Method for detecting blood concentration of NTCP long-acting inhibitor through mass spectrum method
By enzymatically lying to generate characteristic peptides and using LCMS technology, the problem of insufficient sensitivity for blood drug concentration detection of NTCP long-acting inhibitors in the prior art was solved, and high sensitivity and accurate measurement were achieved, meeting the detection needs of pharmacokinetic tests.
Patent Information
- Application Number
- CN202311262838.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-27
- Publication Date
- 2025-03-28
AI Technical Summary
Existing detection methods cannot accurately determine the blood drug concentration of NTCP long-acting inhibitors with high sensitivity and accuracy, especially in pharmacokinetics tests, the detection requirements for polypeptide conjugates at longer time points cannot be met.
The content of the characteristic peptide was detected by enzymatically decomposing the sample using protease, and the concentration of the NTCP long-acting inhibitor peptide was calculated using liquid chromatography mass spectrometry (LCMS) technology, especially the MRM mode of triple tandem quadrupole mass spectrometry (QQQMS).
It has achieved high sensitivity and accurate determination of the blood drug concentration of NTCP long-acting inhibitors, which can meet the detection requirements of long-term post-dose points in pharmacokinetic tests, and overcomes the problems of limited detection sensitivity and inaccurate half-life determination in the prior art.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biological detection technology, and in particular to a method for detecting the blood concentration of a sodium / taurocholate cotransporting polypeptide (NTCP) long-acting inhibitor by mass spectrometry. Background Art
[0002] NTCP is a sodium taurocholate cotransporting polypeptide encoded by the SLC10A1 gene, containing 349 amino acid residues and 9 transmembrane domains, distributed on the basolateral membrane of hepatocytes. Natural NTCP is glycosylated and has a molecular weight of about 55kD; non-glycosylated NTCP has a molecular weight of about 37kD.
[0003] In recent years, reports have suggested that NTCP is a potential therapeutic target for obesity and type 2 diabetes. The main function of NTCP is to take up conjugated bile acids from plasma into hepatocytes in a sodium-dependent manner, playing an important role in the enterohepatic circulation of bile acids and promoting the body's digestion of lipids. NTCP is a common receptor for hepatitis B virus (HBV) and hepatitis D virus (HDV) to invade hepatocytes.
[0004] NTCP long-acting inhibitors form conjugates with serum albumin in vivo. Although there are currently a variety of blood drug concentration detection methods, such as ELISA, MSD (Meso Scale Discovery, electrochemiluminescence detection technology) or quadrupole time-of-flight mass spectrometry (Q-TOF), etc., the detection sensitivity of ELISA and MSD methods is limited, and because the molecular weight of the NTCP long-acting inhibitor polypeptide-serum albumin conjugate is large and the detection sensitivity of the quadrupole time-of-flight mass spectrometry (Q-TOF) is low, it cannot meet the blood drug concentration detection requirements of the polypeptide conjugate at a longer time point after administration in the pharmacokinetic (PK) test, and its half-life cannot be accurately determined. Moreover, after binding, the NTCP long-acting inhibitor polypeptide is also difficult to completely dissociate from the conjugated molecule, which makes it difficult to detect the complete molecule of the NTCP long-acting inhibitor in the plasma sample.
[0005] Therefore, there is an urgent need in the art to find a method for highly sensitive and accurate determination of the blood concentration of NTCP long-acting inhibitors. Summary of the invention
[0006] The invention provides a method for accurately measuring the blood concentration of a long-acting NTCP inhibitor with high sensitivity.
[0007] In a first aspect of the present invention, a method for non-diagnostic and non-therapeutic detection of the content of sodium / taurocholate cotransporting polypeptide (NTCP) long-acting inhibitor polypeptide in a sample is provided, comprising the following steps:
[0008] (Z1) providing a sample containing the NTCP long-acting inhibitor polypeptide;
[0009] (Z2) enzymatically hydrolyzing the sample using a protease to obtain a sample containing characteristic peptides; and
[0010] (Z3) detecting the content of the characteristic peptide, and calculating the concentration of the NTCP long-acting inhibitor polypeptide in the sample according to the content of the characteristic peptide;
[0011] Wherein, the NTCP long-acting inhibitor polypeptide is a polypeptide that can be enzymatically hydrolyzed to form the characteristic peptide, and the protease is an enzyme that can enzymatically hydrolyze the NTCP long-acting inhibitor polypeptide to obtain the characteristic peptide.
[0012] In another preferred embodiment, the characteristic peptide is an amino acid sequence having more than 85% homology with the sequence shown in SEQ ID NO: 1, and the N-terminus of SEQ ID NO: 1 is modified with myristoyl (Myr).
[0013] In another preferred example, the amino acid sequence of the NTCP long-acting inhibitor is as shown in SEQ ID NO: 2 or SEQ ID NO: 3, and the N-terminus of SEQ ID NO: 2 or SEQ ID NO: 3 is modified with Myr.
[0014] In another preferred embodiment, the C-terminus of SEQ ID NO: 2 or SEQ ID NO: 3 may also be modified with a maleimide-substituted C1-C8 acid.
[0015] In another preferred embodiment, the maleimide-substituted C1-C8 acid modification at the C-terminus is a maleimide-substituted C1-C8 acid modification at the C-terminus, and / or a maleimide-substituted C1-C8 acid modification at the 37th amino acid.
