TECH N I CAL FI ELD

5

The present invention relates to a composition comprising Mesna, said composition for use in a method of surgery, a kit comprising Mesna and a method for visualization of a composition comprising Mesna which is applied to tissues. 0 BACKGROUND

There remains a need in the art for improvements in support of the application of Mesna in surgery. 5 Mesna, also known as sodium 2-mercaptoethanesulfonate, is known to be used in surgery for facilitating tissue separation. When Mesna is applied at a cleavage plane, Mesna breaks the molecular bonds between tissue layers. Specifically, Mesna breaks disulfide bonds of polypeptide chains and proteins. Accordingly, Mesna weakens inter-tissues and/or organs adhesion to facilitate dissection along said cleavage0 plane.

US 6143797 A discloses methods of facilitating detaching pathological tissues from healthy tissues with which they have formed adherences and facilitating detachment of healthy tissues from other healthy tissues such as in plastic surgery in which an5 effective amount of sodium 2-mercaptoethanesulfonate (Mesna) is applied to the tissue or tissues.

US 6143797 A shows the problem of not specifying means of monitoring parts of tissues on which Mesna is effectively applied. This could lead to inefficient0 application of Mesna on tissues in terms of inefficiencies in the quantity of Mesna and/or inefficiencies in the locations where Mesna is applied.

The present invention aims to resolve at least aforementioned problem. 5 The invention thereto aims to provide a composition comprising Mesna, wherein the composition further comprises one or more dyes.

SUMMARY OF THE I NVENTI ON I n a first aspect, the invention relates to a com position comprising Mesna, wherein the com position further com prises one or more dyes, according to claim 1 .

5 I n particular, the composition comprising Mesna and one or more dyes is of use in surgery, and more in particular in surgery where Mesna is applied at a cleavage plane between two or more tissues for facilitating tissue separation . The dyes function to stain and, consequently, visualize parts of the tissues on which the Mesna composition is applied. This entails the advantage that a m edical practitioner can0 see the parts where the composition is applied, allowing intuitive and quick ascertaining of a desired application of Mesna to the tissues, or if the application of Mesna should be adj usted, which adj ustm ents can also be directly seen by the visualization brought about by the one or more dyes. The use of a dye allows to visualize whether the m esna solution remains in place for a sufficient duration for5 effective chem ical action . I t also helps visualize the cleavage plane, which is very useful to the surgeon. This is of great im portance for allowing efficient dissection of tissues facilitated by Mesna, and certainly in view of the knowledge that flooding a surgical cavity in order to m ake sure that all tissues to be dissected are wetted by Mesna is not an option because the amount of Mesna which can be dispensed in a0 single surgical procedure is lim ited by toxicity considerations.

Preferred embodim ents of the com position, according to the first aspect of the invention, are described in claims 2 to 5. 5 I n a second aspect, the invention relates to a com position according to the first aspect of the invention for use in a method of surgery, according to claim 6. A preferred embodim ent of the com position for use in a m ethod of surgery, is described in claim 7. 0 I n a third aspect, the invention relates to a kit comprising Mesna, wherein the kit further com prises one or more dyes, according to claim 8. Preferred em bodim ents of the kit, according to the third aspect of the invention, are described in claims 1 0 and 1 1 . 5 I n a fourth aspect, the invention relates to a m ethod for visualization of a composition comprising Mesna which is applied to tissues, according to claim 1 2. Preferred embodim ents of the m ethod for visualization of a composition com prising Mesna which is applied to tissues, according to the fourth aspect of the invention , are described in claims 1 3 and 14.

I n a fifth aspect, the invention relates to a use of a composition according to the

5 first aspect of the invention in a m ethod according to the fourth aspect of the invention, according to claim 1 5.

D ETAI LED D ESCRI PTI ON OF TH E I N VEN TI ON 0 The recitation of num erical ranges by endpoints includes all num bers and fractions subsum ed within that range, as well as the recited endpoints.

