Antagonist-binding polypeptide molecule of human CD200 receptor and its applications

Abstract

The present invention belongs to the field of biomedicine. Specifically, it relates to an antagonist-binding polypeptide molecule of human CD200 receptor (CD200R) and its applications. The amino acid sequences of the polypeptides are as shown in SEQ ID NO.1 to SEQ ID NO.2, wherein the 1st amino acid and the 80th amino acid of SEQ ID NO.1 and SEQ ID NO.2 form a ring through a peptide bond. The cyclic peptides shown in SEQ ID NO.1 and SEQ ID NO.2 can further yield a series of derived small cyclic peptides and linear peptides through a decompression technique. The antagonist-binding polypeptide molecule of human CD200 receptor (CD200R) provided by the present invention can block the interaction between human CD200 ligand and human CD200R, antagonize the immunosuppressive effect (release inhibition) of the CD200 pathway, and can be used to prepare drugs for treating diseases related to the CD200 / CD200R pathway.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, it relates to an antagonist-binding polypeptide molecule of human CD200 receptor (CD200R) and its applications. Background Art

[0002] CD200 (also known as OX2, OX-90, MOX-2) is a cell surface glycoprotein with a molecular weight of 48 kDa. Its gene is located on chromosome 3q13.2 and it belongs to the members of the type I immunoglobulin superfamily. In the central nervous system, it is mainly expressed on neurons and vascular endothelial cells, and is also expressed on cells such as astrocytes, oligodendrocytes and neural stem cells. It is a signal for down-regulating the function of bone marrow cells and can co-stimulate the proliferation of T cells. CD200 has structural similarities with immune checkpoint molecules such as CD47, PD-1, CTLA-4, etc. It contains 2 extracellular immunoglobulin-like domains: a single transmembrane domain and a short cytoplasmic tail without a signal motif.

[0003] CD200R (CD200R1-5 in mice; CD200R1, R2 in humans) also belongs to the members of the type I immunoglobulin superfamily and is a homologous receptor of CD200. It has 2 extracellular immunoglobulin-like domains and a cytoplasmic NPxY motif. CD200R is mainly expressed in myeloid cells and is also detected on lymphoid cells such as natural killer cells (NK) and T cells, and contains a cytoplasmic tail capable of initiating downstream signal cascades.

[0004] The expression of CD200 is relatively conserved among different species, suggesting that the CD200 / CD200R signaling pathway may have important physiological functions. The D200R family has five members, CD200R1-5. Only CD200R1 has a longer intracellular fragment to recruit downstream molecules to exert physiological functions; while other receptors in the CD200R family except CD200R1 are considered to possibly play functions different from CD200R1 through the lysine residues in the transmembrane region and its short intracellular sequence bridged by unknown molecules.

[0005] CD200 is an immune tolerance signaling molecule that plays an important role in maintaining the body's own immune tolerance. The CD200-CD200R interaction can inhibit the expression of pro-inflammatory molecules such as TNF-α, IFN-γ, and inducible nitric oxide synthase, and then protect immune-privileged sites, stem cells, and other important tissue cells from immune damage by regulating the activation threshold of the inflammatory immune response, as well as promote peripheral immune tolerance. In addition, the immunosuppressive effect produced by CD200-CD200R also plays a key role in tissue repair processes, graft rejection reactions, autoimmune diseases, and cancer surveillance. After CD200 binds to CD200R, tyrosine 302 in the NPxY sequence of CD200R can be phosphorylated, activating the Dok2-RasGAP complex, inhibiting Ras-ERK signal transduction, and further inhibiting myeloid cell activation. The interaction between CD200 and CD200R can also effectively inhibit T cell immune responses and the killing activity of NK cells, promote macrophages to secrete the immunosuppressive tryptophan-degrading enzyme indoleamine-2,3-dioxygenase (IDO), and induce the expansion of regulatory T cells, resulting in the conversion of Th1 cytokines to Th2 cytokines, thereby inhibiting the immune response. CD200 / CD200R plays an important role in tumor and non-tumor diseases mainly by regulating immunity and angiogenesis. Thus, CD200 / CD200R plays an important role mainly by regulating immunity and angiogenesis. In addition, CD200 can also promote neurogenesis in the central nervous system and is not related to the immune regulation effect.

