WO1999062395A2

WO1999062395A2 - The use of anticlogging substances and a method for preventing the clogging of medical ventilation tubes

Info

Publication number: WO1999062395A2
Application number: PCT/FI1999/000473
Authority: WO
Inventors: Eeva-Marjatta Salonen
Original assignee: Salonen Eeva Marjatta
Priority date: 1998-06-03
Filing date: 1999-06-01
Publication date: 1999-12-09
Also published as: AU4619099A; FI981257A0; FI981257A; WO1999062395A3

Abstract

The present invention is related to a method for preventing the clogging of medicinal drainage or ventilation tubes, comprising coating the surface of said ventilation tubes with anticlogging substances, especially human serum albumin. The medicinal ventilation tubes are rigid, semirigid and/or elastic tubes, such as tympanostomy tubes, paranasal sinus drainage tubes, thracheostomy tubes, gastric tubes, urinary catheters, wound drains and voice prosthesis tubes made of plastics, silicons, metals, hydroxylapatits, etc. The use of the anticlogging substances for manufacturing surface coated medicinal ventilation tubes for treatment of diseases requiring ventilation and/or drainage is also described.

Description

THE USE OF ANTICLOGGING SUBSTANCES AND A METHOD FOR PREVENTING THE CLOGGING OF MEDICAL VENTILATION TUBES

The Technical Field of the Invention

The present invention is related to methods for preventing the clogging of ventilation tubes used in human and veterinary medicine as well as to the use of anticlogging substances, especially serum albumin for coating said ventilation tubes.

The Background of the Invention

Patients with otitis media with effusion (OME) or patients with problems in the middle ear ventilation are treated with surgical insertion of ventilation tubes (tympanostomy tubes) into the tympanic membrane of the ear. Major problems, following the tympanostomy, arise from the blockage (clogging) of the tube, due to haemmorrhage of the wound in tympanic membrane, chronic ear discharge due to bacterial contamination on the tube surface, serous, mucous or purulent discharges or microbial infections as well as other problems in middle ear, which disturb ventilation.

Surgical treatment involves the insertion of tympanostomy tubes into the tympanic membrane.

The return of hearing loss due to middle ear effusion and otalgia due to acute otitis media in a child with a ventilation tube in si tu is typical of tube clogging. Repeated surgical tube insertations cause great stress to both child and parents. Attempts to open clogged tubes in si tu is very difficult, because cooperation with small children in performing this delicate procedure can be a problem. The use of unblocking substances, such as mucolytic agent and/or antibiotics either systematically or topically has also been suggested, but so far no effective solution has been found. The substances suggested are either not effective enough or have severe side effects. Silicone elastomeric tubes coated with silver oxide have also been suggested and tested, because of its antibiotic effect, but the silver oxide can cause allergic reaction in some patient.

Therefore, there is a clear need to find simple, but still effective methods without deleterious side effects for preventing the clogging of ear ventilation tubes.

When setting up a model system to study the problem the present inventor surprisingly found that the clogging of ventilation tubes could be effectively prevented by coating the tubes with human serum albumin.

Thus, the objective of the present invention was to find a solution to the problem of recurrent tympanostomic tube clogging due to blood clot or middle ear effusion with consequent complications and ailments. A solution to the problem was achievable by the present invention, which is also applicable to other medicinal ventilation systems as defined in the claims.

The Detailed Description of the Invention Definitions

In the present description the terms used generally have the same meaning as in medicinal sciences and biochemistry as set forth in text books and review articles and laboratory handbooks. Some terms are, however, used more extensively and have meaning somewhat deviating from the general use. Some of these terms are defined below.

The term "clogging" means the same as blocking and covers the situations wherein a tube having the task of allowing a free interchange of air or free flowing of a fluid cannot fulfill its task, because the tube is filled with discharges, which prevent or stop the free flow.

