CN115487170A

CN115487170A - Application of pyrogallol in preparation of medicine for preventing or treating influenza virus infection

Info

Publication number: CN115487170A
Application number: CN202211378991.8A
Authority: CN
Inventors: 周倍贤; 杨素珊; 梁月云; 张岳汉; 王茂生
Original assignee: Gaozhou Peoples Hospital
Current assignee: Gaozhou Peoples Hospital
Priority date: 2022-11-04
Filing date: 2022-11-04
Publication date: 2022-12-20

Abstract

The disclosure belongs to the field of medicine, and particularly relates to application of Pyrogallol (PG) in preparation of a medicine for preventing or treating influenza virus infection; the Luminex technology is utilized to prove that pyrogallol has the inhibiting effect on the over-expression of a series of inflammatory genes expressed by human A549 cells induced by influenza virus A/PR8/3/4 (H1N 1); the immunoblotting technology shows that pyrogallol inhibits influenza A virus H1N1 from infecting A549 cells in vitro to activate NF-kB signal channel; the immunofluorescence technology proves that pyrogallol inhibits influenza A virus H1N1 from infecting A549 cells to induce P-P65 to enter the nucleus; meanwhile, in vivo results show that pyrogallol can improve the induction of the increase of the pulmonary index by the influenza virus H1N1 and inhibit the pathological damage of the lung. Therefore, the pyrogallol disclosed by the invention can be developed as a novel anti-influenza drug, and provides a novel drug choice for preventing and treating influenza.

Description

Application of pyrogallol in preparation of medicine for preventing or treating influenza virus infection

Technical Field

The disclosure belongs to the field of medicines, and particularly relates to application of pyrogallol in preparation of a medicine for preventing or treating influenza virus infection.

Background

Influenza a viruses are enveloped RNA viruses, belonging to the orthomyxoviridae family. The acute upper respiratory infectious disease caused by the virus A has strong infectivity and rapid transmission speed, and brings serious threat to public health and serious economic and medical burden to society. Estimated by the world health organization, about 29 to 65 million people die annually from seasonal influenza virus infections. Highly pathogenic avian influenza a viruses, including H5, H7, H10 subtype viruses, are occasionally transmitted to humans from natural wild avian or poultry hosts across species, causing severe respiratory disease and high mortality.

There is increasing evidence that excessive inflammation is closely related to the severity of influenza disease. Up-regulation of proinflammatory mediators (e.g., IL-6, TNF- α, IL-8, IP-10, COX-2, and PGE 2) by highly pathogenic or less pathogenic influenza viruses can lead to mild to severe symptoms and even death. These inflammatory responses influenza virus-infected cytokines can directly damage the alveolar epithelium or damage the epithelial-endothelial barrier by inducing apoptosis, resulting in viral pneumonia, lung injury, or respiratory dysfunction. In addition, further recruitment of inflammatory factors to the site of infection may enhance the severity of influenza virus-induced "cytokine storms," leading to worse clinical outcomes. Treatment of mice infected with lethal influenza virus with immunomodulator plus delayed antiviral drugs can rescue mice infected with lethal influenza virus. The clinical outcome of corticosteroid therapy remains controversial in controlling the intense airway inflammation caused by the virus. The World Health Organization (WHO) does not recommend the use of corticosteroids for the treatment of influenza virus-related inflammation. Therefore, the development of new alternative drugs to modulate excessive inflammation is an urgent need.

Polyphenol compounds are widely present in various types of plants, and have attracted much attention due to their various biological activities, such as anti-inflammatory, anti-tumor, and bacteriostatic effects. It is worth noting that pyrogallol has been reported in the literature to have anti-inflammatory and anti-cancer properties. However, pyrogallol has not been reported for respiratory diseases caused by influenza virus. In this study, we speculate that pyrogallol has a protective effect on influenza virus-mediated lung injury and inflammation.

Disclosure of Invention

Use of pyrogallol or a pharmaceutically acceptable derivative thereof in the manufacture of a medicament for the prevention or treatment of an influenza virus infection in a patient.