[0006] The CD200 inhibitor Samalizumab (ALXN 6000) is a novel recombinant humanized monoclonal antibody against CD200 that can specifically bind to CD200 and block its interaction with CD200R. The preliminary

[0007] results support that Samalizumab can act as an immune checkpoint inhibitor in the treatment of chronic lymphocytic leukemia (CLL), but the current related clinical trials are still in the primary stage and further clinical trial studies are still needed. Chinese Patent CN114728048A discloses an antagonist polypeptide molecule that binds to the human CD200 receptor (CD200R), a composition containing such antagonist polypeptide molecules, and a method for treating cancer using such antagonist polypeptide molecules.

[0008] Compared with therapeutic monoclonal antibodies, the use of peptide inhibitors as immunotherapy has more advantages. Peptides have lower toxicity, higher stability, and higher efficiency. Due to their smaller size, they have unique properties and high targeting precision, which means a lower risk of potential side effects. In addition, peptides can be easily synthesized, modified, and have a lower production cost compared to antibodies, making them a more favorable therapeutic method. Therefore, there is still a need for human CD200R-binding antagonist polypeptide molecules. Summary of the Invention

[0009] To address the deficiencies in the prior art, the present invention provides an antagonist-binding polypeptide molecule of human CD200 receptor (CD200R) and its applications.

[0010] On the one hand, the present invention provides an antagonist-binding polypeptide molecule of human CD200 receptor (CD200R), whose amino acid sequence is as shown in SEQ ID NO.1 to SEQ ID NO.2, and the specific amino acid sequence is shown in Table 1. Among them, the first amino acid and the 80th amino acid of SEQ ID NO.1 and SEQ ID NO.2 form a ring through a peptide bond.

[0011] Table 1 Amino Acid Sequences of the Present Invention

[0012]

[0013]

[0014] Among them, the polypeptide with the amino acid sequence as shown in SEQ ID NO.1 and SEQ ID NO.2 is an 80-ring peptide obtained by detecting the change of intracellular calcium ion concentration from a polypeptide library through high-throughput screening technology. The discovery of this ring peptide includes the dissolution and dilution of the polypeptide library, and screening from the polypeptide library according to the inhibition rate results of the polypeptide by using ELISA and TR-FRET screening methods.

[0015] On the one hand, the present invention provides an antagonist-binding polypeptide molecule of human CD200 receptor (CD200R), and the polypeptide molecule is obtained by decompressing SEQ ID NO.1 and SEQ ID NO.2.

[0016] Preferably, the present invention provides an antagonist-binding polypeptide molecule of human CD200 receptor (CD200R) with the amino acid sequence as shown in SEQ ID NO.3 to SEQ ID NO.11. SEQ ID NO.3 to SEQ ID NO.11 are cyclic peptides, and the specific sequences are shown in Table 1.

[0017] On the one hand, the present invention also provides a polynucleotide molecule, which comprises one or two polynucleotides capable of encoding the antagonist polypeptide molecule of any one of the above-mentioned human CD200R.

[0018] Preferably, the polynucleotide molecule comprises one or two polynucleotides capable of encoding the polypeptide molecules of SEQ ID NO.1 to SEQ ID NO.11.

[0019] On the other hand, the present invention also provides a pharmaceutical composition, which contains (a) a safe and effective amount of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient. The amount of the polypeptide of the present invention is usually 10 micrograms - 100 milligrams per dose, preferably 100 - 1000 micrograms per dose.

[0020] Generally, the therapeutic composition can be made into an injectable form, such as a liquid solution or suspension; it can also be made into a solid form that is suitable for formulation into a solution or suspension, a liquid carrier, before injection.

[0021] Once the composition of the present invention is formulated, it can be administered by conventional routes, including (but not limited to): intratumoral, intramuscular, intravenous, hepatic artery, oral, subcutaneous, intradermal, or topical administration.

