Vacuum blood collection tubes containing protease inhibitors for evaluating contact system activation

Abstract

To solve the problem in which measurement of pathway-specific biomarkers (e.g., 2-chain high molecular weight kininogen) may overestimate the degree of contact system activation that is present within a patient body, unless blood is carefully collected and processed.SOLUTION: Disclosed herein are evacuated blood collection tubes comprising protease inhibitor cocktails in a liquid form and uses thereof for assessing features associated with the contact system in a subject, including the endogenous level of contact system activation, the endogenous level of a drug that targets a component of the contact system during treatment, and / or the immunogenicity of such a drug.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims the benefit of the filing dates of U.S. Provisional Application No. 62 / 204,644, filed on August 13, 2015, and U.S. Provisional Application No. 62 / 214,308, filed on September 4, 2015. The entire contents of each of these referenced applications are incorporated herein by reference.

[0002] Accurately measuring the in - vivo level of contact - system activation using a patient's plasma is difficult because the contact system tends to be activated ex - vivo during blood collection. In particular, blood from patients with certain diseases related to the contact system, such as hereditary angioedema, has a tendency to activate the contact system ex - vivo because the C1 inhibitor, which is a natural inhibitor of this pathway, is insufficient. Therefore, in the measurement of pathway - specific biomarkers (such as double - stranded high - molecular - weight kininogen), if the blood is not carefully collected and processed, the degree of contact - system activation present in the patient's body may be overestimated.

Summary of the Invention

[0003] The present disclosure is based, at least in part, on the development of a non - glass vacuum blood collection tube that contains a protease inhibitor mixture (cocktail) in a liquid formulation that can prevent ex - vivo activation of the contact system during blood collection. Thus, the vacuum blood collection tube described herein enables accurate measurement of the endogenous level of contact - system activation in patients, particularly in patients in whom the natural inhibitor of this pathway (such as C1 inhibitor) is insufficient.

[0004] Thus, one aspect of the present disclosure features a vacuum blood collection tube that contains a liquid formulation that includes a protease inhibitor mixture that substantially does not contain protease inhibitors that are unstable in aqueous solution. This tube may be a non - glass tube. In some embodiments, the tube is made of plastic. In some embodiments, the present vacuum blood collection tube contains any of the 0.5 mL liquid formulations described herein that can be diluted 10 - fold during use.

[0005] In some embodiments, the protease inhibitor mixture in a vacuum blood collection tube described herein comprises at least one serine protease inhibitor (e.g., a plasma kallikrein inhibitor) and at least one cysteine ​​protease inhibitor. In one example, the protease inhibitor mixture comprises EPI-KAL2 (which may be biotinylated) and leupeptin. The amount of EPI-KAL2 may be in the range of 5 to 15 μM in the liquid formulation containing them. Alternatively or in addition, the amount of leupeptin may be in the range of 200 to 300 μM in the liquid formulation.

[0006] In some embodiments, the protease inhibitor mixture described herein may contain at least two serine protease inhibitors, at least one of which is a trypsin inhibitor, such as a soybean trypsin inhibitor. In some examples, the protease inhibitor mixture includes benzamidine, a soybean trypsin inhibitor, leupeptin, and AEBSF. In some examples, the liquid formulation in the vacuum blood collection tube may contain 80–120 mM benzamidine, 1–3 mg / ml soybean trypsin inhibitor, 200–300 μM leupeptin, and / or 10–30 mM AEBSF.

[0007] The liquid formulation in any of the vacuum blood collection tubes described herein may further contain polyblen and EDTA. In some embodiments, any of the liquid formulations described herein may have a pH of 4 to 6 (e.g., 4.5).

[0008] In another embodiment, the Disclosure provides a method for evaluating the endogenous level of contact system activation in a subject. The method comprises (i) collecting blood from the subject into one of the vacuum blood collection tubes described herein, (ii) processing the blood to produce a plasma sample, and (iii) measuring the level of contact system activation in the plasma sample. In some embodiments, the measurement step (step (iii)) can be performed by measuring the level of one or more biomarkers indicating contact system activation. Such biomarkers may include prekallikrein, activated plasma kallikrein (pKal), α2M-pKal complex, activated factor XII, activated factor XI, high molecular weight kininogen (HMWK), and / or bradykinin metabolites. In one example, the one or more biomarkers include cleaved HMWK and / or whole HMWK.

[0009] In yet another aspect, the Disclosure provides a method for evaluating the level of a drug that targets a contact system in a subject. The method includes (i) collecting blood from a subject that has been administered a drug that targets components of a contact system into a vacuum blood collection tube as described herein; (ii) processing the blood to produce a plasma sample; and (iii) measuring the level of the drug in the plasma sample.

[0010] Furthermore, this disclosure provides a method for evaluating the immunogenicity of a contact system-targeting drug, comprising the steps of (i) collecting blood from a subject to which a contact system-targeting drug has been administered into a vacuum blood collection tube as described herein, (ii) processing the blood to produce a plasma sample, and (iii) measuring the level of antibodies that bind to the drug in the plasma sample. Such a method may further include a step of isolating antibodies that bind to the drug from the plasma sample prior to step (iii). In some examples, anti-drug antibodies (ADAs) can be isolated by solid-phase extraction and acid dissociation (SPEAD) assays.

[0011] In any of the methods described herein, the subject may be a human patient who can be treated with a drug that optionally targets a component of the contact system (e.g., plasma kallikrein), such as a drug that specifically targets plasma kallikrein (e.g., the active form of plasma kallikrein) (e.g., an antibody). In some cases, the blood is derived from a human patient with a disease related to the contact system, such as hereditary angioedema (HAE) or idiopathic angioedema. In some cases, the human patient has HAE with normal C1 inhibitor (C1-INH).

