CN110702888A - Liver and kidney clearance detection method for two isomers of Pumei - Google Patents

Abstract

The invention relates to a liver and kidney clearance detection method for two diastereoisomers Gd-A and Gd-B of Pumei, which has the creativity that: the invention firstly discovers that GFR can approximately replace renal clearance of Gd-A and Gd-B, and liver clearance of Gd-A and Gd-B can be obtained by subtracting GFR from respective plasma clearance. The detection method provided by the invention can conveniently and accurately measure the clearance rates of the liver and the kidney of the two isomers of Pumei magnetic resonance, provides a gold standard and an individualized pharmacokinetic index for the Pumei magnetic resonance liver function imaging, and has guiding and reference effects on rational medication and evaluation of the liver and kidney functions.

Description

Liver and kidney clearance detection method for two isomers of Pumei

Technical Field

The invention relates to the technical field of biochemical analysis, in particular to a liver and kidney clearance detection method for two isomers of Pumei.

Background

Disodium gadoxetate (Gd-EOB-DTPA), with trade name promet (Primovist), international non-proprietary name (INN) Gadoxetic acid disodium, is a liver cell specific magnetic resonance contrast agent, was first approved for clinical trials in sweden in 2004, was officially marketed in china in 2011, and has been approved in over 40 countries at present. It is formed by adding fat-soluble ethoxy benzyl (EOB) on gadolinium-meglumine pentetate (Gd-DTPA) molecules, so that the gadolinium-meglumine pentetate has the characteristics of a nonspecific extracellular contrast agent and a hepatobiliary specific contrast agent. The half-life of Gd-EOB-DTPA is about 56 minutes, and liver-gall stage or liver cell specific stage MR imaging can be carried out within 10-40 minutes after injection, in the stage of imaging, the parenchyma of liver cells with normal functions presents high signals due to the intake of contrast agent, and lesion parts present lower or lower signals due to the hypofunction of the liver cells or the lack of the normal liver cells.

The total liver cell uptake rate of Gd-EOB-DTPA (Gd-A and Gd-B, 65:35 w/w) in a healthy human body is about 50%, after being ingested by the liver, the Gd-EOB-DTPA is excreted with bile through the biliary tract, the rest of the Gd-EOB-DTPA is excreted out of the human body with urine through the kidney, when one excretion route is blocked, the other route can be compensated, so that the Gd-EOB-DTPA can be safely used in patients with impaired liver function or kidney function. However, Gd-EOB-DTPA comprises two types of diastereoisomers, Gd-A and Gd-B, and the pharmacokinetics of the two types of isomers in humans, i.e. plasma and liver, kidney clearance, is still unknown. The existing animal experiments suggest that the liver clearance rates of the two isomers are different, and the respective liver and kidney clearance rates of the two isomers are fully known, so that the prior animal experiments have guiding and reference functions on reasonable medication, prevention of toxic and side effects of medicines and evaluation of liver and kidney functions. The respective hepatic and renal clearance rates of the two isomers to be obtained under the existing (conventional) technical conditions can be theoretically achieved by 3 methods, namely 1) the excretion amounts of Gd-A and Gd-B in urine and bile (feces) are respectively monitored after administration, and the renal and hepatic clearance rates are further calculated; 2) after administration, monitoring the plasma concentrations of Gd-A and Gd-B and simultaneously monitoring the excretion amount in urine to obtain a plasma clearance rate and a renal clearance rate, and further subtracting the renal clearance rate from the plasma clearance rate to obtain a liver clearance rate; 3) plasma clearance and liver clearance were obtained by monitoring plasma concentrations of Gd-A and Gd-B and simultaneously monitoring excretion in bile (feces) after dosing, and renal clearance was further obtained by subtracting liver clearance from plasma clearance. Unfortunately, these methods are complicated and have no practical operability, so that no published report for measuring liver and kidney clearance rates of Gd-A and Gd-B exists so far. There is an urgent need for a convenient and practical method for quantitatively measuring the clearance of liver and kidney of these two isomers.