[0022] When the pharmaceutical composition of the present invention is used for actual treatment, pharmaceutical compositions of various different dosage forms can be adopted according to the usage. Preferably, it is an intravenous or hepatic artery pharmaceutical preparation or an intratumoral injection.

[0023] These pharmaceutical compositions can be formulated by conventional methods through mixing, diluting or dissolving, and occasionally adding suitable pharmaceutical additives, such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizing agents, and the formulation process can be carried out in a conventional manner according to the dosage form.

[0024] The pharmaceutical composition of the present invention can also be administered in the form of a sustained-release agent.

[0025] On the other hand, the present invention also provides the use of the above-mentioned polypeptide, polynucleotide or pharmaceutical composition in the preparation of a drug for treating diseases related to the CD200 / CD200R pathway.

[0026] Furthermore, the diseases related to the CD200 / CD200R pathway include solid tumor cancers, hematological malignancies, and neuroendocrine tumor cancers.

[0027] Preferably, the solid tumor cancer can be selected from neuroblastoma, breast cancer, bladder cancer, cervical cancer, colorectal cancer, endometrial cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, liver cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, ovarian cancer, kidney cancer, testicular cancer or thyroid cancer.

[0028] Preferably, the hematological malignancy can be selected from B-cell lymphoma, T-cell lymphoma, leukemia, Hodgkin lymphoma, myeloma, myelodysplastic syndrome or plasmacytoma.

[0029] Preferably, the neuroendocrine tumor cancer can be selected from large cell neuroendocrine carcinoma or pancreatic neuroendocrine carcinoma

[0030] The above-mentioned preparation of a drug for treating a disease related to the CD200 / CD200R pathway can be administered in combination simultaneously, separately or sequentially with one or more immunological drugs.

[0031] The term

[0032] As used herein, the "peptide library" refers to the use of the PICT (Peptide Information Compression Technology) patented technology by Hunan Zhongcheng Quanta Biochemical Co., Ltd. This technology compresses peptide information by biological means, integrating the information of multiple peptides into one peptide, thus achieving a relatively small library capacity containing a large amount of peptide information; a cyclic peptide library containing nearly 73,000 80-amino acid peptides is constructed through the PICT technology. The specific construction method can be referred to in Patent CN201580081102.3 and Patent CN201780089941.9.

[0033] The meaning of "peptide" or "polypeptide" as used herein is well-known to those skilled in the art. Generally, a peptide or polypeptide is composed of two or more amino acids linked by an amide bond, which is formed by the amino group of one amino acid and the carboxyl group of the adjacent amino acid. The polypeptides described herein may contain naturally occurring amino acids or non-naturally occurring amino acids. They can be modified into their analogs, derivatives, functional mimetics, peptidomimetics and other compounds containing at least two amino acids. A polypeptide with a specific amino acid sequence may include modified amino acids and / or additional amino acids, unless the N- and / or C-terminals contain modifications that prevent further addition of amino acids. Such modifications include, for example, acetylation at the N-terminus and / or amidation at the C-terminus.

[0034] The polypeptide of the present invention may be modified, and the modified (usually without changing the primary structure) forms include: chemically derivatized forms of the polypeptide in vivo or in vitro, including but not limited to acetylation, carboxylation, alkylation, acylation, carbamylation. The modification also includes glycosylation, such as those polypeptides produced by glycosylation modification during the synthesis and processing of the polypeptide or further processing steps. Such modification can be accomplished by exposing the polypeptide to enzymes that carry out glycosylation (such as mammalian glycosylating enzymes or deglycosylating enzymes). The modified forms also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that are modified to enhance their proteolytic resistance or optimize their solubility properties.