[0012] In any of the methods described herein, the vacuum blood collection tube may not be the first tube filled with blood from the subject. Alternatively or in addition, the processing step [step(ii)] may be performed within one hour after the blood collection step [step(i)].

[0013] Details of one or more embodiments of the present invention are described below in the “Modes for Carrying Out the Invention.” Other features or advantages of the present invention will become apparent from the following drawings and detailed descriptions of some embodiments, as well as from the appended claims.

[0014] The following drawings form part of this specification and are included to further illustrate specific aspects of this disclosure, which can be better understood by referring to one or more of these drawings together with the detailed descriptions of the specific embodiments provided herein. [Brief explanation of the drawing]

[0015] [Figure 1] This photograph shows that SCAT169 and SCAT153 tubes prevented contact activation when measured by a double-stranded Western blot assay. Adding 10% ellagic acid to plasma activated the contact system, resulting in the conversion of single-stranded HMWK to double-stranded HMWK (see sodium citrate plasma). In contrast, SCAT169 and SCAT153 plasmas contained the same amount of single-stranded HMWK before and after ellagic acid addition. [Figure 2] This graph shows the variability of kininogen cleavage (2-HMWK / cHMWK ratio) based on the sample collection method in plasma from healthy subjects. The clinical site of collection and the type of tube used for collection, including K2EDTA (EDTA), sodium citrate, SCAT169, or P100, are indicated. The data points are grouped from left to right as follows: Site 1: EDTA, Site 2: Citrate, Site 3: Citrate, Site 4: Citrate, Site 1: SCAT169, Site 2: SCAT169, Site 4: SCAT169, Site 5: SCAT169, Site 4: P100. [Figure 3] This graph shows kininogen cleavage (2-HMWK / cHMWK ratio) in plasma collected in SCAT169 tubes from healthy subjects in different clinical settings. The data points are grouped from left to right as follows: Site 1: Commercial supplier, Site 4, Site 5, Sites 4 and 5. [Figure 4] This graph shows kininogen cleavage (2-HMWK / cHMWK ratio) in healthy subjects compared to subjects with type I or type II HAE, nC1-INH type HAE, and idiopathic angioedema (AE). The data points are grouped from left to right as follows: healthy subjects, baseline for type I / II HAE, type I / II HAE attacks, baseline for HAE (nC1-INH), HAE (nC1-INH) attacks, baseline for idiopathic AE, and idiopathic AE attacks. [Figure 5] This graph shows double-stranded HMWK levels in plasma from healthy subjects and patients with HAE. A: Shows the percentage of double-stranded HMWK levels in plasma from healthy subjects. As shown in the graph, clinical setting C did not use an initial discard tube before collecting SCAT169 plasma. B: Shows the percentage of double-stranded HMWK levels in plasma from patients with HAE. [Modes for carrying out the invention]

[0016] This disclosure is based, at least in part, on the development of a vacuum blood collection tube containing a cocktail of protease inhibitors that prevents contact system activation. Careful blood collection and processing are important to accurately assess the endogenous level of contact system activation in patients or healthy volunteers. Accurate assessment of the contact system-related characteristics described herein may be ensured by considering one or more of the following precautions for use. (i) Preferably, the vacuum blood collection tube described herein is not the first tube filled with blood, which may show increased contact system activation due to local trauma following vascular puncture with a needle. (ii) It is best to avoid contact between this blood and the glass (a plastic tube or catheter should be used). (iii) It is better to process the blood into plasma within a short period of time after collection (for example, within about 1 hour). and / or (iv) It is preferable to use a protease inhibitor in the collection tube to stabilize the plasma against in vitro contact activation, which may interfere with the accurate determination of the patient's endogenous state.

[0017] The advantages of the vacuum blood collection tubes described herein include at least (1) the use of non-glass (e.g., plastic) vacuum tubes for standardized and simplified blood collection, (2) the use of liquid formulations containing a cocktail of protease inhibitors to minimize hydrolysis, (3) the optional omission of protease inhibitors that are unstable in aqueous solution (e.g., PPACK II, also known as HD-Phe-Phe-Arg-chloromethyl ketone), and (4) inclusion of a plasma kallikrein inhibitor such as EPI-KAL2 (which may be biotinylated), which in some embodiments gives the tube the ability to contain reagents that enable the detection of activated plasma kallikrein by immunoassay. See, for example, International Publication No. 95 / 21601, the relevant disclosure of which is incorporated herein by reference.

[0018] An unexpected observation from this study was that hemolysis was prevented or reduced by the use of a liquid-form protease inhibitor cocktail. When blood was collected in a vacuum tube containing a lyophilized preparation of the protease inhibitor, a large portion of it lyzed, which could interfere with the measurement of certain samples. However, when blood was collected in a vacuum tube containing a solution of the same protease inhibitor mixture, hemolysis did not occur.

[0019] Therefore, measurements aimed at evaluating the degree of contact system activation in plasma samples processed from blood samples collected in the vacuum tubes described herein can yield more accurate evaluation levels from patients with different diseases. Obtaining an accurate estimate of contact activation can enable the identification of diseases or subsets of patients with different diseases that are potentially mediated by this pathway and are therefore suitable for treatment with contact system inhibitors.

[0020] Furthermore, the use of vacuum blood collection tubes as described herein facilitates the accurate determination of drug levels of therapeutic molecules against activated forms of proteins in contact systems (e.g., plasma kallikrein, FXIIa, and double-stranded kininogen) and / or assessment of immunogenicity. The tubes offer similar advantages to therapeutic molecules targeted to downstream activated proteins of contact system activation that do not require calcium for the production of activated targets (e.g., FXIa and FIXa). The advantages of the tubes primarily apply to biological therapeutic molecules, because the PK and immunogenicity assays used are typically immunoassays that recognize binding sites (e.g., idiotypes in the case of therapeutic antibodies). When therapeutic targets are activated in vitro, they may bind to biological therapeutic molecules present in plasma, thereby interfering with detection in PK and immunogenicity immunoassays. Protease inhibitors in the tubes can prevent target activation. The use of liquid formulations prevents hemolysis, which can interfere with certain laboratory assays.

