CN111202838A

CN111202838A - Application of flagellin derived polypeptide CBLB502 in prevention and treatment of acute lung injury

Info

Publication number: CN111202838A
Application number: CN202010088792.8A
Authority: CN
Inventors: 葛常辉; 杨晓明; 詹轶群; 于淼; 李长燕; 尹荣华
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS; Academy of Military Medical Sciences AMMS of PLA
Priority date: 2020-02-12
Filing date: 2020-02-12
Publication date: 2020-05-29
Anticipated expiration: 2040-02-12
Also published as: CN111202838B

Abstract

The invention discloses application of flagellin derived polypeptide CBLB502 in prevention and treatment of acute lung injury. The invention provides application of CBLB502 protein or related biological materials thereof in preparing products for preventing and/or treating acute lung injury; the related biological material is a nucleic acid molecule capable of expressing the CBLB502 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule. The research of the invention finds that CBLB502 can obviously reduce the tissue damage degree of the hydrochloric acid inhalation lung damage, and reduce the lung wet-dry ratio, the total cell number and the secretory protein amount in alveolar lavage fluid, and the levels of superoxide dismutase SOD and myeloperoxidase MPO of lung tissues. The results indicate that the CBLB502 can be used for preventing and treating the acute lung injury caused by acid substance inhalation, and provides a new idea and a new medicine for treating chemical lung injury and the like caused by haze.

Description

Application of flagellin derived polypeptide CBLB502 in prevention and treatment of acute lung injury

Technical Field

The invention relates to the technical field of biology, in particular to application of a flagellin-derived polypeptide CBLB502 in prevention and treatment of acute lung injury.

Background

The social industrialization process brings serious environmental pollution, and particularly, the haze phenomenon becomes an important public health problem. The lung is directly communicated with the external environment, and is a main organ damaged by haze under the condition of no physical protection. Haze has been shown to cause respiratory disease development and exacerbation, with increased morbidity and mortality. Haze induced lung injury includes physical lung injury (lung injury caused by inorganic non-degradable particulates), chemical lung injury (lung injury caused by soluble components and organic matter, such as acidic lung injury caused by acid mist formed by sulfur dioxide), and biological lung injury (lung infection injury caused by pathogens carried by haze particles). Haze is a complex composition including gaseous, liquid and solid components. The air quality observation results of the environmental protection monitoring center in Beijing City show that the chemical compositions of the Beijing haze PM 2.5 mainly comprise secondary inorganic salts (sulfate, nitrate and ammonium salt), organic matters and the like and unknown components, wherein the secondary inorganic salts account for 42% at most and are important factors causing acidic lung injury. In addition, acid Acute Lung Injury (ALI) can also be caused by aspiration of acid gastric contents, which is a common complication in the clinical perioperative period and the anesthesia period, and severe ALI can finally develop into Acute Respiratory Distress Syndrome (ARDS), the death rate is more than 40%, and the ALI becomes a common critical respiratory disease in the clinical. At present, the main treatment means aiming at ALI/ARDS comprise mechanical ventilation adjuvant therapy, glucocorticoid drug therapy and the like, but the treatment effect is not ideal, and the death rate is high.

At present, hydrochloric acid (HCl) inhalation ALI animal models are generally adopted at home and abroad to simulate the inhalation of haze acidic substances and clinical acidic gastric content inhalation acute lung injury. CBLB502 was first discovered by Americans in 2008, and comes from Salmonella flagellin, and through structural modification, immunogenicity was greatly reduced compared with flagellin, and our studies show that CBLB502 has a very good radioprotective effect (Wangli et al, radioprotective effect of Toll-like receptor 5 agonist CBLB502 protein, J.Ichnological research, 2012, 39 (3): 225).

Disclosure of Invention

The invention aims to provide application of a flagellin-derived polypeptide CBLB502 in prevention and treatment of acute lung injury.

In a first aspect, the present invention claims the use of the CBLB502 protein or related biomaterials for the preparation of a product for the prevention and/or treatment of acute lung injury.