[0016] In another preferred embodiment, the maleimide-substituted C1-C8 acid is 3-maleimidopropionic acid (MPA), 6-maleimidocaproic acid, 5-maleimidopentanoic acid, 4-maleimidobutyric acid, or 4-(N-maleimidomethyl)cyclohexylcarboxylic acid.
[0017] In another preferred embodiment, the maleimide-substituted C1-C8 acid is MPA.
[0018] In another preferred embodiment, the protease is trypsin.
[0019] In another preferred embodiment, in step (Z3), the content of the characteristic peptide is detected by liquid chromatography-mass spectrometry (LCMS).
[0020] In another preferred embodiment, the liquid chromatography-mass spectrometry (LCMS) can be a triple tandem quadrupole mass spectrometry (QQQMS), a tandem quadrupole linear ion trap mass spectrometry (Q-TRAP), a quadrupole time-of-flight mass spectrometry (Q-TOF) or an ion trap-time-of-flight mass spectrometry (IT-TOF).
[0021] In another preferred embodiment, the liquid chromatography-mass spectrometry (LCMS) is a triple tandem quadrupole mass spectrometry (QQQMS).
[0022] In another preferred embodiment, the sample is selected from the following group: biological samples, environmental samples, product sampling; preferably, the sample is a biological sample; more preferably, the sample is a blood sample.
[0023] In a second aspect of the present invention, a method for non-diagnostic and non-therapeutic analysis of the half-life of a long-acting NTCP inhibitor polypeptide in a blood sample is provided, characterized in that the method comprises the following steps:
[0024] (S1) providing a blood sample;
[0025] (S2) mixing the NTCP long-acting inhibitor polypeptide with the blood sample, so that the NTCP long-acting inhibitor polypeptide reacts with plasma albumin in the blood sample;
[0026] (S3) enzymatically hydrolyzing the sample using a protease to obtain a sample containing characteristic peptides; and
[0027] (S4) detecting the content of the characteristic peptide, and calculating the concentration of the NTCP long-acting inhibitor polypeptide in the blood sample based on the content, and calculating the half-life of the NTCP long-acting inhibitor polypeptide based on the concentration;
[0028] Wherein, the NTCP long-acting inhibitor polypeptide is a polypeptide that can be enzymatically hydrolyzed to form the characteristic peptide, and the protease is an enzyme that can enzymatically hydrolyze the NTCP long-acting inhibitor polypeptide to obtain the characteristic peptide.
[0029] In another preferred embodiment, the characteristic peptide has an amino acid sequence with more than 85% homology to the sequence shown in SEQ ID NO: 1, and the N-terminus of SEQ ID NO: 1 is modified with Myr.
[0030] In another preferred example, the amino acid sequence of the NTCP long-acting inhibitor is as shown in SEQ ID NO: 2 or SEQ ID NO: 3, and the N-terminus of SEQ ID NO: 2 or SEQ ID NO: 3 is modified with Myr.
[0031] In another preferred embodiment, the C-terminus of SEQ ID NO: 2 or SEQ ID NO: 3 may also be modified with a maleimide-substituted C1-C8 acid.
[0032] In another preferred embodiment, the maleimide-substituted C1-C8 acid modification at the C-terminus is a maleimide-substituted C1-C8 acid modification at the C-terminus, and / or a maleimide-substituted C1-C8 acid modification at the 37th amino acid.
[0033] In another preferred embodiment, the maleimide-substituted C1-C8 acid is 3-maleimidopropionic acid (MPA), 6-maleimidocaproic acid, 5-maleimidopentanoic acid, 4-maleimidobutyric acid, or 4-(N-maleimidomethyl)cyclohexylcarboxylic acid.
[0034] In another preferred embodiment, the maleimide-substituted C1-C8 acid is MPA.
[0035] In another preferred example, in the method, the step (S4) further includes: continuously detecting the content of the characteristic peptide, and then drawing a change curve according to the change in the concentration of the NTCP long-acting inhibitor polypeptide detected at different times, so as to obtain the drug-time curve of the characteristic peptide, and calculating the half-life of the NTCP long-acting inhibitor polypeptide according to the drug-time curve.
[0036] In another preferred embodiment, in step (S4), the content of the characteristic peptide is detected by liquid chromatography-mass spectrometry (LCMS).
[0037] In another preferred embodiment, the liquid chromatography-mass spectrometry (LCMS) can be a triple tandem quadrupole mass spectrometry (QQQMS), a tandem quadrupole linear ion trap mass spectrometry (Q-TRAP), a quadrupole time-of-flight mass spectrometry (Q-TOF) or an ion trap-time-of-flight mass spectrometry (IT-TOF).
[0038] In another preferred embodiment, the liquid chromatography-mass spectrometry (LCMS) is a triple tandem quadrupole mass spectrometry (QQQMS).
[0039] In another preferred embodiment, the reaction time of step (S2) is 5-60 minutes.
[0040] In another preferred embodiment, in step (Z3), the protease is trypsin.
[0041] In a third aspect of the present invention, a kit is provided, the kit comprising a characteristic peptide having an amino acid sequence having more than 85% homology with the sequence shown in SEQ ID NO: 1, and the N-terminus of SEQ ID NO: 1 is provided with a Myr modification.
