JP2009257899A - Method of detecting infiltration of macrophage and formation degree of blood vessel - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To find out the new relationship of diacetylpolyamine, the infiltration of macrophage and the formation of a blood vessel and to provide new adaptation as the diagnostic marker of diacetylpolyamine by using the relationship. <P>SOLUTION: A method is provided (1) for measuring diacetylpolyamine in a sample and detecting the infiltration of macrophage and the formation of the blood vessel in an animal containing a human being, a method (2) for using the sample originating from a patient during medical treatment and determining an medical treatment effect or the prediction of recuperation or a method (3) for combining the diagnosis of an image with the measurement of diacetylpolyamine in the animal containing the human being to judge the infiltration of macrophage and the formation degree of the blood vessel and determining the medical treatment effect or the prediction of recuperation. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for measuring diacetylpolyamine in a sample and detecting the degree of macrophage infiltration and angiogenesis in animals including humans.

  In cancer, the accumulation of macrophages in the affected area correlates with the malignancy of the cancer, and it is said that the macrophages are involved in the inflammatory reaction and tissue reconstruction in the affected area (Non-patent Document 1). Recent research has revealed that infiltrating macrophages and regions with low oxygen concentration contribute to malignant cancer, and that many vascular tissues are formed in the area where cancer is formed.

  However, in order to count the number of macrophages or blood vessels and to measure the oxygen concentration in tissues, it is necessary to observe invasive and advanced techniques such as biopsy and puncture. The method of diagnosing the malignancy of cancer has not been a realistic method.

  In the process of studying the production mechanism of diacetylpolyamine in cancer cells, the present inventor has found that, surprisingly, diacetylpolyamine is not a substance produced by cancer cells but produced by macrophages. As a result of further studies based on this finding, the production of diacetylpolyamine in macrophages directly reflects the metabolic state of macrophages, particularly the degree of macrophage-derived adverse effects in animals including humans. In addition, it has been confirmed that it is possible to directly detect the deterioration of diseases, disorders, or symptoms in which macrophages such as arteriosclerosis are involved or highly suspected to be involved (Patent Document 1 and Non-Patent Document 2).

JP 2007-225605 A Breast Cancer Res. , 2003, 5: 83-88. Cancer Letters, November 8, 2006; 243 (1): 128-34.

  However, as a result of examining the relationship between macrophages and pathological conditions in more detail, in the growing carcinoma, the state of insufficient angiogenesis is actively maintained, and infiltration of macrophages into the affected area is constantly occurring, It was found that the measurement of diacetylpolyamine reflects the resulting adverse effect. That is, the amount of diacetylpolyamine produced does not vaguely reflect macrophage-derived malignancy, but the degree of angiogenesis that can be a specific index of malignancy, particularly the degree of angiogenesis, and macrophage infiltration It is clear that the degree is quantitatively reflected according to the degree of growth or malignancy of the carcinoma, so it can be a very useful diagnostic marker for the determination of therapeutic effect and prognosis prediction in the pathological stage It has been shown.

  Therefore, an object of the present invention is to find a new relationship between diacetylpolyamine and macrophage infiltration and angiogenesis, and to provide a new application of diacetylpolyamine as a diagnostic marker using this relationship.

  As a result of intensive studies to achieve the above object, the present inventor has found that diacetylpolyamine is produced when the oxygen supply is limited to a certain threshold value or less based on the results of in vitro experiments, and the production amount (D) is It has been found that it is represented by the following formula 1 including three variables.

（式中、Ａは正の定数、ｆは｛体積あたりへの酸素供給速度｝が０以上ある閾値以下の範囲に対して正の値をとる上に凸の関数を示す。）

(In the formula, A is a positive constant, and f is a convex function having a positive value for a range where {the oxygen supply rate per volume} is not less than 0 and not more than a threshold value.)

  On the other hand, the inventor found that (the number of macrophages per volume) and {the rate of oxygen supply per volume} were kept constant during the course of carcinoma growth in the case of carcinoma in vivo. We also found that is proportional to (carcinoma size) only.

  In other words, production of diacetylpolyamine is performed only when the oxygen supply is below a certain value, and the rate of oxygen supply per volume is maintained at a constant value in carcinoma in the living body, An increase in diacetylpolyamine production means that the area of oxygen deficiency is expanding with the growth of the carcinoma. In other words, effective angiogenesis is sufficient to resolve the lack of oxygen supply. It means not done.

