WO2020151666A1 - 血管瘤治疗的生物标记物 - Google Patents

血管瘤治疗的生物标记物 Download PDF

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WO2020151666A1
WO2020151666A1 PCT/CN2020/073250 CN2020073250W WO2020151666A1 WO 2020151666 A1 WO2020151666 A1 WO 2020151666A1 CN 2020073250 W CN2020073250 W CN 2020073250W WO 2020151666 A1 WO2020151666 A1 WO 2020151666A1
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pdgf
treatment
hemangioma
expression level
biological factor
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冉玉平
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四川大学华西医院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • G—PHYSICS
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

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  • the present invention relates to a biomarker for the treatment of hemangioma, in particular to a method and a kit for effectively evaluating and predicting the curative effect of itraconazole in the treatment of hemangioma using biological factors in the blood as a marker, and belongs to the field of biomedicine.
  • Hemangiomas are the most common benign skin tumors in infants and young children, with an incidence of 8%-10%. It usually occurs on the head, face and limbs, and can affect any part of the body. It proliferates rapidly within 1 year of age, and then gradually enters a period of self-remission, which lasts as long as 5-9 years. Although most hemangiomas have a pathophysiological process that resolves spontaneously, they often leave behind pigmentation, telangiectasia, fiber and fatty tissue deposition.
  • Non-invasive treatment includes oral and topical drugs.
  • Oral drugs mainly include glucocorticoids, interferon alpha, vincristine, propranolol, itraconazole, etc.
  • topical drugs mainly include timolol, carteolol, etc. ⁇ Blockers.
  • non-invasive treatment reduces the chances of secondary infections and scars, simple operation, relatively cheap price, and high compliance for children and their families.
  • problems such as long treatment time and side effects of the drug itself.
  • the applicant discovered during the clinical consultation process that most of the family members of children are more willing to choose oral or topical drugs for non-invasive treatment of hemangioma from the beginning.
  • oral or topical drugs are difficult to operate, and oral drugs can only be chosen.
  • the present invention provides a method for evaluating, predicting or optimizing the effectiveness of itraconazole in the treatment of hemangioma, including the steps:
  • the test sample is normal tissue, tumor tissue, cell line, plasma, serum, whole blood, cerebrospinal fluid, lymph fluid, circulating tumor cells, cell lysate, tissue lysate, urine and/or aspirate, preferably , Is serum or tumor tissue;
  • the biological factor is one or more of VEGF-A, PDGF-A, PDGF-D, HES, HEY1, HIF3A, IL-8, TNF-A or FGF.
  • the expression level of the biological factor is the mRNA expression level of the corresponding biological factor in the test sample.
  • the mRNA expression level of one or more of PDGF-A, VEGF-A, HES, HEY1, IL-8, or TNF-A after treatment is up-regulated by a factor greater than or equal to 2, 3, 5, 8, 10, or 20, and more preferably, when the multiple of up-regulation is greater than or equal to 10 or 20, the therapeutic effect is achieved; in other preferred cases, when one of PDGF-D, HIF3A or FGF is treated
  • the biological factor is one or more of PDGF-A, PDGF-D or FGF; more preferably, the biological factor is PDGF-D.
  • the expression level of the biological factor is the protein level of the corresponding biological factor in the test sample.
  • the ratio is greater than or equal to 10%, 30%, 50%. % Or 70%, preferably, when the reduction ratio is greater than or equal to 50% or 70%, the therapeutic effect is achieved.
  • the biological factor is PDGF-D or PDGF-A.
  • the hemangioma patient is an infant.
  • the hemangioma is a proliferative hemangioma.
  • the present invention also provides a detection kit comprising a reagent for measuring the mRNA expression level or protein level of a biological factor in a test sample of a subject, characterized in that the test sample is normal tissue, tumor tissue, cell line , Plasma, serum, whole blood, cerebrospinal fluid, lymphatic fluid, circulating tumor cells, cell lysates, tissue lysates, urine and/or aspirates, preferably serum or tumor tissue, and the biological factor is VEGF -One or more of A, PDGF-A, PDGF-D, HES, HEY1, HIF3A, IL-8, TNF-A or FGF.
  • the detection kit further includes instructions for the purpose selected from the group consisting of: determining whether a patient with hemangioma will respond to itraconazole treatment by the expression level of the biological factor or its change.
  • the expression level or change of the biological factor monitors the disease progression of patients with hemangioma treated with itraconazole, and the biological activity of itraconazole in the patient administered itraconazole is confirmed by the expression level or change of the biological factor, or Its combination.
  • the present invention also provides the use of the detection kit for evaluating, predicting or optimizing the effectiveness of itraconazole in the treatment of patients with hemangioma.
  • the expression level of the biological factor is the mRNA expression level of the corresponding biological factor; preferably, one of PDGF-A, VEGF-A, HES, HEY1, IL-8 or TNF-A after treatment
  • One or more kinds of mRNA expression levels are up-regulated when the difference multiple is greater than or equal to 2, 3, 5, 8, 10 or 20 than before treatment, and more preferably, when the up-regulated multiple difference is greater than or equal to 10 or 20, the therapeutic effect is achieved; preferably Specifically, when the mRNA expression level of one or more of PDGF-D, HIF3A or FGF after treatment is down-regulated by a factor of greater than or equal to 2, 3, 5, 8, 10 or 20, it is more optimally down-regulated When the difference multiple is greater than or equal to 10 or 20, the therapeutic effect is achieved; more preferably, the biological factor is one or more of PDGF-D, HIF3A or FGF; more preferably, the biological factor is PDGF-D .
  • the expression level of the biological factor is the protein level of the corresponding biological factor; preferably, when the protein level of the corresponding biological factor after treatment decreases by greater than or equal to 10%, 30%, 50% Or 70%, the therapeutic effect is achieved; more preferably, the biological factor is PDGF-A or PDGF-D.
  • the hemangioma is a proliferative hemangioma.
  • the hemangioma patient is an infant.
  • the beneficial effects of the present invention are: currently there is no public report about the use of biological factors to effectively evaluate and predict the therapeutic effect of infantile hemangioma.