The related biological material is a nucleic acid molecule capable of expressing the CBLB502 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

The CBLB502 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

In the above protein, the tag is a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In a second aspect, the invention claims the use of the CBLB502 protein or related biomaterials for the preparation of a product; the product has all or part of the following uses:

(B1) reducing the degree of lung tissue damage caused by acute lung injury;

(B2) inhibiting pulmonary edema caused by acute lung injury;

(B3) reducing the extent of alveolar epithelial barrier injury caused by acute lung injury and/or reducing the extent of alveolar epithelial permeability enhancement caused by acute lung injury;

(B4) improving the condition of the reduction of the antioxidant capacity of the lung tissue caused by acute lung injury.

The related biological material is a nucleic acid molecule capable of expressing the CBLB502 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule. The CBLB502 protein is any one of the proteins shown in the above (A1) - (A4).

In the (B3), the alveolar epithelial permeability may be embodied as permeability of alveolar epithelium to cells and/or proteins.

In a second aspect, said use of said product may be prophylactic as well as therapeutic. In a particular embodiment of the invention, in particular prophylactic use.

In a third aspect, the invention claims the use of the CBLB502 protein or related biomaterials in the preparation of products; the product has all or part of the following uses:

(C1) reducing the degree of increase of the lung wet-dry ratio caused by acute lung injury;

(C2) reducing the increase of total cell number in alveolar lavage fluid caused by acute lung injury;

(C3) reducing the increase in total protein content in alveolar lavage fluid caused by acute lung injury;

(C4) improving the content reduction degree of superoxide dismutase in lung tissues caused by acute lung injury;

(C5) reducing the increase of myeloperoxidase content in lung tissue caused by acute lung injury.

In a third aspect, said use of said product may be prophylactic as well as therapeutic. In a particular embodiment of the invention, in particular prophylactic use.

In the first to third aspects, the acute lung injury may be an acidic acute lung injury (i.e., an acidic substance inhalation acute lung injury).

Further, the acidic lung injury may be an acidic acute lung injury caused by haze or an acidic acute lung injury caused by aspiration of acidic gastric contents.

In a particular embodiment of the invention, the acidic lung injury is in particular hydrochloric acid inhalation acute lung injury.

In the foregoing aspects, the "nucleic acid molecule capable of expressing the CBLB502 protein" may be a DNA molecule as described in any one of:

(D1) DNA molecule shown in SEQ ID No. 2;

(D2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (D1) and encodes the CBLB502 protein;

(D3) and (D) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of homology with the DNA sequence limited by (D1) or (D2) and encodes the CBLB502 protein.

In the above nucleic acid molecule, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In each of the foregoing aspects, the product may be specifically a medicament.

In the foregoing aspects, the acute lung injury is acute lung injury in a mammal. The mammal is specifically human or mouse.

In each of the above aspects, the CBLB502 protein may be injected intraperitoneally 0.5h prior to the acute lung injury described above, and the injection amount may be 0.2mg/kg body weight.

The research of the invention finds that CBLB502 can obviously inhibit the symptoms of acid substance inhalation acute lung injury syndrome. According to the invention, an atomization drug delivery system is adopted to deliver hydrochloric acid spray to a mouse, a mouse model simulating haze acid lung injury and stomach content inhalation acute lung injury is established, and the prevention and treatment effect of CBLB502 on hydrochloric acid inhalation lung injury is researched. CBLB502 was found to significantly reduce tissue damage caused by hydrochloric acid inhalation lung injury, reduce the wet-to-dry ratio of the lung, total cell count in alveolar lavage fluid, amount of secreted protein, and levels of superoxide dismutase (SOD) and Myeloperoxidase (MPO) in lung tissue, and the results were statistically different. The results indicate that the CBLB502 can be used as a biotechnology medicament for preventing and treating the acute lung injury caused by acid substance inhalation, and provides a new idea and medicament for treating chemical lung injury and the like caused by haze.

Drawings

FIG. 1 shows the effect of CBLB502 on histopathology of hydrochloric acid-induced lung injury (HE pathological sections).

Figure 2 is a graph of the effect of CBLB502 on lung dry to wet ratio in mice with acute lung injury.