  Similarly, since the number of macrophages per volume in a carcinoma is a constant value, it can be determined that the total number of macrophages in the entire carcinoma also increases according to the degree of growth of the carcinoma.

  Furthermore, in general, the amount of diacetylpolyamine produced in each carcinoma is different, so it is reasonable to assume that the number of macrophages per volume and the rate of oxygen supply per volume have unique values for each carcinoma. It is. Therefore, when the size of a carcinoma can be determined by image diagnosis or the like, the degree of macrophage infiltration and the degree of oxygen supply rate deficiency in individual carcinomas can be relatively evaluated by comparing the amount of diacetylpolyamine in a plurality of carcinomas.

  As described above, the production of diacetylpolyamine reflects the degree of macrophage infiltration and angiogenesis, particularly the degree of angiogenesis, due to the influence of both the size and nature of the cancer area.

  It has been actively studied that the likelihood of macrophage infiltration and hypoxia is contributing to cancer malignancy. Since the amount of diacetylpolyamine reflects the degree of macrophage infiltration and angiogenesis, as described above, measuring the amount of diacetylpolyamine can also be used to determine the therapeutic effect and prognosis of cancer. Can do.

  As described above, the present inventor can easily detect the degree of macrophage infiltration and angiogenesis with respect to the entire carcinoma by measuring diacetylpolyamine in a biological sample, and observe the change over time. Thus, the present invention was completed by finding that it can be used to determine the therapeutic effect and prognosis prediction. Therefore, the present invention is as follows.

(1) A method for measuring the degree of macrophage infiltration and angiogenesis in animals including humans by measuring diacetylpolyamine in a sample.
(2) A method for determining the therapeutic effect and prognosis prediction based on the degree of macrophage infiltration and angiogenesis corresponding to the measured value of diacetylpolyamine using a sample derived from the patient under treatment.
(3) Collect a sample over time from the patient under treatment, measure diacetylpolyamine in the sample, detect the degree of macrophage infiltration and angiogenesis based on the change over time of the obtained measurement value, A method to determine treatment effect and prognosis prediction.
(4) A method for determining the degree of macrophage infiltration and angiogenesis by combining diagnostic imaging and diacetylpolyamine measurement in animals including humans, and determining the therapeutic effect and prognosis prediction.
(5) The method according to any one of (1) to (4) above, wherein the diacetylpolyamine is diacetylspermine and / or diacetylspermidine.
(6) The method according to any one of (1) to (4) above, wherein diacetylpolyamine in the sample is measured by immunoassay.

By using the method of the present invention, it has become possible to detect the degree of macrophage infiltration and blood vessel formation, particularly the degree of insufficient blood vessel formation, in animals including humans.
In particular, by using a sample derived from a patient under treatment and observing the measured value of diacetylpolyamine or its change over time, it is possible to determine the therapeutic effect and prognosis prediction.
In addition, by combining image diagnosis and measurement of diacetylpolyamine, it has become possible for the first time to determine a more accurate therapeutic effect and prognosis prediction.

In the present specification, the following terms have the following meanings.
“Diacetylpolyamine” means diacetylspermine and / or diacetylspermidine, and specific examples thereof include N ¹ , N ¹² -diacetylspermine and N ¹ , N ⁸ -diacetylspermidine.

  “Determination of therapeutic effect” means determination of whether or not the patient's treatment such as surgical operation, irradiation, administration of an anticancer agent, etc. is progressing well in animals including humans.

  “Determination of prognosis prediction” means predicting whether a disease state will be ameliorated or worsened in the future, or whether it will return to normal or death in animals including humans.

  The “sample” is not particularly limited as long as it contains diacetylpolyamine. Specific examples of such samples include urine and serum, and urine samples are particularly suitable for use.

  The “degree of macrophage infiltration” indicates the degree of macrophage infiltration in the affected area.

  “Degree of angiogenesis” refers to the degree to which the oxygen supply rate satisfies the oxygen demand in the affected area, and the degree of lack of angiogenesis refers to the degree to which the oxygen demand is not met. means.

  This is because, considering that the production of diacetylpolyamine is performed when oxygen supply is limited to a certain threshold value or less, the production amount reflects the degree to which the oxygen demand threshold is not satisfied in the tissue or local area such as cancer. Yes. In general, vascular tissue is actively formed in cancer, but it is considered that there is a morphological abnormality peculiar to cancer in the blood vessel, which causes a problem in oxygen supply (Cancer Res., 1999). , 59: 5863-5870), the lack of oxygen supply indicates the extent to which effective blood vessels that can meet local oxygen demand are not formed (the degree of lack of blood vessel formation).