[0035] The polypeptide of the present invention may be a recombinant polypeptide or a synthetic polypeptide. The polypeptide of the present invention may be chemically synthesized or recombinant. Accordingly, the polypeptide of the present invention can be artificially synthesized by conventional methods or produced by recombinant methods. A preferred method is to use liquid-phase synthesis technology or solid-phase synthesis technology. Another method is to produce the polypeptide of the present invention by recombinant technology. By conventional recombinant DNA technology, the polynucleotide of the present invention can be used to express or produce the recombinant polypeptide of the present invention. Since the polypeptide of the present invention is relatively short, it can be considered to concatenate multiple polypeptides together, obtain the expression product after recombinant expression, and then form the required small peptide by methods such as enzymatic cleavage.

[0036] The polypeptides disclosed in the present invention, including their salts, may also exist in their hydrate forms or in forms containing their solvents (such as ethanol, DMSO, etc.) and can be used for crystallization. The compounds disclosed in the present invention can inherently or by design form solvates with pharmaceutically acceptable solvents (including water); thus, the compounds of the present invention include solvated and unsolvated forms.

[0037] As used herein, "human CD200R antagonist" refers to blocking the interaction between human CD200 ligand and human CD200R and antagonizing the immunosuppressive effect (relieving inhibition) of the CD200 pathway. Synonyms of CD200R include CD200R1, OX2R, MOX2R, and HCRTR2.

[0038] As used herein, the term "binding" refers to the molecular interaction between two molecules, such as the molecular interaction between CD200 and CD200R, and the molecular interaction between the polypeptide molecule of the present invention and CD200R.

[0039] As used herein, "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. This term refers to those pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not unduly toxic when administered. Such carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such carriers include (but are not limited to): saline, buffers, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.

[0040] Compared with the prior art, the present invention has the following advantages:

[0041] (1) The polypeptide library used in the present invention has a much larger amount of information on the amino acid sequences of polypeptide compounds than traditional chemically synthesized polypeptide libraries. Each compound in the library is independently produced, and all have been identified by mass spectrometry and accurately weighed, ensuring the accuracy and stability of screening and avoiding the problem of distortion (the actual library capacity is much lower than the theoretical value) of traditional phage libraries and other mixed compound libraries.

[0042] (2) The present invention provides a series of human CD200R antagonist-binding polypeptide molecules, which can block the interaction between human CD200 ligand and human CD200R, antagonize the immunosuppressive effect (release inhibition) of the CD200 pathway, and can be used to prepare drugs for treating diseases related to the CD200 / CD200R pathway, including but not limited to solid tumor cancers, hematological malignancies, and neuroendocrine tumor cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 For the inhibition results of different concentrations of SEQ ID NO.1 and SEQ ID NO.2 by the ELISA method in Example 1;

[0044] Figure 2 For the inhibition results of different concentrations of SEQ ID NO.1 and SEQ ID NO.2 by the TR-FRET method in Example 1;

[0045] Figure 3 For the inhibition results of different concentrations of SEQ ID NO.3 to SEQ ID NO.8 by the ELISA method in Example 2;

[0046] Figure 4 For the inhibition results of different concentrations of SEQ ID NO.9 to SEQ ID NO.11 by the ELISA method in Example 2;

[0047] Figure 5Inhibitory results of different concentrations of SEQ ID NO.3 to SEQ ID NO.8 in the TR-FRET method of Example 2;

[0048] Figure 6 Inhibitory results of different concentrations of SEQ ID NO.9 to SEQ ID NO.11 in the TR-FRET method of Example 2. Specific embodiments

[0049] The present invention can be better understood according to the following embodiments. However, those skilled in the art can easily understand that the content described in the embodiments is only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0050] General screening method:

[0051] (1) ELISA

[0052] That is, enzyme-linked immunosorbent assay, which is a commonly used immunoenzymatic technique. The main method is to adsorb the known antigen or antibody on the surface of the solid-phase carrier, then incubate it with the enzyme-labeled (coupled) antibody or antigen, add the chromogenic agent to develop color, measure the difference in color between the test substance and the standard substance by an enzyme-labeled instrument, and draw an enzyme activity curve to obtain the concentration of the test substance.