The pyrogallol (figure 1) has the following structural formula:

according to one such general method of the art to which the present invention pertains, the above formula (I) of the present invention may form a pharmaceutically acceptable salt thereof with an acid, which may include inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, propionic acid, trifluoroacetic acid, maleic acid, tartaric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; the drug of the present invention may be a drug in which the derivative itself is mixed with a pharmaceutically acceptable diluent, adjuvant and/or carrier, or a drug in which a composition in which the derivative of the present invention or a pharmaceutically acceptable salt, solvate, optical isomer or polymorph thereof is one of activities is mixed with a pharmaceutically acceptable diluent, adjuvant and/or carrier.

Wherein the use of pyrogallol or a pharmaceutically acceptable derivative thereof in a medicament for inhibiting the level of an influenza virus-induced inflammatory factor.

Wherein the inflammatory factors are human A549 cell IL-6, IL-8, TNF-alpha, IP-10, MCP-1, RANTES.

Wherein the pyrogallol or the medicinal derivative thereof is used in the medicine for inhibiting the activation of the cell signaling pathway induced by the influenza virus.

Wherein the use of pyrogallol or a pharmaceutically acceptable derivative thereof in a medicament for inhibiting the induction of nuclear entry of a cell signaling pathway by influenza virus.

Wherein the use of pyrogallol or a pharmaceutically acceptable derivative thereof in a medicament for ameliorating lung lesions caused by influenza virus infection.

Wherein the influenza virus is influenza virus A/PR8/3/4 (H1N 1).

Wherein the cell signaling pathway is human A549 cell signaling pathway NF-kappa B P-P65 signaling pathway.

The medicine of the invention is added with conventional auxiliary materials and can be prepared into various pharmaceutically acceptable dosage forms such as tablets, capsules, oral liquid, pastilles, aerosols, injections, ointments, granules or various sustained and controlled release preparations and the like according to the conventional process; preferably, the preparation is aerosol, injection or oral preparation.

The carriers for the medicaments of the invention are of the usual type available in the pharmaceutical field and include: binders, lubricants, disintegrants, cosolvents, diluents, stabilizers, suspending agents or matrices, and the like. Pharmaceutical formulations may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically), and if certain drugs are unstable under gastric conditions, they may be formulated as enteric coated tablets.

The clinical dosage of the compound of the above formula (1) or a pharmaceutically acceptable salt, solvate, optical isomer or polymorph thereof for a patient may depend on the therapeutic efficacy and bioavailability of the active ingredient in vivo, their metabolic and excretion rates and the age of the adults.

Has the advantages that:

the invention aims to prove that pyrogallol has an inhibiting effect on the over-expression of influenza virus A/PR8/3/4 (H1N 1) induced human A549 cell expression series inflammatory genes; the immunoblotting technology shows that pyrogallol inhibits influenza A virus H1N1 from infecting A549 cells in vitro to activate NF-kB signal channel; the immunofluorescence technology proves that pyrogallol inhibits influenza A virus H1N1 from infecting A549 cells to induce P-P65 to enter the nucleus; meanwhile, in vivo results show that pyrogallol can improve pathological lung injury induced by influenza virus H1N 1. Therefore, the pyrogallol can be developed as a novel anti-influenza drug, and provides a novel drug choice for preventing and treating influenza.

Drawings

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present disclosure, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.

FIG. 1 is a Luminex technology used for detecting the effect of pyrogallol on influenza virus A/PR8/3/4 (H1N 1) to induce human A549 cell to express serial inflammatory genes;

FIG. 2 is a diagram showing the detection of the effect of pyrogallol on the induction of cell signaling pathway activation by influenza virus A/PR8/3/4 (H1N 1) by immunoblotting;

FIG. 3 is a diagram showing that the immunofluorescence technique is used for detecting that pyrogallol induces cell NF-kB signal channel activation entry transcription of influenza virus A/PR8/3/4 (H1N 1);

FIG. 4 is a schematic diagram showing the comparison of pulmonary index of influenza virus mouse lung adapted strain A/FM/1/47 (H1N 1) infected group with that of normal group;

FIG. 5 shows the effect of pyrogallol on lung pathology in influenza A/PR8/3/4 (H1N 1) infected mice using H & E staining technique.