(A) Measurement of diacetylpolyamine The measurement of diacetylpolyamine in a sample is not particularly limited. Among these, an immunoassay is particularly simple and suitable. Regarding the reagents used in the immunoassay and the specific measurement procedures, known literatures (for example, Japanese Patent No. 3770978, WO 2004-81569, J. Cancer Res. Clin. Oncol., 123 (1997), 539-545). J. Biochem. (Tokyo), 132 (2002), 783-788).

(B) Detection of degree of macrophage infiltration and angiogenesis According to the above method, diacetylpolyamine is measured in a sample of a cancer patient, and in particular, the measured value of diacetylpolyamine over time is measured. Based on the change in the measured values obtained, if the change in the direction of increasing diacetylpolyamine is large, the area where the blood vessel formation is not sufficiently performed due to macrophage infiltration and insufficient oxygen supply has expanded. Judgment and it can be judged that the area | region has shrunk | reduced when the change to the direction which diacetyl polyamine decreases is large.

  In addition, when comparing the results of diagnostic imaging, etc. for multiple carcinomas, macrophage infiltration and angiogenesis deficiency are stronger and more qualitatively for carcinomas with relatively large diacetylpolyamine production relative to their size. It can be determined that the cancer is malignant.

(C) Judgment of therapeutic effect and prognosis prediction Diacetylpolyamine is measured, especially the measured value of diacetylpolyamine over time, and based on the change of the measured value, there is a change in the direction of increasing diacetylpolyamine. If it is larger, it is determined that the prognosis is poor or the treatment is not progressing well, and it can be used as one of the judgment materials for whether to perform the treatment again or to perform the treatment different from the conventional one.

  If diacetylpolyamine is flat, there is a possibility of recurrence and metastasis, and it can be determined that it is better to observe the situation carefully. Moreover, when the change in the direction in which diacetylpolyamine decreases is large, it can be determined that the prognosis is good or the treatment is progressing well.

  The determination with diacetylpolyamine as described above is applicable not only to cancer but also to diseases accompanied by angiogenesis accompanied by macrophages, such as fractures and arteriosclerosis. In cancer, a high or increased measured value of diacetylpolyamine meant that the medical condition was not good, but in bone fractures, a moderate increase may indicate a normal healing process. It can be used as an index of seemingly contradictory diagnosis because it can be understood that this measurement value reflects the degree of macrophage infiltration and angiogenesis rather than vaguely reflecting the degree of malignancy. It becomes a very useful method in medical practice.

  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

(Example 1) Relationship between macrophages and diacetylspermine (DAM) production amount Using peritoneal exudate cells (PEC) as macrophages, the relationship between macrophages, that is, the number of PECs, oxygen supply rate, culture days, and DAM production amount was analyzed.

First, RPMI culture solution containing 10% horse serum, 5 mM lactic acid and no glucose was added to the well of a 96-well plate, and PEC was cultured at 37 ° C. with 5% CO ₂ .

The number of PECs per well is 1.0 × 10 ⁵ , 1.5 × 10 ⁵ , 2.0 × 10 ⁵ , 2.5 × 10 ⁵ , and the culture volume is 0.12 ml, 0.14 ml, 0.18 ml 0.25 ml. The amount of DAM in the culture solution on the first day, the second day, the third day, and the fourth day after the start of the culture was measured by a known ELISA method.

  Next, it was examined whether the actual production amount of DAM is expressed by the above formula 1.

  Equation 2, which expresses the amount of DAM produced per well, is based on a model in which components of the mitochondrial citrate cycle that have lost membrane potential are converted to acetyl-CoA in a primary reaction governed by the oxygen supply rate. . However, since the amount accumulated in the culture well was measured, the following equation 2 obtained by converting the equation 1 into an integral form was used. The detailed relationship between Formula 1 and Formula 2 and how to derive it are shown in Reference Examples.

The inner diameter constant well was estimated from the depth of the liquid is proportional to the culture volume, oxygen feed rate to the PEC10 ⁵ per the liquid to a depth, i.e. to be inversely proportional to the culture volume ( (Oxygen supply rate) was calculated as 1 / number of cells (× 10 ⁵ ) / volume of culture solution (ml). Further, (the number of macrophages per well (PEC)) corresponds to (the number of macrophages per volume) in the text, and (the size of carcinoma) in the text is expressed by the number of wells.