[0021] Vacuum blood collection tube containing a protease inhibitor cocktail in a liquid formulation Vacuum blood collection tubes are commonly used in medical practice for the collection of blood samples for various purposes. The tubes described herein may be non-glass tubes containing a liquid formulation comprising a protease inhibitor mixture (protease inhibitor cocktail). In some embodiments, the protease inhibitor cocktail may comprise at least one serine protease inhibitor and at least one cysteine protease inhibitor. The at least one serine protease inhibitor may be a plasma kallikrein inhibitor. Such a protease inhibitor cocktail may comprise multiple (e.g., 2, 3, 4 or 5) serine protease inhibitors, at least one of which may be a trypsin or human plasmin inhibitor. Preferably, the protease inhibitor cocktail described herein substantially does not contain protease inhibitors that are unstable in aqueous solution, i.e., the activity of protease inhibitors that are unstable in aqueous solution is very small relative to the total inhibitory activity of the protease cocktail. In some cases, the amount of protease inhibitor that is unstable in aqueous solution may be less than 5% (w / w), e.g., less than 2%, less than 1% or less than 0.5% of the total protease inhibitors in the cocktail. In some cases, the protease inhibitor cocktail may not contain any protease inhibitors that are unstable in aqueous solution (e.g., an aqueous solution having a pH of 4 - 6). An example of a protease inhibitor that is not stable in aqueous solution is PPACK II, also known as H-D-Phe-Phe-Arg-chloromethyl ketone.

[0022] Table 1 below lists exemplary serine protease inhibitors, cysteine protease inhibitors and trypsin protease inhibitors that can be used to prepare the protease inhibitor cocktails described herein.

Table 1

[0023] In some examples, a protease inhibitor cocktail used to prepare vacuum blood collection tubes may contain at least one (e.g., 1, 2, or 3) serine protease inhibitor, which may also contain at least one (e.g., 1, 2, or 3) trypsin / plasmin inhibitors and at least one (e.g., 1, 2, or 3) cysteine ​​protease inhibitors. Such a protease inhibitor cocktail may contain three serine protease inhibitors (e.g., trypsin / plasmin inhibitors such as benzamidine, AEBSF, and soy trypsin inhibitors) and one cysteine ​​protease inhibitor (e.g., leupeptin).

[0024] In other examples, the protease inhibitor cocktail may include at least one serine protease inhibitor (e.g., a plasma kallikrein inhibitor) and at least one cysteine ​​protease inhibitor (e.g., leupeptin). The plasma kallikrein inhibitor may be EPI-KAL2 (Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, SEQ ID NO: 1), which is a specific plasma kallikrein recombinant protease inhibitor that gives the tube the ability to contain reagents that enable the detection of activated plasma kallikrein, for example, by immunoassay.

[0025] Any of the protease inhibitor cocktails may be dissolved in a suitable solution to form a liquid formulation. The suitable solution may be an acid-citrate-dextrose solution, which may contain trisodium citrate, citric acid, and dextrose. This solution may have a pH value of about 4-6, 4-5, 4.5-5.0, or 4.2-4.7, for example, 4.5. In some embodiments, this solution has a pH value of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. In some embodiments, this solution has a pH value of about 4.5. The liquid formulation may further contain a cationic polymer, such as a hexadimethrin bromide molecule (Polybrene®), which can reduce contact system activation through interaction with a negatively charged surface, and a chelating agent (e.g., EDTA) that can inhibit metalloproteinases.

[0026] The concentration of each protease inhibitor in the cocktail may be 5 or 10 times higher than the final concentration of such inhibitor used to inhibit the corresponding protease, depending on the dilution factor in the implementation. The final concentrations of specific commercially available protease inhibitors are known in the art and can be obtained from the manufacturer's protocol. In some examples, the concentration of EPI-KAL2 may be in the range of 5–15 μM (e.g., 5–10, 7–12 μM, or 10–15 μM). In some embodiments, the concentration of EPI-KAL2 is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or about 15 μM. In some examples, the concentration of leupeptin may be in the range of 200–300 μM (e.g., 200–250, 240–270, or 250–300 μM). In some embodiments, the concentration of leupeptin is approximately 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or approximately 300 μM. In some examples, the concentration of the soy trypsin inhibitor may be in the range of 1–3 mg / ml (e.g., 1–2 or 2–3 mg / ml). In some embodiments, the concentration of the soy trypsin inhibitor is approximately 1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or approximately 3.0 mg / mL. In some examples, the concentration of benzamidine may be in the range of 80–120 mM (e.g., 80–100 or 100–120 mM). In some embodiments, the concentration of benzamidine is approximately 80, 85, 90, 95, 100, 105, 110, 115, or approximately 120 mM. In some examples, the concentration of AEBSF may be in the range of 10–30 mM (e.g., 10–20 or 20–30 mM). In some embodiments, the concentration of AEBSF is approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or approximately 30 mM.

[0027] When using peptide protease inhibitors (e.g., EPI-KAL2), they may be biotinylated according to conventional methodologies. For example, peptide inhibitors may be biotinylated as follows: In short, the peptide inhibitor can be dissolved in a suitable solution such as phosphate-buffered saline (PBS). The freshly prepared sulfo-NHS-LC-biotin can be added to the peptide inhibitor solution and incubated on ice for a suitable period of time. Excess unreactive hydrolyzed biotin can be removed using a spin desalting column. Labeling of the peptide inhibitor can be confirmed by ELISA, and the protein concentration can be measured by, for example, a Bradford assay.