The term“dyes”, as disclosed herein , refers to substances which impart color to a substrate by selective absorption of light. Dyes are soluble and/or go through an5 application process which , at least temporarily, destroys any crystal structure of the color substances. Dyes are retained in the substrate by absorption, solution , and m echanical retention , or by ionic or covalent chem ical bonds. The term “dyes” includes fluorescent and non-fluorescent dyes, which fluorescent dyes include without lim itations fluorophores and which non-fluorescent agents include without0 lim itations pigm ents, chem ilum inescent com pounds, lum inescent compounds and chromophores. The term“fluorophore” as used herein refers to a compound that is inherently fluorescent or demonstrates a change in fluorescence upon binding to a biological compound or m etal ion , i.e. , fluorogenic. Non-lim iting exam ples of fluorophores are fluorescent proteins, inorganic fluorescent pigm ents and organic5 fluorescent dyes. Num erous fluorescent dyes are known to those skilled in the art and are described herein , and include, but are not lim ited to, indocyanine green , riboflavin, riboflavin 5’-phosphate sodium , m ethylene blue, sem iconductor nanocrystals, coumarin , acridine, furan , indole, quinoline, cyanine, benzofuran , quinazolinone, benzazole, borapolyazaindacene and xanthenes, with the latter0 including fluoroscein , rhodam ine, rhodol, rosam ine and derivatives thereof.

Preferably, the dyes are selected from the group consisting of indocyanine green , riboflavin, riboflavin 5’-phosphate sodium , m ethylene blue and fluorescein .

The term “pharm aceutically acceptable solvent”, as disclosed herein, refers to a5 solvent capable of dissolving relevant quantities of Mesna, and preferably capable of dissolving relevant quantities of Mesna in powder form , and also capable of dissolving one or more dyes. Non-lim iting exam ples of pharmaceutically acceptable solvents are water, physiological serum , and water solution comprising up to 0.9 mass/volume percent sodium chloride. Bases, such as sodium hydroxide, may be added to the pharmaceutically acceptable solvent. Preferable, said pharmaceutically acceptable solvent is sterile. Further examples of pharmaceutically acceptable solvents are known to the person skilled in the art.

5

The term “pharmaceutically acceptable buffer”, as disclosed herein, refers to a solution comprising at least one buffer excipient, capable of keeping pH of a composition comprising Mesna and one or more dyes at a nearly constant value. The buffer might be obtained by mixing a solvent with at least one buffer excipient.0 The solvent might be water, might consist of water of high purity (e.g. water for injection as per pharmaceutical standard) and may contain between 0 and 0.9 mass/volume percent of NaCI. The presence of NaCI reduces the side-effects due to chlorine ion depletion. In particular, lack of sufficient chlorine ions may trigger unexpected hyperactivity of some cells such as neurons in brain and in the nerves.5 Pharmaceutically acceptable buffers are known to the person skilled in the art.

According to preferred embodiments, said buffer excipient is selected from the group comprising sodium acetate, acetic acid, glacial acetic acid, ammonium acetate, arginine, aspartic acid, benzoate sodium , benzoate acid, carbonate sodium , bicarbonate sodium, citrate acid, citrate sodium, citrate disodium, citrate trisodium,0 glucono delta lactone, glycine, glycine HCI, histidine, histidine HCI, hydrobromic acid, meglumine, phosphate acid, phosphate monobasic potassium, phosphate dibasic potassium, phosphate monobasic sodium, phosphate dibasic sodium (Na2HP04), tartrate sodium, tartrate acid, tromethamine (tris) or any combination thereof. In preferred embodiments, the concentration of the buffer excipient is of5 from 2 mM to 60 mM, preferably from 2.5 mM to 50 mM, more preferably from 5 to 40 mM, more preferably from 7.5 mM to 35 mM, most preferably from 10 mM to 30 mM. According to a preferred embodiment, the pH of the buffer may be adjusted using pH adjustment agents to reach a desired pH of at least 8.5. Said pH of the buffer allows obtaining a composition comprising Mesna and one or more dyes0 having a pH of from 6 to 8 by only adding solid Mesna in powder form or in lyophilized form, and one or more dyes, to the buffer and without addition of pH adjustment agents. This avoids manipulation of the obtained composition comprising Mesna and one or more dyes, to adjust the pH for instance, thereby providing for the immediate use of the obtained composition. This ensures a5 maximal sterility of the composition and a maximal safety for a patient thanks to the use of a solution with a pH close to physiological pH. According to a preferred embodiment, the buffer comprises phosphate such that the phosphate to Mesna ratio of the composition comprising Mesna is at least 1/500, preferably at least 1/400, more preferably at least 1/300, even more preferably at least 1/250, most preferably at least 1/150, even most preferably at least 1/100. Said ratio is at most 1/1 , preferably at most 1/1.5, more preferably at most 1/2, most preferably 1/3.