[0042] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS
[0043] Figure 1 A typical chromatogram of the 12M characteristic peptide measured using QQQMS in MRM mode is shown.
[0044] Figure 2 The accompanying standard curve 1 for the assay of Example 3 is shown.
[0045] Figure 3 The accompanying standard curve 2 for the assay of Example 3 is shown. DETAILED DESCRIPTION
[0046] After extensive and in-depth research, the inventors unexpectedly discovered a method for more objectively and accurately measuring the concentration of NTCP long-acting inhibitor polypeptides in blood. Specifically, the present invention uses the MRM mode of triple tandem quadrupole mass spectrometry (QQQMS) for quantitative detection of characteristic peptides of NTCP long-acting inhibitor polypeptides, thereby detecting the concentration of NTCP long-acting inhibitor polypeptides in blood. The present invention was completed on this basis.
[0047] the term
[0048] As used herein, the terms "NTCP long-acting inhibitor", "NTCP long-acting inhibitor polypeptide", or "NTCP long-acting inhibitor polypeptide" can be used interchangeably, including NTCP long-acting inhibitor polypeptide 1 (i.e., "12M" or "Compound 12M") and NTCP long-acting inhibitor polypeptide 2 (i.e., "34M" or "Compound 34M").
[0049] The amino acid sequence of NTCP long-acting inhibitor polypeptide 1 is as follows: SEN ID NO: 2 (GTNLSIPNPLGFFPDHQLDPLFRANSNNPDWDLNPNKDHWPEA NKVG), and the N-terminus of SEQ ID NO: 2 is modified with myristoyl (Myr), and the C-terminus of SEQ ID NO: 2 is modified with MPA, also referred to as compound 12M. As an alternative, the amino acid at position 37 of SEQ ID NO: 2 may also be modified with MPA.
[0050] The amino acid sequence of NTCP long-acting inhibitor polypeptide 2 is shown in SEQ ID NO: 3 (GTNLSIPNPLGFFPDHQLDPLFRANSNNPDWDYNPNKDHWPEANKVG), and the N-terminus of SEQ ID NO: 3 is modified with Myr, and the C-terminus of SEQ ID NO: 3 is modified with MPA, also known as compound 34M. As an alternative, the amino acid at position 37 of SEQ ID NO: 3 may also be modified with MPA.
[0051] As used herein, the term "protease" refers to a protease that can enzymatically cleave NTCP long-acting inhibitor polypeptide. In the present invention, the protease used is preferably trypsin.
[0052] As used herein, the term "characteristic peptide" refers to a peptide segment formed after the NTCP long-acting inhibitor polypeptide to be tested is hydrolyzed by a protease. In a preferred embodiment of the present invention, the enzyme fragments (i.e., enzyme fragments 1-23) formed after the NTCP long-acting inhibitor polypeptide is hydrolyzed by trypsin are characteristic enzyme fragments of the NTCP long-acting inhibitor polypeptide to be tested, and the fragments have no source in other species, therefore, they will not be interfered by conventional biological samples, such as inherent components of blood samples, and can be used as detection characteristic peptides.
[0053] In the present invention, “QQQ” refers to triple tandem quadrupole, and “QQQMS” refers to triple tandem quadrupole mass spectrometry.
[0054] The abbreviation "MRM" refers to Multiple Reaction Monitoring.
[0055] The abbreviation "PK" refers to pharmacokinetics.
[0056] Method for detecting the concentration of NTCP long-acting inhibitor polypeptide and albumin conjugate in samples
[0057] The present invention provides a method for detecting the concentration of a NTCP long-acting inhibitor polypeptide and an albumin conjugate in a sample, comprising the following steps:
[0058] (Z1) providing a sample containing the NTCP long-acting inhibitor polypeptide and an albumin conjugate;
[0059] (Z2) enzymatically hydrolyzing the sample using a protease to obtain a sample containing characteristic peptides; and
[0060] (Z3) detecting the content of the characteristic peptide, and calculating the concentration of the NTCP long-acting inhibitor polypeptide and albumin conjugate in the sample based on the content of the characteristic peptide.
[0061] Wherein, the NTCP long-acting inhibitor polypeptide is a polypeptide that can be coupled to albumin and enzymatically hydrolyzed to form the characteristic peptide, and the protease is an enzyme that can enzymatically hydrolyze the NTCP long-acting inhibitor polypeptide to obtain the characteristic peptide; preferably, the characteristic peptide has a sequence as shown in SEQ ID NO: 1 or an amino acid sequence having more than 85% (preferably more than 90%, more preferably more than 95%, and most preferably more than 99%) homology with the sequence as shown in SEQ ID NO: 1.
[0062] The inventor unexpectedly discovered that after a polypeptide (such as a long-acting NTCP inhibitor polypeptide) is combined with a sample containing albumin (such as a blood sample), a complex generated by part of the polypeptide can be enzymatically hydrolyzed by a protease, thereby generating a characteristic peptide as shown in SEQ ID NO: 1 or an amino acid sequence having more than 85% homology (preferably more than 90%, more preferably more than 95%, and most preferably more than 99%) with the sequence shown in SEQ ID NO: 1, and the N-terminus of the SEQ ID NO: 1 is modified by Myr. The inventor found that the characteristic peptide sequence does not exist in other common species (including mice, rats, dogs and humans, etc.), especially in commonly used blood samples, and therefore, it is a characteristic sequence of an albumin-polypeptide complex. By using a method with high precision and high specificity to detect it, the concentration of the albumin-polypeptide complex in the sample can be effectively detected.