[0028] Any of the liquid formulations described herein can be prepared by conventional methods, for example, by dissolving the appropriate components in a suitable solution, and preferably placed in a non-glass vacuum blood collection tube. The tube may be stored at -20°C and may be thawed on ice or at a refrigerated temperature (e.g., about 4°C) within a suitable period before use.

[0029] The usefulness of vacuum blood collection tubes containing liquid-form protease inhibitor cocktails. Using any of the vacuum blood collection tubes described herein, blood samples can be collected from a subject for use in analyzing endogenous characteristics related to the contact system, such as the level of contact system activation, serum levels of drugs that target components of the contact system, and / or the immunogenicity of such drugs. To reduce in vitro activation of the contact system (e.g., activation due to local trauma after needle puncture), the vacuum blood collection tubes described herein do not have to be the first tube filled with blood when blood is collected from the subject. For example, the initial blood from the subject may be collected in the first tube, which may be discarded, and then a vacuum blood collection tube may be used to collect subsequent blood samples that can be used for analysis. The first tube may be a regular blood collection tube used in normal practice.

[0030] After blood collection, the blood sample may be processed to generate a plasma sample within a suitable timeframe (e.g., within 1 hour). The plasma sample can then be subjected to further analysis to evaluate the characteristics related to the contact system from which the initial blood sample was obtained.

[0031] Blood samples may be taken from subjects requiring the analysis described herein. In some cases, such subjects may be human patients who have, are suspected of having, or are at risk of having, a disease related to the contact system. For example, a human patient may have a history of HAE or be at risk of HAE. A human patient may have type I or type II HAE with C1-INH deficiency or production of abnormal C1-INH. Alternatively, a human patient may have type III HAE not associated with C1-INH deficiency. In other cases, a human patient may have a history of idiopathic angioedema or be at risk of idiopathic angioedema. Such human patients may have previously been treated with or are currently being treated with drugs that target components of the contact system (e.g., pKal or FXIIa or high molecular weight kininogen).

[0032] i. Evaluation of the endogenous level of contact system activation In one embodiment, the endogenous level of contact system activation in a subject can be assessed by analyzing a plasma sample described herein, and the subject may be a human patient who has, is suspected of having, or is at risk of having a contact system-related disease (e.g., HAE or idiopathic angioedema). Such a human patient may be receiving treatment for the disease, such as treatment using pKal inhibitors (e.g., anti-pKal antibodies). In another embodiment, such a human patient may not be receiving such treatment. Alternatively, the human subject may be a healthy subject who does not have such a disease.

[0033] The level of contact system activation in a plasma sample can be measured by measuring one or more biomarkers that indicate contact system activation.

[0034] Plasma kallikrein (pKal) is the primary bradykinin-producing enzyme in the circulatory system. Activation of pKal can occur via the contact cascade or factor XIIa, both linked to the disease pathology associated with hereditary angioedema (HAE). Plasma kallikrein circulates primarily as an inactive enzyme precursor called prekallikrein, bound to its substrate, high molecular weight kininogen (HMWK). In response to stimulation, prekallikrein is cleaved to form active plasma kallikrein. This activation of kallikrein can be mediated, for example, by factor XIIa after the activation of FXII to FXIIa, or by effectors in the contact cascade. Approximately 75–90% of circulating prekallikrein is bound to HMWK via inactive-site interactions with domain 6 of HMWK, which hydrolyzes further molecules of HMWK to produce cleaved HMWK and bradykinin. Active plasma kallikrein cleaves HMWK at two sites, releasing bradykinin, a key mediator of pain, inflammation, edema, and angiogenesis. Other cleavage products, such as cleaved kininogen, contain amino acid chains held together by disulfide bonds. Cugno et al., Blood (1997) 89:3213-3218.

[0035] Table 2 below shows example biomarkers that can be used to assess the level of contact system activation in a patient's blood sample (and thus determine whether the patient has elevated levels and / or activity of the contact system, e.g., elevated levels or activity of pKal). [Table 2] JPEG0007829634000005.jpg69158

[0036] One or more biomarkers indicating contact system activation can be analyzed using conventional methods. One particularly preferred assay for qualitative, semi-quantitative, or quantitative detection is immunoassay. Immunoassay is any assay that detects and / or quantifies a target molecule (e.g., a biomarker molecule associated with contact system activation) using a conjugate described herein that specifically binds to the target molecule. The conjugate may be an antibody, and the antibody may be a full-length antibody or its antigen-binding fragment. Immunoassay may be competitive or non-competitive, and may be homogeneous or heterogeneous. For example, immunoassays for detecting contact biomarkers may include enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), chemiluminescence immunoassay (CLIA), counting immunoassay (CIA), immunoenzymometric assay (IEMA) using monoclonal antibodies, enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay, sandwich immunoassay, immunoPCR assay, proximity ligation assay, Western blot assay, or immunoprecipitation assay. Further preferred immunoassays for detecting the biomarkers described herein will be apparent to those skilled in the art. However, this disclosure is not limited to immunoassays, and detection assays not based on antibodies or antigen-binding antibody fragments, such as mass spectrometry, will also be apparent to those skilled in the art as being useful for the detection and / or quantification of contact biomarkers described herein.

[0037] The type of detection assay used for the detection and / or quantification of contact biomarkers described herein depends on several parameters, including the specific context in which the assay is used (e.g., clinical or research use), the type and number of biomarkers to be detected, and the type and number of patient samples performed simultaneously. For example, elevated levels of cleaved kininogen (double-stranded kininogen) can be detected using a Western blot assay in plasma samples taken from HAE patients experiencing acute HAE symptoms or from healthy subjects. While Western blot assays allow for the simultaneous analysis of contact biomarkers in multiple samples, they are limited in the number of biomarkers that can be evaluated simultaneously. Therefore, in some embodiments, when analyzing multiple contact biomarkers described herein in a single sample or multiple samples, assays suitable for such multiplex analysis are preferred. Examples of such assays, but not limited to, include peptide microarrays and lab-on-a-chip assays configured to provide high throughput, and multiplex and rapid assays as an alternative to less scalable immunoassays such as Western blots.