5 In the present text, “Mesna” is used as an abbreviation for sodium-2- mercaptoethane sulfonate. Mesna is a white hygroscopic powder with a characteristic odor. The liquid solution in water is highly sensitive to oxidation and rapidly decomposes on contact with oxygen to form di-Mesna also called disulphide Mesna, which is the inactive degradation product of Mesna. Mesna is known to break0 molecular bonds between tissue layers, thereby facilitating tissue separation.

Specifically, Mesna breaks disulfide bonds of polypeptide chains and proteins, which disulfide bonds are responsible for the adherence of pathological tissues and for the strength of fibrosis. 5 WO 2009/022430 A1 describes a skin whitening agent having an excellent whitening effect, an excellent moisture-retaining action (or moisturizing effect), less side effects and high safety is provided. The skin whitening agent contains a mercaptosulfonic acid or a salt thereof as an effective ingredient. The mercaptosulfonic acid may be a mercaptoalkanesulfonic acid or a dimer thereof, and0 the salt of the mercaptosulfonic acid may be a metal salt (e.g., mesna, and dimesna). The content of the mercaptosulfonic acid or the salt thereof in the skin whitening agent may be about 0.01 to 50% by weight in terms of a solid content. The skin whitening agent is capable of inhibiting melanogenesis. The skin whitening agent may be used in a dosage form for oral administration or external application.5

WO 2015/071910 A1 discloses methods of preventing, ameliorating and/or treating radiation induced gastrointestinal tract injury comprising the administration of a therapeutically effective amount of Mesna to a patient are provided. Methods for preventing, treating and/or ameliorating gastrointestinal tract injury induced by0 radiation therapy, alone or in combination with other therapies for diseases or conditions such as gastrointestinal malignancies, urogenital malignancies, gynecologic malignancies, and osteogenic and other sarcomatous malignancies in which pelvic structures are involved. Auxiliary components such as tablet disintegrants, solubilisers, preservatives, antioxidants, surfactants, viscosity5 enhancers, coloring agents, flavoring agents, pH modifiers, sweeteners or taste- masking agents may be incorporated into a composition according to WO 2015/071910 A1. Peter, C. et al. (2000):“Pharmacokinetics and organ distribution of intravenous and oral methylene blue”, European Journal of Clinical Pharmacology, Springer Verlag, DE, vol. 56, no. 3, pages 247-250, describes a determination of the pharmacokinetics and organ distribution of i.v. and oral methylene blue, which is

5 used to prevent ifosfamide-induced encephalopathy in oncology. The concentration of methylene blue in whole blood was measured using high-performance liquid chromatography in seven volunteers after i.v. and oral administration of 100 mg methylene blue with and without mesna. The distribution of methylene blue in different tissues was measured in rats after intraduodenal and i.v. application. The0 time course of methylene blue in whole blood after i.v. administration showed a multiphasic time course with an estimated terminal half-life of 5.25 h. Following oral administration, the area under the concentration-time curve was much lower (9 nmol/min/ml vs 137 nmol/min/ml). Co-administration of mesna, which could influence distribution by ion-pairing, did not alter the pharmacokinetics. The urinary5 excretion of methylene blue and its leucoform was only moderately higher after i.v. administration (18% vs 28% dose). Intraduodenal administration to rats resulted in higher concentrations in intestinal wall and liver but lower concentrations in whole blood and brain than i.v. methylene blue. Differences in organ distribution of methylene blue are mainly responsible for the different pharmacokinetics after oral0 and i.v. administration. If methylene blue acts in the liver, where ifosfamide is primarily activated to reactive and potentially toxic metabolites, oral and i.v. methylene blue are likely to be equally effective. However, if the site of action is the central nervous system, i.v. methylene blue which results in much higher concentrations in brain seems preferable.