It should be further noted that there is a slow interconversion between Gd-A and Gd-B, i.e. isomerisation, and that due to the inherent equilibrium of isomerisation, the ratio of the two isomers (Gd-A and Gd-B, 65:35 w/w) is relatively constant and independent of pH (5-9) and temperature (25-120 ℃), and that at equilibrium the net transition between Gd-A and Gd-B is zero, and that after entering the body the ratio of Gd-A and Gd-B in plasma changes (65: 35 deviations) due to the different metabolic rates, and there is a net transition between them, which is towards restoration of the original ratio of A and B (65: 35). According to previous studies and our experiments, such net switching speed in the human body is so slow as to be negligible.

Disclosure of Invention

The invention aims to provide a liver and kidney clearance detection method for two isomers Gd-A and Gd-B.

To this end, in a first aspect of the invention, there is provided a method for detecting renal clearance of Gd-EOB-DTBA isomers, Gd-A and Gd-B, comprising detecting Glomerular Filtration Rate (GFR), the renal clearance of Gd-A and the renal clearance of Gd-B both being equal to the glomerular filtration rate.

Further, the method for detecting the glomerular filtration rate comprises the steps of injecting an iodine contrast agent into a subject by intravenous injection, and detecting the plasma clearance rate of the iodine contrast agent, wherein the plasma clearance rate of the iodine contrast agent is the glomerular filtration rate.

Further, the iodine contrast agent is selected from the group consisting of: iopromide or iohexol, preferably iopromide.

Further, the method for detecting renal clearance of Gd-EOB-DTBA isomers Gd-A and Gd-B comprises the following steps of injecting iodine contrast agent into a subject intravenously and respectively detecting injection T₁Post sum T₂Concentration P of iodophor in the latter venous blood sample₁、P₂Calculating the plasma clearance of the iodine contrast agent by using the following formula,the plasma clearance rate is the glomerular filtration rate:

wherein, T is₁Is 40-80min, such as 40, 50, 60, 70, 80min, preferably 60 min;

the T is₂Is 160-200min, such as 160, 170, 180, 190, 200min, preferably 180 min.

Further, after the injection of the iodine contrast agent, the injection of saline is continued to wash the residual agent in the injection path.

The dosage range of the iodine contrast agent is determined according to the following principle, and the lowest dosage is as follows: at the completion of injection T₂Then, the content of the iodine contrast agent in the sample can be effectively detected by HPLC; the highest dosage is: maximum concentration that does not pose a risk of drug side effects. In a preferred embodiment, the amount of iodine contrast agent injected is 185-370 mg/person in iodine.

In a second aspect of the invention, there is provided a method for detecting liver clearance of Gd-EOB-DTBA isomers Gd-A and Gd-B, comprising:

i) respectively detecting the plasma clearance rates of Gd-EOB-DTBA isomers Gd-A and Gd-B;

ii) detecting the renal clearance of the Gd-EOB-DTBA isomer according to the renal clearance detection method of the invention;

iii) subtracting the renal clearance from the plasma clearance of Gd-A to obtain the hepatic clearance of Gd-A, and subtracting the renal clearance from the plasma clearance of Gd-B to obtain the hepatic clearance of Gd-B.

Further, the method for detecting the plasma clearance rate of Gd-EOB-DTBA isomers Gd-A and Gd-B comprises the following steps:

the subjects were treated by intravenous injection of Gd-EOB-DTBA, and the injection T was detected separately₁Post sum T₂Analyzing the concentration of Gd-A and Gd-B in the other venous blood sample to obtain the plasma clearance rate of Gd-A and the plasma clearance rate of Gd-B;

wherein, T is₁Is 40-80min, such as 40, 50, 60, 70, 80min, preferably 60 min;

the T is₂Is 160-200min, such as 160, 170, 180, 190, 200min, preferably 180 min.

Further, the plasma clearance of the isomers Gd-A and Gd-B is measured simultaneously with the glomerular filtration rate.

Preferably, the order of injecting the iodine contrast agent and the Gd-EOB-DTBA is not limited, and the Gd-EOB-DTBA can be injected after the iodine contrast agent is injected first, or the Gd-EOB-DTBA is injected before the iodine contrast agent is injected, or the iodine contrast agent and the Gd-EOB-DTBA are injected simultaneously.