  • the biomarkers of the present invention are of great significance for determining the curative effect of infantile hemangioma, evaluating the prognosis, and guiding the course of medication.
  • Figure 1 The trend of changes in serum VEGF and PDGF-AA levels in infants and young children before and after treatment.
  • Figure 2 The mRNA expression profile of infant hemangioma treated with itraconazole and the top 20 related biological processes, cell components, molecular functions and pathways with significant changes.
  • HemEC infantile hemangioma
  • DMSO control Red dots indicate up-regulated mRNA
  • green dots indicate down-regulated mRNA (fold change>2.0).
  • Figure 3 PI3K/Akt/mTOR signaling in infantile hemangioma is inhibited by itraconazole in vitro.
  • (a) Detection of PDGF-D mRNA expression in HemEC treated with 10 ⁇ M itraconazole for 48 hours by reverse transcription PCR.
  • (b) The expression of PDGF-D protein in HemEC treated with 10 ⁇ M itraconazole for a specified time, detected by immunoblotting.
  • (c) Check the phosphorylation levels of p-Akt, p-p70S6K, p-4E-BP1 and T-Akt in HemEC treated with 10 ⁇ M itraconazole for 2 hours, 24 hours and 48 hours, respectively.
  • FIG. 4 Comparison of the effects of itraconazole and PDGFR- ⁇ inhibitor CP-673451 on HemECs of infantile hemangioma.
  • (c) The effect of 10 ⁇ M itraconazole and 10 ⁇ M CP-673451 on HemEC was determined by tube format. Scale bar 0.1mm.
  • the methods and techniques of the present invention are usually performed according to traditional methods known in the art, unless otherwise specified.
  • the nomenclature, experimental methods and techniques related to biology, pharmacology, and medicine and medicinal chemistry described herein are known and commonly used in the art.
  • the chemical synthesis method, chemical analysis method, pharmaceutical preparation method, blending method and transmission method, and detection or testing method all adopt standard technology.
  • the term "effective dose”, also commonly referred to as “therapeutically effective dose”, refers to any amount of a drug as described below, which when used alone or in combination with another therapeutic agent, can promote the regression of the disease.
  • the regression of the disease is manifested as a decrease in the severity of disease symptoms, an increase in the frequency and duration of the asymptomatic period, or prevention of disorder or disability caused by the disease.
  • the "effective dose” of the drug of the present invention also includes the “prophylactic effective dose”.
  • the “prophylactic effective dose” is any amount of the drug as described below, when the amount of the drug is administered alone or in combination with another therapeutic agent. It can inhibit the occurrence or recurrence of the disease when the patient is at risk of disease or suffers from the recurrence of the disease.
  • the effective in vivo dosage and specific administration method will vary according to the type, weight and age of the mammal being treated, the specific compound used and the specific purpose of using these compounds.
  • Those skilled in the art can determine the effective dosage level (ie, the dosage level necessary to achieve the desired effect) according to conventional pharmacological methods.
  • the human clinical application of the product starts from a lower dosage level, and then the dosage level is continuously increased until the desired effect is achieved.
  • acceptable in vitro studies can be used by existing pharmacological methods to establish useful doses and routes of administration of the compositions identified by this method.
  • the "drug” is any chemical substance recognized in the art as a biological, physiological or pharmacologically active substance.
  • Drugs are also called “therapeutics", examples of which are described in known references (such as Merck Index, Physicians Desk Reference, and The Pharmacological Basis of therapeutics), and they include (but are not limited to) drugs, vitamins, and minerals Supplements, substances used to treat, prevent, diagnose, cure or alleviate diseases or ailments, substances or prodrugs that affect the structure or function of the body, which have biological activity or stronger activity when placed in a physiological environment.
  • Various forms of therapeutic drugs can be used, wherein when administered to a subject, the composition can be released from the subject into adjacent tissues or fluids.
  • the drug in the present invention refers to itraconazole, including various pharmaceutical preparations or pharmaceutical compositions using itraconazole as an active ingredient.
  • hemangioma refers to congenital benign tumors or vascular malformations commonly found in the skin and soft tissues formed by the proliferation of hemangioblasts during the embryonic period. It is more common at birth or shortly after birth. The remaining embryonic hemangioblasts and active endothelioid germs invade adjacent tissues to form endothelioid cords, which are connected to the remaining blood vessels to form hemangioma after tubeization. The blood vessels in the tumor form a system of its own and are not connected to the surrounding blood vessels. Hemangiomas can occur throughout the body, most of which occur in facial skin, subcutaneous tissues and oral mucosa, such as tongue, lips, floor of mouth and other tissues, and a few occur in jaw bones or deep tissues.
  • vascular endothelial cells of hemangioma have proliferative properties, and the natural course of the disease can be divided into a proliferative phase, a stable phase and a regression phase.
  • hemangioma can be divided into capillary hemangioma, cavernous hemangioma, mixed hemangioma and cranial hemangioma.
  • PDGF platelet-derived growth factor
  • PDGF-A platelet-derived growth factor
  • PDGF-B PDGF-C
  • PDGF-D PDGF protein encoded by four genes.
  • PDGF is produced by a discrete cell population and is secreted in the form of disulfide-bonded homodimers or heterodimers, including PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD and PDGF-AB.
  • PDGF dimers act mainly in a paracrine manner by binding to PDGF receptors.
  • PDGFR- ⁇ and PDGFR- ⁇ which can simultaneously form heterodimers and homodimers.
  • Ligand binding promotes receptor dimerization, autophosphorylation and activation of multiple downstream intracellular signal cascades, thereby stimulating actin filament rearrangement, disruption of gap junction communication, initiation of gene transcription and cell survival.
  • test sample may be any biological material isolated from the body of a patient or subject, such as normal tissue, tumor tissue, cell line, plasma, serum, whole blood, cerebrospinal fluid, lymph fluid, circulating tumor cells, cells Lysates, tissue lysates, urine and aspirates; preferably, the test sample is derived from normal tissue, tumor tissue, cell lines, circulating tumor cells, serum, plasma or whole blood; more preferably, the test sample is derived from serum, Tumor tissue.
