WO2018150186A1

WO2018150186A1 - Tissue-adhesive materials

Info

Publication number: WO2018150186A1
Application number: PCT/GB2018/050408
Authority: WO
Inventors: Ian Thompson; Shannon SOUTHALL; David Mandley; Iain Johnson
Original assignee: Tissuemed Limited
Priority date: 2017-02-15
Filing date: 2018-02-15
Publication date: 2018-08-23
Also published as: GB2561947A; GB2561947B; GB201802484D0

Abstract

A tissue-adhesive polymer intended for application to internal and external surfaces of the body for therapeutic purposes, comprises vinyl pyrrolidone-derived units and acrylate-derived units containing at least one ester group and a tissue-reactive group. Compositions including freeze-dried compositions containing said polymer provide a tissue-contacting layer in tissue-adhesive articles, for example sheets having one or more layers. Liquid compositions containing said polymer provide hydrogels with useful tissue-sealing properties.

Description

Tissue-adhesive materials

This invention relates to a tissue-adhesive polymer intended for application to internal and external surfaces of the body for therapeutic purposes. In particular, the present invention relates to a tissue-adhesive polymer comprising vinyl

pyrrolidone-derived units and acrylate-derived units containing at least one ester group and a tissue-reactive group.

The invention also relates to a process for the preparation of the polymer, more specifically a process for the preparation of the polymer as a freeze-dried product. Also described are sheets comprising a solvent-cast or freeze-dried tissue- contacting layer comprising the polymer, liquid formulations comprising the polymer that form tissue-adhesive hydrogels upon reaction with crosslinkable reactants, and implantable medical devices coated with the polymer.

The invention further relates to a novel monomer.

The invention further relates to freeze-dried tissue-adhesive polymers and polymer compositions comprising a polymer that contains tissue-reactive functional groups. It relates to articles comprising said freeze-dried polymer or polymer composition, particularly articles comprising sheets of said freeze-dried polymer composition coated with non-adhesive polymer films, and to implantable medical devices coated with the freeze-dried polymer composition and methods for their manufacture.

There is considerable interest in the use, for a number of surgical or other

therapeutic applications, of materials that adhere to biological tissues, eg as an alternative or adjunct to the use of mechanical fasteners such as sutures, staples etc. These adhesive materials may be in the form of sheets, films, gels or liquids and may be either self-adhesive (by either chemical or physical means) or require energy (eg light) to promote chemical reaction.

Tissue-adhesive polymers of N-vinyl pyrrolidone, acrylic acid and acrylic acid-N- hydroxysuccinimide (NHS) ester are known. These materials, when formulated into sheets, offer strong adhesive performance via a combination of electrostatic forces (eg hydrogen bonding and/or van der Waals forces) and covalent bonding (reaction of NHS-ester functionality with nucleophilic groups present on proteinaceous surfaces to form covalent bonds).

WO2004/087227 discloses a polymer of vinyl pyrrolidone and acrylic acid NHS ester.

WO2006/013337 discloses a film comprising a preformed and crosslinked matrix that is formed from one or more polymers, at least one polymer being a terpolymer of vinyl pyrrolidone, acrylic acid and acrylic acid NHS ester. The film adheres to tissue and is described for use as the tissue-contacting layer of a tissue-adhesive sheet or a coating on an implantable medical device.

WO2007/088402 discloses a multilamellar tissue-adhesive sheet with a

tissue-contacting layer that comprises a terpolymer of vinyl pyrrolidone, acrylic acid and acrylic acid NHS ester.

The in vivo resorption period of polymers of N-vinyl pyrrolidone, acrylic acid and acrylic acid NHS ester may be 6 months or more. In certain applications it is desirable to use a tissue-adhesive polymer that takes a shorter time to be completely resorbed in vivo.

A further limitation of prior art polymers of N-vinyl pyrrolidone, acrylic acid and acrylic acid NHS ester is their poor solubility in aqueous media. Solubility in organic solvents limits their use to thin films and coatings. Other product formats are desirable, for instance freeze-dried articles or liquid formulations, which would be particularly advantageous for applications such as wound healing, sealing large areas of tissue, sealing air leaks, haemostasis and the prevention of post-surgical adhesions in open and minimally invasive surgery.

Thus, there remains a need for biocompatible and biodegradable polymers that bond effectively to tissue but are resorbed quickly in vivo (relative to known polymers of N-vinyl pyrrolidone, acrylic acid and acrylic acid NHS ester). For biocompatibility and to enable a broader range of product and delivery formats, it would be advantageous for the polymer to be soluble in aqueous media.

There have now been devised improvements to the tissue-adhesive materials of the general type described above, and to related materials, that overcome or

substantially mitigate the above-mentioned and/or other disadvantages of the prior art.

According to a first aspect of the invention, there is provided a tissue-adhesive polymer comprising: a) vinyl pyrrolidone-derived units of general formula (I):

(I) where R¹ , R², R³, R⁴, R⁵ and R⁶ independently represent H, optionally substituted C1-6 alkyl, optionally substituted C1-6 alkoxy or halo; and acrylate-derived units of general formula (II):

wherein

A is -OC(=O)-CH₂CH2- or a single bond,

R⁷ is -H or -CH3, and

X is a tissue-reactive group.

If any of R¹, R², R³, R⁴, R⁵ or R⁶ is substituted C1-6 alkyl or substituted C1-6 alkoxy, substituents that may be present include C1-6 alkyl, -OH, -OR⁹, -COOH

and -C(O)OR⁹, where R⁹ is C1-6 alkyl.

Preferably, the polymer is completely resorbed in vivo within about 1 -12 months depending on composition and molecular weight. By "complete resorption" we mean that the polymer is broken down by hydrolysis, enzymatic action or otherwise, in vivo, and absorbed, metabolised or otherwise removed to the extent that the polymer is no longer detectable by eye or histological analysis. During resorption, wound healing restores the normal morphology and therefore, over time, the cellular environment around the site of the polymer becomes clinically unremarkable.

Resorption may be complete within about 9 months. Resorption may be complete within about 6 months. Resorption may be complete within about 4.5 months.

Resorption may take at least one month to complete, at least 2 months to complete or at least 3 months to complete. For example, the polymer may take between about 1 and 12 months to be resorbed, between about 2 and 9 months to be resorbed, between about 3 and 6 months to be resorbed, or between about 3 and 4.5 months to be resorbed in vivo.

The polymer of the present invention comprises vinyl pyrrolidone-derived units of general formula (I) (also referred herein as unit (I)). These units may be

incorporated into the polymer using the equivalent N-vinyl-2-pyrrolidone, ie an N- vinyl-2-pyrrolidone monomer of general formula (III):

where R¹ , R², R³, R⁴, R⁵ and R⁶ independently represent H, optionally substituted C-1 -6 alkyl, optionally substituted C1 -6 alkoxy or halo.

The polymer may be prepared by including the N-vinyl-2-pyrrolidone monomer of general formula (III) in the polymerisation reaction mixture in an amount of about 50 to 97.5 mol %, in an amount of about 70 to 95 mol %, in an amount of about 80 to 95 mol % or in an amount of about 85 to 90 mol %. The polymer may be prepared by including the N-vinyl-2-pyrrolidone monomer of general formula (III) in the

polymerisation reaction mixture in an amount of about 90 mol %.

The term "mol %" is the mole fraction for the monomeric component in question, in the above case the N-vinyl-2-pyrrolidone of general formula (III), multiplied by 100 (ie the number of moles of that monomeric component divided by the total number of moles of monomer in the reaction mixture, multiplied by 100).

In preferred embodiments, the polymer comprises a vinyl pyrrolidone-derived unit of general formula (I) wherein R¹ , R², R³, R⁴, R⁵ and R⁶ independently represent H, in which case the starting monomer is N-vinyl-2-pyrrolidone (NVP). Polyvinyl pyrrolidone) is water-soluble and therefore it is believed that the

incorporation of vinyl pyrrolidone-derived units (I) increases the water solubility of the polymer, its overall solubility depending on the proportion of those units in the polymer, the nature of the R¹, R², R³, R⁴, R⁵ and R⁶ substituents and the solubility of the other units in the polymer. The vinyl pyrrolidone-derived units also provide the polymer with its initial contact adhesion or "tack" by forming hydrogen bonds and van der Waals interactions between its amide residues and the tissue surface.

The polymer of the present invention also comprises an acrylate-derived unit of general formula (II) (also referred herein as unit (II)). Unit (II) may be incorporated into the polymer by polymerisation of the appropriate acrylate monomer (IV):

wherein

A is -OC(=O)-CH₂CH2- or a single bond;

R⁷ is -H or -CH3; and

X is a tissue-reactive group.

The polymer of the invention will generally comprise a polyolefin-type chain, referred to herein as the polymer "backbone" with pendant groups determined by the particular monomers (III) and (IV) that are used.

By "tissue-reactive functional group" we refer to groups that are chemically reactive towards the tissue to which the polymer is, in use, applied, or which exhibit increased reactivity to tissue. For example, the tissue-reactive functional groups on unit (II) confer adhesive properties to the polymer by reacting with nucleophilic groups on proteinaceous surfaces, for example amines on a tissue surface, and forming covalent bonds.

Tissue-reactive functional groups that may be of utility in the present invention are any functional groups capable of reaction (under the conditions prevalent when the polymer is applied to tissue, ie in an aqueous environment and without the application of significant amounts of heat or other external energy) with functional groups present at the surface of the tissue to form covalent bonds. As proteins commonly contain thiol and primary amine moieties, and tissues usually contain an abundance of protein, the functional groups present at the surface of the tissue include thiol and amine groups, and tissue-reactive functional groups therefore include groups reactive to thiol and/or amine groups. Examples are: imido ester, p-nitrophenyl carbonate, N-hydroxysuccinimide (NHS) ester, epoxide, isocyanate, acrylate, vinyl sulfone, orthopyridyl-disulfide, maleimide, aldehyde, and

iodoacetamide.

In some embodiments, X is -C(=O)-OR⁸, where -OR⁸ is a labile group that renders the carbonyl group reactive to tissue, for instance by the formation of an amide bond between the carbonyl and an amine at the tissue surface.

R⁸ may be, for example, imidyl, p-nitrophenyl or N-hydroxysuccinimidyl.

When A is -OC(=O)-CH₂CH₂- and R⁷ is -H, and X is -C(=O)-OR⁸, monomer (IV) is mono(2-acryloyloxyethyl) succinate-R⁸ (referred to herein as MAES-R⁸):

CH2=CH-C(=O)O-CH2CH2-OC(=O)-CH2CH2-C(=O)OR⁸ (MAES-R⁸)

When A is a single bond and R⁷ is H, and X is -C(=O)-OR⁸, monomer (IV) is 2- carboxyethyl acrylate-R⁸ (referred to herein as CEA-R⁸):

CH2=CH-C(=O)O-CH₂CH2-C(=O)OR⁸ (CEA-R⁸)

When A is -OC(=O)-CH₂CH₂- and R⁷ is -CH₃, and X is -C(=O)-OR⁸, monomer (IV) is mono(2-methacryloyloxyethyl) succinate-R⁸ (referred to herein as MMES-R⁸):

CH2=C(CH₃)-C(=O)O-CH2CH2-OC(=O)-CH2CH2-C(=O)OR⁸ (MMES-R⁸) When A is a bond and R⁷ is H, and X is -C(=O)-OR⁸, monomer (IV) is 2-carboxyethyl methacrylate-R⁸ (referred to herein as CEMA-R⁸).

Thus, when monomer (IV) is MAES-R⁸, the free carboxyl group on the succinate of the MAES is derivatised or "activated" with R⁸ so as to form a tissue-reactive functional group.

In some embodiments, the carboxyl groups may be converted to

N-hydroxysuccinimide (NHS) esters. As used herein, the term NHS is intended to encompass not only N-hydroxysuccinimide itself, but also derivatives thereof in which the succinimidyl ring is substituted. An example of such a derivative is

N-hydroxysulfosuccinimidyl and salts thereof, particularly the sodium salt. In some preferred embodiments, X is -C(=O)-OR⁸, R⁸ is NHS, and therefore the acrylate- derived unit (II) is:

wherein

A is -OC(=O)-CH2CH2- or a single bond; and R⁷ is -H or -CH₃.

In some embodiments, wherein A is -OC(=O)-CH₂CH2-, R⁷ is -H, X is -C(=O)-OR⁸ and R⁸ is NHS, the acrylate-derived unit (II) is

and the corresponding acrylate monomer (IV) is NHS-functionalised MAES ("MAES- NHS").

We believe that NHS-functionalised MAES is a novel compound and therefore, in a further aspect of the invention, there is provided mono(2-acryloyloxyethyl)succinate N-hydroxysuccinimide ester.