[0063] In the present invention, the preferred protease is trypsin.
[0064] The method can be used to detect any polypeptide that can complex with albumin and be enzymatically hydrolyzed to form the characteristic sequence, which in a specific embodiment of the present invention is an NTCP long-acting inhibitor polypeptide; preferably, the NTCP long-acting inhibitor polypeptide has a sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3, and the N-terminus and / or amino acid at position 37 of SEQ ID NO: 2 or SEQ ID NO: 3 are modified by Myr.
[0065] In the above method, there is no particular limitation on the method for detecting the concentration of characteristic peptides, and methods known in the art for detecting the concentration of characteristic peptides or proteins can be used, such as competitive ELISA or MSD (electrochemiluminescence detection technology). Since the sample concentration is relatively low in some embodiments, a high-precision and high-specificity test method is required. Therefore, a preferred detection method is liquid chromatography-mass spectrometry (LCMS), such as triple tandem quadrupole mass spectrometry (QQQMS), tandem quadrupole composite linear ion trap mass spectrometry (Q-TRAP), quadrupole time-of-flight mass spectrometry (Q-TOF) or ion trap-time-of-flight mass spectrometry (IT-TOF; more preferably, the detection method of the detection is preferably triple tandem quadrupole mass spectrometry (QQQMS).
[0066] The sample is not particularly limited and can be a biological sample, an environmental sample, a product sample, etc. In a preferred embodiment, the sample is a biological sample; more preferably, the sample is a blood sample. In another preferred embodiment, the sample is a mammalian blood sample; wherein the mammal is a human or other non-human mammal (e.g., a rat blood sample or a beagle blood sample).
[0067] The method of the present invention can also be used to analyze the half-life of polypeptides in blood samples. Specifically, the method comprises the following steps:
[0068] (S1) providing a blood sample;
[0069] (S2) mixing the polypeptide with the blood sample, so that the polypeptide reacts with plasma albumin in the blood sample;
[0070] (S3) enzymatically hydrolyzing the sample using a protease to obtain a sample containing characteristic peptides; and
[0071] (S4) detecting the content of the characteristic peptide, and calculating the concentration of the polypeptide in the blood sample based on the content, and calculating the half-life of the polypeptide based on the concentration;
[0072] Wherein, the polypeptide is a polypeptide that can be coupled to albumin and enzymatically hydrolyzed to form the characteristic peptide, and the protease is an enzyme that can enzymatically hydrolyze the NTCP long-acting inhibitor polypeptide to obtain the characteristic peptide; preferably, the characteristic peptide has an amino acid sequence with 85% homology to the sequence shown in SEQ ID NO: 1, and the amino acid sequence of SEQ ID NO: 1 is: GTNL SIPNP LGFFP DHQLD PLFR, and the N-terminus of SEQ ID NO: 1 is modified by Myr.
[0073] In a preferred embodiment, in the method, the step (S4) further includes: continuously detecting the content of the characteristic peptide, and then drawing a change curve according to the change of the polypeptide concentration detected at different times, so as to obtain the drug-time curve of the characteristic peptide, and calculating the half-life of the polypeptide according to the drug-time curve.
[0074] In another preferred embodiment, in the method, the reaction time of step (S2) is 5-60 minutes.
[0075] The main advantages of the present invention include:
[0076] (a) The NTCP long-acting inhibitor polypeptide of the present invention is coupled to albumin, and there is no free state. This method is more specific than the ELISA method and is more suitable for detecting longer fragments of polypeptides. It has been determined through research that the characteristic peptide segment is derived from the enzymatic cleavage of the conjugate. After the blank plasma and the blank plasma are treated with the same enzymatic cleavage, no such characteristic peptide segment is detected. Therefore, the inherent components of plasma will not interfere with the test results. It overcomes the limited detection sensitivity of the ELISA method and the MSD method existing in the prior art; and because the molecular weight of the NTCP long-acting inhibitor polypeptide-serum albumin conjugate is large, the detection sensitivity of the quadrupole time-of-flight mass spectrometry (Q-TOF) is low, and it cannot meet the requirements for the blood drug concentration detection of the polypeptide conjugate at a longer time point after administration in the pharmacokinetic (PK) test, so that the half-life of the NTCP long-acting inhibitor polypeptide can be accurately determined.
[0077] (b) High sensitivity: This method uses the classic MRM mode for data acquisition, which can remove various interferences to the greatest extent. Experiments have shown that the LLOQ (Lower Limit of Quantification) detected by this method can reach 1 ng / ml (based on the blood drug concentration of 12M peptide), which is thousands of times more sensitive than the competitive ELISA method or MSD method (electrochemiluminescence detection technology), greatly improving the detection sensitivity.