  • the method for removing the sample is well known in the art, and it can be removed from the subject, for example, by biopsy, for example, by needle biopsy, core needle aspiration biopsy or fine needle aspiration biopsy, endoscopic biopsy or surface Biopsy; or through venipuncture, whole blood, plasma or serum samples are collected and further processed according to standard techniques.
  • the sample is subjected to method steps covering the measurement of the expression level of biological factors.
  • the expression level of biological factors mainly refers to the expression level of mRNA and the level of protein.
  • Example 1 Observation of the clinical efficacy of itraconazole in the treatment of infantile hemangioma and the effect of itraconazole on the serum VEGF and PDGF-AA levels in children with hemangioma
  • a total of 17 children were enrolled in this study, including 6 males and 11 females, aged 1-12 months (average 5.4 months), weighing 5-10.5 kg, 17 samples were collected before treatment, 17 samples after treatment, and the interval before and after treatment The time is 13-56 days (average 31 days), and the total dose of itraconazole orally is 390-2240 mg (average 1076 mg).
  • the total follow-up time ranges from 0.5 to 6 months.
  • 12 were proliferative infantile hemangioma
  • 7 cases of proliferative hemangioma showed a trend of regression during the follow-up period, and the regression rate was 58.3% (7/12).
  • Serum VEGF vascular endothelial cell growth factor
  • the serum VEGF concentration of the children before treatment was 214.82 ⁇ 135.58pg/mL, after treatment it was 390.81 ⁇ 649.18pg/mL, after treatment it increased by 175.98 ⁇ 551.17pg/mL compared with before treatment (of which VEGF increased in 8 patients, 9 patients The patient declined), and the T test analysis showed that the difference between the two groups was not statistically significant (p>0.05).
  • 4 had an increase in VEGF level and 3 had a decrease.
  • Serum PDGF-AA platelet-derived growth factor
  • the serum PDGF-AA concentration of the children before treatment was 52879.92 ⁇ 31435.65 pg/mL, after treatment it was 26289.34 ⁇ 26095.77 pg/mL, after treatment, it decreased by 26590.57 ⁇ 35627.71 pg/mL (2 patients increased, 15 decreased) , After T test analysis, the difference between the two groups is statistically significant (p ⁇ 0.05).
  • the PDGF-AA level increased in 1 (14.3%) and decreased in 6 (85.7%) after treatment.
  • Itraconazole, ketoconazole and propranolol were purchased from Sigma-Aldrich.
  • CP-67345 was purchased from MedChem Express.
  • EOMA was obtained from the Vascular Biology Project of Dr. Catherine Butterfield, Department of Surgery, Children's Hospital, Harvard Medical School (Boston, USA). Cultured in RPMI 1640 (GIBCO) containing 10% fetal bovine serum and penicillin/streptomycin/glutamine. After obtaining written informed consent for the use of infant hemangioma specimens from parents, we collected the hyperplastic tissue of infant hemangioma. They have undergone surgical treatment of hemangioma.
  • the tissue blocking method of hyperplastic IH tissue successfully cultured hemangioma endothelial cells (HemEC) in vitro. It was then cultured in RPMI 1640 (GIBCO) containing 10% fetal bovine serum, 10 ng/ml basic fibroblast growth factor and penicillin/streptomycin/glutamine. All cells were cultured in an incubator containing 5% CO 2 at 37°C.
  • the cells were seeded in a 96-well plate, and treatment with itraconazole or CP-673451 was started the next day. After 72 hours of incubation, MTS analysis (Promega, Madison, WD) was performed according to the manufacturer's instructions, and GraphPad Prism software (version 6) was used to fit a normalized cell viability curve.
  • the cells were treated with itraconazole or CP-673451 for 48 hours. Using a Canto flow cytometer (Becton Dickinson, Franklin Lakes, NJ), according to the manufacturer's instructions, apoptotic cells were analyzed by Annexin V/PI staining (Annexin V FITC and PI kit, Thermo Scientific). In addition, the nuclei of EOMA and HemEC were stained with Hoechst33258 at a concentration of 5 ⁇ g/ml at 37°C for 5 minutes. The cells were washed to remove unbound dye, and then cell viability was observed by fluorescence microscope (Olympus).
  • HemECs were seeded in 96-well plates on the surface of Matrigel at a density of 1.5 ⁇ 10 cells/well.
  • the cells were treated with 0.1% DMSO, itraconazole, propanol or CP-673451. After 3 hours of treatment, 6 randomly selected microscope fields (magnification 200 times) were taken. The length of the manifold and the density of the capillary network were evaluated by Axiovision software (The Math Works, Inc., Natick, Massachusetts).
  • the HemEC of 2 infants was treated with 10 ⁇ M itraconazole or 0.1% DMSO for 48 hours. According to the manufacturer's instructions, total mRNA was extracted for mRNA array analysis of Agilent human (V2) gene expression microarray (8 ⁇ 60K chip).
  • the primer sequence is as follows: PDGF-D, 5'-CCCAGGAATTACTCGGTCAA-3'(F) (SEQ ID NO.: 1) and 5'-ACAGCCACAATTTCCTCCAC-3'(R) (SEQ ID NO.: 2); ACTB, 5' -GGACTTCGAGCAAGAGATGG-3'(F) (SEQ ID NO.: 3) and 5'-AGCACTGTGTTGGCGTACAG-3'(R) (SEQ ID NO.: 4).
  • p-p70S6K product number 9272
  • p-4E-BP1-S65(9451) PDGFR (3169), p-PDGFR-B (3161) and B-actin (4970) were purchased from Cell Signaling Technologies;
  • PDGF-D was purchased from R&D Systems (product number AF1159).
  • the two groups were compared by Student's t-test, and multiple groups were compared by unmatched one-way analysis of variance with Tukey's correction.
  • the average value of each column in GraphPad Prism was compared with the average value of each other column (version 6: La Jolla. CA).
  • the top 20 significantly enhanced biological process gene ontology terms (Figure 2c) and the top 20 significantly enhanced cell component gene ontology terms ( Figure 2d) were determined respectively.
  • a positive number means an upward adjustment
  • a negative number means a downward adjustment
  • the difference multiple is expressed by the absolute value of the value in the table.