MAES-NHS may be prepared by the derivatisation of the carboxylic acid functionality of MAES with NHS utilising dicyclohexylcarbodiimide as a coupling agent in a suitable dry solvent, using techniques known to those skilled in the art. It follows that polymers are novel, which polymers comprise the acrylate-derived unit:

In US201 1/152455, the N-hydroxysuccinimide acrylate ester monomers, mono-(2- methacryloyloxyl)-ethyl succinate N-hydroxysuccinimide ester and 2-carboxyethyl acrylate N-hydroxysuccinimide ester, are described. These monomers are used in the manufacture of synthetic polymeric cell culture surfaces suitable for the culture of difficult-to-culture cells, including undifferentiated embryonic stem cells.

US201 1 /275154 also describes acrylate monomers with an N-hydroxysuccinimide moiety for use in the preparation of a synthetic cell culture surface to support the growth of cells including undifferentiated embryonic stem cells.

We believe this is the first time MAES has been incorporated in a polymer. In particular, we believe this is the first time MAES has been incorporated in a tissue- adhesive polymer for medical applications such as haemostasis, wound healing, joining, sealing and reinforcing tissue, or the delivery of therapeutic agents. Thus, according to a further aspect of the invention, there is provided the use of MAES in the preparation of tissue-adhesive polymers for medical use.

The speed at which the polymer is resorbed will depend on a variety of factors, including rate of hydration, the ease at which it breaks down or degrades into fragments in vivo and the molecular weight of those fragments. Monomer (IV) is used to incorporate degradable, adhesive side-chains into the polymer. When using MAES or MMES, two ester groups are included in each side chain. When using CEA or CMEA, one ester group is included in each side chain. These ester groups facilitate degradation of the polymer and therefore the use of MAES or MMES is generally preferred as they contain two degradable ester linkages. In the most preferred embodiments, monomer (IV) is MAES-R⁸.

The polymer may be prepared by including monomer (IV) in the polymerisation reaction mixture in an amount of about 2.5 to 50 mol %, in an amount of about 5 to 30 mol %, in an amount of about 5 to 20 mol % or in an amount of about 10 to 15 mol %. The polymer may be prepared by including monomer (IV) in the

polymerisation reaction mixture in an amount of about 10 mol %.

In some embodiments of the present invention, the tissue-adhesive polymer may also comprise an ester-containing unit of general formula (V):

)

Ester-containing units of general formula (V) may be incorporated into the polymer backbone by adding a cyclic ketene acetal that undergoes free radical ring-opening during polymerisation. 2-Methylene-1 ,3-dioxepane (MDO) is a cyclic ketene acetal that is particularly suitable, introducing ester-containing units of the following formula into the polymer backbone:

Thus, MDO introduces additional degradable ester groups within the polymer backbone and can therefore be added to increase the speed at which the polymer is resorbed in vivo.

A cyclic ketene acetal may be included in the polymerisation reaction mixture in an amount of up to about 20 mol %, up to about 15 mol %, or up to about 10 mol %. A cyclic ketene acetal may be included in the polymerisation reaction mixture in an amount of about 0 to about 20 mol %, in an amount of about 5 to 20 mol %, in an amount of about 5 to 15 mol % or in an amount of about 10 to 15 mol %.

In accordance with literature reports, polymerisation reactions to incorporate MDO were found by the present inventors to be inefficient. Typically, only 10-50 % of the MDO in the starting monomer feed was incorporated into the final polymer

composition, and of that only a portion with the ester in the backbone of the polymer, rather than as a pendent side-chain. Incorporation was improved under more concentrated reaction conditions, at higher temperatures and with shorter feed times. Typically, reactions at high solid content were required to achieve sufficient incorporation of the ester-containing units into the polymer backbone.

In some embodiments, the tissue-adhesive polymer may also comprise acrylic acid- derived units of general formula

which may be incorporated by including acrylic acid in the polymerisation mixture in an amount up to about 20 mol %. The use of acrylic acid provides the polymer with additional initial adhesive tack due to van der Waals forces and/or hydrogen bonding between the available carboxylic acid moiety on the acrylic acid and the surface of the tissue. However, the inclusion of acrylic acid reduces the solubility of the polymer in water and may cause or at least contribute to inflammation on the tissue surface with which the polymer is in contact. Thus, the decision whether to include acrylic acid, and in what amount, will depend on the balance of properties required for the intended application of the polymer.

Acrylic acid may be included in the polymerisation reaction mixture in an amount of up to about 20 mol %, up to about 15 mol %, or up to about 10 mol %. Acrylic acid may be included in the polymerisation reaction mixture in an amount of about 0 to about 20 mol %, in an amount of about 5 to 20 mol %, in an amount of about 5 to 15 mol % or in an amount of about 10 to 15 mol %.

In some embodiments, the monomers are polymerised in the presence of a crosslinker to form a crosslinked polymer. Crosslinking improves the cohesive strength of the polymer and may therefore improve its performance, yet the polymer remains biodegradable because of its degradable ester groups. In some

embodiments, the crosslinker itself contains degradable ester groups. In preferred embodiments, the crosslinker is a PEG-diacrylate of general formula (VII):

where n is 1 -1000 in which case the polymer will contain crosslinking units of general formula VIII:

where n is 1 -1000

In some preferred embodiments, n may be 1 -100. In some preferred embodiments, n may be 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. For instance, n may be 3.

A crosslinker may be included in the polymerisation reaction mixture in an amount of about 0 to 5 mol %, about 1 to 4 mol %, or about 2 to 3 mol %. For instance, a crosslinker may be included in an amount of about 2.5 mol %.

Use of the PEG-diacrylate crosslinker of general formula (VII) incorporates polyethylene glycol (PEG) moieties into the polymer. However, in some

embodiments, the tissue-adhesive polymers of the invention are not prepared with a PEG-diacrylate crosslinker. In some embodiments, the tissue-adhesive polymers of the invention do not contain PEG moieties.

According to a further aspect of the invention there is provided a method for the manufacture of a polymer comprising vinyl pyrrolidone-derived units of general formula (I) and acrylate-derived units of general formula (II), which method comprises polymerisation of one or more vinyl pyrrolidone monomers of general formula (III) and one or more acrylate monomers of general formula (IV). Solution free-radical polymerisation is a suitable method of making the polymer of the present invention: monomers (III) and (IV) may be polymerised in a suitable solvent in the presence of a free radical initiator using techniques known to those skilled in the art. Thus, polymerisation may be carried out in a solvent. The optional additional monomers (eg MDO, acrylic acid and/or crosslinker) may be added to the reaction vessel as an initial charge or fed continuously with monomers (III) and (IV).

Any solvent that solubilises all the reagents and the final polymer may be suitable. The preferred solvent is DMSO.

The molecular weight of the polymer and the distribution of monomer units can be adjusted by altering the ratio of monomers to initiator, feed rates, reaction

temperature and other experimental parameters, in order to optimise the

performance, rate of resorption and other material characteristics. Thus a wide variety of polymers are possible.

By "distribution" of units we refer to the pattern of monomer units along the backbone (ie main chain) of the polymer. The distribution is "even" if the monomer units occur in a regular, substantially alternating pattern, whilst being distributed uniformly throughout the length of the polymer. The distribution may be described as "blocky" if significant numbers of the same monomer unit occur together, ie as blocks. For example, a polymer of the invention containing about 90 mol % vinyl pyrrolidone- derived units of general formula (I) and about 10 mol % acrylate-derived units of general formula (II) will have an "even" distribution if the local ratio of 9:1 is substantially the same throughout the polymer.

Since monomers (III) and (IV) react at different rates, the distribution of units (I) and (II) in the polymer may be altered by adjusting the feed time of monomers (III) and (IV) in the polymerisation reactive mixture. In particular, a more even distribution may be achieved by slowing down the rate of addition (increasing the feed time, ie a "starve-fed" polymerisation). Starve-fed polymerisation allows the monomers in the vessel to react before more are added, and is a well-known technique for controlling the distribution of monomers with different activities in a polymer. A feed time of at least 6 hours may be required to generate a polymer with a relatively even distribution of units (I) and (II) using the monomers NVP and MAES- NHS. Polymers in which units (I) and (II) are more evenly dispersed have been shown to have significantly better adhesive performance than corresponding blocky polymers prepared using shorter feed times.

It has been observed that the greater the loading of vinyl pyrrolidone-derived units (I) in the polymer, the greater its solubility in water. Conversely, it appears that increasing amounts of acrylate-derived units (II) make the polymer less water- soluble. If the polymer is too hydrophobic it will not be readily wetted when it is placed on the tissue surface, and therefore the contact area and the attractive forces between the polymer and the tissue surface will not be maximised. Similarly, if the polymer is in a film or sheet format, contact with the tissue surface will be reduced if it is too hydrophobic and it will not bond to the tissue surface as effectively. For that reason, the loading of acrylate-derived units (II) in the polymer may be limited to approximately 15 mol %.

When the reaction solvent is DMSO, it has been found that a suitable means of recovering the polymer from the reaction mixture is, surprisingly, freeze-drying (lyophilisation). Standard methods of recovery known in the art (eg precipitation or vacuum evaporation) may be unsatisfactory because the polymer is soluble in many of the solvents that are normally used for recovery (eg water, hexane or diethyl ether), particularly when the reaction solvent is also present. As a result of this solubility, it is difficult to retain the low molecular weight fractions of the polymer in the precipitate. For example, when precipitation of a polymer made from N-vinyl pyrrolidone and MAES-NHS monomers is attempted, it is typical to observe that polyvinyl pyrrolidone)-rich and/or low molecular weight fractions are solubilised, leaving poly(MAES-NHS)-rich and/or high molecular weight fractions behind.

Lyophilisation of the reaction mixture provides a better method of recovering all fractions of the polymer as a solid. The utility of freeze-drying was unexpected both for isolation of the tissue-adhesive polymer and subsequent processing.

Freeze-drying was not considered in relation to the tissue-adhesive polymers previously described by the inventors (such as the terpolymer of vinyl pyrrolidone, acrylic acid and acrylic acid NHS ester), because those polymers are not soluble in water and it is usual to freeze-dry from an aqueous solution.

For example, the exemplified compositions described in WO2004/087227 contain powdered albumin (porcine or human serum albumin) and a powdered tissue- adhesive polymer. The albumin is obtained in powder form by freeze-drying a buffered aqueous solution of the protein. However, the tissue-adhesive polymer, which is insoluble in water, is manufactured using precipitation and then ground to a powder. The freeze-dried albumin and precipitated tissue-adhesive polymer powders are mixed and compressed into a tissue adhesive sheet. Freeze-drying was only considered for the water-soluble component.

Adjustments to the composition of the tissue-adhesive polymers previously described by the inventors can render them soluble in water (eg increasing the vinyl pyrrolidone content). Moreover, the tissue-adhesive polymer according to the first aspect of the present invention is advantageously soluble in water.

Nevertheless, freeze-drying from an aqueous solution would not be expected to be successful, because tissue-reactive functional groups are susceptible to hydrolysis and therefore tissue-adhesive polymers would degrade on contact with or when dissolved in water, certainly within the time span required for freeze-drying.

The need to avoid premature hydrolysis of tissue-reactive functional groups is acknowledged in WO2013/053753 and WO201 1 /079336.

WO2013/053753 discloses a haemostatic composition comprising a biocompatible polymer in particulate form and a crosslinker comprising tissue-reactive groups (such as NHS-ester groups). The crosslinker is a hydrophilic polymeric component such as PEG-NHS.

WO2013/053753 suggests that it is important that the composition is manufactured such that the reactive groups of the hydrophilic crosslinker are retained and are able to react once the composition is applied to a wound. To achieve that,

WO2013/053753 teaches that the crosslinker may be (a) processed in an aqueous medium at a very low pH; (b) melted and then sprayed or printed onto the surface of the matrix; (c) a dry form of the hydrophilic crosslinker may be sprinkled onto the matrix; or (d) a solution of the hydrophilic crosslinker in an inert organic solvent (eg dry ethanol, dry acetone or dry dichloromethane) may be used to apply the crosslinker to the matrix.

WO201 1/079336 also discloses a haemostatic composition comprising crosslinkable components that contain tissue-adhesive functional groups. It refers to a composite sponge comprising a porous matrix of a biomaterial, such as collagen, and a first and second crosslinkable component. The first crosslinkable component is a multi- nucleophilic alkylene oxide and the second crosslinkable component a multi- electrophilic polyalkylene oxide. The Examples include collagen sponges that are treated with an acidic solution (pH 3.0, hydrochloric acid) of

pentaerythritolpoly(ethyleneglycol)ether tetrasuccinimidyl glutarate and

pentaerythritolpoly(ethyleneglycol)ether tetra-thiol and then freeze-dried.