[0038] In some cases, plasma samples can be placed in multiwell microplates in or without the presence of a pKal inhibitor and / or a contact system activator. This mixture can be incubated in the presence of a pKal-labeled peptide substrate on ice for a suitable period (e.g., 2 minutes), and the activation reaction can be stopped by adding a Corn Trypsin Inhibitor (CTI) to the mixture. This mixture can be diluted if necessary, and the proteolytic activity can be measured by measuring the level of the fluorescent peptide substrate. The results obtained from such assays can be relied upon to determine the endogenous level of contact system activation of the subject from which the plasma sample was obtained. These results can also be relied upon to determine the inhibitory activity of the pKal inhibitor if one is used.

[0039] ii. Evaluation of endogenous levels of drugs that target the contact system Another aspect of this disclosure relates to the use of vacuum blood collection tubes described herein for determining the level of a drug targeting a component of a contact system. Drug levels are required to evaluate pharmacokinetic parameters. For example, if a contact system is activated in vitro in a plasma sample collected to determine the amount of a plasma kallikrein inhibitor (e.g., DX-2930) in plasma, excess activated plasma kallikrein may bind to the drug, thereby interfering with its detection in the assay. Using any of the vacuum blood collection tubes described herein, the drug level in a sample collected, for example, from a subject can be more accurately estimated.

[0040] To carry out this method, plasma samples derived from subjects (e.g., human patients) treated with a drug that targets a component of the contact system (e.g., pKal) may be prepared from blood samples collected in a vacuum blood collection tube as described herein, according to the method described herein. Drug levels in the plasma samples can be measured according to normal procedures. In some cases, drug levels can be measured by immunoassay, such as the immunoassay described herein.

[0041] iii. Evaluation of the immunogenicity of drugs that target the contact system Another aspect of this disclosure relates to the use of vacuum blood collection tubes for determining the immunogenicity of biological inhibitors against components of a contact system (e.g., pKal). For example, it is common practice to develop immunogenicity assays that can measure antibodies against a drug ("ADA") in the presence of excess drug in circulating plasma. Such a need for an immunogenicity assay capable of measuring ADA is certainly present in the case of therapeutic monoclonal antibodies that may have half-lives of several weeks and high drug levels in the circulating system. To overcome interference from excess drug in the sample, techniques are employed to separate anti-drug antibodies from the drug. Such anti-drug antibodies can be isolated by solid-phase extraction and acid dissociation (SPEAD), in which the biotinylated form of the drug is incubated with a plasma sample for an extended period (usually overnight), and then the biotinylated drug capable of binding to the anti-drug antibody is isolated using a streptavidin-coated plate. This plate is then treated with acid to release the anti-drug antibody. The released antibody may be coated directly onto another assay plate for detection. If no protease inhibitor is present in the collection tube, the contact system becomes activated in vitro, potentially leading to the production of active plasma kallikrein. After the acid release and recoating steps described above, both active pKal and anti-drug antibodies bind to the plate surface. Anti-drug antibodies are typically detected using labeled drugs, but these drugs, if present, can also bind to active pKal, resulting in a false-positive signal in the ADA assay.

[0042] This false-positive signal can be prevented by using vacuum blood collection tubes containing the protease inhibitor cocktail described herein.

[0043] General technology In carrying out the present invention, unless otherwise specified, the use of prior arts in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology is within the scope of the skill of those skilled in the art. Such techniques are found in "Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press," "Oligonucleotide Synthesis (MJ Gait, ed., 1984)," "Methods in Molecular Biology (Humana Press)," "Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press," "Animal Cell Culture (RI Freshney, ed., 1987)," "Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press," "Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths and DG Newell, eds., 1993-8) J. Wiley and Sons," and "Methods in "Enzymology (Methods in Enzymology) (Academic Press, Inc.)", "Handbook of Experimental Immunology (Edited by DM Weir and CC Blackwell)", "Gene Transfer Vectors for Mammalian Cells (Edited by JM Miller and MP Calos, 1987)", "Current Protocols in Molecular Biology (F."M. Ausubel et al. (eds., 1987)", "PCR: The Polymerase Chain Reaction (Mullis et al. (eds., 1994)", "Current Protocols in Immunology (JE Coligan et al. (eds., 1991)", "Short Protocols in Molecular Biology (Wiley and Sons, 1999)", "Immunobiology (CA Janeway and P. Travers, 1997)", "Antibodies (P. Finch, 1997)", "Antibodies: a practical approach (D. Catty. (eds., IRL Press, 1988-1989)", "Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean (eds., Oxford University Press, 2000)", "Using antibodies: a laboratory This is thoroughly explained in literature such as "Manual (Use of Antibodies, Laboratory Manual)" (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)) and "The Antibodies" (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995).

[0044] Without further details, those skilled in the art will likely be able to make the most of the present invention based on the above description. Accordingly, the following specific embodiments should be construed as merely illustrative and in no way limit the remainder of this disclosure. All publications referenced herein are incorporated by reference for the purposes or subjects referred to herein. [Examples]

[0045] Example 1: Preparation of a protease inhibitor cocktail to prevent contact system activation

[0046] A cocktail of protease inhibitors was developed to prevent contact system activation. Vacuum plastic tubes were used for standardized and simplified blood collection.