[0053] (2) Time-resolved fluorescence resonance energy transfer technology (Time-resolved fluorescence resonance energy transfer, TR-FRET)

[0054] It is a non-radiative energy transfer phenomenon between two fluorophores with specific spectral properties. To facilitate the understanding of the occurrence of FRET, the emission spectrum of one fluorophore (i.e., the "donor") must overlap with the excitation spectrum of the second fluorophore (i.e., the "donor"). When the donor is excited by the incident light, the energy can be transferred to the acceptor through long-range dipole-dipole interactions, resulting in the emission of fluorescence by the acceptor. FRET can only occur when the donor and the acceptor are close enough (less than 10 nm). When using a fluorescent donor with a long excitation half-life (up to 1500 μs) such as the rare earth metal Europium (abbreviation Eu) or Terbium (abbreviation Tb), a time delay (50 - 150 μs) is allowed between the donor excitation and the acceptor emission recording to avoid interference from other fluorescent signals. This technology is called Time Resolved (TR) FRET, TR-FRET, and is also called Homogeneous Time Resolved Fluorescence (HTRF). Its principle can be referred to the Assay Guidance Manual.

[0055] As a method for screening CD200R antagonist peptides, the test is based on time-resolved fluorescence energy transfer on a microphotographic plate reader. The CD200R antagonist peptide and its target CD200R are labeled with two fluorophores, with Streptavidin-Eu as the fluorescence donor and Goat Anti-Human IgG Fc-Alexa Fluor647 as the fluorescence acceptor. The interaction between the two molecules will bring the two fluorophores close to each other and cause energy transfer between them, so that they emit special fluorescence at a specific wavelength. By measuring this specific wavelength on a microphotographic plate reader, potential CD200R antagonist peptides can be screened in a very short time.

[0056] The above detection method combining TR-FRET and antibody technology has the following advantages: (1) the antibody is selective, specific, and has a high signal-to-noise ratio; (2) the autofluorescence of the compound itself is effectively avoided from interfering with the experiment; (3) the fluorescence signal is stable for a longer time and does not need to be detected immediately; (4) the reaction is homogeneous and easy to quantify in high throughput.

[0057] Example 1 Screening of CD200R antagonist 80 cyclic peptides

[0058] Dissolution of peptide library: Place the 96-well deep-well plate of peptide library in a centrifuge at 4000rpm for 2-3 minutes. Use an automatic dispenser to add 200μL / well of ultrapure water to the 96-well deep-well plate. Seal with a silicone cover and place in a 95℃ water bath for 5 minutes. Note: At this time, the peptide concentration is about 50μM. Place the dissolved 96-well plate peptide in a centrifuge at 4000rpm for 2-3 minutes.

[0059] Peptide library dilution: The dissolved peptides were transferred to a 384-well plate using a workstation and diluted to 10 μM using loading buffer (Tris-Hcl buffer, pH 7.4).

[0060] Among them, the peptide library was self-made by Hunan Zhongsheng Quanpeptide Biochemical Co., Ltd.

[0061] ELISA screening

[0062] (1) Coating: Dilute CD200 to 0.125 μg / mL with coating solution, add 25 μL to each well of a 384-well plate, seal the plate with a sealing film, and place at 4°C for overnight coating.

[0063] (2) Washing: Add 100uL of washing solution to each well, shake for 5 minutes, aspirate the washing solution, tap on paper to completely drain the liquid. Repeat 5 times.

[0064] (3) Blocking: Add 100 μL of blocking solution to each well and incubate at 37 °C for 2 hours for blocking.

[0065] (4) Washing the plate: Add 100 μL of washing solution to each well, shake for 5 minutes, aspirate the washing solution, and gently tap on the paper to completely drain the liquid. Repeat 3 times.

[0066] (5) Sample addition: Dilute CD200 R-biotin-conjugated to 0.5 μg / mL with diluent, premix it with SA-HRP (diluted 1:5000) at a ratio of 1:1 for 1 - 2 hours, add 12.5 μL of the premixed solution to the 384-well plate, and then add 12.5 μL of diluent / peptide solution. Incubate at 37 °C for 1 hour.