5

WO 98/16213 A1 discloses topical formulations containing sodium 2- mercaptoethanesulfonate (MESNA), the use of MESNA for the preparation of pharmaceutical formulations intended to be used in surgical procedures that involve the dissection of tissues of the use of MESNA in said surgical procedures.

0

WO2017/157670 A1 describes a device for delivering a Mesna formulation to tissues and/or organs. The device comprises a first chamber comprising Mesna in solid form, a second chamber comprising a buffer and at least one outlet for delivering the Mesna formulation, said outlet is in fluid communication with at least one of the5 chambers. The chambers are separated from each other by at least one disruptable separation means and are in fluid communication with each other upon disruption of said separation means thereby forming the Mesna formulation. The pH of the buffer comprised in the second chamber is at least 8.5. The invention further provides a process for the preparation of a Mesna formulation.

US 4 191 176 A discloses an enzymatic intralenticular cataract treatment for

5 removal of nuclear cortical and subcapsular regions of the cataractous lens through enzymatic digestion thereof which comprises introduction of a concentrated solution of a trypsin enzyme into the nuclear and cortical regions of a cataractous lens, and after enzymatic digestion removing the liquefied cataractous material. The procedure allows subsequent removal of the nuclear, cortical and subcapsular0 portions of a cataractous lens through a very tiny incision in the eye and lens capsule, leaving all other structures within the eye intact. Bovine and porcine trypsins are preferred. According to US 4 191 176 A, for an enzyme containing solution injected into the lens, the distribution pattern of the injected fluid may be observed by incorporating a soluble, inert dye such as fluorescein into the injection5 fluid in amounts of 0.01 -1.0% w/v.

US 5273 751 A describes compositions for killing undifferentiated epithelial cells during cataract surgery on an eye to prevent posterior capsule clouding after the surgery and to a method for performing cataract surgery on an eye including0 injecting a cell-killing substance between the anterior capsule and the natural lens prior to removing the natural lens from the eye. The cell-killing substance is preferably an acid or base adjusted aqueous solution having a pH in the range between about 1.0 to below 6.5 or about above 7.5 to 14.0; or a hypotonic solution having a salinity less than 0.9% or a pH adj usted hypotonic solution having a salinity5 less than 0.9% and a pH either below 6.5 or above 7.5. The compositions of US 5 273751 A also incorporate a viscoelastic material, a dye or a mixture thereof, in combination with the cell-killing substance. According to US 5273 751 A, for the purpose of visually determining that cell-killing substance has completely filled the pocket and expanded all portions of the anterior capsule segment, a dye is admixed0 with the cell-killing substance prior to or after injection of the cell-killing substance between the inner surface of the anterior capsule and the lens.

In a first aspect, the invention relates to a composition comprising Mesna, wherein the composition further comprises one or more dyes.

5

In particular, the composition comprising Mesna and one or more dyes is of use in surgery, and more in particular in surgery where Mesna is applied at a cleavage plane between two or more tissues for facilitating tissue separation. The dyes function to stain and, consequently, visualize parts of the tissues on which the Mesna composition is applied. This entails the advantage that a medical practitioner can see the parts where the composition is applied, allowing intuitive and quick ascertaining of a desired application of Mesna to the tissues, or if the application of

5 Mesna should be adjusted, which adjustments can also be directly seen by the visualization brought about by the one or more dyes. The use of a dye allows to visualize whether the mesna solution remains in place for a sufficient duration for effective chemical action. It also helps visualize the cleavage plane, which is very useful to the surgeon. This is of great importance for allowing efficient dissection of0 tissues facilitated by Mesna, and certainly in view of the knowledge that flooding a surgical cavity in order to make sure that all tissues to be dissected are wetted by Mesna is not an option, because the amount of Mesna which can be used in a single surgical procedure is limited by toxicity considerations. 5 In a preferred embodiment, the invention relates to a composition according to the first aspect of the invention, wherein the one or more dyes are selected from the group consisting of indocyanine green, riboflavin, riboflavin 5’-phosphate sodium, methylene blue and fluorescein. 0 Indocyanine green is a fluorescent cyanine dye with a peak spectral absorption at about 800 nm, i.e. it is a green-colored dye. For use in combination with Mesna, indocyanine green shows the advantage of acting as a reductant, thus not reacting with Mesna which is prone to oxidation. Besides, the green color has the advantage of being especially visible on tissues.