  • Itraconazole significantly reduces the level of PDGF-D, thereby inhibiting the activation of PDGFR- ⁇ and inhibiting its downstream effectors.

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Abstract

一种血管瘤治疗的生物因子，以及通过该生物因子的表达水平对伊曲康唑治疗婴幼儿血管瘤疗效进行有效评估和预测的方法和试剂盒，该生物因子为VEGF-A、PDGF-A、PDGF-D、HES、HEY1、HIF3A、IL-8、TNF-A或FGF中的一种或几种。该方法和试剂盒对婴幼儿血管瘤疗效判定、预后评估和用药疗程指导有重要意义。

Description

血管瘤治疗的生物标记物 技术领域
本发明涉及血管瘤治疗的生物标记物，具体地，涉及使用血液中生物因子作为标记物对伊曲康唑治疗血管瘤疗效进行有效评估和预测的方法和试剂盒，属于生物医药领域。
背景技术
血管瘤是婴幼儿最常见的皮肤良性肿瘤，其发生率为8％-10％。好发于头面部和四肢，可累及身体任何部位，在1岁以内快速增殖，随后逐渐进入自行消退期，持续时间长达5-9年。尽管多数血管瘤有自行消退的病理生理过程，但常遗留色素沉着、毛细血管扩张、纤维和脂肪组织沉积。10％-20％患儿瘤体随年龄增长而增大，甚至出现如溃疡、出血、感染等严重并发症，导致容貌损毁、功能丧失等后果：面部中央、气道、皮肤皱褶、会阴和肛周等容易摩擦或受汗液、尿液、粪便浸渍区域一旦溃疡形成后很难自愈；位于眼睑、结膜的血管瘤可影响视力导致视弱、散光及致盲；位于呼吸道则会导致呼吸障碍，影响心肺功能；部分血管瘤可导致Kasabach-Merritt综合征(卡萨巴赫-梅里特综合征)、充血性心力衰竭等而危及生命；皮肤血管瘤严重影响形象，导致患儿出现自卑、内向、自闭、极端性格等一系列心理障碍。
治疗血管瘤的有创治疗技术涉及到包括手术切除、染料脉冲激光、介入、冷冻、电灼等，因对手术医师临床技能、手术条件、仪器设备、患儿及家属的依从性、费用和方便程度要求不一，且容易继发感染、遗留瘢痕，具有一定副作用或局限性。无创治疗包括口服和外用药物，口服药物主要有糖皮质激素、α干扰素、长春新碱、普萘洛尔、伊曲康唑等，外用药物主要有噻吗洛尔、卡替洛尔等β受体阻滞剂。无创治疗优点在于减少了继发感染、遗留瘢痕的几率，操作简单，价格相对便宜，患儿及家属依从性高。但存在治疗时间长，药物本身副作用等等问题。申请人在临床接诊过程中发现，多数患儿家属更愿意从一开始先选择口服或者外用药物这些无创方法治疗血管瘤。一些特殊部位例如眼睑、鼻腔内、外阴、肛周部位的血管瘤，手术或外用药物均操作困难，只能选择口服药物。
长期使用口服药物所产生的副作用，例如对肝肾功能、心功能的影响是家属主要担心的问题。若顾及药物副作用，过早停药，可能导致皮损复发和反弹。因此，找到一些与血管瘤消退相关的生物标记因子，根据生物标记来评估治疗有效或无效，何时停药，是医生、家属都迫切关注的问题，目前尚无此类生物标记报道。
发明内容
本发明提供了一种评估、预测或优化伊曲康唑对血管瘤治疗有效性的方法，包括步骤：
a)获取来自血管瘤病人的测试样品，检测所述测试样品中生物因子的表达水平；
b)所述血管瘤病人接受有效剂量的伊曲康唑的治疗；
c)比较治疗前后，所述生物因子的表达水平的变化，当所述生物因子的表达水平上调或者下调时，达到治疗效果。
所述测试样品为正常组织、肿瘤组织、细胞系、血浆、血清、全血、脑脊髓液、淋巴液、循环肿瘤细胞、细胞裂解物、组织裂解物、尿和/或抽吸物，优选地，为血清或肿瘤组织；所述生物因子为VEGF-A、PDGF-A、PDGF-D、HES、HEY1、HIF3A、IL-8、TNF-A或FGF中的一种或几种。
在一些实施例中，所述生物因子的表达水平为相应生物因子在所述测试样品中的mRNA表达水平。
在一些优选例中，当治疗后PDGF-A、VEGF-A、HES、HEY1、IL-8或TNF-A中的一种或多种的mRNA表达水平较治疗前上调差异倍数大于或等于2、3、5、8、10或20时，更优地，上调差异倍数大于或等于10或20时，达到治疗效果；在另一些优选例中，当治疗后PDGF-D、HIF3A或FGF中的一种或多种的mRNA表达水平较治疗前下调差异倍数大于或等于2、3、5、8、10或20时，较优地，下调差异倍数大于或等于10或20时，达到治疗效果；较优地，所述生物因子为PDGF-A、PDGF-D或FGF中的一种或几种；更优地，所述生物因子为PDGF-D。
在另外一些实施例中，所述生物因子的表达水平为相应生物因子在测试样品中的蛋白水平，优选地，当治疗前后相应生物因子的蛋白水平下降比例大于或等于10％、30％、50％或70％时，较优地，下降比例大于或等于50％或70％时，达到治疗效果。更优地，所述生物因子为PDGF-D或PDGF-A。
在一些实施例中，所述血管瘤病人为婴幼儿。
在一些实施例中，所述血管瘤为增殖期血管瘤。
本发明还提供了一种检测试剂盒，包含用于测量受试者测试样品中生物因子的mRNA表达水平或蛋白水平的试剂，其特征在于，所述测试样品为正常组织、肿瘤组织、细胞系、血浆、血清、全血、脑脊髓液、淋巴液、循环肿瘤细胞、细胞裂解物、组织裂解物、尿和/或抽吸物，优选地，为血清或肿瘤组织，所述生物因子为VEGF-A、 PDGF-A、PDGF-D、HES、HEY1、HIF3A、IL-8、TNF-A或FGF中的一种或几种。
在一些实施例中，所述检测试剂盒还包含用于选自下述的目的的说明书：通过所述生物因子表达水平或其变化测定血管瘤病人是否将响应伊曲康唑治疗，通过所述生物因子表达水平或其变化监控伊曲康唑治疗的血管瘤病人的疾病进展，通过所述生物因子表达水平或其变化证实在施用伊曲康唑的病人中伊曲康唑的生物活性，或其组合。
本发明还提供了所述检测试剂盒用于评估、预测或优化伊曲康唑对血管瘤病人治疗有效性的用途。