FIG. 3 is a graph of the effect of CBLB502 on the total number of alveolar lavage fluid cells in mice with acute lung injury.

FIG. 4 is a graph of the effect of CBLB502 on total alveolar lavage fluid protein in mice with acute lung injury.

Figure 5 is a graph of the effect of CBLB502 on SOD expression in lung tissue of acute lung injury mice.

FIG. 6 is a graph of the effect of CBLB502 on MPO expression in lung tissue of mice with acute lung injury.

In each figure, denotes P < 0.05; denotes P < 0.01.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The flagellin-derived polypeptide CBLB502 used in the examples below was prepared by the military medical institute radiology research, military scientific institute. It can be prepared according to methods known in the art, and specific methods for its preparation can be found in the patent application "a method for preparing a recombinant bacterial flagellin-derived polypeptide and identifying its activity" with application No. 201110257680.1, or from the literature: wanepi et al, radioprotective effect of the Toll-like receptor 5 agonist CBLB502 protein, journal of international pharmaceutical research, 2012, 39 (3): 225. the amino acid sequence of the CBLB502 protein is shown in SEQ ID No.1, and the corresponding coding gene sequence is shown in SEQ ID No. 2.

Example 1 Effect of CBLB502 on histopathology of hydrochloric acid inhalational Lung injury

6-8 weeks C57BL/6 mice, 15 in total, were randomly divided into 3 groups as follows: control group, model administration group. Each group had 5.

Model set (HCl): the drug is sprayed into the trachea of a mouse by an atomized spray drug delivery system, and the hydrochloric acid water solution with pH1.5 is sprayed into the trachea of the mouse to cause acute lung injury of the mouse, and the drug is delivered according to the weight of 2 ml/kg.

Model dosing group (HCl + CBLB 502): injecting CBLB502 protein of 0.2mg/kg body weight into abdominal cavity, spraying hydrochloric acid aqueous solution of pH1.5 into trachea of mouse after 0.5h via atomized spray administration system to cause acute lung injury, and administering according to body weight of 2 ml/kg.

Control group (Con): the water solution is sprayed into the trachea of the mouse by an atomized spray administration system, and the administration is carried out according to the weight of 2 ml/kg.

At 24h post-treatment or dosing, groups of mice were sacrificed and lung tissue was collected for HE pathological section analysis.

The results are shown in FIG. 1. Displaying: the lung tissue structure of the control group is clear, and no lesion is seen. The local lung tissue interstitium in the HCl treatment group is subjected to diffuse lymphocyte infiltration, and the alveolar space of the obvious part of local inflammation is slightly thickened. The lung interstitial diffuse inflammatory cell infiltration of the HCL + CBLB502 treatment group is realized, inflammatory cells mainly comprise lymphocytes, and a small amount of neutrophils are locally visible. The results show that the HCl treatment in the model group causes acute lung injury, and the administration in the model group can obviously reduce the lung injury.

Example 2 Effect of CBLB502 on Lung Dry-to-Wet ratio in mice with acute Lung injury

C57BL/6 mice at 6-8 weeks, 20 in total, were randomly divided into the following 2 groups: model group and model administration group. Each group had 10.

After the model group treatment or model administration group administration, each group of mice was sacrificed at 0h and 24h, respectively, 5 mice per group at each time point, lung tissues were collected, the wet weight of the lung tissues was weighed, the lung tissues were dried in an oven at 60 ℃ for 72h to a constant weight, the dry weight of the lung was weighed, the ratio of the wet weight/dry weight of the lung was calculated, and the degree of edema of each group of lung tissues was evaluated.

The results are shown in FIG. 2. As can be seen, the HCl + CBLB502 treatment group had a significantly lower wet to dry weight ratio than the HCl group, indicating that CBLB502 treatment inhibited the degree of pulmonary edema.

Example 3 Effect of CBLB502 on the Total number of alveolar lavage fluid cells in mice with acute Lung injury

After the administration of the model group treatment or the model administration group, the mice were sacrificed at 6h and 24h, respectively, 5 mice/group at each time point, the trachea was exposed with scissors, 500. mu.l of precooled PBS was flushed into the lungs through the trachea and timed for 1min, and then recovered and flushed 3 times in a row. The recovered alveolar lavage fluid was centrifuged at 1500rpm for 10min at 4 ℃ and then the cells were resuspended for total cell count.