5

As a fluorescent dye, riboflavin provides a yellow to orange-yellow coloring to substrates stained with it. For use in combination with Mesna, riboflavin shows the advantage of acting as a reductant, thus not reacting with Mesna which is prone to oxidation.

0

Riboflavin 5’-phosphate sodium is the phosphate sodium salt of riboflavin. Like riboflavin, riboflavin 5’-phosphate sodium provides a yellow to orange-yellow coloring to substrates stained with it. Like riboflavin, for use in combination with Mesna, riboflavin 5’-phosphate shows the advantage of acting as a reductant, thus5 not reacting with Mesna which is prone to oxidation. Methylene blue is a fluorescent thiazine dye with a blue color. Methylene blue acts as an oxidant, yet by intelligent selection of relative amounts of methylene blue in respect of Mesna, oxidation of Mesna by methylene blue can be rendered negligible.

5 Fluorescein is a red-colored fluorophore which appears purple under UV light. When applying UV light, fluorescein is thus well-suited for visualizing a composition comprising Mesna on tissues.

In a preferred embodiment, the invention relates to a composition according to the0 first aspect of the invention, wherein the composition comprises one or more pharmaceutically acceptable solvents and/or one or more pharmaceutically acceptable buffers, the one or more solvents and/or one or more buffers comprising from 0.2 weight per volume percent to 30 weight per volume percent, more preferably from 0.4 weight per volume percent to 25 weight per volume percent,5 more preferably from 2 weight per volume percent to 22 weight per volume percent, yet even more preferably from 3 weight per volume percent to 14 weight per volume percent, yet even more preferably from 4 weight per volume percent to 6 weight per volume percent of Mesna and from 0.0001 weight per volume percent to 30 weight per volume percent, more preferably from 0.002 weight per volume percent0 to 10 weight per volume percent, more preferably from 0.005 weight per volume percent to 1 weight per volume percent, even more preferably from 0.008 weight per volume percent to 0.5 weight per volume percent and even more preferably from 0.01 weight per volume percent to 0.1 weight per volume percent of the one or more dyes.

5

Said weight percentages of the one or more dyes are large enough to allow sufficient staining and clear visualization of tissues on which a composition comprising Mesna and one or more dyes is applied while not too large so that excessive use or wastage of the one or more dyes occurs or that undesired damage to the tissues would take0 place because of the one or more dyes.

When used in surgery, said weight percentages of Mesna are high enough for facilitating tissue separation due to breakage of molecular bonds between tissue layers by Mesna, in particular by Mesna breaking disulfide bonds of polypeptide5 chains and proteins which disulfide bonds are responsible for the adherence of pathological tissues and for the strength of fibrosis, and are low enough for minimizing the risk of undesired tissue separation of surrounding tissues which are not to be separated. In addition, said weight percentages of Mesna and of the one or more dyes allow for complete dissolving in the one or more pharmaceutically acceptable solvents and/or one or more pharmaceutically acceptable buffers, and resulting solutions comprising Mesna and the one or more dyes according to said weight percentages show good stability properties.

In a preferred embodiment, the invention relates to a composition according to the first aspect of the invention, wherein the one or more pharmaceutically acceptable solvents are selected from the group comprising water, preferably sterile water, and a solution of up to 0.9 mass/volume percent of NaCI in water, preferably a sterile solution of up to 0.9 mass/volume percent of NaCI in water.

In a preferred embodiment, the invention relates to a composition according to the first aspect of the invention, wherein a solution of Na ₂ HPC> ₄ in water or a solution of Na2HP04 and NaCI in water is selected as pharmaceutically acceptable buffer. More preferably, the buffer is an aqueous solution of 10 mM Na2HPC>4 or an aqueous solution of 10 mM Na2HPC>4 and 75 mM NaCI. Preferably, the water is water of high purity (e.g. water for injection as per pharmaceutical standard) and may contain between 0 and 0.9 mass/volume percent of NaCI. Preferably, the water is sterile.