Both WO2013/053753 and WO201 1 /079336 disclose the use of an acid (acetic acid or hydrochloric acid) to lower the pH and inhibit hydrolysis of the tissue-reactive functional groups, which are NHS-ester groups. In the experience of the present inventors this is undesirable because a large proportion of the acid will be extracted under vacuum during the freeze-drying process, meaning that some hydrolysis is inevitable. Also, any acid remaining after processing will result in a product that has a residual low pH, which will reduce crosslinking reactivity.

WO2002/034304 describes a freeze-dried polymeric matrix in the form of a sheet, patch or film suitable for application to moist surfaces of the body. The matrix comprises a naturally occurring or synthetic polymerisable and/or crosslinkable material that supports wound healing (eg albumin or carboxymethyl cellulose

(CMC)), and a synthetic polymer having bioadhesive properties (eg

polyvinylpyrrolidone (PVP)), such properties enabling the matrix to adhere to underlying tissue by means of ionic and/or hydrogen bonding. Aqueous solutions of the polymerisable and/or crosslinkable material and the synthetic polymer having bioadhesive properties are cast in layers and heated to partially or fully crosslink the materials and evaporate the water. The crosslinked layer or laminate is then freeze- dried. It is notable that the synthetic polymer having bioadhesive properties does not contain tissue-reactive functional groups capable of reaction with functional groups present at the surface of the tissue to form covalent bonds.

DMSO is not commonly used as a solvent for freeze-drying due to its low vapour pressure and high boiling point (189°C). Surprisingly, the inventors have discovered that it is possible to freeze-dry tissue-adhesive polymers containing tissue-reactive functional groups using a dry, non-aqueous solvent such as DMSO.

The absence of water in the anhydrous DMSO reaction solvent enables tissue- adhesive polymers to be freeze-dried without degradation of the tissue-reactive functional groups. Care must be taken to avoid water contamination throughout all stages of the process to prevent hydrolysis of the NHS-ester chemistry. This may be achieved by drying the DMSO using 3A molecular sieves and minimising the time that frozen DMSO-sheets are open to air.

The solid freeze-dried polymer may be milled to form a powder and the powder may be delivered in that form, producing a crosslinked hydrogel upon hydration or contact with moist tissue surfaces. It may be delivered as a single component or may be mixed with other ingredients such as reaction promotors (eg buffer salts) or secondary reactive species (eg synthetic or natural species that contain amine or thiol moieties, for instance albumin), all in powder form. Alternatively, the powder (or powder mixture) may be compressed to form a sheet (or the tissue-contacting layer of a multilayer sheet) or a three-dimensional device (eg plug or pellet). The powder may also be used to coat a sheet or medical device.

For the applications of particular interest to the applicant, the powdered, freeze-dried polymer is typically processed further.

The polymer may be delivered in liquid format by dissolving the powdered, freeze- dried polymer in aqueous solution. The polymer solution may be mixed with a second aqueous solution containing nucleophilic materials at the surface of the tissue or shortly before application, to form a tissue-adhesive crosslinked hydrogel.

Alternatively, the powdered, freeze-dried polymer may be dissolved in a solvent and cast as a film or a layer in a sheet having one or more layers.

The powdered, freeze-dried polymer may be dissolved in a dry, non-aqueous solvent and freeze-dried to form a freeze-dried matrix in the form or a sheet or other article. The freeze-dried matrix has a high porosity and high surface area for fluid

absorption.

The tissue-adhesive polymer of the invention may be suitable, in any of these formats, for application to both internal and external tissue surfaces of the body, ie it may be applied topically to the exterior of the body (ie to the skin) or to internal tissue surfaces such as surfaces of internal organs during surgical procedures.

Tissue adhesive powder

The solid polymer may be milled to form a powder and may be delivered in that form, as a single component or in admixture with other particulate components (eg other materials having tissue-reactive functional groups and buffer materials). There are clear advantages of delivering the polymer as a powder, in particular the fact that a powder formulation adheres to the tissue surface and does not spread unduly. On contact with the tissue surface the formulation becomes hydrated, thereby causing reaction between the tissue-reactive functional groups and the underlying tissue surface. Such reactions between the tissue-reactive functional groups and the underlying tissue result in high adhesion between the formulation and the tissue surface. Reaction may also take place between the tissue-reactive functional groups and the other components of the formulation to form a strong, flexible and

tissue-adherent gel. The powder absorbs physiological fluids (as a consequence of application onto exuding tissue surfaces), and any additional solutions used to hydrate the formulation following application (such fluids can be commonly used solutions used in surgical irrigation), becoming gelatinous and adherent to the tissue surfaces, and thereby providing an adhesive sealant, haemostatic and pneumostatic function.

In addition, a powdered formulation is essentially inactive until hydrated by contact with the tissue surface, so the shelf-life may be considerable.

The polymer may also be delivered in powder form by compressing the powder into sheets, using a compressed powder layer in a multilayer sheet, or by compressing the powder to form another three-dimensional article, such as a plug.

Liquid formulations that form tissue-adhesive hydrogels

The aqueous solubility of the polymer according to the first aspect of the invention allows the polymer to be delivered in a liquid format, for instance in a syringe or spray. A tissue-adhesive crosslinked hydrogel may be formed in situ by preparing an aqueous solution of the polymer (eg dissolving the powdered freeze-dried product in water) and mixing that solution containing the polymer with a second aqueous solution containing nucleophilic materials (ie materials having nucleophilic groups for instance amines or thiols). The solutions may be delivered separately and mixed in situ or they may be mixed shortly before delivery. The functional lifespan of the tissue-adhesive solution is typically up to 6 hours due to hydrolysis of the NHS-ester groups. The rate of hydrolysis may be slowed by decreasing the pH to 4 or below. However, this also inhibits reactivity towards the nucleophilic component in the second solution (eg amine) and proteinaceous tissue surfaces. The second solution containing nucleophilic materials may be a solution of synthetic or natural material. Suitable examples include poly(ethylene imine (PEI), 2-armed or 4-armed poly(ethylene glycol)-amine, natural or recombinant albumin, trypsine, or poly(ethylene glycol)-thiol. A chromophore may be included in one or more of the solutions to aid visualisation. A spray may provide a useful format for delivering either the separate solutions or a pre-mixed solution.

Liquid formulations may be applied to the surface of a tissue at a surgical site via open or minimally invasive surgical techniques. The crosslinked hydrogel product may be used, for example, to adhere tissues, seal (eg achieve haemo or pneumostatsis), join or occlude tissues, or to deliver an active to a target tissue.

This is believed to be the first disclosure of a liquid composition containing a tissue- adhesive polymer comprising vinyl pyrrolidone-derived units of general formula (I).

Thus, in a further aspect of the invention, there is provided a liquid composition for application to the surface of a tissue comprising a tissue-adhesive polymer, which polymer comprises vinyl pyrrolidone-derived units of general formula (I):

where R¹ , R², R³, R⁴, R⁵ and R⁶ independently represent H, optionally substituted C1-6 alkyl, optionally substituted C1-6 alkoxy or halo.

If any of R¹ , R², R³, R⁴, R⁵ or R⁶ is substituted Ci-e alkyl or substituted Ci-e alkoxy, substituents that may be present include C1-6 alkyl, -OH, -OR⁹, -COOH

and -C(O)OR⁹, where R⁹ is Ci-e alkyl. Tissue-adhesive solvent-cast sheet

The tissue-adhesive polymer of the invention may be dissolved in an appropriate solvent in order to be cast as a film or as a layer in a sheet having one or more layers. In some preferred embodiments the polymer is obtained as a freeze-dried product, powdered, dissolved in solvent and cast as a single-layered film or sheet. The resulting film or sheet may be perforated.

A multilayer sheet can be prepared, for example by solvent-casting one or more layers of the tissue-adhesive polymer of the claimed invention with layers of one or more other synthetic or natural polymers. The layers may alternate or may not. The layers of one or more other synthetic or natural polymers may be structural layers that may comprise a non-adhesive polymer. Thus, according to a further aspect of the invention there is provided a multilamellar, tissue-adhesive sheet comprising a structural layer or laminate comprising a non-adhesive polymer, and a tissue- adhesive layer comprising a tissue-adhesive polymer, which tissue-adhesive polymer comprises: a) vinyl pyrrolidone-derived units of general formula (I)

where R¹ , R², R³, R⁴, R⁵ and R⁶ independently represent H, optionally substituted C1-6 alkyl, optionally substituted C1-6 alkoxy or halo; and acrylate-derived units of general formula (II)

wherein

A is -OC(=O)-CH₂CH2- or a single bond,

R⁷ is -H or -CH3, and

X is a tissue-reactive group.

and -C(O)OR⁹, where R⁹ is C1-6 alkyl.

The individual layers may be cast sequentially from volatile solvent on top of each other.

Non-adhesive polymers that may be used in the structural layer or laminate are preferably degradable polymers and may be synthetic or naturally-occurring materials. Suitable synthetic non-adhesive polymers include biodegradable aliphatic polyesters, for instance poly(glycolide), poly(L-lactide), poly(D-lactide), poly(DL- lactide), poly(caprolactone) and copolymers thereof in any ratio of monomers. In some preferred embodiments, the structural layer or laminate comprises poly(DL- lactide-co-glycolide) (50/50 molar ratio) (PLGA) or poly(L-lactide-co-caprolactone) (70/30 molar ratio) (PLC). Examples of naturally-occurring materials that may be suitable for use in the structural layer or laminate are collagen and chitosan.

The function of the structural layer or laminate may be to provide a non-adhesive backing and/or to increase the strength of the sheet. A tissue-adhesive sheet having a non-adhesive coating will adhere only to the target tissue and not to surrounding tissues (eg the pleural or peritoneal wall). Such a non-adhesive coating will typically have a thickness of about 4-50 μιη.

To aid visualisation and sheet orientation, the sheet may also incorporate a surface marking comprising a visible chromophore, for example FD&C Blue No 1 or

Methylene Blue.

The sheet may have the polymer of the present invention on one or both surfaces (ie be single- or double-sided). In some embodiments a single- or double-sided sheet intended to seal/join two opposing tissue surfaces may be perforated to allow tissue- ingrowth. The perforations may be square or circular holes. The dimensions of the perforations may be, but are not limited to, a width or diameter of about 1 to 10 mm.

By the term "sheet" is meant an article with a thickness that is considerably less than its other dimensions. The sheet may have an overall thickness of about 0.01 to 1 mm, typically 25-50 μιη.

The sheet may be produced with, or subsequently cut to, dimensions from a few square millimetres to hundreds of square centimetres.

Tissue-adhesive lyophilised matrix

The tissue-adhesive polymer according to the first aspect of the invention may be dissolved in a dry, non-aqueous solvent and freeze-dried to form sheets or other articles. In the most preferred embodiments, the polymer is prepared in DMSO, isolated by freeze-drying and powdered. Then the powdered freeze-dried product may be re-dissolved in a dry, non-aqueous solvent, with or without other components, poured into a mould and freeze-dried again to form the final freeze- dried polymer composition.

It is necessary for the non-aqueous solvent to be capable of fully dissolving the tissue-adhesive polymer. The solution must be able to be frozen and the solvent must be capable of being removed by sublimation in the freeze-drying (lyophilisation) process. Preferably the non-aqueous solvent is dimethyl sulfoxide (DMSO).

The tissue-adhesive polymer of the invention may be freeze-dried in combination with one or more additional components, included for example to alter the structure, flexibility and strength, colour or adhesive properties, or to introduce therapeutic agents, such as anti-inflammatories, anti-infective agents or clotting agents. For instance, in order to increase the structural integrity of the freeze-dried product and generate a scaffold-type structure, the tissue-adhesive polymer may be freeze-dried in combination with a structural component such as poly(lactide-co-glycolide) (PLGA) or other biodegradable aliphatic polyester.

In general, the additional component(s) must also be soluble in a dry non-aqueous solvent.

Thus, in a further aspect of the invention, there is provided a freeze-dried

composition comprising a tissue-adhesive polymer according to the first aspect of the invention.

The polymer composition may substantially comprise the tissue-adhesive polymer of the present invention. By this is meant that the polymer composition may consist entirely or substantially of the tissue-adhesive polymer, the polymer comprising more than 80%, more than 90%, more than 95% or more than 99% by weight of the polymer composition.