在一些实施例中，所述生物因子的表达水平为相应生物因子的mRNA表达水平；优选地，当治疗后PDGF-A、VEGF-A、HES、HEY1、IL-8或TNF-A中的一种或多种的mRNA表达水平较治疗前上调差异倍数大于或等于2、3、5、8、10或20时，更优地，上调差异倍数大于或等于10或20时，达到治疗效果；优选地，当治疗后PDGF-D、HIF3A或FGF中的一种或多种的mRNA表达水平较治疗前下调差异倍数大于或等于2、3、5、8、10或20时，更优地，下调差异倍数大于或等于10或20时，达到治疗效果；更优地，所述生物因子为PDGF-D、HIF3A或FGF中的一种或几种；更优地，所述生物因子为PDGF-D。
在另一些实施例中，所述生物因子的表达水平为相应生物因子的蛋白水平；优选地，当治疗后相应生物因子的蛋白水平较治疗前下降比例大于或等于10％、30％、50％或70％时，达到治疗效果；更优地，所述生物因子为PDGF-A或PDGF-D。
在一些实施例中，所述血管瘤为增殖期血管瘤。
在一些优选实施例中，所述血管瘤病人为婴幼儿。
本发明的有益效果是：目前尚无任何公开报道使用生物因子对婴幼儿血管瘤治疗疗效进行有效评估和预测的生物标记。而本发明所述的生物标记对婴幼儿血管瘤疗效判定、预后评估和用药疗程指导有重要意义。
附图说明：
图1治疗前后婴幼儿血清中VEGF与PDGF-AA水平变化趋势。
图2用伊曲康唑处理的婴儿血管瘤的mRNA表达谱及显著变化的前20个相关生物过程、细胞组分、分子功能及途径。(a)用伊曲康唑(10μM)和DMSO对照处理的来自婴儿血管瘤内皮细胞(HemEC)之间的mRNA表达水平的散点图。红点表示上调的mRNA，绿点表示下调的mRNA(倍数变化>2.0)。(b)分层聚类显示伊曲康唑 和对照之间可区分的mRNA表达模式。在HemEC中，在伊曲康唑处理后，总共172个mRNA被上调(倍数变化>2.0)，819个mRNA被下调(倍数变化>2.0)。与伊曲康唑治疗的HemEC中差异表达显著的mRNA对应的，前20个生物过程的基因本体论术语(c)，前20个细胞组分的基因本体论术语(d)，前20个分子功能的基因本体论术语(e)，和前20个途径术语(f)。
图3婴儿血管瘤中的PI3K/Akt/mTOR信号传导在体外被伊曲康唑抑制。(a)通过逆转录PCR检测，用10μM伊曲康唑处理48小时的HemEC中PDGF-D mRNA的表达。(b)通过免疫印迹检测得到的，用10μM伊曲康唑处理指定时间的HemEC中PDGF-D蛋白的表达。(c)在用10μM伊曲康唑分别处理2小时，24小时和48小时的HemEC中检查p-Akt，p-p70S6K，p-4E-BP1和T-Akt的磷酸化水平。(d)用0.3μM或1μM伊曲康唑处理2小时和24小时的小鼠血管瘤内皮细胞(EOMA)中的p-Akt，p-p70S6K，p-4E-BP1和β-肌动蛋白水平。
图4伊曲康唑和PDGFR-β抑制剂CP-673451对婴儿血管瘤HemECs的影响的比较。(a)HemECs用指定浓度的伊曲康唑或CP-673451处理72小时，然后通过MTS测定法测定细胞活力。(b)用3μM伊曲康唑和5μM CP-673451处理48小时后，对HemEC进行Annexin V/PI染色。(c)通过管形式测定，10μM伊曲康唑和10μM CP-673451对HemEC的作用。比例尺＝0.1mm。(d)通过蛋白质印迹检测得到的，用10μM伊曲康唑或10μM CP-673451处理48小时的HemEC中p-PDGFR-β，p-Akt，p-p70S6K，p-4E-BP1，PDGFR-β和T-Akt的磷酸化水平。
具体实施方式：
本发明的方法与技术通常依据本领域已知的传统方法进行，除非另有说明。与本文中描述的生物学、药理学、及医学与医药化学相关的命名法，及实验方法与技术是本领域已知且常用的。化学合成法、化学分析法、医药制法、调配法与传送法，及检测或测试法均采用标准技术。
除非另有说明，否则本文中所使用的科学与技术术语应具有那些本领域普通技术人员通常理解的含义。但下列术语具有如下定义：
术语“有效剂量”，通常也称为“治疗有效剂量”，是指药物的任何如下所述的量，当单独使用或与另一种治疗剂组合使用该量的药物时，可促进疾病消退，疾病消退表现为疾病症状的严重度降低、无疾病症状期的频率和持续时间增加、或者防止由患病导致的障碍或失能。本发明药物的“有效剂量”也包括“预防有效剂量”，“预防有效剂 量”是药物的任何如下所述的量，当将该量的药物单独施用或者与另一种治疗剂组合施用于具有发生疾病的风险或者遭受疾病复发的病人时，可抑制疾病的发生或复发。
如对本领域所属技术人员显而易见地，有效的体内给药剂量及具体的给药方式会根据所治疗的哺乳动物种类、体重和年龄，所使用的具体化合物及使用这些化合物的具体目的而变化。本领域所属技术人员根据常规的药理学方法可确定有效剂量水平(即达到所需效果所必需的剂量水平)。通常，产物的人体临床应用从较低的剂量水平开始，随后不断提高剂量水平直到达到所需的效果。可选择地，可通过现有的药理学方法采用可接受的体外研究来建立本方法鉴定的组合物的有用剂量和给药途径。
所述“药物”，是本领域认可的，作为生物、生理或药理活性物质的任何化学物质。药物也被称为“治疗剂”，其实例在已知参考文献中有描述(如Merck Index、Physicians Desk Reference和The Pharmacological Basis of therapeutics)，并且其包括(但不限于)药物、维生素、矿物质补充剂、用于治疗、预防、诊断、治愈或缓解疾病或病痛的物质、影响身体结构或功能的物质或前药，当将其置于生理环境后具有生物活性或活性更强。可使用各种形式的治疗药物，其中当给予受试者之后，组合物就能够从受试者中释放至邻近组织或流体中。本发明中的药物指伊曲康唑，包括以伊曲康唑为有效成分的各种药物制剂或药物组合物。