The term "anticlogging substances" means any substances, which are characterized by a strong adherence to surfaces and which simultaneously repel other biological substances, especially proteinic substances, as well as cells. Typical examples of such substances are proteins such as albumins and especially useful for ventilation tubes used in human medicine is human serum albumin and in veterinary it is preferable to use autologous serum albumins, i.e. bovine serum albumin for bovines, etc., but other proteins with similar properties can be used. Proteins with the desired adhering and repelling properties can be prepared synthetically, semisynthetically or by recombinant DNA techniques. For human applications the most preferred embodiment is the production of human serum albumin by recombinant DNA techniques . Because only very small amounts are required to coat the tubes and the serum albumins can be used as solution with very low concentration, e.g. 0.001 mg/ml or less, it is both industrially and economically feasible to use highly purified serum albumin.

The term "medicinal drainage or ventilation tubes" means tympanostomy tubes, paranasal sinus drainage tubes, thracheostomy tubes, gastric tubes, urinary catheters, wound drains and voice prosthesis tubes etc. They comprise rigid, semirigid, elastic tubes or devices provided with a hole. The tubes are preferably made of plastics, silicons, metals, hydroxylapatits, not even the use of glass is excluded, even if it is not the preferred embodiment.

The term "diseases requiring ventilation and/or drainage" means health disturbances or diseases, which can be treated or alleviated with ventilation tubes. Such diseases are for example otitis media, disturbances of middle ear ventilation and tracheostomy . But also catheters, such as urinary catheters, voice prosthesis and wound dressings are incorporated in this term.

The General Description of the Invention

The invention relates to a method for preventing clogging of medicinal ventilation tubes by coating with anticlogging substances commercially available ventilation tubes, e.g. such as tympanostomic tubes or grommets used in the ears of patients following surgical treatment of otitis media with effusion or in the treatment of disturbances/problems in middle ear ventilation. All these applications are characterized by the use of purified human serum albumin as a coating, and/or anticlogging substance, which enables the prevention of other proteins, cellular material or microbes from binding on tube material and subsequently from clogging the ventilation tubes, e.g. the ear ventilation tubes, which are placed in the ear of patients with OME or patients with problems in middle ear ventilation.

The method for preventing the blocking of medicinal ventilation tubes, comprises coating the surface of said ventilation tubes with anticlogging substances, such as albumins available e.g. as human serum albumin or bovine serum albumin. For human medicinal application human serum albumin naturally is the preferred embodiment.

Typical examples of medicinal ventilation tubes are tympanostomy tubes or grommets, thracheostomy tubes, catheters and voice prosthesis, etc. Said tubes or devices are commercially available in a multitude of different forms suitable for different applications. The ventilation tubes are rigid, semirigid, elastic tubes generally made of plastics, silicons, metals, materials of hydroxylapatit, but other substances with the desired flexible, elastic, semirigid or rigid consistence are in no way excluded from the scope of the present invention. Said ventilation tubes include tubes (tympanostomy tubes, paranasal sinus drainage tubes, tracheostomy tubes, gastric tubes, urinary catheters, wound drains or voice prosthesis as well as any other devices used in treatment of diseases requiring ventilation or drainage.

The invention was tested by setting up a model system wherein fibronectin, one of the most adhesive glycoproteins known was used to mimick the effluents of ear. Since increased levels of fibronectin, fibrinogen and fibrin are present in atherosclerotic plaques (Stenman, S., et al . (1980), Acta Med. Scand. (Suppl.) 642, 165-170), in experiments below fibronectin was chosen as a model protein to study the binding of the cellular/plasma protein to albumin-coated ear ventilation tubes and the results were compared to the fibronectin binding on commercially available uncoated ear ventilation tubes.

Fibronectin is a high molecular weight adhesive glycoprotein, also called "cell glue", which is present in plasma and other body fluids in soluble form and in insoluble form in connective tissue matrices and in association with basement membranes (Vaheri, A. et al . (1985) Proc . Finn. Dent. Soc . 84, 13-18; Mosher, D.F. and Proctor, R.A. (1980), Science 209, 927-929 , 1989) . Characteristics of fibronectin include its multiple reactions with other proteins and bacteria, e.g. Staphylo- coccus aureus (Mosher and Proctor, 1980) .