The tissue-adhesive polymer of the invention may comprise at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or at least about 80% by weight of the polymer composition. The tissue-adhesive polymer may comprise less than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or less than about 10% by weight of the polymer composition. The tissue-adhesive polymer may comprise between about 10% and 90%, between about 20% and 80%, between about 30% and 70%, or between about 40% and 60% by weight of the polymer composition. The

tissue-adhesive polymer may comprise about 50% by weight of the polymer composition.

The freeze-dried polymer composition may consist solely of the tissue-adhesive polymer or may be a homogenous mixture of the tissue-adhesive polymer with one or more additional components. For example, a visible light-absorbing chromophore may be added to the solution of polymer in non-aqueous solvent before freeze- drying to give the freeze-dried polymer composition a distinguishing colour.

Examples of suitable chromophores include Methylene Blue (methylthioninium chloride) and FD&C Blue No 1 .

Additional components may be added to alter the structure, flexibility or strength of the freeze-dried composition, or to alter its structural integrity or gelation

characteristics upon hydration.

The tissue-adhesive polymer may be freeze-dried in combination with a structural component, for example poly(lactide) or poly(lactide-co-glycolide) (PLGA), in order to increase the structural integrity of the freeze-dried product so that it retains an open structure upon hydration, rather than forming a continuous gel, and acts more like a scaffold. The physical characteristics of the structural component (for instance its tensile strength or flexibility) will affect the same characteristics in the freeze-dried product, and can be increased by increasing the proportion of structural component in the composition.

The tissue-adhesive polymer and the structural polymer may be a homogenous single freeze-dried layer or two or more freeze-dried layers of the separate polymers conjoined during manufacture.

The structural component may comprise more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more than about 80 % by weight of the polymer composition. The structural component may comprise less than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or less than about 10% by weight of the polymer composition. The structural component may comprise between about 10% and 90%, between about 20% and 80%, between about 30% and 70%, or between about 40% and 60% by weight of the polymer composition. The structural component may comprise about 50% by weight of the polymer composition.

The product that is created and retained following freeze-drying has a more open structure compared, for example, to a solid homogenous film or sheet comprising the same polymer, and therefore it has a higher porosity and higher surface area for fluid absorption. This generally leads to an improvement in moisture uptake and wettability, and consequently the rate of adhesion between the polymer and the tissue and the haemostatic properties of the polymer may be improved. The density and porosity of the freeze-dried polymer composition are determined by the concentration of polymer solids dissolved in solution prior to freeze-drying. A concentrated solution will lead to a stiff, dense matrix with a relatively low porosity. Conversely, a dilute solution will lead to a flexible matrix with a relatively high porosity. Precise control over the physical appearance and characteristics of the freeze-dried polymer composition to suit a particular application is thus possible.

The freeze-dried polymer composition is also advantageous because it is essentially inactive until hydrated upon and following contact with the tissue surface, so its shelf-life may be considerable, for example more than six months when stored appropriately at room temperature.

The freeze-dried polymer composition may be described as a "matrix" in view of its more open structure. However, the tissue-adhesive polymer rapidly becomes a gelatinous and sticky hydrogel when it hydrates and reacts with a tissue surface and/or proteinaceous fluids. This is in contrast to the haemostatic collagen matrices described in the prior art which are described as "sponges" or "pads" and act as scaffolds, retaining the open structure of the dried matrix upon hydration rather than gelling. The freeze-dried polymer composition according to the invention may be entirely synthetic, or substantially so, being free or substantially free of materials of human or animal, particularly mammalian, origin, eg wherein such materials account for less than 1 %, more preferably less than 0.5 % or less than 0.1 % of the polymer composition. One situation in which a relatively small proportion of material of human or animal origin may be present is where that material takes the form of one or more therapeutically active agents that are included in the formulation and are of such origin.

In most embodiments the freeze-dried polymer composition does not contain biological material, eg material that occurs naturally in a living being. Examples of biological materials include collagen, gelatin, albumin, haemoglobin, fibrinogen, fibrin, casein, fibronectin, elastin, keratin, laminin, and polysaccharides such as glycosaminoglycan, starch, cellulose, dextran, hemicellulose, xylan, agarose, alginate and chitosan. In currently preferred embodiments, the freeze-dried polymer composition does not contain cellulose. In currently preferred embodiments, the freeze-dried polymer composition does not contain gelatin. In currently preferred embodiments, the freeze-dried polymer composition does not contain albumin.

In some embodiments, the freeze-dried polymer composition does not contain a modified polysaccharide such as carboxymethyl cellulose (CMC).

The freeze-dried polymer composition of the present invention is prepared by freeze- drying the polymer from a dry non-aqueous solution. The process must be undertaken in the absence of water to ensure that the tissue-reactive functional groups are not hydrolysed.

Thus, according to a further aspect of the invention there is provided a method for the manufacture of a freeze-dried polymer composition comprising the tissue- adhesive polymer according to the first aspect of the invention, which method comprises the steps of:

a) preparing a solution comprising a tissue-adhesive polymer in a dry, non-aqueous solvent; and b) freeze-drying the solution to yield the freeze-dried polymer composition.

In most preferred embodiments, the dry, non-aqueous solvent is DMSO.

The freeze-dried polymer composition of the invention may be in the form of a sheet. By the term "sheet" is meant that the freeze-dried polymer composition has a thickness that is considerably less than its other dimensions. The freeze-dried sheet may have an overall thickness of about 0.05 to 10 mm, typically 0.5 to 5 mm. The sheet may be produced with, or subsequently cut to, dimensions from a few square millimetres to hundreds of square centimetres.

Thicker, three-dimensional structures are also envisaged. Thus, the freeze-dried polymer composition may take the form of a plug or pellet, which may be used to seal or fill cavities and holes in the body. Such plugs may be formed with any suitable shape, eg generally cylindrical, ellipsoidal or cuboidal. Another suitable three-dimensional structure that is envisaged is a cylindrical filament that may be used for securing other devices in place, in the manner of a suture. The freeze-dried polymer composition may be formed in a three-dimensional structure by using the appropriately shaped freeze-drying mould or by grinding the freeze-dried product to a powder and compressing to form the required shape. Structures having more complex shapes may also be produced, for example by using shaped moulds.

Examples include pre-formed connectors, eg for the end-to-end or end-to-side anastomotic apposition and closure of vessels, fasteners such as staples or barbed pins for holding tissues together, or fixing plugs to be fitted, for example, into holes in bone to provide anchorages for mechanical fasteners such as screws or for dental crowns.

Optionally, the freeze-dried tissue-adhesive polymer composition of the present invention may be used as the tissue-contacting component in a tissue-adhesive article that contains two or more different components. Thus, according to a further aspect of the invention, there is provided a tissue-adhesive article comprising a freeze-dried polymer composition according to the invention. In preferred embodiments, the tissue-adhesive article of the invention is a multilamellar sheet having two or more layers. In preferred embodiments, the multilamellar sheet includes a layer or laminate comprising a non-adhesive polymer to provide a non-adhesive backing and/or increase the strength of the sheet. Most preferably, the non-adhesive material is a synthetic polymer. Examples of suitable polymers include biodegradable polyesters such as poly(glycolide), poly(L-lactide), poly(D-lactide), poly(DL-lactide), poly(caprolactone) and copolymers thereof in any ratio of monomers. In some preferred embodiments, the non-adhesive polymer is poly(DL-lactide-co-glycolide) (PLGA) or poly(L-lactide-co-caprolactone) (PLC). The non-adhesive polymer may be cast from a solvent to provide a thin, continuous film, or several thin layers may be cast to produce a laminate.

For example, if it is not desirable for a surface of a sheet of the freeze-dried polymer composition to adhere to tissue when in use, that surface may be coated with a layer or laminate of non-adhesive polymer. A tissue-adhesive sheet having a non- adhesive coating will adhere only to the target tissue (to which the underside of the sheet is applied) and not to surrounding tissues (eg the pleural or peritoneal wall). The non-adhesive coating may include a visibly-absorbing chromophore in the form of a logo or marking to enable identification of the non-tissue-contacting surface of the sheet. Examples of suitable chromophores include Methylene Blue and FD&C Blue No 1 . The thickness of the non-adhesive coating may be between about 4μιη to Ι ΟΟμιη. The coating may be a single layer or may be built up of several thin layers, for example to facilitate incorporation of a logo.

Alternatively, a layer or laminate comprising a non-adhesive polymer may be used to form a structural layer within a multilamellar sheet.

For example, the multilamellar sheet may consist of a layer or laminate comprising a non-adhesive polymer with a layer of tissue-adhesive freeze-dried polymer composition on each side (ie a double-sided product). In some embodiments a double-sided multilamellar sheet or a single-layer sheet of the freeze-dried polymer composition intended to seal/join two opposing tissue surfaces may be perforated to allow tissue-ingrowth. The perforations may be square or circular holes. The dimensions of the perforations may be, but are not limited to, a width or diameter of about 1 to 10 mm.

Multilamellar sheets may also be prepared from alternating layers that comprise tissue-adhesive polymer and non-adhesive polymer. The layers comprising tissue- adhesive polymer may all be freeze-dried layers (ie layers of freeze-dried polymer composition comprising a tissue-adhesive polymer containing tissue-reactive functional groups), or may include layers of tissue-adhesive polymer that are cast from solvent. In preferred embodiments, the multilamellar sheet has a tissue- contacting layer of the freeze-dried polymer composition according to the invention, and alternative solvent-cast layers comprising non-adhesive polymer and tissue- adhesive polymer.

Other components that may be useful in the tissue-adhesive articles of the present invention include absorbent pads, scaffolds, layers of therapeutic material or biodegradable layers containing therapeutic agents. For example, the freeze-dried polymer composition of the invention may provide the tissue-contacting surface of an article that comprises an absorbent pad.

Alternatively, the freeze-dried polymer composition may be used to coat a substrate. In particular, the invention may find application in the provision of an adhesive coating to an implantable medical device. Tissue-adhesive articles are envisaged in which at least part of the external surface of an implantable medical device is coated with a freeze-dried polymer composition according to the invention. A solution containing the tissue-adhesive polymer may be applied to a device, frozen and subsequently lyophilised to generate a freeze-dried coated medical device.

It will be appreciated by those skilled in the art that the method described herein for preparing freeze-dried polymer compositions comprising the tissue-adhesive polymer of the invention, can be used to prepare freeze-dried polymer compositions comprising other tissue-adhesive polymers. Using this method it is possible to freeze-dry both water-insoluble and water-soluble tissue-adhesive polymers without degradation of the tissue-reactive functional groups during the freeze-drying process. Thus, according to a further aspect of the invention, there is provided a method for the preparation of a freeze-dried polymer composition comprising a tissue-adhesive polymer containing tissue-reactive functional groups, which method comprises the steps of: a) preparing a solution comprising a tissue-adhesive polymer containing tissue-reactive functional groups in a dry, non-aqueous solvent; and

b) freeze-drying the solution to yield the freeze-dried polymer composition.

Tissue-adhesive polymers containing tissue-reactive functional groups that can be freeze-dried in accordance with the present invention may also contain vinyl pyrrolidone-derived units. By vinyl pyrrolidone-derived units is meant repeat units in the polymer that result from the addition of vinyl pyrrolidone or a derivative thereof as a monomer in the polymerisation reaction mixture. For example, vinyl pyrrolidone- derived units according to general formula (I):

where R¹ , R², R³, R⁴, R⁵ and R⁶ independently represent H, optionally substituted C-1 -6 alkyl, optionally substituted C1-6 alkoxy or halo. If any of R¹ , R², R³, R⁴, R⁵ or R⁶ is substituted Ci-e alkyl or substituted Ci-e alkoxy, substituents that may be present include C1-6 alkyl, -OH, -OR⁹, -COOH

and -C(O)OR⁹, where R⁹ is Ci-e alkyl.

The present invention is believed to be the first disclosure of the freeze-drying of a polymer that contains vinyl-pyrrolidone units using DMSO.

Thus, according to a further aspect of the invention there is provided a method for freeze-drying a polymer containing vinyl pyrrolidone-derived units of general formula (I), which method comprises:

a) preparing a solution comprising the polymer in dimethyl sulfoxide (DMSO); and b) freeze-drying.

Tissue-adhesive coating

The tissue-adhesive polymer of the present invention may be coated onto the external surface of a preformed implantable medical device in order to render the device adhesive.

The implantation of devices in the body is commonplace in surgical procedures, and many such devices are known. However, implanted devices can become dislodged from the site of application, leading to a failure of the device to perform its intended function and/or other complications (such as inflammation, migration, tissue trauma, pain, post-surgical adhesions, fistula formation, seroma formation, haematoma and recurrence of tissue defect). Serious complications may necessitate further surgical intervention.

