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

[0014]

EXAMPLES (1) Preparation of anti-VEGF polyclonal antibody A fusion protein (GST-V) of isolated human VEGF cDNA with glutathione S-transferase (GST)
EGF was produced in Escherichia coli, and a rabbit anti-VEGF polyclonal antibody was prepared using the obtained protein as an antigen according to a conventional method. The serum of the rabbit with an increased antibody titer was separated, and an IgG fraction of a rabbit anti-VEGF polyclonal antibody was obtained by anion exchange column chromatography.

(2) Enzyme Labeling of Anti-VEGF Polyclonal Antibody A part of the IgG fraction is digested with pepsin to prepare F (ab ') 2, and then peroxidase (horseradish) by the hinge method.
And peroxidase-labeled rabbit anti-VEG
An F polyclonal antibody was obtained.

(3) Method of Measuring Serum VEGF Concentration Serum VEGF concentration was measured by an enzyme immunoassay as shown below. Anti-VEGF polyclonal antibody (5
(μg / ml) was spread on a 96-well plate at 100 μl / well, left at 4 ° C. overnight, and then washed four times with 0.1% bovine serum albumin (BSA) and PBS. 1% BS
A, blocking with 0.1 M sodium chloride, 0.1% sodium azide, 0.1 M sodium carbonate buffer pH 6.5 (4 hours at 37 ° C), 1% BSA, 0.4%
Gelatin, 1 mM magnesium chloride, 20 mM sodium ethylenediaminetetraacetate, 0.1 M sodium chloride,
Serum diluted with 3 in 50 mM sodium phosphate buffer pH 7.0 (sample dilution) containing 0.1% sodium azide or standard VEGF dissolved in the same sample dilution was added and left at room temperature for 1 hour. After washing 6 times with 0.1% BSA and PBS, 100 µl / well of peroxidase-labeled anti-VEGF polyclonal antibody was added and reacted at room temperature for 1 hour. After washing 8 times with 0.1% BSA and PBS again,
125% (w / v) orthophenylenediamine, 0.0
100% 15% hydrogen peroxide, 0.2M Tris (hydroxymethyl (aminomethane) -citrate buffer (pH 5.2)
Each μl / well was added and reacted at room temperature for 30 minutes.
After 2N sulfuric acid was added at 100 μl / well to stop the reaction, the absorbance at 490 nm relative to the absorbance at 650 nm was measured using a plate reader (M-Vmax, Molecular Devices).
Manufactured by the company).

(4) Serum VEGF concentrations in SCT patients with VOD and non-VOD patients were measured by the above-mentioned method in 20 healthy subjects and VOD in SCT patients.
Serum EGF of 6 patients with VOD and 40 patients without VOD
The concentration is shown in FIG. The mean and standard deviation in each case were 52.6 pg /
ml, 14.8 pg / ml, and 772.1 pg / ml and 1213.9 pg / m, respectively, in patients with VOD.
l, 12.7 pg / in each patient without VOD
ml, 42.6 pg / ml, and the VEGF concentration in the serum of the patient with VOD significantly (p <0.001) higher than that of a healthy person or a patient without VOD.

(5) Results of Measurement of Serum VEGF in Six Patients with VOD After SCT The clinical course and serum VEGF concentration of six VOD patients in SCT measured by the above method are shown in FIGS. VEG after SCT in all six SCT patients
The F concentration significantly increased. Further, after SCT, the VEGF concentration became high in accordance with the clinical course of VOD (ascites retention; ascites, jaundice; jaundice).

From the above results, the following results were shown by SCT. (1) VEGF concentration in serum can be used to predict the onset of VOD before transplantation. (2) VEGF in serum serves as a clinical marker for diagnosis and treatment of VOD after transplantation. (1) It becomes a therapeutic agent for VOD after transplantation by suppressing the amount of VEGF with an antibody or the like.

[0020]

According to the present invention, VEGF in human serum
By measuring the concentration, it is possible to predict central hepatic vein occlusion after hematopoietic stem cell transplantation, and the present invention is useful as a test method for determining a clinical diagnosis or a treatment method.

[Brief description of the drawings]

FIG. 1 shows VEGF concentrations in serum of healthy individuals and patients after hematopoietic stem cell transplantation.

FIG. 2 shows the clinical course and serum VEGF concentration of patients with central hepatic vein occlusion after hematopoietic stem cell transplantation.

FIG. 3 shows the clinical course and serum VEGF concentration of patients with central hepatic vein occlusion after hematopoietic stem cell transplantation.

FIG. 4 shows the clinical course and serum VEGF concentration of patients with central hepatic vein occlusion after hematopoietic stem cell transplantation.

FIG. 5 shows the clinical course and serum VEGF concentration of patients with central hepatic vein occlusion after hematopoietic stem cell transplantation.

FIG. 6 shows the clinical course and serum VEGF concentration of a patient with central hepatic vein occlusion after hematopoietic stem cell transplantation.

FIG. 7 shows the clinical course and serum VEGF concentration of patients with central hepatic vein occlusion after hematopoietic stem cell transplantation.

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Claims (5)

[Claims] 1. A method for examining central hepatic vein occlusion after hematopoietic stem cell transplantation, comprising measuring the concentration of vascular endothelial cell growth factor / vascular permeability factor (VEGF / VPF) in human serum. 2. The hepatic central vein after transplantation of hematopoietic stem cells according to claim 1, wherein the method for measuring the concentration of vascular endothelial cell growth factor / vascular permeability factor (VEGF / VPF) is a labeled immunoassay. How to test for obstruction. 3. A method for measuring the concentration of vascular endothelial cell growth factor / vascular permeability factor (VEGF / VPF), comprising the step of using a compound that specifically reacts with vascular endothelial cell growth factor / vascular permeability factor (VEGF / VPF). The method for examining central hepatic vein occlusion after hematopoietic stem cell transplantation according to claim 1 or 2, wherein the method is performed using the method. 4. A compound which specifically reacts with vascular endothelial cell growth factor / vascular permeability factor (VEGF / VPF), wherein the antibody or receptor binds to vascular endothelial cell growth factor / vascular permeability factor (VEGF / VPF). The method for testing central hepatic vein occlusion after hematopoietic stem cell transplantation according to claim 3, which is a body. 5. A therapeutic agent for central hepatic vein occlusion after hematopoietic stem cell transplantation, comprising a preparation containing an antibody against vascular endothelial cell growth factor / vascular permeability factor (VEGF / VPF).