术语“血管瘤(hemangioma)”是指由胚胎期间成血管细胞增生而形成的常见于皮肤和软组织内的先天性良性肿瘤或血管畸形，多见于婴儿出生时或出生后不久。残余的胚胎成血管细胞，活跃的内皮样胚芽向邻近组织侵入，形成内皮样条索，经管化后与遗留下的血管相连而形成血管瘤，瘤内血管自成系统，不与周围血管相连。血管瘤可发生于全身各处，其中大多数发生于颜面皮肤、皮下组织及口腔黏膜、如舌、唇、口底等组织，少数发生于颌骨内或深部组织。血管瘤的血管内皮细胞具有增殖特性，自然病程可分为增生期、稳定期和消退期。根据组织学结构与临床表现，血管瘤又可分为毛细血管瘤、海绵状血管瘤、混合型血管瘤和蔓状血管瘤。
本发明所述生物因子的代码及其中英文名称对照见表1。
表1 生物因子代码及其中英文名称对照表
Figure PCTCN2020073250-appb-000001
Figure PCTCN2020073250-appb-000002
术语“PDGF”，即血小板衍生生长因子，是一种生长因子，可以调控细胞的生长和分化，且在血管新生上扮演重要角色，未控制的血管新生常常导致癌症。存在由四个基因编码的四个已知的PDGF蛋白，PDGF-A，PDGF-B，PDGF-C和PDGF-D。PDGF由离散的细胞群体产生，并以二硫键连接的同二聚体或异二聚体形式分泌，包括PDGF-AA，PDGF-BB，PDGF-CC，PDGF-DD和PDGF-AB。PDGF二聚体通过结合PDGF受体主要以旁分泌的方式起作用。有两种已知的PDGF受体，PDGFR-α和PDGFR-β，它们可以同时形成异二聚体和同二聚体。配体结合促进受体二聚化，自磷酸化和激活下游的多个细胞内信号级联反应，从而刺激肌动蛋白丝重排，间隙连接通讯的破坏，基因转录的起始和细胞存活。
所述“测试样品”可以是从病人或受试者身体分离的任何生物材料，例如正常组织、肿瘤组织、细胞系、血浆、血清、全血、脑脊髓液、淋巴液、循环肿瘤细胞、细胞裂解物、组织裂解物、尿和抽吸物；优选地，测试样品源自正常组织、肿瘤组织、细胞系、循环肿瘤细胞、血清、血浆或全血；更优选地，测试样品源自血清、肿瘤组织。用于取出样品的方法是本领域众所周知的，并且它可以例如通过活组织检查从受试者中取出，例如通过穿刺活检、空芯针穿刺活检或细针抽吸活检、内窥镜活检或表面活检；或者通过静脉穿刺，收集全血、血浆或血清样品，并且根据标准技术进一步加工。从病人或受试者中获得测试样品后，对样品实施涵盖测量生物因子表达水平的方法步骤。在本发明中，生物因子表达水平主要指mRNA表达水平和蛋白水平。
实施例1 伊曲康唑治疗婴幼儿血管瘤临床疗效观察及伊曲康唑对血管瘤患儿血清中VEGF及PDGF-AA水平的影响
门诊收集婴幼儿血管瘤患者17例，由患儿父母或法定监护人签署知情同意书后予 以口服伊曲康唑5mg/kg/d，疗程2周至12周。从治疗起每4周记录其皮损变化情况(瘤体面积、颜色变平、变淡等)，从临床角度判断和评价治疗效果。采集血管瘤患儿服药前、服药4周、服药12周的血液样本17例，通过ELISA法检测血清中VEGF、PDGF-AA水平的变化，并进行定量分析。用SPSS软件对统计数据分析。研究结果参见图1。
本研究共纳入17名患儿，男6例，女11例，年龄1-12月(平均5.4月)，体重5-10.5kg，治疗前收集样本17个，治疗后样本17个，治疗前后间隔时间为天13-56天(平均31天)，内服伊曲康唑总剂量390-2240mg(平均1076mg)。总共随访时间为0.5-6月不等。这17例中，有12例为增殖期婴幼儿血管瘤，其中7例增殖期血管瘤在随访期间呈消退趋势，消退率为58.3％(7/12)。
(1)治疗前后血清VEGF(血管内皮细胞生长因子)检测(图1A)。
治疗前患儿血清VEGF浓度为214.82±135.58pg/mL，治疗后为390.81±649.18pg/mL，治疗后比治疗前上升175.98±551.17pg/mL(其中8名患者治疗后VEGF升高，9名患者下降)，经T检验分析，二组之间的差异不具有统计学意义(p>0.05)。治疗有效的7例增殖期血管瘤患儿中，治疗后VEGF水平上升有4人，下降有3人。
(2)治疗前后血清PDGF-AA(血小板衍生生长因子)检测(图1B)。
治疗前患儿血清PDGF-AA浓度为52879.92±31435.65pg/mL，治疗后为26289.34±26095.77pg/mL，治疗后比治疗前下降26590.57±35627.71pg/mL(其中2名患者上升，15名下降)，经T检验分析，二组之间的差异具有统计学意义(p＜0.05)。治疗有效的7例增殖期血管瘤患儿中，治疗后PDGF-AA水平上升有1人(14.3％)，下降有6人(85.7％)。
因此，以上17例婴幼儿血管瘤患儿在伊曲康唑治疗前后，血清中VEGF水平变化差异无统计学意义，而血清中PDGF-AA水平呈下降趋势，差异具有统计学意义。而在治疗有效的增殖期婴幼儿血管瘤中，PDGF-AA水平下降与血管瘤消退有密切相关性。由于PDGF-AA是PDGF-A的二聚体，PDGF-AA的变化反应PDGF-A蛋白水平的变化，因而可知PDGF-A蛋白水平的下降与血管瘤消退有密切相关性。
实施例2 伊曲康唑治疗血管瘤的分子机制
(1)材料与方法
试剂和细胞培养
伊曲康唑，酮康唑和普萘洛尔购自Sigma-Aldrich。CP-67345从MedChem Express购买。EOMA获自哈佛医学院的儿童医院外科系(美国波士顿)的Catherine Butterfield 博士的血管生物学计划。在含有10％胎牛血清和青霉素/链霉素/谷氨酰胺的RPMI 1640(GIBCO)中培养。从父母那里获得使用婴儿血管瘤标本的书面知情同意书后，我们收集了婴儿血管瘤的增生组织。他们接受过血管瘤外科手术治疗。用增生的IH组织的组织阻断方法成功培养了体外婴儿血管瘤内皮细胞(hemangioma endothelial cell，HemEC)。然后在含有10％胎牛血清，10ng/ml碱性成纤维细胞生长因子和青霉素/链霉素/谷氨酰胺的RPMI 1640(GIBCO)中进行培养。将所有细胞在37℃含5％CO 2孵箱中培养。
细胞活力测定
为了进行细胞活力分析，将细胞接种在96孔板中，并于第二天伊曲康唑或CP-673451开始处理。孵育72小时后，根据制造商的说明进行MTS分析(Promega，Madison，WD)，并使用GraphPad Prism软件(版本6)拟合归一化的细胞活力曲线。