Sutures, staples or other forms of mechanical fastener are commonly used to attempt to fix the device more securely in position. However, fixation is often difficult to achieve, and moreover, such approaches introduce further drawbacks.

Methods for modifying current implantable devices to render them self-adhesive (or more self-adhesive, as the case may be), present clear advantages. The tissue-adhesive polymer of the present invention may be used to modify a wide variety of implantable devices. One group of implantable devices that are

particularly suitable are graft products, intended principally for implantation to join or seal tissues, to reinforce weakened soft tissue and/or to assist the repair of internal wounds. The modification of proprietary graft products by application of the polymer significantly improves the placement, positioning and adhesion characteristics with no significant change in the handling or flexibility of the product.

Furthermore, non-absorbable constructs, including but not limited to tantalum, graphene, stainless steel and titanium, in the form of sheets, meshes, clothes, wires and complex shapes such as pacemakers may be coated.

If the surface of the device is smooth, eg a device having a smooth surgical steel exterior, then full encapsulation of the product may be required, in order that the coating remains adequately attached during use.

It is preferred to use devices having a surface that is not perfectly smooth, such that the coating may wholly or partially encapsulate parts of the device or may fill interstices in the device thereby aiding physical attachment of the coating.

So, for instance, the polymer of the present invention may be particularly useful in the coating of mesh-type products, fibrous products, fabrics or the like.

There may be some crosslinking reaction between the polymer coating and the medical device to which it is applied, particularly if the device comprises a

biomaterial having groups that react with the tissue-reactive functional groups on the polymer, eg the tissue-reactive groups on the acrylate-derived units of general formula (II). Delivery of actives

The polymer according to the invention may also be used for the delivery of one or more therapeutically active agents to the tissue surface to which the polymer is applied. The agent(s) will be slowly released, either by diffusion or as the polymer degrades over time. The agent(s) may be incorporated into the polymer during its manufacture, eg by admixture with the monomer(s) and any other ingredients prior to polymerisation. Alternatively, the agent(s) may be attached to a component of the polymer after the polymer is manufactured. For example, in a multilamellar, tissue- adhesive sheet comprising a structural layer of material or laminate that comprises one or more synthetic polymers, and a tissue-contacting layer of material that comprises the tissue-adhesive polymer, one or more therapeutically active agents may be covalently bonded to the tissue-contacting surface of the tissue-adhesive polymer. However, in other embodiments, the sheet may be substantially free of therapeutically active agents.

By "therapeutic agent" is meant any pharmaceutically active substance or its prodrug, or a salt or solvate of a pharmaceutically active substance. By

"substantially free" is meant in this context that the polymer does not contain anything that may be considered to be a therapeutic agent, or that it contains something that may be considered to be a therapeutic agent, but in such low amounts that it would have no significant therapeutic effect. By "prodrug" is meant any structural derivative of a therapeutic agent which is chemically transformed within the body to exert its pharmacological or therapeutic action. For example, an ester of a therapeutic compound containing a carboxy group may be convertible by hydrolysis in vivo to the active molecule.

Suitable therapeutic agents, which are exemplary and are not meant to be limiting in any way, include analgesics (eg endorphins), anaesthetics, anti-infective agents (eg gentamicin, bacitracin, aciclovir), antineoplastics (eg doxorubicin, bleomycin), antiinflammatory agents (eg celecoxib), angiogenic agents (eg vascular epithelial growth factor, fibroblast growth factor), anti-angiogenic agents (eg endostatin), growth promoters (eg vascular epithelial growth factor, fibroblast growth factor), haemostatic agents (eg antifibrinolytics, blood coagulation factors, fibrinogen and vitamin K), and therapeutic monoclonal antibodies (eg basiliximab, trastuzumab).

The local, sustained delivery of anti-cancer agents is of particular interest, eg for residual/non-resectable colorectal and pancreatic cancers. Exemplary anti-cancer agents include doxorubicin.

The incorporation of therapeutic agents to improve and/or increase the rate of wound healing and reduce infections, for both external and internal applications, is also of particular interest.

Non-limiting examples of anti-infective agents include antibacterial, antifungal, antiviral and anti-parasitic agents.

By "anti-infective agent" is meant any agent that is capable of acting against infections, by killing infective micro-organisms and/or inhibiting the spread of an infective micro-organism. Infective micro-organisms include bacteria, parasites, yeast, moulds, fungi, viruses, prions and viroids.

Anti-infective agents suitable for use in the present invention may be drugs, such as antibiotics or antifungals.

Examples of antimicrobial or antibacterial compounds are triclosan, neomycin, clindamycin, polymyxin, bacitracin, benzoyl peroxide, tetracylines such as

doxycycline or minocycline, sulfa drugs such as sulfacetamide, penicillins, cephalosporins such as cephalexin, and quinolones such as lomefloxacin, olfoxacin or trovafloxacin.

Antiviral compounds that may be incorporated include acyclovir, oseltamivir, and penciclovir.

Antifungal compounds include farnesol, clotrimazole, ketoconazole, econazole, fluconazole, calcium or zinc undecylenate, undecylenic acid, butenafine hydrochloride, ciclopirox olaimine, miconazole nitrate, nystatin, sulconazole, and terbinafine hydrochloride.

However, due to increasing concerns about antibiotic and antifungal drug-resistance, the use of alternative anti-infective agents may be preferred. Thus, the anti-infective agent may comprise metal ions that have anti-infective properties, for instance silver, gold or copper ions.

In some embodiments, the polymer of the invention may be used to deliver or pick up cells or cellular components.

Abbreviations

AAc acrylic acid

AIBN azo-iso-butyronitrile

CEA 2-carboxyethyl acrylate

CEA-NHS 2-carboxyethyl acrylate N-hydroxysuccinimide ester

CEMA 2-carboxyethyl methacrylate

DCC dicyclohexylcarbodiimide

DCM dichloromethane

DCU dicyclohexylurea

DMSO dimethyl sulfoxide

DPBS Dulbecco's Phosphate-Buffered Saline

MAES mono(2-acryloyloxyethyl) succinate

MAES-NHS mono(2-acryloyloxyethyl) succinate N-hydroxysuccinimide ester

MeOH methanol

MDO 2-methylene-1 ,3-dioxepane

MMES mono(2-methacryloyloxyethyl) succinate

NHS N-hydroxysuccinimide

NVP N-vinyl-2-pyrrolidone

RO reverse osmosis

PEI poly(ethylene imine)

PEG polyethylene glycol PEG-diacrylate polyethylene glycol diacrylate

PLGA poly(lactide-co-glycolide)

PLC poly(lactide-co-caprolactone)

Description of the Drawings

Figure 1 illustrates the synthesis of mono(2-acryloyloxyethyl) succinate

N-hydroxysuccinimide ester (MAES-NHS).

Figure 2 illustrates the synthesis of poly(NVP-co-(MAES-NHS)).

Figure 3 illustrates the synthesis of poly(NVP-co-(MAES-NHS)-co-MDO).

Figure 4 illustrates the synthesis of poly(NVP-co-(MAES-NHS)-co-AAc).

Figure 5 illustrates the synthesis of poly(NVP-co-(MAES-NHS)-co-PEG-diacrylate).

Figure 6 illustrates the synthesis of poly(NVP-co-(MAES-NHS)-co-MDO-co-PEG- diacrylate).

Figure 7 illustrates a representative tissue-adhesive sheet (not to scale) as described in Example 4.

Figure 8 illustrates a representative tissue-adhesive article that is a sheet of lyophilised matrix with a barrier film (not to scale), as per Example 1 1 .

Figure 9 illustrates a representative tissue-adhesive article that is a sheet of lyophilised matrix with a multilamellar barrier film (not to scale), as per Example 12. Examples

Example 1

Synthesis of mono(2-acryloyloxyethyl) succinate N-hydroxysuccinimide ester (MAES-NHS)

The reaction is shown schematically in Figure 1 .

In a reaction flask MAES (100.0 g, 0.463 mol) and NHS (53.3 g, 0.463 mol) were dissolved with stirring in DCM (300 mL) and cooled to approximately 10^QC by immersing in an ice bath. To this was added a solution of DCC (95.5 g, 0.463 mol) dissolved in DCM (100 mL) dropwise with stirring over a period of 1 hour maintaining a temperature between 10-20^QC. Following addition, the solution was left stirring for a minimum of 48 hours at room temperature during which period a white precipitate (DCU) formed.

At the completion of the reaction the DCU was removed by vacuum filtration. The majority of the solvent was then removed by evaporation under reduced pressure which gave rise to further DCU formation. The mixture was repeatedly diluted with DCM, filtered and concentrated several times until a clear yellow solution resulted. This solution was then added to diethyl ether (1600 mL) to liberate a white powder. The powder was washed with additional diethyl ether (1600 mL), milled and dried under vacuum at 20^QC for >48 hours. Recovered yield 82 to 99 g (57 to 68 mol %).

Characterisation

¹ H NMR (Bruker Avance, 400 MHz, de-DMSO): δ 6.34 (dd, J = 17.4, 1 .4 Hz, 1 H), 6.18 (dd, J = 17.2, 10.4 Hz, 1 H), 5.96 (dd, J = 10.4, 1 .2 Hz, 1 H), 4.36-4.22 (m, 4 H) 2.94 (t, 2 H), 2.80 (s, 4 H), 2.70 (t, 2 H).

FT-IR (Nicolet iS10 diamond crystal ATR): IR (neat) 2949, 1819, 1787, 1733, 1636, 1616 cm^"1. UV-Vis (Helios β, 0.1 M NH₄OH aq): AMAX 260 nm, NHS content 36.7 wt % (theory = 37 wt %).

LC-MS (Waters 2695 Separations Module equipped with 996 PDA and Quattro Ultima TQ MS, 1 img/mL in MeOH, mobile phase 0.1 % formic acid in water (A) and MeCN (B) operated in a gradient from 90 % A to 90 % B over 30 minutes (10 minute hold), 5 μιη C1 8 column, positive electrospray MS): retention time 1 6.54 mins,

[M+H]+ M/z 314 (theory = 31 3 + 1 = 314).

GC-FID (Agilent 6890, 0.1 img/mL in EtOH and 1 μΙ_ splitless injection, 0.25 mm fused silica capillary column, 0.25 μιη 5 % phenylpolysiloxane phase): retention time 1 7.9 min, purity by peak area >99 %.

Example 2

Synthesis of PVP-co-MAES-NHS general procedure

The reaction is shown schematically in Figure 2.

In a reaction flask DMSO (30.6 imL) was heated to 80^QC with stirring and

deoxygenated using nitrogen for 30 minutes at 1 L/min. A monomer solution containing NVP (20.0 g, 0.180 mol), MAES-NHS (6.26 g, 0.020 mol) and DMSO (20 imL) was prepared. An initiator solution containing AIBN (0.0821 g, 5.0x10^"4 mol, [monomer]/[initiator] = 400, 0.25 mol % based on monomers) and DMSO (5 imL) was prepared. The polymerisation utilised a monomer feed time of 6 hours and a simultaneous initiator feed time of 6.5 hours. At the completion of monomer and initiator feeds the reaction flask was maintained at 80^QC with stirring and nitrogen flow until a total polymerisation time of 24 hours was achieved.

The polymer was isolated by lyophilisation to remove DMSO yielding a white crystalline solid. This was milled and purified by multiple washes/extractions in diethyl ether and dried under vacuum at 40^QC for >48 hours. Recovered yield 22 to 24 g (84 to 91 wt %). Characterisation

Solution viscosity (Brookfield DV-III, spindle 40, 50 rpm, 25 ^QC): 37.4 cP (10 % w/v DMSO), 7.8 cP (1 0 % w/v 1 .0M NH₄OH).

Aqueous GPC (Viscotek GPC Max, 50 img/mL in 0.1 M NH₄OH, mobile phase 0.2M sodium nitrate, flow rate 0.8 imL/min, 2x30cm A6000M Viscotek GPC columns, Rl detection, conventional calibration using PVP standards 1 kDa to 3500kDa): Mn 74,000 PDI 4.0 (0.1 M NH₄OH). H NMR (Bruker Avance, 400 MHz, de-DMSO): δ 4.3-4.1 (br, MAES-NHS, 4 H), 3.9- 3.5 (br, PVP, 1 H), 3.2-3.0 (br, PVP, 2 H), 2.94 (br, MAES-NHS, 2 H), 2.81 (br, MAES- NHS, 4 H), 2.70 (br, MAES-NHS, 2 H), 2.4-1 .2 (br PVP, 6 H). Incorporation of MAES- NHS determined from integrals and corrected for number of protons = 0.1 08/0.952 = 1 0.2 mol % (theory = 1 0 mol %).