The results are shown in FIG. 3. As can be seen, the total number of cells in the alveolar lavage fluid of the HCl + CBLB502 treatment group is significantly lower than that of the HCl group, which indicates that the CBLB502 treatment can significantly inhibit the damage degree of the mouse alveolar epithelial barrier and reduce the infiltration degree of the alveolar epithelium to the cells.

Example 4 Effect of CBLB502 on Total protein of alveolar lavage fluid from mice with acute Lung injury

The control group, model group and model administration group mice were sacrificed at 24h, the trachea was exposed with scissors, 500. mu.l of precooled PBS was flushed into the lungs through the trachea and timed for 1min, and then recovered and continuously flushed 3 times. The recovered alveolar lavage fluid was centrifuged at 1500rpm at 4 ℃ for 10min, and 50. mu.l of the alveolar lavage fluid was aspirated to determine the total protein concentration.

The results are shown in FIG. 4. As can be seen, the total protein content in the alveolar lavage fluid of the HCl + CBLB 502-treated group is significantly lower than that of the HCl group, indicating that the CBLB502 treatment can significantly inhibit the degree of protein penetration of the alveolar epithelium.

Example 5 Effect of CBLB502 on SOD expression in Lung tissues of mice with acute Lung injury

After the model group treatment or model administration group administration, each group of mice was sacrificed at 0h and 24h, 5 mice/group at each time point, lung tissues were collected, the weight of the lung tissues was accurately weighed, homogenized, and absorbance values were measured at 450nm with an ultraviolet spectrophotometer, according to the SOD detection test kit (beijing bi yunnan biotechnology).

SOD detection process:

(1) sample preparation

An appropriate amount of lung tissue sample was taken and homogenized by adding 4 ℃ pre-cooled PBS. The homogenate is then centrifuged at 4 ℃ and the supernatant is taken as the sample to be tested.

(2) Preparation of WST-8/enzyme working solution

An appropriate amount of WST-8/enzyme working solution was prepared in 160. mu.l per reaction. 151. mu.l of SOD detection buffer, 8. mu.l of WST-8 and 1. mu.l of enzyme solution were mixed uniformly to prepare 160. mu.l of WST-8/enzyme working solution. And preparing a proper amount of WST-8/enzyme working solution according to the number of samples (including standard products) to be detected.

(3) Preparation of reaction starting working solution

And (3) melting the reaction starting solution (40X) in the kit, uniformly mixing, diluting according to the proportion that 39 mul of SOD detection buffer solution is added into every 1 mul of reaction starting solution (40X), and uniformly mixing to obtain the reaction starting working solution. And preparing a proper amount of reaction starting working solution according to the number of samples (including standard products) to be detected.

(4) SOD standard substance preparation

The SOD standard substance is diluted by the diluent to the following series of concentrations: 200U/ml, 100U/ml, 50U/ml, 20U/ml, 10U/ml, 5U/ml and 2U/ml. In the subsequent tests, 20. mu.l of each sample can be taken and the reference sample can be tested.

(5) Sample assay

The sample wells and various blank control wells were set using a 96-well plate with reference to table 1. And the sample to be tested and the other various solutions were added in sequence as in table 1. Adding reaction starting working solution, mixing well, incubating at 37 deg.C for 30min, and measuring absorbance (A) at 450 nm.

TABLE 1 reagent addition procedure

(6) Calculation of Total SOD Activity in samples

Percent inhibition ═ a [ ("a_{Blank control 1}-A_{Blank control 2})-(A_{Sample (I)}-A_{Blank control 3})]/(A_{Blank control 1}-A_{Blank control 2})×100％。

Definition of SOD enzyme activity unit: when the percentage of inhibition in the above reaction is 50%, the SOD enzyme activity in the reaction system is defined as one enzyme activity Unit (Unit).

The results are shown in FIG. 5. As can be seen from the figure, the SOD activity in the lung tissue of the HCl + CBLB502 treatment group is obviously higher than that of the HCl group and is close to the normal level, which shows that the antioxidant capacity of the lung tissue can be obviously improved by the CBLB502 treatment.