[0047] Hydrolysis was prevented in the liquid formulation by using a cocktail of the following two types of protease inhibitors. 1) A 10x protease inhibitor cocktail A: SCAT169 A 5 mL total volume vacuum plastic tube containing 100 mM benzamidine dissolved in acid-citrate-dextrose (100 mM trisodium citrate, 67 mM citric acid, and 2% dextrose, pH 4.5), 400 μg / mL polyblen, 2 mg / mL soy trypsin inhibitor, 20 mM EDTA, 263 μM leupeptin, and 20 mM AEBSF (4-(2-aminoethyl)benzenesulfonyl hydrochloride fluoride) (0.5 ml). 2) 10x protease inhibitor cocktail B: SCAT153 A 5 ml total volume vacuum plastic tube containing 10 μM biotinylated EPI-KAL2 dissolved in acid-citrate-dextrose (100 mM trisodium citrate, 67 mM citric acid, and 2% dextrose, pH 4.5), 400 μg / mL polyblen, 20 mM EDTA, and 263 μM leupeptin (0.5 ml).

[0048] Biotinylated EPI-KAL2 was included in protease inhibitor cocktail B (SCAT153). SCAT169 tubes (special coagulation assay tubes, formulation 169) and SCAT153 tubes (special coagulation assay tubes, formulation 153) were stored at 2-8°C.

[0049] Example 2: SCAT169 and SCAT153 tubes prevent contact system activation as shown by double-stranded Western blot assay.

[0050] Plasma collected in either a SCAT169 or SCAT153 tube inhibited contact system activation induced by the in vitro addition of ellagic acid, a well-known contact system activator, when measured by Western blot analysis using conversion from single-stranded to double-stranded HMWK (Figure 1).

[0051] The observed ellagic acid-induced contact system activation was compared among plasma samples collected in three different blood collection tubes: sodium citrate tubes (standard tubes used in clinical chemistry laboratories for coagulation measurements), SCAT169 tubes, and SCAT153 tubes.

[0052] Single-stranded HMWK was essentially completely consumed in the sample activated by ellagic acid in a sodium citrate tube, and the appearance of double-stranded HMWK was detected.

[0053] In contrast, single-stranded HMWK was conserved in ellagic acid-activated plasma of SCAT169 and SCAT153 tubules.

[0054] These results provide evidence that the SCAT169 and SCAT153 tubes are effective in preventing in vitro contact system activation that may occur during plasma sample collection and processing.

[0055] Example 3: The SCAT169 and SCAT153 protease inhibitor cocktail prolonged plasma clotting time.

[0056] For three different donor samples, plasma coagulation times were measured in samples processed using three different blood collection tubes: sodium citrate tubes, SCAT169 tubes, and SCAT153 tubes (Table 1).

[0057] As indicated by prothrombin and activated partial thromboplastin, the coagulation time was prolonged in samples containing SCAT159 and SCAT153 compared to sodium citrate. The results are shown in Table 3. [Table 3]

[0058] Example 4: Use of vacuum blood collection tubes to evaluate the endogenous level of contact system activation in human subjects

[0059] Testing plasma biomarkers for contact system activation is difficult due to activation caused by carelessness during blood collection and processing. In this study, we investigated the usefulness of specialized blood collection tubes (SCAT159 and SCAT153) in evaluating the levels of cleaved high molecular weight kininogen (cHMWK) in plasma from healthy subjects and subjects with type I / II hereditary angioedema (HAE) and idiopathic angioedema, i.e., HAE with normal C1-INH (HAEnC1, also known as nC1-INH), as follows.

[0060] To avoid artificial activation of the contact pathway during blood sample collection, this study used standardized blood collection techniques and custom-made tubes containing protease inhibitors. Blood samples were collected from healthy subjects and subjects with the aforementioned disease (in the sedated and recurrent phases of the disease), and the proportion of cHMWK was evaluated using a Western blot assay. These blood samples were placed in SCAT159 or SCAT153 tubes (5 mL total volume, 0.5 mL x 10 protease inhibitor cocktail) using a catheter equipped with a butterfly needle system. These blood samples were then processed to produce plasma samples within one hour of collection.

[0061] Plasma samples in SCAT tubes were analyzed by simple Western blotting (SBHD) and Western blotting (TGA) to determine the levels of cleaved kininogen (double-stranded kininogen) in the plasma samples according to the method disclosed, for example, in International Publication No. 2015 / 061183.

[0062] In clinical settings 1-5, plasma was collected from healthy subjects in plastic collection tubes containing various anticoagulants, protease inhibitors, or protein stabilizers, and then processed to form plasma. Specifically, the tube types were K2EDTA, sodium citrate, SCAT169, or P100 (BD Biosciences). As shown in Figure 2, tubes that were found to minimize in vitro contact system activation contained protease inhibitors, and these tubes were either P100 or SCAT169 tubes, which are 5 mL volume plastic vacuum blood collection tubes containing a 0.5 mL 10-fold concentrated mixture consisting of 100 mM benzamidine, 400 μg / mL polyblen, 2 mg / mL soy trypsin inhibitor, 20 mM EDTA, 263 μM leupeptin, and 20 mM AEBSF dissolved in acid-citrate-dextrose (100 mM trisodium citrate, 67 mM citrate, and 2% dextrose, pH 4.5). A sampling method using a tube containing a protease inhibitor cocktail prevented an increase in cHMWK when assaying plasma from healthy volunteers.

[0063] The results obtained from this study show that in healthy subjects, cHMWK levels remain stable at room temperature (RT) for at least 24 hours after blood collection using custom-made tubes (<5%), but cHMWK levels increase (12%) in plasma samples obtained from commercial suppliers. This highlights the importance of optimizing blood collection techniques when investigating contact pathway activation.