FT-IR (Nicolet iS1 0 diamond crystal ATR): IR (neat) 2949, 1 813, 1 783, 1 737, 1 666 cm^-1.

UV-Vis (0.1 M NH₄OH aq): AMAX 260 nm, NHS content 8.3 wt % (theory = 8.8 wt %). Example 3

Synthesis of alternative polymer formulations

The reactions are shown schematically in Figures 3-6.

Alternative polymers were prepared utilising the same experimental procedure described in Example 2. By changing the reaction temperature, feed times and/or including different combinations of monomers (in the monomer feed) the properties and characteristics of the final polymer could be readily adjusted. Table 1 - Monomer feed ratios and reaction conditions

Table 2 - Polymer characterisation data

Polymer Viscosity Viscosity GPC NHS NHS MDO MDO

Reference DMSO 0.1 M Analysis content theoretical content theoretical

(CP) NH₄OH 0.1 M (mol %) (mol %) (mol %) (mol %)

(cP) NH₄OH

Mn (PDI)

LB0261 -156 517.1 32.2 - - 8.76 - -

LB0261 -150 371 .6 28.6 - - 8.76 - -

LB0261 -152 320.5 30.3 - 7.21 8.76 - -

LB0261 -154 192.7 25.0 - 7.94 8.76 - -

LB0261 -176 200.5 27.5 - - 8.76 - -

LB0261 -180 88.5 17.7 - 7.51 8.76 - - 121 ,000 8.76 - -

LB0265-065 139.6 27.5 (6.3) 7.68

LB0264-062 146.1 20.9 - 7.52 8.76 - -

74,000 8.76 - -

LB0265-069 37.4 7.8 (4.0) 7.30

LB0264-066 25.5 7.9 - 8.93 8.76 - -

LB0265-083 267.4 78.6 - 7.49 8.31 - -

LB0265-088 109,000 5.2 9.09

100.3 15.7 8.24 8.06

(4.7)

LB0265-090 44,000 5.7 16.67

35.4 3.9 8.52 7.46

(3.3)

LB0265-177 275.3 37.4 - 8.01 8.65 - -

Viscosity was measured in DMSO (undegraded) and following hydrolysis by NH₄OH (degraded) for a series of polymers prepared from NVP and MAES-NHS using different feed times: 1 , 1 .5, 3, 6, 8 and 16 hours.

Increasing monomer feed time reduces the viscosity of the polymer (in DMSO). This may be due to changes in distribution of MAES-NHS-derived units along the polymer backbone: MAES-NHS will have a disproportionate effect on viscosity due to its bulky structure. A more even (and therefore spaced-out) distribution of the bulky MAES-NHS-derived units is thought to reduce entanglement, resulting in a lower viscosity.

When subjected to hydrolysis in NH₄OH the viscosities across the series are similar, demonstrating that the backbone molecular weights are broadly equivalent.

Increasing the reaction temperature generally decreases the viscosity both in DMSO (undegraded) and NH₄OH (degraded).

The use of MDO to incorporate caprolactone units into the polymer is determined by NMR analysis, in particular the ring-opened ester methylene CH2 multiplet at δ 3.99 ppm is useful for semi-quantification of incorporation. MDO undergoes free-radical ring-opening during polymerisation to form additional ester bonds in the polymer backbone. However, the reaction is inefficient and the quantity in the final polymer is much lower than the starting monomer feed. It is suspected that the majority of the MDO either degrades or forms low molecular weight oligomers, which are then extracted from the polymer during the washing stage in preference to being incorporated.

Following hydrolysis, the viscosity and molecular weight of polymers of the present invention made with MDO are lower on a like-for-like basis compared to those with no MDO. This offers additional confirmation that ester bonds have been successfully incorporated into the polymer backbone.

In the following Examples the general term "polymer" refers to a polymer according to Table 1 .

Example 4

Preparation of tissue adhesive sheet (multilayer)

A multilamellar tissue-adhesive sheet is shown schematically in Figure 7. The sheet comprises a structural laminate and a tissue-contacting layer.

The structural laminate has the form of:

a) a first layer 1 of PLGA;

b) a second layer 2 of polymer according to Table 1 above;

c) a third layer 3 of PLGA; and

d) a fourth layer of polymer.

The tissue-contacting layer 4 is conjoined to the third layer 3 and comprises a polymer. The second and fourth layers may comprise the same polymer or different polymers.

The first and third layers 1 ,3 each have a thickness of approximately 4 μιη, and the second layer 2 a thickness of approximately 5 μιη. The tissue-contacting layer 4 has a thickness of approximately 22 μιη. The sheet is prepared as follows:

1 .1 Preparation of solutions

Three solutions are prepared as follows:

Solution A is 10g PLGA dissolved in 100ml DCM.

Solution B is 22.5g polymer dissolved in 100ml DCM/MeOH 15/4.

Solution C is a viscous printing ink comprised of polymer (2.25 g, 22.5 % w/v) and

FD&C Blue 1 (or Methylene Blue) (0.09 g, 0.9 % w/v) dissolved in RO water (10 mL).

1 .2 Casting of layer 1

Solution A is cast onto a release substrate such as silicone-backed release paper using a device referred to as a K bar. The film is dried for 30 minutes at

20° C/atmospheric pressure. The film is not removedfrom the release substrate.

1 .3 Casting of layer 2

Solution B is cast onto Layer 1 using a K bar. The film is dried for 30 minutes at 20° C/atmospheric pressure. The film is not removedfrom the release substrate.

1 .4 Application of logo

Solution C is printed by screen printing onto the surface of Layer 2 to form an alphanumeric trade/visualisation logo.

1 .5 Casting of layer 3

Solution A is cast onto Layer 2 using a K bar. The film is dried for 30 minutes at 20° C/atmospheric pressure. The film is not removedfrom the release substrate.

1 .6 Casting of layer 4

Solution B is cast onto Layer 3 using a K bar. The film is dried for 30 minutes at 20 ^QC/atmospheric pressure. The film is then peeled from the release substrate and dried for 1 12 hrs at 45 ^QC at 0.1 mbar. 1 .7 Cutting out

The product is cut to size using specially designed cutters.

1 .8 Sterilisation

The dried, cut down product is subjected to heat/pressure to flatten and then is stored in a foil pouch to maintain sterility and exclude moisture. The product is then gamma sterilised at 25-40 kGy.

The product is a clear opaque film with the logo visible throughout. Example 5

Alternative preparations of tissue adhesive sheet

Using the procedure described in Example 4 alternative formulations can be readily prepared.

Table 3 - Tissue adhesive sheet formulations

Example 6

In vitro characterisation of tissue adhesive sheets of the invention

Using the procedure described in Example 4, the polymers referenced in Table 1 were used to prepare tissue-adhesive sheets with the following layer thicknesses: Layer 1 :10 μιη, Layer 2:5 μιη, Layer 3:4 μιη, Layer 4:22.5 μιη. Adhesive performance was assess quantitatively and qualitatively, and the results are shown in Table 4.

Quantitative adhesive performance: A section of the sheet was applied onto a suitable section of freshly excised porcine liver with moderate pressure for 60 seconds. After 5 minutes, the sample was immersed in DPBS for a further 5 minutes. The energy of adhesion was quantified using a Zwick universal testing machine.

Qualitative adhesive performance: A section of the sheet was applied onto a suitable section of freshly excised porcine liver with moderate pressure for 60 seconds. After 5 minutes, the sample was immersed in DPBS for a further 30 minutes. The performance was evaluated blind by three assessors using a scoring system from 0 (no adhesion) to 5 (strong adhesion, liver could be lifted off the bench).

Increasing the monomer feed time from 1 hour to 16 hours gives an improvement in performance, despite the reduction in viscosity (molecular weight) due a more favourable even distribution of MAES-NHS moieties along the polymer backbone.

Incorporation of MDO at the lower level (10 mol %) marginally reduces performance. Higher levels (20 mol %) have a larger effect.

Increasing the reaction temperature (to decrease molecular weight) has negligible effect on performance.

Incorporation of acrylic acid improved qualitative performance.

Incorporation of a crosslinker improved both quantitative and qualitative

performance. Table 4 - Tissue adhesive multilayer sheet in vitro performance

Physical characteristics 10 χ 50 mm samples of the sheets listed in Table 4 were evaluated using a Zwick universal testing machine. Samples were evaluated both dry and after submersion in DPBS for 10 minutes. Typical results (depending on thickness of individual layers): Tensile strength (dry) 20-40 MPa (wet) 5-25 MPa, FMAX (dry) 5-20 N (wet) 1 .5-5 N, e-modulus (dry) 0.8-1 .5 GPa (wet) 0.3-1 .2 GPa.

Example 7

In vivo pre-clinical acute performance of a tissue adhesive sheet

Example tissue adhesive sheets were evaluated as surgical sealants in a range of preclinical models. The sheet was cut to a suitable size to the site of the injury allowing for at least a 1 cm overlap onto non-injured tissue. All samples showed strong adhesion, sealing and haemostatic properties, as shown in Table 5. The samples achieved a "pass" if the leak (blood, air or CSF) was controlled. Neurosurgery - dural closure (pig): 1 .5-2 cm sutured durotomy, CSF leak observed, 1 .5 x 2.5 cm tissue adhesive sheet applied with 60 seconds moderate pressure using a damp swab.

Lung surgery - pneumostasis (pig): 5 mm punch biopsy to lung, air and blood leak observed, 5 ^χ 5 cm tissue adhesive sheet applied with 90 seconds moderate pressure using a damp swab.

General surgery - haemostasis (pig): 5 mm punch biopsy to liver, medium-heavy blood leak (including arterial and venous components) observed, 5 ^χ 5 cm tissue adhesive sheet applied with 60 seconds moderate pressure using a damp swab.

General surgery - haemostasis (pig): 2 cm end resection of spleen, medium blood leak observed, 5 ^χ 5 cm tissue adhesive sheet wrapped around end of spleen and applied with 60 seconds moderate pressure using a damp swab.

General surgery - haemostasis (rabbit): 3 mm punch biopsy to liver, medium-heavy blood leak observed, 5 ^χ 5 cm tissue adhesive sheet applied with 60 seconds moderate pressure using a damp swab.

Table 5 - Tissue adhesive multilayer sheet in vivo performance

Neurosurgery Pneumostasis Haemostasis

Polymer (Pig) Durotomy (Pig) lung 5mm (Pig) (Pig) (Rabbit)

Reference biopsy liver spleen liver 3mm

5mm resection biopsy biopsy

LB0261 -152 - - - - Pass

LB0261 -154 - - - - Pass

LB0265-065 Pass Pass Pass - Pass

LB0265-069 Pass Pass Pass Pass^† Pass

LB0265-088 - - - - Pass

LB0265-090 - - - - Pass † A sheet containing a thinner barrier layer (Layer 1 , 4μιη) was found to give improved conformability around the resection.

Example 8

In vitro measurement of degradability

Biodegradation and resorption may be determined by the concentration of

hydrolysable ester groups in the tissue-adhesive polymer and the molecular weight of the degraded fragments.

In vitro tests were carried out to assess the potential of different polymers to degrade into fragments and to quantify the molecular weight of those fragments. Base hydrolysis was used to cleave ester groups and liberate the polymer backbone and molecular weight fragments, which were evaluated by gel permeation

chromatography or viscosity analysis. By comparing the viscosity of solutions in DMSO (no hydrolysis) and 1 .0M NH₄OH (complete hydrolysis) the efficacy of degradation was quantified. This test was particularly useful for measuring the effect of MDO incorporation.

In vivo pre-clinical rate of degradation/resorption of a tissue adhesive sheet

In addition to the presence of hydrolysable ester groups, when the polymer is applied to a tissue surface or contacts other bodily fluids, an amide bond forms (via reaction of the tissue-reactive functional groups, such as NHS esters, with proteinaceous amine groups), which generates a crosslinked hydrogel entity. The degradation and resorption of the post-application crosslinked species was evaluated in vivo using a standard subcutaneous implantation preclinical model. Histology reports were prepared after 14 and 28 days, and 3, 4.5 and 6 months to assess the amount of polymer material remaining and the tissue response.

Example tissue adhesive sheets were evaluated in a standard rat subcutaneous implant model to evaluate local tissue response and determine the rate of degradation/resorption. A 1 1 cm piece of the sheet was implanted into an identifiable subcutaneous pocket on back of a rat, with each rat receiving 4 test articles in separate pockets. Subjects were recovered for 14 and 28 days, and 3, 4.5 and 6 months.