Example 6 Effect of CBLB502 on MPO expression in Lung tissues of mice with acute Lung injury

The level and activity of Myeloperoxidase (MPO) directly reflect the function and activation state of neutrophils, and is an important index for evaluating the severity of inflammation.

After the treatment of the model group or the administration of the model administration group, each group of mice was sacrificed at 0h and 24h, 5 mice/group per time point, lung tissues were collected, the weight of the lung tissues was accurately weighed, homogenized, and absorbance values were measured at 460nm with an ultraviolet spectrophotometer, according to the instructions of the MPO test kit (Nanjing institute of biomedical engineering).

Definition of MPO enzyme Activity Unit: the hydrogen peroxide in the reaction system at 37 ℃ per gram of lung tissue wet sheet is decomposed by 1 mu mol to be 1 enzyme activity unit.

The results are shown in FIG. 6. As can be seen, the MPO activity in the lung tissue of the HCl + CBLB502 treated group is significantly lower than that of the HCl group and is close to the normal level, which indicates that the CBLB502 treatment can significantly inhibit the hydrochloric acid-induced activation of neutrophils and reduce the inflammatory reaction degree.

<110> military medical research institute of military science institute of people's liberation force of China

Application of <120> flagellin derived polypeptide CBLB502 in prevention and treatment of acute lung injury

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Claims

Use of a CBLB502 protein or a related biomaterial thereof in the manufacture of a product for the prevention and/or treatment of acute lung injury;

the related biological material is a nucleic acid molecule capable of expressing the CBLB502 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the CBLB502 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
Application of CBLB502 protein or its related biological material in preparing products; the product has all or part of the following uses:

(B1) reducing the degree of lung tissue damage caused by acute lung injury;

(B2) inhibiting pulmonary edema caused by acute lung injury;

(B3) reducing the extent of alveolar epithelial barrier injury caused by acute lung injury and/or reducing the extent of alveolar epithelial permeability enhancement caused by acute lung injury;

(B4) improving the condition of the reduction of the antioxidant capacity of lung tissues caused by acute lung injury;

the related biological material is a nucleic acid molecule capable of expressing the CBLB502 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the CBLB502 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
3. Use according to claim 2, characterized in that: in the step (B3), the permeability of the alveolar epithelium is expressed by permeability of the alveolar epithelium to cells and/or proteins.
4. Use according to claim 2 or 3, characterized in that: said use of said product is a prophylactic use and/or a therapeutic use.
Application of CBLB502 protein or its related biological material in preparing products; the product has all or part of the following uses:

(C1) reducing the degree of increase of the lung wet-dry ratio caused by acute lung injury;

(C2) reducing the increase of total cell number in alveolar lavage fluid caused by acute lung injury;

(C3) reducing the increase in total protein content in alveolar lavage fluid caused by acute lung injury;

(C4) improving the content reduction degree of superoxide dismutase in lung tissues caused by acute lung injury;

(C5) reducing the increase of myeloperoxidase in lung tissue caused by acute lung injury;

the related biological material is a nucleic acid molecule capable of expressing the CBLB502 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the CBLB502 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
6. Use according to claim 5, characterized in that: said use of said product is a prophylactic use and/or a therapeutic use.
7. Use according to any one of claims 1 to 6, characterized in that: the acute lung injury is acidic acute lung injury.
8. Use according to claim 7, characterized in that: the acidic lung injury is acidic acute lung injury caused by haze or acidic acute lung injury caused by mistaken inhalation of acidic gastric contents.
9. Use according to any one of claims 1 to 8, wherein: the "nucleic acid molecule capable of expressing the CBLB502 protein" is a DNA molecule described in any one of the following items:

(D1) DNA molecule shown in SEQ ID No. 2;

(D2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (D1) and encodes the CBLB502 protein;

(D3) and (D) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of homology with the DNA sequence limited by (D1) or (D2) and encodes the CBLB502 protein.
10. Use according to any one of claims 1 to 9, characterized in that: the product is a medicament.