[0078] In this way, even at low dosages, reliable data on drug half-life can be obtained. Because the 1-23 peptide segment is a characteristic peptide of the compound to be detected, the mass spectrometry detection process is not interfered by other proteins or peptides in the blood, and can objectively reflect the changes in the blood drug concentration of the compound to be detected.
[0079] (c) Wide quantitative range: This method can use a double calibration curve to extend the quantitative range to 1 ng / ml to 40 μg / ml, effectively covering the blood drug concentration range for intravenous and subcutaneous injections; each calibration curve has at least 6 concentration levels, and the correlation coefficient is greater than 0.99.
[0080] (d) High accuracy: The recovery rate of this method can reach 100% ± 15% from 1 ng / ml to 40 μg / ml, meeting the requirements of the Chinese Pharmacopoeia 2020 ChP <9012> and the requirements of the EMA Guideline on bioanalytical method validation.
[0081] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.
[0082] Example 1. Preparation of enzymatic hydrolysate of NTCP long-acting inhibitor polypeptide
[0083] 1.1 Preparation of test samples
[0084] 1.1.1 Preparation of a series of standard plasma solutions
[0085] Take a reference substance with a calibrated content, and quantitatively add the polypeptide reference substance to be tested into blank commercial Beagle dog plasma within a concentration range of 1 ng / ml to 40 μg / ml, and incubate at 37°C for 10-15 minutes to allow the polypeptide to fully couple with plasma albumin to prepare a series of standard plasma sample solutions, which are then frozen for storage.
[0086] 1.1.2 Preparation of the calibration curve and the enzymatic hydrolyzed samples of the samples to be tested
[0087] Take out the frozen plasma sample, thaw it at room temperature and mix it, then take an appropriate amount of plasma sample, add trypsin solution, incubate at 37℃ for 4 hours, add an appropriate amount of 0.1% TFA aqueous solution in the enzyme digestion solution at a ratio of 2:1 (v / v), terminate the reaction, and complete the preparation of characteristic peptides. A series of standard plasma solutions are subjected to enzyme digestion reaction in the same way to obtain enzyme digestion samples containing target peptides.
[0088] 1.2 Sample pretreatment
[0089] Take an appropriate amount of plasma sample solution and a series of standard plasma solutions after enzyme digestion, add methanol: water (80:20, v / v) at a ratio of 1:1 (v / v), vortex, centrifuge, take an appropriate amount of supernatant for solid phase extraction, and collect 10 μl of the eluate for mass spectrometry analysis.
[0090] Example 2. Screening of characteristic peptides and their specificity studies
[0091] Characteristic peptides were screened from the enzymatic hydrolysate of Example 1 and detected by mass spectrometry, as follows:
[0092] 2.1 Mass spectrometry parameters
[0093] Electrospray ionization source was used in positive ion mode. Q1 m / z 936 (3 + ion), Q2 m / z 1006.1 (2 fragments of amino acids 8-23 of the target peptide + ion).
[0094] 2.2 Data processing
[0095] The peak area of the extracted ion chromatogram from m / z936→1006 was used for the concentration of the series of standard plasma solutions, and a weighted least squares regression was performed according to 1 / x^2 to establish a calibration curve. The calibration curve R was not less than 0.99, and included at least 6 concentration levels. The recovery rate of each concentration level should be within 100%±15%. The blood drug concentration of the plasma sample was calculated using the accompanying calibration curve that met regulatory requirements.
[0096] 2.3 Sample preparation
[0097] The simulated plasma sample with 40 μg / ml standard addition was prepared in the same manner as in 1.1.1 of Example 1 and incubated at 37° C. for 120 hours. After the peptide was fully coupled to plasma albumin and fully interacted with the plasma contents, full scan analysis was performed using QTOF MS.
[0098] 2.4. Test results
[0099] No other peptide information (such as 1-25, 1-26...1-50) that could generate the target peptide 1-23 (SEQ ID NO: 1) after enzymatic digestion was detected in the plasma samples.
[0100] In addition, Blast analysis showed that fragment 1-23 was located on the hepatitis B virus pre-S1 antigen (HBV PreS1), and this sequence could not be found in other species (including mice, rats, and humans). The target peptide was not detected in the blank plasma of rats and Beagle dogs, indicating that this peptide segment does not exist in blank plasma. In addition, this peptide segment was not detected in the enzyme digestion product of blank plasma.
[0101] In addition, the 1-23 peptide segment of compound 12M (NTPC long-acting inhibitor polypeptide 1) and plasma protein coupling samples were analyzed by primary spectrum data, and no 1-23 peptide segment of compound 12M was detected; nor was any degradation fragment information that may be cleaved into 1-23 peptide segments, such as 1-25, 1-26...1-47 information, detected. However, after the NTCP inhibitor was added to the plasma and cleaved by enzyme, the above peptide segments could be detected. Therefore, the peptide segment with a length of 1-23 is a peptide fragment hydrolyzed from the coupling of compound 12M and plasma protein after enzyme cleavage. The peptide fragment has good specificity and can be used as a characteristic peptide for detecting NTCP long-acting inhibitor polypeptide 1.
[0102] Example 3. Study on the accuracy of the QQQ mass spectrometry method for analyzing the blood concentration of NTCP long-acting inhibitor polypeptide 1
[0103] 3.1 Detection of blood concentration of NTCP long-acting inhibitor peptide 1 by QQQMS
[0104] 3.1.1 Detection methods
[0105] Instrument model: AB Scix 7500+ QQQMS.