The results are summarised in Table 6. The results from this study demonstrate that the tissue adhesive sheets visibly resorb within approximately 3 months (this includes both components of the device - PLGA and polymer - and do so without any detrimental or clinically significant local tissue response. In some formulations, microscopically small fragments of material remain along with a low level cellular response. By 6 months all material from all formulations had been completely resorbed and the cellular response has returned to normal/non-clinically relevant levels. There is a correlation with molecular weight of the polymer: the lower molecular weight materials demonstrate a slightly faster rate of cellular infiltration and resorption.

Table 6 - Degradation/resorption profile of tissue adhesive sheets.

Macroscopic scores: 0 (no resorption/degradation) to 5 (complete visible resorption).

Microscopic scores: 0 (no resorption/degradation and/or very little cellular infiltration) to 5 (complete microscopic resorption and/or cellular response returned to normal). Example 9

Preparation and evaluation of a tissue-adhesive hydrogel

Following the synthetic procedure outlined in Examples 1 and 2, additional polymers were prepared covering a range of MAES-NHS loadings. It was found that decreasing MAES-NHS content and/or molecular weight increases water solubility.

Table 7 - Preparation and characterisation of polymers for tissue-adhesive hydrogel formulation

Mixing an equal volume of an aqueous solution of polymer with a second aqueous solution containing nucleophilic materials gave a crosslinked hydrogel. For these studies PEI (2000 MW) was chosen as the crosslinker molecule, however broadly similar results were observed with other amine or thiol-containing molecules either synthetic (eg 2-armed or 4-armed PEG-amine) or natural (eg albumin).

Table 8 - Gel time experiments (30 μΙ_ solution 1 + 30 μΙ_ solution 2)

Solution 1 Solution 2

Polymer Concentration PEI Ratio of Gel Time Hydrogel Reference % w/v Concentration actives formation

% w/v

LB0265-035 5 5 1 :1 10-20 sec Grainy liquid 10 0.1 100:1 10-20 sec Grainy

LB0265-035 liquid

0.25 40:1 < 3 sec Strong

0.5 20:1 < 3 sec Strong

1 10:1 < 3 sec Strong

2.5 4:1 < 3 sec Medium

5 2:1 < 3 sec Medium

10 1 :1 < 3 sec Weak, grainy

LB0265-038 5 5 2:1 < 3 sec Weak, grainy

10 1 :1 < 3 sec Weak, grainy

LB0265-038 10 5 1 :1 < 3 sec Weak, grainy

10 1 :2 < 3 sec Weak, grainy

5 0.25 20:1 10-20 sec Grainy

LB0264-1 14 liquid

0.5 10:1 < 3 sec Grainy liquid

0.75 6.67:1 < 3 sec Grainy liquid

1 5:1 < 3 sec Grainy liquid

10 0.25 40:1 < 3 sec Grainy liquid

0.5 20:1 < 3 sec Medium

0.75 13:3:1 < 3 sec Medium

1 10:1 < 3 sec Strong

15 0.25 60:1 < 3 sec Medium

0.5 30:1 < 3 sec Medium

0.75 20:1 < 3 sec Medium

The terms "weak", "medium" and "strong" refer to the relative solidity of the resulting hydrogel. A "strong" hydrogel was the most preferred, but the "medium" hydrogels still had sufficient integrity to be viable and useful products.

Table 9 describes examples of the application of tissue-adhesive hydrogel mixtures in a preclinical assessment. The formulations were applied by the use of a dual syringe and mixer spray tip to deliver a stream which crosslinked rapidly on contact with the tissue surface and formed an effective seal on both a durotomy and liver injury.

Table 9 - Preclinical assessment of tissue-adhesive hydrogel formulations

Example 10

Preparation of single layer co-mixed (homogenous) freeze-dried tissue- adhesive sheets

A series of solutions comprising various mixtures of polymer according to Table 1 and PLGA in DMSO were prepared according to Table 10. The solutions were poured into 5 x 5 x 0.3 cm moulds, frozen and the DMSO removed by lyophilisation to liberate sheets with dimensions equivalent to the volume of solution poured into the mould. The sheets were washed in diethyl ether to remove traces of DMSO and dried under vacuum at 40 °C for >48 hours. Table 10 - Preparation of co-mixed tissue-adhesive freeze-dried sheets

A freeze dried layer of polymer alone was too fragile to be handled. Blending in a structure component, in this case PLGA, enabled the preparation of sheets. The characteristics of the sheets were defined by the ratio of polymer to PLGA and the overall concentration of the materials in solution.

Example 11

Preparation of a tissue-adhesive sheet comprising a freeze-dried sheet and barrier layer

A two-layer tissue-adhesive sheet is shown schematically in Figure 8.

The sheet has the form of:

a) a solvent cast first layer 1 1 of PLGA and;

b) a freeze-dried second layer 12 of polymer.

A solution of PLGA (10 % w/v) in DCM was prepared. A small quantity of Methylene Blue was added to provide blue colouration. The solution was cast onto a release substrate such as silicone-backed release paper affixed to a glass sheet using a 10Ομιη gauge K bar. When the solvent evaporated this resulted in a 10μιη dry film of PLGA containing a blue tint. The film was dried for 30 minutes at

20°C/atmospheric pressure and 16 hours at 40°C/redi_Ded pressure. The film was not removed from the release substrate.

A mould was placed over the PLGA film and a solution comprising polymer according to Table 1 (5 % w/v) in DMSO was poured into the mould, frozen and the DMSO removed by lyophilisation to liberate a sheet with dimensions equivalent to the volume of solution poured into the mould. The sheet was washed in diethyl ether to remove traces of DMSO and dried under vacuum at 40 °C for >48 hours.

The resultant article had a blue PLGA barrier layer on one surface.

Example 12

Preparation of a tissue-adhesive sheet containing a multilaminate freeze-dried sheet, barrier layer and logo

A multilamellar tissue-adhesive sheet is shown schematically in Figure 9. The sheet comprises a structural laminate and a tissue-contacting layer as follows: a) a solvent cast first layer 21 of PLGA;

b) a solvent cast second layer 22 of polymer;

c) a solvent cast third layer 23 of PLGA; and

d) a freeze-dried fourth layer 24 of PLGA; and

e) a freeze-dried fifth layer 25 of polymer.

The tissue-contacting fifth layer 25 is conjoined to the fourth layer 24 and comprises tissue-adhesive polymer. The second layer also comprises tissue-adhesive polymer.

The first and third layers 21 , 23 each have a thickness of approximately 10μιη, and the second layer 22 a thickness of approximately 3 μιη. A logo is applied to the second layer 22. The fourth layer 24 has a thickness of approximately 600 μιη. The tissue-contacting layer 25 has a thickness of approximately 200 μιη. The sheet was prepared as follows:

Preparation of solutions

Solution A was 10g (10 % w/v) PLGA dissolved in 100ml DCM.

Solution B was 7.5g (7.5 % w/v) polymer dissolved in 100ml DCM/MeOH 15/4.

Solution C was a viscous printing ink comprised of polymer (2.25 g, 22.5 % w/v) and

Methylene Blue (0.09 g, 0.9 % w/v) dissolved in RO water (10 imL).

Solution D was 1 g (1 % w/v) PLGA dissolved in 100ml DMSO.

Solution E was 2.5g (2.5% w/v) polymer dissolved in 100ml DMSO.

Casting of first layer

Solution A was cast onto a release substrate such as silicone-backed release paper affixed to a glass sheet using a Ι ΟΟμιτι gauge K bar. When the solvent evaporated this resulted in a 10μιη dry film of PLGA. The film was dried for 30 minutes at 20°C/atmospheric pressure. The film was not removed from the release substrate.

Casting of second layer

Solution B was cast onto the first layer using a 40μιη K bar. When the solvent evaporated it resulted in a 3μιη dry film of A. The film was dried for 30 minutes at 20°C/atmospheric pressure. The laminate was not removed from the release substrate.

Application of logo

Solution C was printed by screen printing onto the surface of the second layer to form an alpha-numeric trade/visualisation logo.

Casting of third layer

Solution A was cast onto the second layer using a Ι ΟΟμιτι K bar. When the solvent evaporated this resulted in a 10μιη dry film of PLGA. The film was dried for 30 minutes at 20°C/atmospheric pressure and 16 hours d 40°C/reduced pressure. The laminate was not removed from the release substrate. Freeze drying of fourth layer

A polypropylene mould 50mm x 50mm x 5mm was affixed onto the third layer using double sided adhesive tape. The glass sheet with the laminate (first, second and third layers) and the mould affixed was then chilled to -20^QC. Solution D (15ml) was poured into the mould. The glass sheet was returned to the freezer for at least 1 hour.

Freeze drying of fifth layer

Solution E (5ml) was poured into the mould onto the frozen fourth layer. The glass sheet was returned to the freezer for at least 1 hour.

The DMSO from the fourth and fifth layers was removed by lyophilisation to liberate a sheet with dimensions equivalent to the mould. The sheet was washed in diethyl ether to remove traces of DMSO and dried under vacuum at 40°C for >48 hours.

Cutting out

The sheet was cut to size using specially designed cutters. Sterilisation

The dried, cut down product was subjected to pressure to flatten it and then stored in a foil pouch to maintain sterility and exclude moisture. The product was then gamma-sterilised at 25-40 kGy.

The resultant article had a blue logo sandwiched between the PLGA barrier layer and freeze-dried adhesive layer.

The resultant article was haemostatic to a bleeding liver punch biopsy in an in vivo preclinical assessment. Example 13

In vitro haemostatic performance

The haemostatic properties of the polymer were demonstrated by mixing either the dry milled powder or a solution in saline with porcine plasma. Results are summarised in Table 1 1 .

Table 1 1 - Example haemostatic properties

Component 1 Component 2

Polymer Concentration Porcine Plasma Ratio of Gelation

Reference % w/v actives

LB0264-1 14 Dry powder Neat 1 :10 Yes

LB0264-1 14 5 % in saline Neat 1 :1 Yes

1 :3 Yes

Claims

Tissue-adhesive polymer comprising:

vinyl pyrrolidone-derived units of general formula (I)

wherein A is -OC(=O)-CH2CH2- or a single bond,

R⁷ is -H or -CH3, and

X is a tissue-reactive group.

2. The polymer of any preceding claim, wherein R¹ , R², R³, R⁴, R⁵ and R⁶ represent H.

3. The polymer of any preceding claim, wherein the vinyl pyrrolidone-derived units of general formula (I) are present at about 50 to 97.5 mol %, about 70 to 95 mol %, about 80 to 95 mol % or about 85 to 90 mol %.

4. The polymer of any preceding claim, wherein R⁷ is H.

5. The polymer of any one of Claims 1 -3, wherein R⁷ is -CH3.

6. The polymer of any preceding claim, wherein A is -OC(=O)-CH2CH2-.

7. The polymer of any one of Claims 1 -5, wherein A is a bond.

8. The polymer of any one of Claims 1 -4 or 6, wherein R⁷ is H and A

9. The polymer of any preceding claim, wherein X is selected from the group consisting of imido ester, p-nitrophenyl carbonate, N-hydroxysuccinimide (NHS) ester, epoxide, isocyanate, acrylate, vinyl sulfone, orthopyridyl-disulfide, maleimide, aldehyde, and idoacetamide.

10. The polymer of any preceding claim, wherein X is -C(=O)-OR⁸.

1 1 . The polymer of Claim 10, wherein R⁸ is imidyl, p-nitrophenyl or N- hydroxysuccinimidyl (NHS).

12. The polymer of Claim 10 or Claim 1 1 , wherein the acrylate-derived units of general formula (II) comprise:

13. The polymer of any preceding claim, wherein the acrylate-derived units of general formula (II) are present at about 2.5 to 15 mol %, about 5 to 10 mol % or about 7 to 10 mol %.

14. The polymer of any preceding claim, which further comprises ester-containing units of general formula (V):

15. The polymer of any preceding claim, which further comprises acrylic acid-derived units of general formula (VI):

16. The polymer of any preceding claim, which is crosslinked using a crosslinker.

17. The polymer of Claim 15, wherein the crosslinker contains ester groups.

18. The polymer of Claim 16, wherein the crosslinker is PEG-diacrylate.

19. The polymer of any preceding claim, which further comprises a chromophore.

20. The polymer of any preceding claim, which further comprises one or more therapeutically active agents.

21 . The polymer of Claim 20, wherein the one or more therapeutically active agents are selected from the group consisting of analgesics, anaesthetics, anti- infective agents, antineoplastics, anti-inflammatory agents, angiogenic agents, anti- angiogenic agents, growth promoters, haemostatic agents, therapeutic monoclonal antibodies and anti-cancer agents.

22. The polymer of Claim 20 or 21 , wherein the one or more therapeutically active agents are incorporated into the polymer during manufacture.