[0064] The effects of kininogen cleavage in plasma collected in SCAT169 tubes from healthy subjects in two different clinical settings (sites 4 and 5) and a commercial supplier (site 1) were also evaluated (Figure 3). The commercial supplier collected blood samples directly into SCAT169 tubes without using initial discard tubes.

[0065] Kininogen cleavage was also evaluated in blood samples collected in SCAT169 tubes from subjects with angioedema (type I / II HAE, nC1-INH type HAE, and idiopathic AE). Plasma was collected from healthy subjects or subjects with various types of angioedema during both sedation (basal) and relapse (attack) periods, and the amount of cHMWK in each disease state was measured. In subjects with HAE (n=21), the percentage of cHMWK was clearly elevated at baseline compared to healthy controls (n=26), but not in plasma from subjects with idiopathic angioedema (n=4) or HAEnC1 (n=5) (Figure 4). This suggests a limit to plasma kallikrein activity, but does not rule out the role of contact pathway activation during acute attacks in these disorders.

[0066] Plasma samples evaluated for cHMWK detection are susceptible to contact system activation stimulated by the collection method and the tube. Therefore, this study provides an improved plasma sample collection method for evaluating contact system activation that can prevent HMWK cleavage in vitro.

[0067] Example 5: Use of vacuum blood collection tubes to evaluate the endogenous level of contact system activation in human subjects

[0068] We evaluated the usefulness of specialized blood collection tubes (SCAT159 and SCAT153) for assessing levels of cleaved high molecular weight kininogen (cHMWK) in plasma from healthy subjects and subjects with type I / II hereditary angioedema (HAE).

[0069] In short, blood samples were collected from healthy subjects and patients with HAE during the disease sedation (basal) and relapse (attack) phases. The percentage of double-stranded HMWK in plasma was detected using a Western blot assay. Plasma samples were collected from randomized patients with type I / II HAE in a phase 1b multicenter double-blind study who received two subcutaneous doses of anti-pKal antibody (DX-2930) on days 0 and 15 in adjacent groups of 30, 100, 300 or 400 mg or placebo. Blood samples were obtained before and after anti-pKal antibody (DX-2930) administration on days 1, 8, 22, 64, 92 and 120.

[0070] As shown in Figure 5A, the percentage of double-stranded HMWK levels in plasma from healthy subjects collected from three different clinical sites (A, B, and C) differed depending on the collection method and type of tube used. Samples collected using sodium citrate tubes had higher levels of double-stranded HMWK compared to samples collected in SCAT169 tubes. Notably, samples collected directly into SCAT169 tubes and without using a discard tube before this tube containing a protease inhibitor cocktail from clinical site C had higher levels of double-stranded HMWK compared to samples collected using a discard tube.

[0071] Similarly, as shown in Figure 5B, the percentage of double-stranded HMWK levels in plasma from HAE samples was higher in sodium citrate tubes compared to plasma collected in SCAT169 tubes, which is likely due to exogenous activation of the contact system related to plasma collection and processing.

[0072] This study demonstrates the advantages of using blood collection tubes containing the protease inhibitor cocktail described herein and provides an improved method for collecting plasma samples for evaluating contact system activation that can prevent in vitro contact system activation abnormalities (e.g., those indicated by HMWK cleavage).

[0073] Example 6: Use of vacuum blood collection tubes to evaluate plasma drug levels

[0074] Blood is collected from HAE patients treated with DX-2930 and healthy controls using routine procedures and placed in collection tubes. After the initial blood sample is placed in one or more collection tubes, blood samples of 5 mL or less are placed in SCAT159 or SCAT153 tubes. The blood samples are then processed to generate plasma samples within one hour of collection.

[0075] The amount of DX-2930 in SCAT plasma samples is measured using a standard immunoassay, such as ELISA. In short, a Fab version of the anti-idiotype monoclonal antibody against DX-2930 is coated onto the surface of a 96-well plate overnight, and the plate is washed repeatedly to remove unbound anti-idiotype Fab molecules. SCAT plasma samples are then added to this plate and incubated at room temperature for 2-3 hours. The plate is washed several times, and a biotinylated IgG version of the anti-idiotype monoclonal antibody against DX-2930 is added, followed by incubation and washing. Horseradish peroxidase-conjugated streptavidin is then added to the plate. After 30 minutes of incubation, the plate is washed again, and the signal released by the dye is examined. The intensity of this signal corresponds to the amount of DX-2930 in the plasma sample.

[0076] Example 7: Use of vacuum blood collection tubes to evaluate the immunogenicity of a drug

[0077] As disclosed above, plasma samples from HAE patients treated with DX-2930 are prepared from blood samples collected in SCAT159 or SCAT153 tubes. Anti-DX-2930 antibodies in the plasma samples are isolated by solid-phase extraction and acid dissociation (SPEAD).

[0078] In short, a plasma sample is incubated with biotinylated DX-2930 overnight. This mixture is then placed on a streptavidin-coated plate to capture any biotinylated DX-2930 that, if present, binds to the anti-DX-2930 antibody in the plasma sample. The anti-DX-2930 antibody is then released by acid treatment and directly coated onto a Meso Scale Discovery (MSD) plate. Ruthenium-labeled DX-2930 is added to the MSD plate, and the presence of the anti-DX-2930 antibody is detected by measuring the electrochemiluminescence signal.

[0079] Other Embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced with another feature that serves the same, equivalent, or similar purpose. Thus, unless expressly otherwise specified, each disclosed feature is merely an example of a general set of equivalent or similar features.

[0080] Those skilled in the art can easily identify the essential characteristics of the present invention from the above description and can make various modifications and alterations to the present invention without departing from its spirit and scope, and adapt them to various uses and conditions. Accordingly, other embodiments are also included in the claims.

[0081] Equivalents and range Those skilled in the art will recognize many equivalents of the specific embodiments of this disclosure described herein, or can verify this by ordinary experimental methods alone. The scope of this disclosure is not limited to the above description, but rather to the claims attached.