Fibronectin has its role in cell migration and anchorage through its cell-binding site, in chemotaxis and opsonization and in the coagulation system; it binds to fibrin, collagen and components of the plasminogen activation system such as plasminogen and its activators (Salonen, E-M et al . (1985), J. Biol. Chem. 260, 12302-12307.). Fibronectin is also known for its sensitivity to several proteinases, including plasmin, and the degradation of fibronectin results in loss of the integrity of the cellular matrix (Vartio et al . (1981,), J. Biol. Chem. 256, 471-477 ; Vaheri et al . (1985), In: Fibrosis, Ciba Foundation Symposium 114, Pitman, London, pp 111-126.

Being the foremost abundant protein in serum, albumin was chosen for a model protein in studies on the effect of protein coating of ear ventilation tubes to prevent other proteins, cellular material and microbes from binding on commercially available ear ventilation tubes. An extremely small amount of serum albumin, e.g. 0.001 mg/ml or less, is sufficient to fully coat the surfaces of the tubes

The invention is described in more detail in the following nonlimiting examples. Based on these examples and the results it is evident for one skilled in the art to recognize further applications and to manufacture further embodiments of the present invention.

Example 1

Immobilization of human serum albumin on ear ventilation tubes

A volume of 200 μl of purified human serum albumin (HSA) or bovine serum albumin (BSA) (0.01-10 mg/ml), purchased from Sigma, MO, USA, was immobilized on ear ventilation tube (Xomed, FL, USA) , the tube material being either titan, C-flex, silicone, fluoroplastic or silver-oxide-coated silicone, in 0.01 M PBS (phosphate-buffered saline), pH 7.4, at room temperature overnight . The unbound albumin was removed by three washes with the washing buffer (10 M PBS containing 0.15 M NaCl, air-dried and stored at +4°C, if not immediately used. A coating concentration of 100 μg/ml was later used for the albumin coating.

Example 2

Binding of purified plasma fibronectin on albumin coated ear ventilation tubes 1. Fibronectin (200 μg in a volume of 200 μl of Tris-buffer, pH 7.4 containing 10 mM EDTA) was radiolabelled with 0.5 mCi of 125 i_n iodogen tubes (10 mg iodogen/ml CHCI3) using a 3 min incubation and mixing according to a standard procedure . The free iodide in radiolabelled fibronectin preparation was removed using G-25 gel chromatography column (PD-10; Pharmacia, Sweden), precoated with PBS containing 0.5% BSA (50ml) and washed with PBS (100 ml) before the use. The radiolabelled fibronectin was applied into the column and the fractions 6, 7 and 8, 0.5 ml each, containing the 125j_fibronectin were collected and pooled. The specific activity of the radio- labelled fibronectin preparation was 300 000 cpm/100 ng (lμl) fibronectin.

2. The ⁱ²⁵I-fibronectin (800 ng in a volume of 200 μl) in 0.1 M PBS, pH 7.4 was incubated with ear ventilation tube, coated with albumin as above or without coating, in a Minisorp tube

(Nunc, Roskilde Denmark), and incubated on an end-over-end shaker at room temperature overnight .

3. Unbound fibronectin was washed ten times with 10 mM PBS, pH 7.4.

4. The ear ventilation tubes were counted for radioactivity in a gamma counter (Wallac, Turku, Finland) .

Some of the results of the test are indicated in Table 1. The results were made as duplicate tests and the mean values have been calculated.

The Table shows that the albumin coating of the ear ventilation tubes significantly reduced the binding of radiolabelled fibronectin. FN-binding assay - HSA post coating

Tube added added bound mean bound material cpm ng cpm ng

Titan 1.337.000 800 119769

Titan 1.337.000 800 124775 122272 73

Titan + HSA 1. .337. 000 800 20994

Titan + HSA 1. 337. 000 800 18329 19661 11

C-flex 1. ,337. 000 800 190655 C-flex 1. ,337. 000 800 149688 170172 101

C-flex + HSA 1. .337. ,000 800 48259 C-flex + HSA 1. .337. ,000 800 54812 51536 31

Silicone 1. .337. .000 800 123185 Silicone 1. .337. .000 800 128266 125726 75

Silicone + HSA 1. .337. .000 800 43394 Silicone + HSA 1, .337. .000 800 32010 37702 23

Fluoroplastic 1, .337. .000 800 148325 Fluoroplastic 1, .337. .000 800 114934 131629 78

1 .337, .000 800 63514

Fluoroplastic + HSA 1 .337, .000 800 43655 53585 32

Silicone-AgO 1 .337 .000 800 128092 Silicone-AgO 1 .337 .000 800 194276 161184 96

Silicone-AgO + HSA 1 .337 .000 800 21853 Silicone-AgO + HSA 1 .337 .000 800 28561 25207 15

Example 3

Binding of unlabelled purified fibronectin on albumin-coated ear ventilation tubes.