23. The polymer of Claim 20 or 21 , wherein the one or more therapeutically active agents are bound to a component of the polymer after manufacture of the polymer.

24. The polymer of any one of Claims 1 -18, wherein the polymer is substantially free of any therapeutically active agents.

25. A monomer for use in a tissue-adhesive polymer, which is

mono(2-acryloyloxyethyl)succinate N-hydroxysuccinimide ester) (MAES-NHS).

26. Tissue-adhesive polymer comprising the acrylate-derived unit:

27. Use of mono(2-acryloyloxyethyl)succinate (MAES) in a tissue-adhesive polymer for medical use.

28. The use as claimed in Claim 27, wherein the MAES component is

functionalised to render it capable of reaction with functional groups on the surface of tissue.

29. The use as claimed in Claim 28, wherein the MAES component is

MAES-NHS.

30. Method for the manufacture of the polymer of any one of Claims 1 -24, which method comprises:

(a) polymerisation of N-vinyl-2-pyrrolidone monomers of general formula (III):

where R¹ , R², R³, R⁴, R⁵ and R⁶ independently represent H, optionally substituted Ci-6 alkyl, optionally substituted C-i-6 alkoxy or halo; and acrylate monomers of general formula (IV):

CH2=CR⁷-C(=O)O-CH₂CH2-A-C(=O)-OR⁸ (IV) wherein

A is -OC(=O)-CH₂CH2- or a single bond;

R⁷ is -H or -CH3; and

X is a tissue-reactive group.

31 . The method of Claim 30, wherein the polymerisation is carried out in DMSO.

32. The method of Claims 30 or 31 , wherein R¹ , R², R³, R⁴, R⁵ and R⁶

represent H.

33. The method of any one of Claims 30-32, wherein the vinyl pyrrolidone monomers of general formula (III) are added in an amount of about 50 to 97.5 mol %, about 70 to 95 mol %, about 80 to 95 mol % or about 85 to 90 mol %.

34. The method of any one of Claims 30-33, wherein R⁷ is H.

35. The method of any one of Claims 30-33, wherein R⁷ is -Chte.

36. The method of any one of Claims 30-35, wherein A is -OC(=O)-CH2CH2-.

37. The method of any one of Claims 30-35, wherein A is a bond.

38. The method of any one of Claims 30-33, wherein R⁷ is H and A

39. The method of any one of Claims 30-38, wherein X is selected from the group consisting of imido ester, p-nitrophenyl carbonate, N-hydroxysuccinimide (NHS) ester, epoxide, isocyanate, acrylate, vinyl sulfone, orthopyridyl-disulfide, maleimide, aldehyde, and idoacetamide.

40. The method of Claim 39, wherein X is -C(=O)-OR⁸.

41 . The method of Claim 40, wherein R⁸ is imidyl, p-nitrophenyl or N- hydroxysuccinimidyl (NHS).

42. The method of Claim 40, wherein the one or more acrylate monomers of general formula (IV) comprise mono(2-acryloyloxyethyl)succinate

N-hydroxysuccinimide ester) (MAES-NHS).

43. The method of any one of Claims 30-42, wherein the acrylate monomer is added in an amount of about 2.5 to 15 mol %, about 5 to 10 mol % or about 7 to 10 mol %.

44. The method of any one of Claims 30-43, wherein a cyclic ketene acetal is included as an additional monomer in polymerisation step (a).

45. The method of Claim 44, wherein the cyclic ketene acetal is added in an amount of about 0 to about 20 mol %, in an amount of about 5 to 20 mol %, in an amount of about 5 to 15 mol % or in an amount of about 10 to 15 mol %.

46. The method of Claim 44 or 45, wherein the cyclic ketene acetal is 2- methylene-1 ,3-dioxepane (MDO).

47. The method of any one of Claims 30-45, wherein acrylic acid is included as an additional monomer in polymerisation step (a).

48. The method of Claim 47, wherein acrylic acid is added in amount of about 0 to about 20 mol %, in an amount of about 5 to 20 mol %, in an amount of about 5 to 15 mol % or in an amount of about 10 to 15 mol %.

49. The method of any one of Claims 30-48, wherein a crosslinker is included in polymerisation step (a).

50. The method of Claim 49, wherein the crosslinker contains ester groups.

51 . The method of Claim 50, wherein the crosslinker is PEG-diacrylate.

52. The method of any one of Claims 49-51 , wherein the crosslinker is added in an amount of about 0 to 5 mol %, about 1 to 4 mol %, or about 2 to 3 mol %.

53. The method of any one of Claims 30-52, wherein the polymerisation is carried out in DMSO, which further comprises:

(b) lyophilisation to recover the polymer from the solvent.

54. The method of Claim 53, which further comprises:

(c) milling of the lyophilised polymer to produce a powder.

55. The polymer of any one of Claims 1 -24 in the form of a freeze-dried solid.

56. The polymer of Claim 55 in the form of a freeze-dried powder.

57. Tissue-adhesive formulation comprising the polymer of Claim 56 and optionally a particulate buffer material.

58. Hydrogel prepared in situ by mixing a first aqueous solution comprising the tissue-adhesive polymer of any one of Claims 1 -24 and a second aqueous solution comprising nucleophilic groups.

59. The hydrogel of Claim 58, wherein the second aqueous solution comprising nucleophilic groups is a solution comprising PEI, 2-armed or 4-armed poly(ethylene glycol)-amine, or natural or recombinant albumin.

60. The hydrogel of Claims 58 or 59, wherein the first and/or second aqueous solutions contain a chromophore.

61 . Multilamellar, tissue-adhesive sheet comprising a structural layer of material or laminate comprising a non-adhesive polymer, and a tissue-contacting layer comprising the polymer of Claims 1 -24.

62. The sheet of Claim 61 , wherein the non-adhesive polymer is selected from PLGA and PLC.

63. The sheet of Claim 61 or Claim 62, which is a structural layer of material or laminate comprising a non-adhesive polymer, with a tissue-adhesive layer comprising the polymer of Claims 1 -24 on both sides of the structural layer.

64. The sheet of any one of Claims 61 -63, which is perforated.

65. A freeze-dried polymer composition comprising the tissue-adhesive polymer of any one of Claims 1 -24.

66. The freeze-dried polymer composition as claimed in Claim 65, which comprises a structural component.

67. The freeze-dried polymer composition as claimed in Claim 66, wherein the structural component is a non-adhesive synthetic polymer.

68. The freeze-dried polymer composition as claimed in Claim 67, wherein the structural component is poly(lactide-co-glycolide) (PLGA).

69. The freeze-dried polymer composition as claimed in any one of Claims 65-68, which comprises at least 10% by weight of tissue-adhesive polymer.

70. The freeze-dried polymer composition as claimed in any one of Claims 65-69, which does not contain a biological material.

71 . The freeze-dried polymer composition as claimed in Claim 70, which does not contain cellulose, albumin and/or gelatin.

72. The freeze-dried polymer composition as claimed in any one of Claims 65-71 , which is in the form of a sheet.

73. The freeze-dried polymer composition as claimed in Claim 72, wherein the sheet has an overall thickness of about 0.05 to 10 mm.

74. The freeze-dried polymer composition as claimed in any one of Claims 65-71 , which is in the form of a plug, pellet or other three-dimensional shape.

75. A tissue-adhesive article comprising the freeze-dried polymer composition as claimed in any one of Claims 65-74.

76. The article as claimed in Claim 75, which is a multilamellar sheet comprising a layer of the freeze-dried polymer composition and a layer or laminate comprising a non-adhesive polymer.

77. The article as claimed in Claim 76, wherein the non-adhesive polymer is poly(lactide), poly(lactide-co-caprolactone) (PLC) or poly(lactide-co-glycolide) (PLGA).

78. The article as claimed in Claim 76 or Claim 77, wherein the layer or laminate comprising a non-adhesive polymer is a solvent-cast layer or a freeze-dried layer.

79. The article as claimed in Claim 78, wherein the layer or laminate comprising a non-adhesive polymer is a solvent-cast layer with a thickness between about 4μιη and Ι ΟΟμιη.

80. The article as claimed in Claim 78, wherein the layer or laminate comprising a non-adhesive polymer is a freeze-dried layer with a thickness between about 10Ομιη and 1000μηΊ.

81 . The article as claimed in any one of Claims 76 to 80, which is a multilamellar sheet that consists of a layer or laminate comprising a non-adhesive polymer with a layer of freeze-dried polymer composition on each side.

82. The article as claimed in any one of Claims 76 to 81 , which is perforated.

83. The article as claimed in Claim 76, which has a tissue-contacting layer of freeze-dried polymer composition, one or more solvent-cast layers comprising a non- adhesive polymer, one or more solvent-cast layers comprising a tissue-adhesive polymer, and optionally one or more freeze-dried layers comprising a non-adhesive polymer.

84. The article as claimed in Claim 75, which comprises an implantable medical device, at least part of the external surface of which bears a coating comprising the freeze-dried polymer composition as claimed in any one of Claims 65 to 71 .

85. A method for the preparation of the freeze-dried polymer composition or article of any one of Claims 65-84, which method comprises the steps of: a) preparing a solution comprising a tissue-adhesive polymer containing tissue- reactive functional groups in a dry, non-aqueous solvent; and

b) freeze-drying the solution to yield the freeze-dried polymer composition.

86. The method as claimed in Claim 85, wherein the dry non-aqueous solvent is dimethyl sulfoxide (DMSO).

87. The method as claimed in Claim 85 or Claim 86, wherein the solution comprising the tissue-adhesive polymer containing tissue-reactive functional groups in a dry, non-aqueous solvent comprises one or more additional components selected from chromophores, therapeutic agents and structural components.

88. The method as claimed in Claim 87, wherein the solution comprises a chromophore selected from Methylene Blue (methylthioninium chloride) and FD&C Blue No 1 .

89. The method as claimed in Claim 87, wherein the solution comprises a therapeutic agent selected from analgesics, anaesthetics, anti-infective agents, antineoplastics, anti-inflammatory agents, angiogenic agents, anti-angiogenic agents, growth promoters, clotting agents, therapeutic monoclonal antibodies, and anti-cancer agents.

90. The method as claimed in Claim 89, wherein the solution comprises a therapeutic agent selected from the group consisting of anti-inflammatory agents, anti-infective agents and clotting agents.

91 . The method as claimed in Claim 87, wherein the solution comprises a structural component.

92. The method as claimed in Claim 91 , wherein the structural component is a non-adhesive synthetic polymer.

93. The method as claimed in Claim 92, wherein the structural component is poly(lactide-co-glycolide) (PLGA).

94. Method for the preparation of a freeze-dried polymer composition comprising a tissue-adhesive polymer containing tissue-reactive functional groups, which method comprises the steps of:

a) preparing a solution comprising a tissue-adhesive polymer containing tissue- reactive functional groups in DMSO; and

b) freeze-drying the solution to yield the freeze-dried polymer composition.

95. The method as claimed in Claim 94, wherein the tissue-reactive functional groups are selected from the group consisting of imido ester, p-nitrophenyl carbonate, N-hydroxysuccinimide (NHS) ester, epoxide, isocyanate, acrylate, vinyl sulfone, orthopyridyl-disulfide, maleimide, aldehyde, iodoacetamide and

combinations thereof.

96. Freeze-dried polymer composition comprising a tissue-adhesive polymer comprising tissue-reactive functional groups and vinyl pyrrolidone-derived units of general formula (I):

where R¹ , R², R³, R⁴, R⁵ and R⁶ independently represent H, optionally substituted Ci-6 alkyl, optionally substituted C-i-6 alkoxy or halo.

97. A method for freeze-drying the polymer of Claim 96, which method comprises: a) preparing a solution comprising the polymer in dimethyl sulfoxide (DMSO); and b) freeze-drying the solution to yield the freeze-dried polymer composition.

98. Method for freeze-drying a polymer containing vinyl pyrrolidone-derived units, which method comprises:

a) preparing a solution comprising the polymer in dimethyl sulfoxide (DMSO); and b) freeze-drying the solution to yield the freeze-dried polymer composition.

99. Device suitable for implantation in the human or animal body, which device carries on at least part of the external surface thereof a coating comprising a tissue-adhesive polymer of Claims 1 -24.

100. Method of haemostasis, wound healing, joining, sealing or reinforcing tissue, which method comprises the application to a tissue surface of a tissue-adhesive polymer as claimed in any one of Claims 1 -24, a powder formulation as claimed in Claim 56 or 57, a hydrogel as claimed any one of Claims 58-60, a tissue-adhesive sheet as claimed in any one of Claims 61 -64, a freeze-dried polymer composition or article as claimed in any one of Claims 65-84, or a device as claimed in Claim 99.