凋亡分析
用伊曲康唑或CP-673451处理细胞48小时。使用Canto流式细胞仪(Becton Dickinson，Franklin Lakes，NJ)，根据制造商的说明，通过膜联蛋白V/PI染色(Annexin V FITC和PI试剂盒，Thermo Scientific)分析凋亡细胞。此外用浓度为5μg/ml的Hoechst33258在37℃下将EOMA和HemEC的核染色5分钟。洗涤细胞以除去未结合的染料，随后通过荧光显微镜(Olympus)观察细胞活力。
血管生成测定
将HemECs以1.5×10细胞/孔的密度接种在Matrigel表面上的96孔板中。用0.1％DMSO、伊曲康唑、丙醇或CP-673451处理细胞。处理3小时后，拍摄了6个随机选择的显微镜视野(放大200倍)。总管长度和毛细管网络的密度通过Axiovision软件(马萨诸塞州内蒂克市The Math Works，Inc.)进行评估。
HemECs mRNA阵列和数据分析
用10μM伊曲康唑或0.1％DMSO处理2例婴儿的HemEC 48小时。根据制造商的说明，提取总mRNA进行安捷伦人类(V2)基因表达微阵列(8×60K芯片)的mRNA阵列分析。
逆转录PCR
从伊曲康唑处理的HemEC中提取总RNA。按照制造商的说明，使用Trizol试剂(Invitrogen，15596-026)用伊曲康唑或0.1％DMSO作为对照处理。引物序列如下：PDGF-D，5'-CCCAGGAATTACTCGGTCAA-3'(F)(SEQ ID NO.:1)和 5'-ACAGCCACAATTTCCTCCAC-3'(R)(SEQ ID NO.:2)；ACTB，5'-GGACTTCGAGCAAGAGATGG-3'(F)(SEQ ID NO.:3)和5'-AGCACTGTGTTGGCGTACAG-3'(R)(SEQ ID NO.:4)。
蛋白质印迹
参照参考文献(Zheng et al.,2009)使用以下抗体进行蛋白质印迹：p-p70S6K(产品编号9272)，pAKT-T308(9275)，T-AKT(4691)，p-4E-BP1-S65(9451)，PDGFR(3169)，p-PDGFR-B(3161)和B-肌动蛋白(4970)购自Cell Signaling Technologies；PDGF-D购买自R&D Systems(产品编号AF1159)。
统计分析
定量数据表示为n＝3±标准差(S.D.)的平均值。两组通过学生t检验进行比较，多组通过不匹配的单因素方差分析与Tukey校正进行比较，使用GraphPad Prism中每列的平均值与其他每列的平均值进行比较(版本6：La Jolla.CA)。
(2)伊曲康唑作用的潜在靶向基因及生物因子
申请人通过使用全基因组基因表达谱(GEO登录号：GSE123108)探索了伊曲康唑在体外婴儿HemEC中的潜在靶向基因。进行散点图和层次聚类分析以鉴定差异表达的mRNA(参见图2)。HemECs mRNA阵列和数据分析来自2名婴儿患者的HemEC用10μM伊曲康唑或0.1％DMSO处理48小时。根据制造商的说明，用Agilent人类(V2)基因表达微阵列(8×60K芯片)提取总mRNA用于mRNA阵列分析。
根据MAS生物功能注释系统手册，分别确定了前20个显著增强的生物过程基因本体论术语(GO terms)(图2c)，前20个显著增强的细胞成分基因本体论术语(图2d)，前20个显著增强的分子功能基因本体论术语(图2e)和前20个显著增强的途径术语(图2f)。
同时发现PDGF-A、VEGF-A、HES、HEY1、IL-8或TNF-A这些生物因子对应的基因在差异表达的mRNA中上调显著，差异倍数大于2；PDGF-D、HIF3A或FGF这些生物因子对应的基因在差异表达的mRNA中下调显著，差异倍数大于2，具体差异情况见表2：
表2 表达差异显著的基因及其对应生物因子名称
Figure PCTCN2020073250-appb-000003
Figure PCTCN2020073250-appb-000004
注：以上差异倍数中，正数表示上调，负数表示下调，差异倍数以表中数值的绝对值表示。
(3)伊曲康唑显著降低了PDGF-D水平，从而抑制了PDGFR-β的活化并抑制了其下游效应物。
体外婴儿血管瘤内皮细胞(HemEC)在伊曲康唑处理后PDGF-D蛋白质水平被显著抑制(图3a、b)。进一步使用蛋白质印迹分析了PI3K/Akt/mTOR途径的活性，该途径是PDGF/PDGF受体-β(PDGFR-β)信号传导的重要下游和涉及婴儿血管瘤的关键信号传导途径。如图3c所示，发现伊曲康唑处理诱导Akt(p-Akt)磷酸化水平降低，并且Akt/mTOR，p70S6激酶(p-p70S6K)和4E结合蛋白1的两个下游效应物(p-4E-BP1)，在HemEC(图3c)和EOMA(图3d)细胞中。
(4)伊曲康唑对HemEC的活性与CP-673451(PDGFR-β抑制剂)类似
为了证实伊曲康唑是否可以通过抑制PDGF-D/PDGFR-β信号传导来抑制婴儿血管瘤，比较了HemEC中伊曲康唑与PDGFR-β抑制剂CP-673451的活性,结果表明，伊曲康唑和CP-673451均抑制细胞增殖(图4a)，促进细胞凋亡(图4b)，减少血管生成(图4c)，抑制磷酸化PDGFR-β及其下游效应物，包括p-Akt，p-p70S6K和HemEC中的p-4E-BP1(图4d)。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范 围。
参考文献：
Zheng B,Jeong JH,Asara JM,Yuan Y-Y,Granter SR,Chin L,et al.Oncogenic B-RAF negatively regulates the tumor suppressor LKB1to promote melanoma cell proliferation.Mol Cell 2009；33:237-47.