[0067] (6) Washing the plate: Add 100 μL of washing solution to each well, shake for 5 minutes, aspirate the washing solution, and gently tap on the paper to completely drain the liquid. Repeat 3 times.

[0068] (7) Substrate addition: Add 25 μL of substrate chromogenic solution (TMB Kt: Mix A and B at a ratio of 1:1) to each well, shake for 5 minutes, and incubate in the dark at 37 °C for 30 minutes until the color develops to the desired depth.

[0069] (8) Termination of reaction: Add 25 μL of 1 M HCl to each well.

[0070] (9) Detection: Measure the absorbance at OD450 immediately after adding hydrochloric acid to terminate the reaction.

[0071] TR-FRET screening

[0072] Verify a large-scale solid peptide library using the TR-FRET screening method. Add different concentrations of test peptides, 4 nM CD200R, 8 nM CD200, the fluorescent donor Streptavidin-Eu, and the fluorescent acceptor Goat Anti-Human IgG Fc-Alexa Fluor 647 to the 384-well plate in sequence. After incubating at room temperature for 2 hours, detect the TR-FRET signal. Positive control: Without peptides, only containing 4 nM CD200R, 8 nM CD200, the fluorescent donor Streptavidin-Eu, and the fluorescent acceptor Goat Anti-Human IgG Fc-Alexa Fluor 647; Negative control: Without peptides, only containing one component of 4 nM CD200R and 8 nM CD200 or neither, and the fluorescent donor Streptavidin-Eu and the fluorescent acceptor Goat Anti-Human IgG Fc-Alexa Fluor 647. Calculate the inhibition rate.

[0073] Through repeated experiments on the initially screened polypeptides, it was finally determined that the samples of SEQ ID NO.1 and SEQ ID NO.2 screened from the self-made 80-cyclic peptide library had relatively high inhibition rates, and then concentration-dependent verification was carried out. The inhibition rate was calculated and graphed using GraphPad. The experimental results are shown in Table 2, and the concentration-response curves are as Figure 1 , Figure 2 shown.

[0074] Table 2 Screening results of 80-cyclic peptides of CD200R antagonists

[0075]

[0076] Example 2 Screening of CD200R antagonist polypeptides

[0077] Using the internal cyclic peptide decompression technology, the amino acid sequences of the 80-cyclic peptides SEQ ID NO.1 and SEQ ID NO.2 screened in Example 1 were analyzed and disassembled, and 10 - 80 linear peptides or cyclic peptides with different amino acid sequences were designed. The 10 - 80 linear peptides or cyclic peptides with different amino acid sequences designed were screened according to the screening process in Example 1, and the IC50 of the active linear peptides or cyclic peptides among the 10 - 80 different amino acid sequences designed was verified.

[0078] After the IC50 verification of the 10 - 80 different amino acid sequences designed, further concentration-dependent verification of the active polypeptides was carried out. The experimental results are shown in Table 3, and the concentration-response curves are as Figures 3 to 6 shown.

[0079] Table 3 Screening results of CD200R antagonist polypeptides

[0080]

[0081]

[0082] The present invention provides an idea and method for inhibiting human CD200R antagonist polypeptides and their applications. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. A human CD200R antagonist-binding polypeptide molecule, characterized in that its amino acid sequence is as shown in SEQ ID NO.2, SEQ ID NO.6 or SEQ ID NO.8, and the 1st amino acid and the 80th amino acid of the amino acid sequence shown in SEQ ID NO.2 form a ring through a peptide bond; the 1st amino acid and the 33rd amino acid of the amino acid sequence shown in SEQ ID NO.6 or SEQ ID NO.8 form a ring through a peptide bond.

2. A polynucleotide molecule, characterized in that the polynucleotide molecule is a polynucleotide capable of encoding the human CD200R antagonist-binding polypeptide molecule according to claim 1.

3. A pharmaceutical composition, which contains (a) a safe and effective amount of the human CD200R antagonist-binding polypeptide molecule according to claim 1 or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient.

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