Detailed Description

The technical solutions in the embodiments of the present disclosure will be described clearly and completely with reference to the drawings in the embodiments of the present disclosure, and it is obvious that the embodiments described are only some embodiments of the present disclosure, rather than all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments disclosed herein without inventive step, are intended to be within the scope of the present disclosure.

EXAMPLE one preparation of Phyllanthus emblica pyrogallol Compound

1. Experimental materials:

1. reagent: emblic leafflower fruit, 95% ethanol, petroleum ether, ethyl acetate and n-butyl alcohol

2. The experimental steps are as follows:

taking 10kg of emblic leafflower fruit, cutting, heating and refluxing the cut emblic leafflower fruit by 95 percent ethanol for 3 times, decompressing and concentrating the extracting solution, and drying to obtain 600g of extract. Dispersing the extract in appropriate amount of distilled water, sequentially extracting with ethyl acetate and n-butanol, and concentrating to obtain two parts: 180g of crude ethyl acetate and 480g of n-butyl alcohol; the crude ethyl acetate fraction was then soaked with petroleum ether, subjected to solid-liquid extraction and rotary evaporation under reduced pressure to obtain 50g of petroleum ether fraction and 140g of ethyl acetate fraction. The ethyl acetate fraction (130 mg) was eluted through silica gel column chromatography (300 to 400 mesh) in a gradient of chloroform-methanol (100 → 0) and analyzed by TLC to combine 7 fractions (fr.1 to 7), and through repeated separation by silica gel column chromatography, gel column chromatography and recrystallization, a white powder was obtained, which was identified as pyrogallol with a purity of 98.4%.

Example II Luminex technology for detecting the effect of pyrogallol on influenza virus A/PR8/3/4 (H1N 1) to induce human A549 cell to express serial inflammatory genes

1. Experimental materials:

1. reagent: a549 cell, DMEM/F12 culture medium, fetal bovine serum and Bio-Plex suspension chip detection kit

2. Consumable material: pipette, six-hole plate, gun head and 1.5EP tube

2. The experimental steps are as follows:

1) A549 cells at 5X 10 ⁶ The cells are planted in a 6-pore plate in density and are placed in an incubator for culture;

after the cells are adhered to the wall, removing the original culture medium, adding PBS to wash for two times, and then adding a serum-free DMEM/DF12 (1:1) culture medium containing influenza virus A/PR8/3/4 (H1N 1) to adsorb the cells for 2 hours; washing the cells 2 times with PBS removes unadsorbed virus;

2) A normal group, an influenza virus H1N 1-infected group (model group), an H1N1+ pyrogallol-dried group (10. Mu.g/mL, 20. Mu.g/mL, 30. Mu.g/mL), and a pyrogallol-treated group alone (20. Mu.g/mL) were set. No drug intervention was added to the influenza virus H1N1 infected group (model group); in the pyrogallol-only treatment group, A549 cells of the pyrogallol-only treatment group are not adsorbed by viruses;

3) After 24 hours of drug intervention, supernatants from each group were collected and transferred to 1.5mLEP tubes. Centrifugation was carried out at 13000rpm x 15min in a 4 ℃ precooled centrifuge. And after centrifugation, taking the supernatant, subpackaging in small parts, and freezing and storing in a-80-degree refrigerator for use in detection.

4) Detecting the content of inflammatory factors in cell culture supernatant by using a Bio-plex suspension chip technology:

(1) a balance kit: and taking out the Bio-Plex suspension chip detection kit, and placing the kit at room temperature to restore the normal temperature balance. At the same time, the cells were removed from the culture and thawed slowly on ice.

(2) Setting a detection plate: and setting the positions of the standard sample hole, the blank hole and the sample hole on the detection plate.

(3) Adding 50 mu L of detection antibody into each hole of the detection plate and adding corresponding samples into the detection plate according to the kit instructions; and incubated for 30min at 300rpm in a 96-well plate shaker.

(4) After incubation, the test plate was washed and reporter antibody was added and incubated for 10min on a 96-well plate shaker at 300 rpm.