Further, after the injection of the iodine contrast agent and Gd-EOB-DTBA, the injection of saline was continued to wash the residual agent in the injection path.

In a specific embodiment, the method for detecting the plasma clearance of the Gd-EOB-DTBA isomers Gd-A and Gd-B comprises the following steps:

subject was treated by intravenous injection of an iodine contrast agent and Gd-EOB-DTBA, respectively, at completion of the injection of T₁And T₂Thereafter, a blood sample was obtained from the other vein and analyzed for plasma clearance of Gd-A, plasma clearance of Gd-B, plasma clearance of the iodine contrast agent; the plasma clearance of the iodine contrast agent is the renal clearance of Gd-A and Gd-B; subtracting the renal clearance from the plasma clearance of Gd-A to obtain the liver clearance of Gd-A; subtracting the renal clearance from the plasma clearance of Gd-B to obtain the liver clearance of Gd-B;

wherein, T is₁Is 40-80min, such as 40, 50, 60, 70, 80min, preferably 60 min;

the T is₂Is 160-200min, such as 160, 170, 180, 190, 200min, preferably 180 min.

The plasma clearance rate of the iodine contrast agent, the plasma clearance rate of Gd-A and the plasma clearance rate of Gd-B can be detected by adopting a double plasma method or a single plasma method.

The detection method of the double plasma method comprises injecting iodine contrast agent and Gd-EOB-DTBA into the vein of a subject, respectively obtaining a blood sample from the other vein after completing injection for about 60min and 180min, analyzing the plasma drug concentration by High Performance Liquid Chromatography (HPLC), and calculating the plasma clearance rate of Gd-A, the plasma clearance rate of Gd-B and the plasma clearance rate of the iodine contrast agent according to the following formulas,

d is the dose administered, T₁And T₂Blood sampling time (min), P₁And P₂Are respectively T₁And T₂Plasma drug concentration at time.

The single plasma method differs from the double plasma method in that a blood sample is drawn in a single time, i.e., about 120min after completion of the injection. The invention preferably uses a double plasma method for detection.

The invention provides a new method for respectively detecting the clearance rate of the liver and the kidney of two diastereoisomers of Gd-EOB-DTBA, and has the advantages of simple and convenient operation and high accuracy. The invention discovers for the first time that the renal clearance rates of two diastereoisomers Gd-A and Gd-B of Gd-EOB-DTBA are respectively equal to the glomerular filtration rate, so that the renal clearance rates of Gd-A and Gd-B can be obtained by detecting the glomerular filtration rate. Based on this, because the plasma clearance is the sum of the drug clearance of the liver and kidney, the liver clearance of Gd-A is obtained by subtracting the glomerular filtration rate from the plasma clearance of Gd-A, and the liver clearance of Gd-B is obtained by subtracting the glomerular filtration rate from the plasma clearance of Gd-B. The detection method provided by the invention can conveniently and accurately measure the clearance rates of the liver and the kidney of the two isomers of Pumei magnetic resonance, provides a gold standard and an individualized pharmacokinetic index for the Pumei magnetic resonance liver function imaging, and has guiding and reference effects on rational medication and evaluation of the liver and kidney functions.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:

FIG. 1 is a schematic diagram of a process for detecting liver and kidney clearance of Gd-A and Gd-B;

FIG. 2 is a Bland-Altman plot comparing RCL-GdA and RCL-GdB to GFR.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

EXAMPLE 1 determination of plasma clearance of Gd-A and Gd-B by the double plasma method

The study was approved by the review board and written informed consent was obtained from all patients. During the period from 7 months in 2017 to 2 months in 2018, we studied 41 patients (mean age 41.8 ± 12.1 years, range 30-73 years) and performed data analysis. Of these, 33 had diffuse cirrhosis, 29 patients had hepatitis b cirrhosis, and 4 had alcoholic cirrhosis. 19 of 33 patients with diffuse cirrhosis had diameters greater than 2 cm; 18 of 33 patients with diffuse liver disease were classified as Child-Pugh class A and 15 as class B. There were 8 non-cirrhosis observers, each with at least one hepatic nodule or mass greater than 1cm in diameter.