[0106] The present invention uses an accompanying standard curve (standard curve) to prove the accuracy of the method of the present invention. The standard curve preparation method is the same as 1.1.1 and 1.1.2 in Example 1. The synthesized and calibrated NTCP long-acting inhibitor polypeptide 1 (SEQ ID NO: 2) is accurately added to rat or Beagle dog plasma to prepare a 1ng / ml-40μg / ml simulated standard plasma solution, and after incubation at 37°C for 10min in vitro, 50μl of each is added with 0.2μg / μl Trypsin enzyme at a volume ratio of 1:1 for enzyme digestion at 37°C for 4h, and then 50μl 0.1% TFA aq. is added to terminate the enzyme digestion reaction to obtain an enzyme digestion sample.
[0107] After the digested sample was re-thawed at room temperature, it was treated with solid phase extraction (SPE) to remove the plasma interfering substances such as extra proteins and lipids in the sample, and the sample concentration was tested in MRM mode of QQQMS. The prepared concentration was fitted to the standard curve with the characteristic ion pair peak area, and the sample concentration was calculated by the external standard method. Then the determination was carried out according to the method of 1.1.2 in Example 1. The standard plasma sequence concentration covers the concentration range of the actual blood sample, and the same digestion and clearance steps are used to correct the errors that may be introduced during the plasma sample processing.
[0108] According to the concentration range of the actual sample, two standard curves are fitted according to the following requirements: each standard curve contains at least 6 concentration levels, and the recovery rate (accuracy) of each concentration point is 85-115% of the standard addition amount, and the correlation coefficient of each standard curve is not less than 0.99. The accuracy of the measurement results is guaranteed by "calibration" of the accompanying standard curve.
[0109] The detection results of the two standard curves are as follows Figure 2-3 As shown in Table 1-2, Figure 2 The calibration equation is y = 2312.14618x + 202.72979 (r = 0.99766, r 2 =0.99532)(weighted: 1 / x^2), Figure 3 The calibration equation is y = 1373.68413x + 6.56424e5 (r = 0.99459, r 2 =0.98922)(weighted: 1 / x^2).
[0110] Table 1
[0111] Sample name Sample type Actual concentration Calculate concentration Recovery rate % NTCP long-acting inhibitor peptide 1-1-1ng standard 1 1.01 100.63 NTCP long-acting inhibitor peptide 1-2-5ng standard 5 4.90 98.03 NTCP long-acting inhibitor peptide 1-3-25ng standard 25 23.63 94.54 NTCP long-acting inhibitor peptide 1-4-50ng standard 50 49.11 98.22 NTCP long-acting inhibitor peptide 1-5-250ng standard 250 249.50 99.81 NTCP long-acting inhibitor peptide 1-6-500ng standard 500 569.50 113.9 NTCP long-acting inhibitor peptide 1-7-1000ng standard 1000 948.80 94.88
[0112] Table 2
[0113] Sample name Sample type Actual concentration Calculate concentration Recovery rate % NTCP long-acting inhibitor peptide 1-6-500ng standard 500 480.80 96.17 NTCP long-acting inhibitor peptide 1-7-1000ng standard 1000 1119.00 111.93 NTCP long-acting inhibitor peptide 1-8-2500ng standard 2500 2231.00 89.22 NTCP long-acting inhibitor peptide 1-10-25000ng standard 25000 24660.00 98.62 NTCP long-acting inhibitor peptide 1-11-40000ng standard 40000 41620.00 104.06
[0114] from Figure 2-3 It can be seen that each standard curve includes at least 6 concentration levels; and the accuracy of each concentration point is 85-115% of the standard addition amount, and the standard plasma outside this range is excluded from the standard curve fitting; the correlation coefficient of each standard curve is not less than 0.99.
[0115] 3.1.2 Test results
[0116] The results show that:
[0117] a. The recovery rate of the MRM mode of QQQMS for the blood drug concentration detection value of 1ng / ml-40000ng / ml (40μg / ml) NTCP long-acting inhibitor polypeptide 1 is between 89.22% and 113.9%, which meets the acceptable range of recovery rate (% Recovery) of 80%-120%.
[0118] b. The MRM mode of QQQMS can detect the blood concentration of NTCP long-acting inhibitor peptide 1 with a sensitivity of up to 1 ng / ml.
[0119] At the rat standard plasma LLOQ (1 ng / ml), the characteristic peptide of compound 12M was determined by this method. The results are as follows: Figure 1As shown in the figure, the chromatographic peak at 4.243 min is the characteristic peptide signal of compound 12M. Quantitative analysis can be performed based on the peak area.
[0120] 3.2 ELISA method to detect the blood concentration of NTCP long-acting inhibitor peptide 1
[0121] 3.2.1 Detection methods
[0122] The present invention adopts ELISA method to detect the blood concentration of NTCP long-acting inhibitor polypeptide 1, and the specific steps include:
[0123] 1) Plate coating: Add 100 μL of coating solution to the wells of a 96-well blank ELISA plate, attach the sealing film and incubate at 2-8°C overnight (16-23h).