(5) As shown in FIG. 1, the prognosis of pyrogallol shows that the levels of inflammatory factors (IL-6, IL-8, TNF-. Alpha., IP-10, MCP-1, RANTES) induced by influenza virus were significantly suppressed. Suggesting that pyrogallol intervention may play a role in treating influenza infection by inhibiting the induction of excessive inflammatory responses by influenza infection. (in comparison with the normal group, ^### p<0.001, compared with the H1N1 group ^* p<0.05， ^** p<0.01， ^*** p<0.001)。

Example three immunoblotting techniques to detect the Effect of pyrogallol on influenza A/PR8/3/4 (H1N 1) Virus induced activation of cell signaling pathway

1. Experimental materials:

1. reagent: rabbit anti-human p-p65 antibody, rabbit anti-human GADPH antibody, goat anti-rabbit secondary antibody, cell lysate RIPA, protease inhibitor cocktail, PMSF

2. Consumable material: PVDF membrane, pipette, six-hole plate, gun head, 1.5EP tube and Whatman3M filter paper

2. The experimental steps are as follows:

1) A549 cells at 5X 10 ⁶ The cells are planted into a 6-hole plate in density and are placed in an incubator for culture;

after the cells adhere to the wall, removing the original culture medium, adding PBS to wash twice, and then adding a serum-free DMEM/DF12 (1:1) culture medium containing influenza virus A/PR8/3/4 (H1N 1) to adsorb the cells for 2 hours; washing the cells 2 times with PBS removes unadsorbed virus;

2) A normal group, an influenza virus H1N 1-infected group (model group), an H1N1+ pyrogallol-treated group (10. Mu.g/mL, 20. Mu.g/mL, 30. Mu.g/mL) and a pyrogallol-alone treated group (20. Mu.g/mL) were set. No drug intervention was added to the influenza virus H1N1 infected group (model group); in the pyrogallol-only treatment group, A549 cells of the pyrogallol-only treatment group are not adsorbed by viruses;

3) After 24 hours of drug intervention, cell culture supernatant was discarded and washed twice with cold PBS; after washing, 200. Mu.l of cell lysate RIPA (containing the protease inhibitor cocktail, which is added to the cell lysate RIPA at a volume ratio (V/V) of 1 to 100 and PMSF was added to a final concentration of 10. Mu.M) was added to extract total cell protein.

4) The cell lysate was transferred to a 1.5mL EP tube and placed in a 4 ℃ pre-chilled centrifuge for 13000rpm × 15min centrifugation;

5) After centrifugation, the protein concentration of the total cell extract was determined according to the BCA protein assay kit instructions: adding 10 mu L/well of standard substance and sample (2 multiple wells) into a 96-well plate, then adding 200 mu L/well of BCA detection working solution into each well (preparing working solution A: B =1 50), uniformly mixing, incubating for 30min at 37 ℃, and measuring the OD value at the absorbance of 572nm by using a microplate reader. And (6) drawing a standard curve, and converting the OD value of the sample into concentration according to the standard.

6) Preparation of SDS polyacrylamide gel:

preparation 10% SDS polyacrylamide gel electrophoresis separation gel:

mixing, pouring into a separating glass plate, and adding ddH into the upper layer after pouring ₂ Sealing the liquid level of the separation gel, and waiting for 30min for the condensation of the separation gel;

7) Configuration 5% SDS polyacrylamide gel electrophoresis concentrate:

after fully and uniformly mixing, pouring concentrated glue between the separation glass plates, pouring a Bi Charu comb, and waiting for 30min for the separation glue to coagulate;

8) Denatured protein: mixing 5 Xloading buffer with each group of samples (20 μ g) at a volume to mass ratio (V/V) (μ L/. Mu.L) (1:4)), and denaturing the protein in a 95 ℃ metal heater for 10min;

9) Loading and electrophoresis: pulling out a comb and adding samples one by one; setting a Bio-Rad vertical electrophoresis apparatus for 90V constant voltage electrophoresis, and stopping electrophoresis when a bromophenol blue indicator reaches the bottom of the separation gel;

10 Film transfer: PVDF membrane was activated beforehand with methanol (15 min soaking). After electrophoresis, the gel was removed. The filter plates were stacked in order of Whatman3M filter paper-gel-PVDF membrane-Whatman 3M filter paper and placed on the wet-rotor plate. In a cold storage, the membrane is rotated for 2h at a constant current of 380 mA;