Further in formula 2, obtained experimentally, PEC10 ⁵ Up DAM production amount per the (equivalent to citric acid content in the mitochondria) and 113.4, the oxygen supply rate upper limit DAM production occurs 5.59 Dependence on the oxygen supply rate of citrate lyase that produces acetyl CoA used for DAM production is 1.04, and the values are used by applying each value.

  As a result, as shown in FIG. 1, it was clarified that the predicted value of the DAM production amount by the simulation indicated by the solid line and the actual measurement value represented by each point agree very well. That is, the actual production of DAM in macrophages can be calculated and applied by the simulation of Formula 2, and it is confirmed that the predicted value of the DAM production amount calculated by Formula 1 is almost the same as the actual DAM production amount. We were able to.

(Example 2) Relationship between tumor size and diacetylspermine production amount Balb / c mice were inoculated with Colon 26, a cancer cell line, and after 14 days, the size of cancer was measured every 3 days, and urine The amount of medium DAM was also measured by ELISA, and the relationship between them was shown in FIG. The urinary DAM concentration was corrected by the creatinine concentration (measured by the enzyme method). The size of the cancer was calculated as length (cm) × width ² (cm ² ) × 1/2.

  As shown in FIG. 2, in the growth phase of cancer, DAM production is proportional only to (the size of the carcinoma), and the number of macrophages per volume and the oxygen supply rate per volume are almost constant. It was strongly speculated. That is, the constants that have the number of macrophages per volume and the rate of oxygen supply per volume are that the two variables are controlled by the interaction between the cancer side, host side, or both. Is considered to be the cause.

  Therefore, based on the fact that the number of macrophages per volume and the oxygen supply rate are constant, the relative size of carcinoma can be evaluated by measuring the amount of DAM produced, It is also possible to quantitatively determine the degree of macrophage infiltration and the degree of lack of effective angiogenesis (degree of lack of oxygen supply) as the total amount according to the size of the carcinoma.

  In addition, when the size of multiple carcinomas has been clarified by diagnostic imaging, etc., the number of macrophages per volume and the degree of lack of angiogenesis are relatively evaluated by combining DAM production. It is possible to accurately evaluate the relative malignancy of carcinoma.

(Reference example)
In this model, citrate leaked from mitochondria that lost membrane potential is converted to acetyl CoA by citrate lyase, and further, it reacts with monoacetylspermine by an unknown acetylase to produce DAM. The necessary ATP concentration was proportional to the oxygen supply rate [sO ₂ ], and the leaked citric acid was consumed only in this reaction, and the citric acid concentration [cit] decreased in the primary reaction.

First, consider the amount of DAM produced per 10 ⁵ macrophages. Assuming that the proportionality constant is A and the time (day) is t, Equation 3 is established for the citric acid concentration [cit].

ただし、下の漏出したクエン酸の式とあわせるため５．５９で除しておいた。

However, it was divided by 5.59 to match the leaked citric acid formula below.

Solving this differential equation with the first leaked citric acid as [cit] ₀ ,

が導かれる。

Is guided.

The leaked citric acid is 0 when [sO ₂ ] at which DAM is produced is an upper limit (= lower limit at which no DAM is produced, and this value is substituted because it is 5.59 in actual measurement), and when [sO ₂ ] is 0 ( [Cit] ₀ can be expressed as follows because it is considered to be expressed by a linear expression related to [sO ₂ ], which is a total citrate concentration [TC] (per 10 ⁵ macrophages).

All of the produced acetyl-CoA is converted to DAM ([TC] was verified in the experiment of Example 1 to match the maximum amount of DAM produced by extrapolation. Below, 113. 4), so that [dam] accumulated in the well (per 10 ⁵ macrophages) is

となる。

It becomes.

By the way, the proportional constant A contains the reaction constant of citrate lyase, but since this enzyme is known to be activated by modification with ATP (produced by supplying oxygen), Looking at the dependency on [sO ₂ ],

であった。

Met.

  Substituting Equation 7 into Equation 6 and multiplying the number of macrophages per well by c gives the amount of DAM / well DAM / well as shown in Equation 8 below. Since Formula 8 and Formula 2 are the same, Formula 2 in Example 1 was derived.

Next, Equation 1 is derived. Consider an event per volume of carcinoma that corresponds to the analysis in one well in Example 1. In the analysis in the well, the accumulated amount was obtained, but when measuring the urine sample, the produced DAM is excreted one after another, so the measured value represents the production rate, that is, the differentiation with time of citric acid reduction. It will be. If a certain macrophage has infiltrated the carcinoma at time T (T is a negative number), the production rate of DAM per 10 ⁵ macrophages at sampling time 0 is calculated as follows.