In a second aspect, the invention relates to a composition according to the first aspect of the invention for use in a method of surgery. Preferably, said method of surgery comprises the step of separation of tissue layers along a cleavage plane between said tissue layers, wherein the separation of tissue layers is facilitated by applying the composition comprising Mesna and one or more dyes to said cleavage plane. Due to the incorporation of one or more dyes, the Mesna composition is especially suited for visualizing parts of tissues on which Mesna is applied, allowing fine-tuning of surgery involving dissection of tissue layers facilitated by application of Mesna.

In a third aspect, the invention relates to a kit comprising Mesna, wherein the kit further comprises one or more dyes.

The kit can be used to allow a user, such as a medical practitioner or an assisting person, to manufacture a composition comprising Mesna and one or more dyes shortly prior to the intended use of the composition comprising Mesna. This minimizes the risk of Mesna oxidation and maximizes Mesna activity for applications of facilitating tissue separation.

In a preferred embodiment, the invention relates to a kit according to the third aspect of the invention, wherein the one or more dyes are selected from the group consisting of indocyanine green, riboflavin, riboflavin 5’-phosphate sodium, methylene blue and fluorescein. For the properties, advantages and technical effects of these dyes is referred to the above description.

In a preferred embodiment, the invention relates to a kit according to the third aspect of the invention, wherein Mesna is in powder form and the kit further comprises one or more pharmaceutically acceptable solvents and/or one or more pharmaceutically acceptable buffers.

A kit comprising one or more dyes and Mesna in powder form next to one or more pharmaceutically acceptable solvents and/or one or more pharmaceutically acceptable buffers allows one or more dyes and Mesna in powder form to be dissolved in the one or more pharmaceutically acceptable solvents and/or one or more pharmaceutically acceptable buffers shortly before using the resulting Mesna solution, thus minimizing the risk of Mesna oxidation and maximizing Mesna activity for facilitating tissue separation, and also minimizing the risk of degradation of the one or more dyes when dissolved in the one or more pharmaceutically acceptable solvents and/or one or more pharmaceutically acceptable buffers.

Preferably, the one or more pharmaceutically acceptable solvents are selected from the group comprising water, preferably sterile water, and a solution of up to 0.9 mass/volume percent of NaCI in water, preferably a sterile solution of up to 0.9 mass/volume percent of NaCI in water.

Preferably, a solution of Na ₂ HPC> ₄ in water or a solution of Na ₂ HPC> ₄ and NaCI in water is selected as pharmaceutically acceptable buffer. More preferably, the buffer is an aqueous solution of 10 mM Na2FIPC>4 or an aqueous solution of 10 mM Na2FIPC>4 and 75 mM NaCI. Preferably, the water is water of high purity (e.g. water for injection as per pharmaceutical standard) and may contain between 0 and 0.9 mass/volume percent of NaCI. Preferably, the water is sterile.

In a preferred embodiment, the invention relates to a kit according to the third aspect of the invention, wherein the kit further comprises a fluorescence surgical goggle. Any goggle suitable for making fluorescence visual to a user, as known in the state of the art, m ay be selected as the fluorescence surgical goggle. According to an em bodiment, the fluorescence surgical goggle comprises an excitation light source, and preferably a m ulti-wavelength excitation light source, a cam era equipped with a fluorescence filter for collecting fluorescence while elim inating interference from the excitation light, a head mount display, and a com puter configured for image processing and/or other im aging processing accessories. A graphical program m ing software program m ay be used to fuse a background im age with fluorescence em ission captured by the camera, and to proj ect to the head mount display for real-tim e image guidance. A fluorescence surgical goggle provides the advantage of being able for excitation of fluorescent dyes by directing a light source with a wavelength , equal to an optimal excitation wavelength of the fluorescent dye, to the fluorescent dye, which goggle is sim ultaneously able to visualize the fluorescence selectively to a user, preferably a m edical practitioner, wearing the goggle. According to a preferred em bodim ent, the kit com prises Mesna, one or more fluorescent dyes, a fluorescence surgical goggle, and optionally one or more pharmaceutically acceptable solvents and/or optionally one or more pharmaceutically acceptable buffers. Accordingly, a user, preferably a medical practitioner, of the goggle can selectively see the fluorescent dye without interferences of naturally occurring colors of the tissues. Preferably, the one or more fluorescent dyes are selected from the group comprising of indocyanine green , riboflavin, riboflavin 5’-phosphate sodium , m ethylene blue and fluorescein .