11 Sealing: after the membrane transfer was completed, 5% skim milk powder (5% mil/TBST) was added to block the PVDF membrane for 2h;

12 Antibody incubation: preparation 5% BSA/TBST, 1: diluting rabbit anti-human p-p65 antibody, rabbit anti-human p65 antibody, and rabbit anti-human GADPH antibody at a ratio of 1000% in BSA (5%); placing the mixture in a refrigerator at 4 ℃ for overnight incubation;

13 Secondary antibody incubation: after overnight, taking out the PVDF membrane and recovering to room temperature; washing the membrane with TBST for 3 times, 5 min/time, adding horseradish peroxidase (HRP) -conjugated goat anti-rabbit secondary antibody, and incubating at room temperature for 1h.

14 Development): removing the secondary antibody and washing the membrane for 3 times and 5 min/time by using TBST (tert-butyl ether-tert-butyl ether); adding ECL immunofluorescence chemiluminescence developer for development;

15 Analysis and collation of results.

As shown in figure 2, after the result is collated, pyrogallol obviously inhibits the activation of an NF-kappa B P-P65 signal channel of a human A549 cell signal channel induced by influenza A/PR8/3/4 (H1N 1).

Example four immunofluorescence assays for the Effect of pyrogallol on the Induction of P-P65 Nuclear penetration by influenza Virus A/PR8/3/4 (H1N 1)

1. Experimental materials:

1. reagent: rabbit anti-human P-P65 antibody and fluorescein FITC coupled goat anti-rabbit secondary antibody

2. Consumable material: 48 pore plates, sterilized 9mm glass cell slide, PBS, gun heads, 1.5mL of EP tube II and experiment steps:

1) Placing a 9mm glass cell slide in a 48-pore plate, and adding PBS to wash and moisten the cell slide;

2) A549 cells were harvested from 100cm ² Digested in a Petri dish and adjusted to a density of 1X 10 ⁵ The cell density of (a); adding 500 mu L of cell suspension into a 48-hole culture plate with cell slide, and placing the culture plate in an incubator for 12 hours;

3) After the cells are attached to the wall, the original culture medium is discarded, PBS is added for washing twice, and then DMEM/DF12 (1: 1) Adsorbing cells for 2 hours by using a culture medium; washing the cells 2 times with PBS removes unadsorbed virus;

4) Three groups are set, respectively: normal group, influenza virus H1N1 infected group (model group), H1N1+ pyrogallol dried group (30 μ g/mL). No drug intervention was added to the influenza virus H1N1 infected group (model group);

5) After the cells were placed in an incubator and incubated for 24 hours, the cells were fixed for 10 minutes by adding 4% paraformaldehyde (dissolved in PBS, pH 7.4), and the cells were washed 3 times with ice PBS for 5 minutes each;

6) A permeable nuclear membrane: incubating the sample with 0.25% Triton X-100 for 10 minutes; cells were washed 3 times with PBS for 5 minutes each;

7) Cells were incubated with 5% bsa for 30min to block non-specific binding of antibody;

8) Antibody diluted in 1% BSA, placed in wet box at 4 ℃ overnight incubation;

9) Primary antibody was removed and cells were washed 3 times with ice PBS for 5 minutes each;

10 Adding fluorescein FITC coupled secondary antibody, and incubating for 1 hour at room temperature; cells were washed 3 times with ice PBS for 5 minutes each;

11 Add DAPI to stain nuclei for 10min, wash cells 3 times with ice PBS for 5min each;

12 Sealing with glycerol, and photographing under confocal microscope to store the result;

15 Analysis and sorting of results.

The result is collated and shown in figure 3, the pyrogallol obviously inhibits the induction of the human A549 cell signaling pathway NF-kappa B P-P65 into the nucleus by influenza A/PR8/3/4 (H1N 1).