For each patient, 1ml of iodine contrast agent (iopromide, 370mg iodine per ml; Bayer Schering, Berlin, Germany) was injected intravenously, followed by 20ml of saline solution. Gd-EOB-DTPA (Gd-A and Gd-B, 65:35 w/w; Bayer Healthcare, Berlin, Germany) was then injected through the same intravenous route at a rate of 2ml/s, administered in an amount of 181.43mg/ml X10 ml per person. Thereafter, the injection of 20ml of saline was continued to wash the contrast agent remaining in the injection path.

After 1h and 3h, respectively, 3ml of anticoagulated blood sample was obtained from another antecubital vein. Each blood sample was centrifuged at 1500-fold gravity for 15min to separate the plasma for HPLC analysis.

The chromatographic conditions for Gd-EOB-DTPA were the same as those in the previous study (Ogawa J, Yokota A, Araki T, ethyl. quantitative evaluation of biological metabolism of gadoxtate, a magnetic resonance imaging contrast agent, via geological isomer transport system in rates. biological Drug dispersions.2014; 35(6) 362-71.doi:10.1002/bdd.1907)

The chromatographic conditions of iopromide are that a separation column: diamonsil C18(2) column, column temperature 25 ℃, eluent: 2 per mill of 20 percent methanol-containing acetic acid solution, the flow rate is 1.0mL/min, and the detection wavelength is 254 nm.

For a patient, the plasma clearance of Gd-A (PCL-GdA) and Gd-B (PCL-GdB) were calculated as follows:

the content of Primei is 181.43mg/ml × 10ml

Gd-a (65% of pemetrexed) 181.43 × 10 × 1000 × 0.65(μ g) 1178288(μ g)

Gd-B (35% of sompo @) 181.43 × 10 × 1000 × 0.35(μ g) ═ 636012(μ g)

The dosage of iopromide is as follows: 370mg/ml × 1ml ═ 370 × 1 × 1000 ═ 370000(μ g)

3ml of blood was collected at 1h and 3h after administration, and plasma concentrations of Gd-A, Gd-B and iopromide measured by HPLC are shown in Table 1, respectively:

TABLE 1

	Gd-A	Gd-B	Iopromide
				1h(μg/ml)	21.43	27.35	7.94
3h(μg/ml)	7.99	10.09	3.91

The plasma clearance rate of the drug can be calculated by substituting the amount of the injected drug and the plasma drug concentrations of 1h and 3h according to the following calculation formula of a pharmacokinetic single-exponential model,

d is the dose administered, T₁And T₂Blood sampling time (min), P₁And P₂Are respectively T₁And T₂Plasma drug concentration at time. The plasma clearance rate (PCL-GdA) of Gd-A is calculated to be 276.06ml/min, the plasma clearance rate (PCL-GdB) of Gd-B is calculated to be 117.39ml/min, and the plasma clearance rate (namely GFR) of iopromide is calculated to be 110.31 ml/min.

The results of the plasma drug clearance tests on 41 patients are shown in table 2,

TABLE 2

Example 2 detection of renal clearance of Gd-A and Gd-B

During the test of example 1, urine from the patient was collected between 1h and 3h after injection of Gd-EOB-DTPA and a sample of the urine was subjected to HPLC analysis under the same conditions as in example 1. The Gd-a and Gd-B concentrations in the urine were determined and multiplied by the volume of the urine, i.e. the amount of Gd-a and Gd-B excreted by the urine during the double plasma sampling of example 1 was calculated.