1. The ear ventilation tubes (titanium and C-flex), purchased from Xomed, MO, USA, were coated with 5 mg/ml purified bovine serum albumin in 10 mM PBS, followed by an overnight incubation at room temperature. The unbound BSA was removed by washing ten times with 10 mM PBS, pH 7.4, containing 0.05% Tween 20. The uncoated tubes served as controls.

2. Albumin-coated ear ventilation tubes and uncoated control tubes were incubated with purified human fibronectin, in a normal plasma concentration of 300 μg/ml , in PBS, pH 7.4 overnight at +37 °C.

3. The unbound fibronectin was removed by washes as above.

4. The ear ventilation tubes were treated with Laemmli's sample buffer, pH 6.8, incubated at 96°C, centrifuged at 12000 rpm and run under reducing conditions in a 10 % SDS-poly- acrylamide gel electrophoresis (SDS-PAGE; Laemmli, U.K.

(1970), Nature 227, 680-685.

5. The proteins from the gel were transferred to a nitrocellulose sheet in an immunoblotting procedure (Towbin, H. et al (1979). Proceedings of the National Academy of Sciences, USA 76, 4350-4354), and the fibronectin stained with horseradish peroxidase-labelled anti-human fibronectin antibodies

(DAKO, Copenhagen, Denmark) .

The results clearly showed that albumin coating significantly reduced the binding of fibronectin on ear ventilation tubes. Example 4

The analysis of protein content of ear ventilation tubes, inserted through the tympanic membrane, clogged with secretory fluid and extruded.

Ear ventilation tubes, clogged with ear effusion fluid, were treated with Laemmli ' s sample buffer, heated for seven min at +96°C and run in 10 % SDS-PAGE under reducing conditions. The gel was stained with Coomassie brilliant blue for total protein staining, and visually recorded.

The results show, that the strong protein staining was obtained over the lane. Proteins comigrating with the purified human fibronectin, immunoglobulin G (IgG) or albumin standards, were strongly stained with enzyme- labelled anti-human fibronectin, IgG or albumin antibodies, respectively, suggesting, that the dried protein material contained fibronectin, immunoglobulin G, albumin and many other unidentified cellular or serum proteins.

Claims

1. A method for preventing the clogging of medicinal ventilation tubes, comprising coating the surface of said ventilation tubes with anticlogging substances, with surface adhering and biological substance repelling properties such as albumins .

2. The method of claim 1, wherein the anticlogging substance is an autologous serum albumin.

3. The method of claim 1, wherein the anticlogging substance is human serum albumin.

4. The method of claim 1, wherein the medicinal ventilation tubes are selected from a group consisting of tympanostomy tubes, paranasal sinus drainage tubes, thracheostomy tubes, gastric tubes, urinary catheters, wound drains and voice prosthesis .

5. The method of claim 2 wherein the medicinal ventilation tubes are rigid, semirigid, elastic tubes made of plastics, silicons, metals and/or hydroxylapatits .

6 The use of anticlogging substances, with surface adhering and biological substance repelling properties, such as albumins, for coating the surface of medicinal ventilation tubes, such as tympanostomy tubes, paranasal sinus drainage tubes, thracheostomy tubes, gastric tubes, urinary catheters, wound drains and voice prosthesis for treatment of diseases requiring ventilation and/or drainage.

7. The use of anticlogging substances with surface adhering and biological substance repelling properties, such as albumin, for manufacturing medicinal ventilation tubes, such as tympanostomy tubes, paranasal sinus drainage tubes, thracheostomy tubes, gastric tubes, urinary catheters, wound drains and voice prosthesis for treatment of diseases requiring ventilation or drainage.