Example Effect of pentapyrogallol on Lung pathology in influenza Virus murine Lung adapted Strain A/FM/1/47 (H1N 1) nose drops infected mice

1) 20 BALB/c mice of SPF grade 4-6 weeks old, purchased from the centre of medical laboratory animals in Guangdong province, were randomly divided into four groups (average five per group), each of which was: a normal group, an influenza virus mouse lung adaptive strain A/FM/1/47 (H1N 1) infection group (a model group), an H1N1+ low pyrogallol dry control group (20 mg/kg/d), and an H1N1+ high pyrogallol dry control group (40 mg/kg/d);

2) Pyrogallol (20 mg/kg/d,40 mg/kg/d) was administered intragastrically two days in advance;

3) Freezing and thawing influenza virus mouse lung adapted strain A/FM/1/47 (H1N 1), and diluting with serum-free DMEM/F12 to 5LD ₅₀ After the mice are anesthetized by 10% chloral hydrate (mass/volume ratio) (M/V), a flu infection model is prepared by dripping 50 mu L of the medicine into a nose; dripping equivalent DMEM/F12 into the normal group and the same method;

4) Preparing an influenza infection model, anesthetizing a mouse by 10% chloral hydrate on the 7 th day, dissecting and taking a lung to determine a lung index (the lung index = lung weight (g)/body weight (g) × 100), and evaluating the body weight of the mouse and the change of the lung index; and dissecting and taking the lung, fixing with paraformaldehyde, embedding paraffin, slicing, H & E staining, observing lung lesions of the mouse under a microscope, and taking a picture.

5) The results are shown in FIG. 4, in which the lung index of the influenza virus mouse lung adapted strain A/FM/1/47 (H1N 1) infected group is obviously increased and has obvious difference compared with the normal group (the ^### p<0.001 ); the lung index of the pyrogallol-depleted group was statistically different from that of the virus group ( ^* p<0.05， ^** p<0.01)。

6) The lung pathology results are shown in FIG. 5, in which influenza virus mouse lung adapted strain A/FM/1/47 (H1N 1) infects a large amount of inflammatory cells in the lung to infiltrate, and the alveolar structure is destroyed; the pyrogallol intervention group significantly improved lung lesions caused by H1N1 infection.

In the description herein, references to the description of "one embodiment," "an example," "a specific example," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the disclosure. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The foregoing illustrates and describes the general principles, principal features, and advantages of the present disclosure. It will be understood by those skilled in the art that the present disclosure is not limited to the embodiments described above, which are presented solely for purposes of illustrating the principles of the disclosure, and that various changes and modifications may be made to the disclosure without departing from the spirit and scope of the disclosure, which is intended to be covered by the claims.

Claims

1. Use of pyrogallol or a pharmaceutically acceptable derivative thereof in the manufacture of a medicament for the prevention or treatment of an influenza virus infection in a patient.

2. The use according to claim 1, wherein the pyrogallol is in the form of a pharmaceutically acceptable salt.

3. The use of claim 1, wherein the use of pyrogallol or a pharmaceutically acceptable derivative thereof in a medicament for inhibiting the level of an influenza virus-induced inflammatory factor.

4. The use as claimed in claim 3, wherein the inflammatory factor is IL-6, IL-8, TNF- α, IP-10, MCP-1, RANTES of human A549 cells.

5. Use according to claim 1, wherein the pyrogallol or a pharmaceutically acceptable derivative thereof is used in a medicament for inhibiting the activation of an influenza virus-induced cell signaling pathway.

6. Use according to claim 1, wherein the pyrogallol or a pharmaceutically acceptable derivative thereof is used in a medicament for inhibiting an influenza virus-induced cell signaling pathway.

7. The use of claim 1, wherein the pyrogallol or the pharmaceutically acceptable derivative thereof is used for the improvement of lung pathology caused by influenza virus infection.

8. The use according to any one of claims 1 to 7, wherein the influenza virus is influenza A/PR8/3/4 (H1N 1).

9. The use of any one of claims 5 to 7, wherein the cell signaling pathway is the human A549 cell signaling pathway NF- κ B P-P65 signaling pathway.

10. Use according to any one of claims 1 to 7, wherein the pyrogallol or a pharmaceutically acceptable derivative thereof is administered in a dosage form suitable for releasing the pyrogallol or a pharmaceutically acceptable derivative thereof in the respiratory system of the patient.