[0124] 2) Blocking: Shake out the liquid in the wells and pat dry on absorbent paper. Add 300 μL of blocking buffer to each well, apply the sealing film and incubate at room temperature for 2.5 hours.
[0125] 3) Sample dilution and incubation: Prepare 12M stock solution with a concentration of 3 mg / mL using whole plasma, incubate at 37°C for 30 min, and then dilute to the corresponding concentration using sample diluent. The sample diluent is 5% plasma and contains 0.6 μM Biotin-labeled compound 12M. After blocking, discard the liquid in the wells, pat dry with absorbent paper, add 100 μl / well of the diluted sample, incubate at room temperature for 1.5 h, and wash the plate 3 times with 300 μl / well of DPBST after incubation.
[0126] 4) Streptavidin incubation: Add 100 μl / well of streptavidin (1:2000 dilution) into the microwells. Incubate at room temperature for 1 hour. Wash the plate three times with DPBST after the incubation.
[0127] 5) Color development: Add TMB working solution into the microwells at 200 μL / well and incubate at room temperature for 20 min.
[0128] 6) Stop: Add 50 μL / well of stop solution into the microwells. Within 15 minutes after the stop solution is added, use a microplate reader to read the OD value at 450 / 630 nm.
[0129] 3.2.2 Test results
[0130] The test results are shown in Table 3.
[0131] Table 3
[0132]
[0133]
[0134] The test results show that:
[0135] a. The recovery rate of the competitive ELISA method for the blood drug concentration detection values of 25μg / ml-500μg / ml NTCP long-acting inhibitor polypeptide 1 was between 102.35% and 107.84%, meeting the acceptable range of recovery rate (%Reovcery) of 80%-120%; the coefficient of variation (Coefficient of Variation, CV) was in the range of 1.04%-9.26%, meeting the acceptable range of CV% of ±15%.
[0136] b. The competitive ELISA method has a sensitivity of 25 μg / ml for detecting the blood concentration of NTCP long-acting inhibitor polypeptide 1.
[0137] 3.3 Comparison of the accuracy of ELISA and QQQMS detection methods
[0138] The above test results are compared, and the results are shown in Table 4.
[0139] Table 4
[0140]
[0141] From the table above we can see that:
[0142] 1. The recoveries of the MRM mode of QQQMS and the competitive ELISA method for the detection results of compound 12M at 40μg / ml and 25μg / ml both met the acceptable range of 80%-120%, meeting the regulatory requirements for biological sample detection.
[0143] 2. The MRM mode of QQQMS and the competitive ELISA method were used to detect the blood drug concentration of compound 12M 40μg / ml and 25μg / ml. The coefficients of variation between the detection results of the two methods were 2.68% and 2.62%, respectively, which met the acceptable range of CV% value of ±15% and met the regulatory requirements for biological sample testing.
[0144] in conclusion:
[0145] 1. The MRM mode of QQQMS for the detection of compound 12M blood concentration is highly consistent with the competitive ELISA method, and the results are reliable. This method can be used to detect the blood concentration of compound 12M.
[0146] 2. The detection sensitivity of the MRM mode of QQQMS for the blood concentration of compound 12M reached 1 ng / ml, which was much higher than the detection sensitivity of the competitive ELISA method of 25 μg / ml.
[0147] discuss
[0148] The present invention uses the characteristic peptide after trypsin cleavage to study the drug-time curve of compound 12M. On the one hand, it covers the two states of free compound 12M and compound 12M-conjugate, and the detection result is more objective; on the other hand, it greatly improves the sensitivity. The lowest limit of quantification (LLOQ) of the characteristic peptide QQQ detection can reach 1 ng / ml, which is thousands of times more sensitive than the competitive ELISA method or MSD method (electrochemiluminescence detection technology).
[0149] In this way, even at low dosages, reliable data on the half-life of the drug can be obtained. Because the 1-23 peptide segment is a characteristic peptide of compound 12M, the mass spectrometry detection process is not interfered by other proteins or peptides in the blood, and can objectively reflect the changes in the blood concentration of compound 12M.
[0150] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.
Claims
1. A non-diagnostic and non-therapeutic method for detecting the content of a sodium / taurocholate cotransporting polypeptide (NTCP) long-acting inhibitor polypeptide in a sample, characterized in that: The following steps are involved: (Z1) providing a sample containing the NTCP long-acting inhibitor polypeptide; (Z2) using a protease to enzymatically hydrolyze the sample to obtain a sample containing characteristic peptides; and (Z3) detecting the content of the characteristic peptide, and calculating the concentration of the NTCP long-acting inhibitor polypeptide in the sample according to the content of the characteristic peptide; Wherein, the NTCP long-acting inhibitor polypeptide is a polypeptide that can be enzymatically hydrolyzed to form the characteristic peptide, and the protease is an enzyme that can enzymatically hydrolyze the NTCP long-acting inhibitor polypeptide to obtain the characteristic peptide.
2. The method according to claim 1, characterized in that The characteristic peptide has an amino acid sequence with more than 85% homology to the sequence shown in SEQ ID NO: 1, and the N-terminus of SEQ ID NO: 1 is modified with myristoyl.