The graph was plotted in Excel, with the horizontal axis representing the time of blood sample collection and the vertical axis representing the plasma concentrations of Gd-A and Gd-B measured by the double plasma method. For each isomer, an exponential curve connecting two points (1h and 3h) in the coordinates was obtained using a curve fitting function of Excel, and the renal clearance of Gd-A (RCL-GdA) and Gd-B (RCL-GdB) were calculated by integrating the area under the curve (AUC) and dividing the urine volume by AUC, respectively. The results of the renal clearance measurements of Gd-A, Gd-B in 41 patients are shown in Table 3 (together with the GFR values obtained in the measurements of example 1 are shown in the Table):

TABLE 3

Comparing RCL-GdA and RCL-GdB with GFR by a Bland-Altman plot, as shown in FIG. 2, it can be seen from FIG. 2 that the 95% confidence interval (95% CI) of the difference between GFR and RCL-GdA is 12.5mL/min, indicating that the maximum deviation between the two is about 12.6% of GFR; the 95% confidence interval for the difference between GFR and RCL-GdB was 12.4mL/min, indicating a maximum deviation between the two of about 12.5% of GFR. And in 41 patients there was no significant difference between RCL-GdA, RCL-GdB and GFR (one-way repeated measures analysis of variance: F2.616, P0.097), correlation between RCL-GdA and GFR: r 0.95, P < 0.001, correlation between RCL-GdB and GFR: r is 0.94 and P is less than 0.001. It can be concluded that the renal clearance of Gd-A and Gd-B, respectively, equals GFR.

Example 3 calculation of liver clearance of Gd-A and Gd-B

This example calculated liver clearance of Gd-A (HCL-GdA) and Gd-B (HCL-GdB). Calculated as follows:

RCL-GdA—GFR＝HCL-GdA；

the results of the liver clearance calculation of Gd-A, Gd-B in 41 patients, calculated from RCL-GdB-GFR ═ HCL-GdB, are shown in Table 4.

TABLE 4

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims (9)

1. A method for detecting renal clearance of Gd-EOB-DTBA isomers Gd-A and Gd-B is characterized by comprising the step of detecting glomerular filtration rate, wherein the renal clearance of Gd-A and the renal clearance of Gd-B are both equal to the glomerular filtration rate.

2. The method of claim 1, wherein the method of measuring the glomerular filtration rate comprises measuring the plasma clearance of an iodine contrast agent by intravenous injection of the iodine contrast agent into the subject, wherein the plasma clearance of the iodine contrast agent is the glomerular filtration rate.

3. The detection method of claim 2, wherein the iodine contrast agent is selected from the group consisting of: iopromide or iohexol.

4. The detection method as claimed in claim 2, wherein the injection amount of the iodine contrast agent is 185-370 mg/person in terms of iodine.

5. The detection method according to any one of claims 1 to 4, comprising the step of separately detecting the injection T by intravenous injection of an iodine contrast agent into the subject₁Post sum T₂Concentration P of iodophor in the latter venous blood sample₁、P₂Calculating the plasma clearance of the iodine contrast agent, namely the glomerular filtration rate, by using the following formula:

wherein, T is₁Is 40-80 min;

the T is₂Is 160-.

6. The assay of claim 5, wherein after the injection of the iodine contrast agent, the injection of saline is continued to wash residual reagent in the injection pathway.

7. A method for detecting liver clearance of Gd-EOB-DTBA isomers Gd-A and Gd-B is characterized by comprising the following steps:

i) respectively detecting the plasma clearance rates of Gd-EOB-DTBA isomers Gd-A and Gd-B;

ii) detecting renal clearance of Gd-A and Gd-B according to the detection method of any one of claims 1 to 6;

iii) subtracting the renal clearance from the plasma clearance of Gd-A to obtain the hepatic clearance of Gd-A, and subtracting the renal clearance from the plasma clearance of Gd-B to obtain the hepatic clearance of Gd-B.

8. The detection method according to claim 7, comprising the steps of:

the subjects were treated by intravenous injection of Gd-EOB-DTBA, and the injection T was detected separately₁Post sum T₂Analyzing the concentration of Gd-A and Gd-B in the other venous blood sample to obtain the plasma clearance rate of Gd-A and the plasma clearance rate of Gd-B;

wherein, T is₁Is 40-80 min;

the T is₂Is 160-.

9. The assay of claim 8 wherein the plasma clearance of the isomers Gd-a and Gd-B is measured simultaneously with the glomerular filtration rate.

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