（式９）
見やすくする為にパラメータはウェル解析のままにした。別の値が入ることもありうるが本質的な違いはない。

(Formula 9)
For clarity, the parameters were left as well analysis. There may be other values, but there is no essential difference.

  By the way, macrophages in carcinomas should have a certain distribution with respect to the time of infiltration. The amount of time handled in this simulation is approximately several days, and is sufficiently small in the course of human cancer, so that the carcinoma is in a steady state and can be treated as having no change in (size or) macrophage configuration. Considering this, the following two types of distribution are considered.

(A) First, in the case where c × 10 ⁵ macrophages having an infiltration period of L days or less are present uniformly (this means that the macrophages disappear as soon as it becomes longer than L days, which is somewhat unrealistic. The total DAM production rate is

（式１０）
と算出される。整理して定数部分を改めてＡとおけば

(Formula 10)
Is calculated. If you sort out the constant part again and put A

とあらわされる。

It is expressed.

(B)
Next, if the macrophage with infiltration time T (T is a negative number) decreases with an exponential function of half-life M days (more realistic), the overall production rate is

と算出される。ただし、

Is calculated. However,

で除して細胞数を規格化した。整理して定数部分を改めてＡとおけば

The number of cells was normalized by dividing by. If you sort out the constant part again and put A

とあらわされる。

It is expressed.

  In both Expression 11 and Expression 14, the part other than c × A indicates the function f in Expression 1. Since Equations 11 and 14 show the amount of DAM production per volume, that is, in both cases (A) and (B), it can be seen that the DAM production rate in the entire carcinoma is expressed in the form of Equation 1.

  Further, in both cases (A) and (B), simulation was performed by substituting numerical values, and as a result, it was found that the oxygen supply rate became convex upward (FIG. 3). Since two similar macrophage distribution simulations draw similar curves, it was estimated that the function f in Equation 1 is a convex function even in the actual distribution.

FIG. 1 shows how the production of diacetylspermine (DAM) by peritoneal exudate cells (PEC) is represented by Formula 2. The graphs A, B, C, and D in the figure show the number of PECs per well as 1.0 × 10 ⁵ , 1.5 × 10 ⁵ , 2.0 × 10 ⁵ , and 2.5 × 10 ⁵ , respectively. Represents the case. The vertical axis represents the amount of DAM production, and the horizontal axis represents the amount of the culture solution (reflecting the oxygen supply rate). The solid line shows the simulation value of the DAM production amount with respect to the oxygen supply rate for each culture day based on the derived formula, and each point shows the actually measured DAM production amount. FIG. 2 shows the relationship between cancer size and urinary DAM production. The vertical axis represents the amount of DAM production in urine corrected by the amount of creatinine, and the horizontal axis represents the volume of the carcinoma. FIG. 3 shows the result of simulation by substituting a numerical value that Expressions 11 and 14 are upward convex functions. The figure of X represents the simulation result in the case of (A) of the third embodiment, and the figure of Y represents the simulation result in the case of (B) of the third embodiment. The horizontal axis represents the oxygen supply rate [sO ₂ ], and the vertical axis represents the calculated DAM production simulation value. In addition, the respective points and solid lines indicate that the macrophage infiltration period L (day) in Example 3 (A) is X in 0.1 and the macrophage half-life M (day) in Example 3 (B) is 0.1 in Y, respectively. The simulation results are shown assuming 1 day, 1 day, 2 days, and 5 days.

Claims (6)

A method for measuring the degree of macrophage infiltration and angiogenesis in animals including humans by measuring diacetylpolyamine in a sample. A method of using a sample from a patient under treatment and determining the prediction of treatment effect and prognosis based on the degree of macrophage infiltration and angiogenesis corresponding to the measured value of diacetylpolyamine. Samples are collected over time from patients undergoing treatment, diacetylpolyamine in the samples is measured, and the degree of macrophage infiltration and angiogenesis is detected based on changes over time in the obtained measurement values. A method for determining prognosis. A method of determining the degree of macrophage infiltration and angiogenesis by combining diagnostic imaging and diacetylpolyamine measurement in animals, including humans, and determining the therapeutic effect and prognosis prediction. The method according to any one of claims 1 to 4, wherein the diacetylpolyamine is diacetylspermine and / or diacetylspermidine. The method according to any one of claims 1 to 4, wherein diacetylpolyamine in the sample is measured by immunoassay.

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