3. The method according to claim 2, characterized in that The amino acid sequence of the NTCP long-acting inhibitor is as shown in SEQ ID NO: 2 or SEQ ID NO: 3, and the N-terminus of SEQ ID NO: 2 or SEQ ID NO: 3 is modified with myristoyl; preferably, the C-terminus of SEQ ID NO: 2 or SEQ ID NO: 3 is modified with a maleimide-substituted C1-C8 acid.
4. The method according to claim 3, characterized in that The maleimide-substituted C1-C8 acid modification at the C-terminus is a maleimide-substituted C1-C8 acid modification at the C-terminus, and / or a maleimide-substituted C1-C8 acid modification at the amino acid at position 37; Preferably, the maleimide-substituted C1-C8 acid is 3-maleimidopropionic acid, 6-maleimidohexanoic acid, 5-maleimidopentanoic acid, 4-maleimidobutyric acid or 4-(N-maleimidomethyl)cyclohexylcarboxylic acid, and the maleimide-substituted C1-C8 acid is preferably 3-maleimidopropionic acid.
5. The method according to any one of claims 1 to 4, characterized in that The protease is trypsin; preferably, the sample is selected from the group consisting of biological samples, environmental samples and product sampling; preferably, the sample is a biological sample; more preferably, the sample is a blood sample.
6. The method according to any one of claims 1 to 5, characterized in that In step (Z3), the content of the characteristic peptide is detected by liquid chromatography-mass spectrometry (LCMS); preferably, the liquid chromatography-mass spectrometry (LCMS) is a mass spectrometry method selected from the following groups: triple tandem quadrupole mass spectrometry (QQQMS), tandem quadrupole composite linear ion trap mass spectrometry (Q-TRAP), quadrupole time-of-flight mass spectrometry (Q-TOF) and ion trap-time-of-flight mass spectrometry (IT-TOF).
7. A method for non-diagnostic and non-therapeutic analysis of the half-life of a long-acting NTCP inhibitor polypeptide in a blood sample, characterized in that: The method comprises the following steps: (S1) providing a blood sample; (S2) mixing the NTCP long-acting inhibitor polypeptide with the blood sample, so that the NTCP long-acting inhibitor polypeptide reacts with the plasma albumin in the blood sample, preferably for 5-60 minutes; (S3) enzymatically hydrolyzing the sample using a protease to obtain a sample containing characteristic peptides; and (S4) detecting the content of the characteristic peptide, and calculating the concentration of the NTCP long-acting inhibitor polypeptide in the blood sample according to the content of the characteristic peptide, and calculating the half-life of the NTCP long-acting inhibitor polypeptide according to the concentration; Wherein, the NTCP long-acting inhibitor polypeptide is a polypeptide that can be enzymatically hydrolyzed to form the characteristic peptide, and the protease is an enzyme that can enzymatically hydrolyze the NTCP long-acting inhibitor polypeptide to obtain the characteristic peptide; preferably, the characteristic peptide has an amino acid sequence with more than 85% homology to the sequence shown in SEQ ID NO: 1, and the N-terminus of the SEQ ID NO: 1 is provided with a myristoyl modification.
8. The method according to claim 7, characterized in that The amino acid sequence of the NTCP long-acting inhibitor is as shown in SEQ ID NO: 2 or SEQ ID NO: 3, and the N-terminus of SEQ ID NO: 2 or SEQ ID NO: 3 is modified with myristoyl; preferably, the C-terminus of SEQ ID NO: 2 or SEQ ID NO: 3 is modified with a maleimide-substituted C1-C8 acid.
9. The method according to claim 8, characterized in that The maleimide-substituted C1-C8 acid modification at the C-terminus is a maleimide-substituted C1-C8 acid modification at the C-terminus, and / or a maleimide-substituted C1-C8 acid modification at the amino acid at position 37; Preferably, the maleimide-substituted C1-C8 acid is 3-maleimidopropionic acid, 6-maleimidohexanoic acid, 5-maleimidopentanoic acid, 4-maleimidobutyric acid, 4-(N-maleimidomethyl)cyclohexylcarboxylic acid, and the maleimide-substituted C1-C8 acid is preferably 3-maleimidopropionic acid.
10. The method according to any one of claims 7 to 9, characterized in that In the method, the step (S4) further comprises: continuously detecting the content of the characteristic peptide, and then drawing a change curve according to the change in the concentration of the NTCP long-acting inhibitor polypeptide detected at different times, thereby obtaining a drug-time curve of the polypeptide, and calculating the half-life of the NTCP long-acting inhibitor polypeptide according to the drug-time curve.
11. The method according to any one of claims 6 to 8, characterized in that In step (S4), the content of the characteristic peptide is detected by liquid chromatography-mass spectrometry (LCMS); preferably, the liquid chromatography-mass spectrometry (LCMS) is a mass spectrometry method selected from the following group: triple tandem quadrupole mass spectrometry (QQQMS), tandem quadrupole composite linear ion trap mass spectrometry (Q-TRAP), quadrupole time-of-flight mass spectrometry (Q-TOF) and ion trap-time-of-flight mass spectrometry (IT-TOF).
12. A kit, characterized in that: The kit comprises a characteristic peptide having an amino acid sequence with more than 85% homology to the sequence shown in SEQ ID NO: 1, and the N-terminus of SEQ ID NO: 1 is modified with myristoyl.