Claims (15)

  1. 一种评估、预测或优化伊曲康唑对血管瘤治疗有效性的方法，其特征在于，包括步骤：
    a)获取来自血管瘤病人的测试样品，检测所述测试样品中生物因子的表达水平；
    b)所述血管瘤病人接受有效剂量的伊曲康唑的治疗；
    c)比较治疗前后，所述生物因子的表达水平的变化，当所述生物因子的表达水平上调或者下调时，达到治疗效果。
    所述测试样品为正常组织、肿瘤组织、细胞系、血浆、血清、全血、脑脊髓液、淋巴液、循环肿瘤细胞、细胞裂解物、组织裂解物、尿和/或抽吸物，优选地，为血清或肿瘤组织；所述生物因子为VEGF-A、PDGF-A、PDGF-D、HES、HEY1、HIF3A、IL-8、TNF-A或FGF中的一种或几种。
  2. 如权利要求1所述的方法，其特征在于，所述生物因子的表达水平为相应生物因子在所述测试样品中的mRNA表达水平。
  3. 如权利要求2所述的方法，其特征在于，
    a)当治疗后PDGF-A、VEGF-A、HES、HEY1、IL-8或TNF-A中的一种或多种的mRNA表达水平较治疗前上调差异倍数大于或等于2、3、5、8、10或20时，达到治疗效果；或者
    b)当治疗后PDGF-D、HIF3A或FGF中的一种或多种的mRNA表达水平较治疗前下调差异倍数大于或等于2、3、5、8、10或20时，达到治疗效果。
  4. 如权利要求2所述的方法，其特征在于，所述生物因子为PDGF-D、HIF3A或FGF中的一种或几种，优选地，所述生物因子为PDGF-D。
  5. 如权利要求1所述的方法，其特征在于，所述生物因子的表达水平为相应生物因子在测试样品中的蛋白水平，优选地，当治疗后相应生物因子的蛋白水平较治疗前下降大于或等于10％、30％、50％或70％时，达到治疗效果。
  6. 如权利要求5所述的方法，其特征在于，所述生物因子为PDGF-A。
  7. 如权利要求1～6任一项所述的方法，其特征在于，所述血管瘤病人为婴幼儿。
  8. 如权利要求1～6任一项所述的方法，其特征在于，所述血管瘤为增殖期血管瘤。
  9. 一种检测试剂盒，包含用于测量受试者测试样品中生物因子的mRNA表达水平或蛋白水平的试剂，其特征在于，所述测试样品为正常组织、肿瘤组织、细胞系、血浆、血清、全血、脑脊髓液、淋巴液、循环肿瘤细胞、细胞裂解物、组织裂解物、尿 和/或抽吸物，优选地，为血清或肿瘤组织，所述生物因子为VEGF-A、PDGF-A、PDGF-D、HES、HEY1、HIF3A、IL-8、TNF-A或FGF中的一种或几种。
  10. 如权利要求9所述的检测试剂盒，其特征在于，还包含用于选自下述的目的的说明书：通过所述生物因子表达水平或其变化测定血管瘤病人是否将响应伊曲康唑治疗，通过所述生物因子表达水平或其变化监控伊曲康唑治疗的血管瘤病人的疾病进展，通过所述生物因子表达水平或其变化证实在施用伊曲康唑的病人中伊曲康唑的生物活性，或其组合。
  11. 如权利要求9～10任一项所述的检测试剂盒，其特征在于，所述生物因子为PDGF-A或PDGF-D。
  12. 权利要求9～11任一项所述的检测试剂盒用于评估、预测或优化伊曲康唑对血管瘤病人治疗有效性的用途。
  13. 如权利要求12所述的用途，其特征在于，所述生物因子的表达水平为相应生物因子的mRNA表达水平；优选地，当治疗后PDGF-A、VEGF-A、HES、HEY1、IL-8或TNF-A中的一种或多种的mRNA表达水平较治疗前上调差异倍数大于或等于2、3、5、8、10或20时，达到治疗效果，或者当治疗后PDGF-D、HIF3A或FGF中的一种或多种的mRNA表达水平较治疗前下调差异倍数大于或等于2、3、5、8、10或20时，达到治疗效果。
  14. 如权利要求12所述的用途，其特征在于，所述生物因子的表达水平为相应生物因子的蛋白水平；优选地，当治疗后相应生物因子的蛋白水平较治疗前下降比例大于或等于10％、30％、50％或70％时，达到治疗效果。
  15. 如权利要求12所述的用途，其特征在于，所述血管瘤为增殖期血管瘤。
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