[0082] In the claims, the articles “one (a),” “one (an),” and “the foregoing” may mean one or more unless otherwise clearly stated or the context indicates otherwise. A claim or statement containing “or” between one or more components of a group is deemed to satisfy the condition if, unless otherwise clearly stated or the context indicates otherwise, one, two or more, or all of the components of the group are present in, used in, or otherwise related to a given product or process. This disclosure includes embodiments in which strictly one component of a group is present in, used in, or otherwise related to a given product or process. This disclosure includes embodiments in which two or more, or all, of the components of a group are present in, used in, or otherwise related to a given product or process.

[0083] Furthermore, this disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim dependent on another claim can be modified to include one or more limitations in any other claim that depends on the same basic claim. Where elements are listed as enumerated, for example in Markush group form, each subgroup of those elements is also disclosed, and any element can be removed from that group. In general, where this disclosure or an aspect of this disclosure is referred to as including certain elements and / or features, it should be understood that certain embodiments of this disclosure or an aspect of this disclosure consist of, or essentially consist of, such elements and / or features. For the sake of clarity, those embodiments are not specifically described herein in these words. Note that the words “comprising” and “containing” are non-limiting and also allow for the inclusion of further elements or processes. Where a scope is given, the endpoint is included. Furthermore, unless otherwise specified, or unless it is clear from the context and the understanding of those skilled in the art, any value expressed as a range may, unless the context clearly indicates otherwise, be any specific value or subrange within the range defined in the different embodiments of this disclosure, up to one-tenth of the lower limit of that range.

[0084] This application references various issued patents, published patent applications, journal articles, and other documents, all of which are incorporated herein by reference. In the event of any conflict between any of the incorporated references and this specification, this specification shall prevail. Furthermore, any particular embodiment of this disclosure that falls within the scope of the prior art may be expressly excluded from one or more of the claims. Such embodiments may be excluded even if the exclusion is not expressly stated herein, as they are considered to be known to those skilled in the art. Any particular embodiment of this disclosure may be excluded from any claim for any reason, whether or not it relates to the existence of the prior art.

[0085] Those skilled in the art will recognize, or can verify by conventional experimental methods, many equivalents of the specific embodiments described herein. The scope of these embodiments described herein is not limited to the foregoing, but rather to the claims provided in the appendix. Those skilled in the art will see that various modifications and alterations can be made to this description without departing from the spirit or scope of the disclosure as defined in the following claims.

Claims

1. A vacuum blood collection tube containing a liquid formulation comprising a protease inhibitor mixture consisting solely of EPI-KAL2 and leupeptin.

2. The vacuum blood collection tube according to claim 1, wherein the tube is a non-glass tube.

3. The vacuum blood collection tube according to claim 1 or claim 2, wherein the tube is a plastic tube.

4. The vacuum blood collection tube according to any one of claims 1 to 3, wherein the liquid formulation comprises 5 to 15 μM of EPI-KAL2 and 200 to 300 μM of leupeptin.

5. The vacuum blood collection tube according to claim 4, wherein the liquid formulation comprises 10 μM EPI-KAL2 and 263 μM leupeptin.

6. The vacuum blood collection tube according to any one of claims 1 to 5, wherein the EPI-KAL2 is biotinylated.

7. The vacuum blood collection tube according to any one of claims 1 to 6, wherein the liquid preparation further comprises polyblen and EDTA.

8. The vacuum blood collection tube according to any one of claims 1 to 7, wherein the liquid preparation has a pH of 4 to 6.

9. The vacuum blood collection tube according to claim 8, wherein the liquid preparation has a pH of 4.

5.

10. The vacuum blood collection tube according to any one of claims 1 to 9, wherein the tube contains 0.5 mL of the liquid preparation.

11. The vacuum blood collection tube according to any one of claims 1 to 10, wherein the protease inhibitor mixture substantially does not contain protease inhibitors that are unstable in aqueous solution.

12. (i) A step of processing blood collected in a vacuum blood collection tube according to any one of claims 1 to 11 to produce a plasma sample, (ii) A step of measuring the level of contact system activation in the plasma sample A method for evaluating the endogenous level of contact system activation in a subject, including the following.

13. The method according to claim 12, wherein in step (i), the vacuum blood collection tube is not the first tube to be filled with blood from the subject.

14. The method according to claim 12 or claim 13, wherein step (i) is performed within one hour after blood is collected from the subject.

15. The method according to any one of claims 12 to 14, wherein the subject is a human subject.

16. The method according to claim 15, wherein the human subject is a human patient having a disease related to the contact system.

17. The method according to claim 16, wherein the disease is hereditary angioedema (HAE) or idiopathic angioedema.

18. The method according to claim 17, wherein the human patient has a HAE having normal C1-INH.

19. The method according to any one of claims 15 to 18, wherein the human subject is treated with a drug that targets the components of the contact system.

20. The method according to claim 19, wherein the component of the contact system is plasma kallikrein.

21. The method according to claim 20, wherein the drug inhibits active plasma kallikrein.

22. The method according to claim 21, wherein the drug is an antibody that binds to activated plasma kallikrein.

23. The method according to claim 22, wherein the antibody is DX-2930.

24. The method according to any one of claims 12 to 23, wherein step (ii) is performed by measuring the level of one or more biomarkers indicating contact system activation.

25. The method according to claim 24, wherein the one or more biomarkers are selected from the group consisting of prekallikrein, activated plasma kallikrein (pKal), α2M-pKal complex, activated factor XII, activated factor XI, high molecular weight kininogen (HMWK), and bradykinin metabolites.

26. The method according to claim 25, wherein the one or more biomarkers include cleaved HMWK and / or whole HMWK.

Images