I n a fourth aspect, the invention relates to a method for visualization of a com position com prising Mesna which is applied to tissues, comprising the step of applying the com position comprising Mesna to two or more tissues, wherein the com position further comprises one or more dyes for staining parts of the two or more tissues where the composition has been applied.

I n particular, the method for visualization of a composition com prising Mesna which is applied to tissues is of use in surgery, and more in particular in surgery where Mesna is applied at a cleavage plane between two or more tissues for facilitating tissue separation . The dyes function to stain and, consequently, visualize parts of the tissues on which the Mesna composition is applied. This entails the advantage that a m edical practitioner can see the parts where the composition is applied, allowing intuitive and quick ascertaining of a desired application of Mesna to the tissues, or if the application of Mesna should be adj usted, which adj ustm ents can also be directly seen by the visualization brought about by the one or more dyes. The use of a dye allows to visualize whether the mesna solution remains in place for a sufficient duration for effective chemical action. It also helps visualize the cleavage plane, which is very useful to the surgeon. This is of great importance for allowing efficient dissection of tissues facilitated by Mesna, and certainly in view of the knowledge that flooding a surgical cavity in order to make sure that all tissues to be dissected are wetted by Mesna is not an option, because the amount of Mesna which can be used in a single surgical procedure is limited by toxicity considerations.

In a preferred embodiment, the invention relates to a method for visualization of a composition comprising Mesna which is applied to tissues according to the fourth aspect of the invention, wherein the one or more dyes are selected from the group consisting of indocyanine green, riboflavin, riboflavin 5’-phosphate sodium, methylene blue and fluorescein. For the properties, advantages and technical effects of these dyes is referred to the above description.

In a preferred embodiment, the invention relates to a method for visualization of a composition comprising Mesna which is applied to tissues according to the fourth aspect of the invention, wherein the one or more dyes are one or more fluorescent dyes and wherein the method further comprises the step of providing a fluorescence surgical goggle to enable visualization of the fluorescent dyes through the goggle. Accordingly, a user, preferably a medical practitioner, of the goggle can selectively see the fluorescent dye without interferences of naturally occurring colors of the tissues. Preferably, the one or more fluorescent dyes are selected from the group comprising of indocyanine green, riboflavin, riboflavin 5’-phosphate sodium, methylene blue and fluorescein.

In a fifth aspect, the invention relates to a use of a composition according to the first aspect of the invention in a method according to the fourth aspect of the invention. Accordingly, all the technical achievements and advantageous characteristics of the composition according to the first aspect of the present invention are combined with those of the method according to the fourth aspect of the present invention.

The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention. EXAMPLES

Examples 1 -90: Compositions comprising Mesna and selected dves

5 Examples 1-90 relate to compositions comprising Mesna and selected dyes. Each composition according to Examples 1-90 comprises Mesna and a selected dye. In Examples 1-90, weight percentages of Mesna between the different compositions vary from 2% by weight to 22%. The compositions according to Examples 1-15, Examples 16-30, Examples 31-45 and Examples 46-60 are shown in Tables 1-4,0 respectively. The compositions according to Examples 61-75 and Examples 76-90 are equal to the ones of Examples 1-15 and Examples 31 -45, respectively, with the difference that water is replaced by a solution of 0.9 mass/volume percent of NaCI in water. When used in surgery, concentrations of Mesna from 2 weight per volume percent to 22 weight per volume percent, as shown in Examples 1-90, are high5 enough for facilitating tissue separation due to breakage of molecular bonds between tissue layers by Mesna, in particular by Mesna breaking disulfide bonds of polypeptide chains and proteins which disulfide bonds are responsible for the adherence of pathological tissues and for the strength of fibrosis, and are low enough for minimizing the risk of undesired tissue separation of surrounding tissues0 which are not to be separated. In fact, flooding a surgical cavity in order to make sure that all tissues to be dissected are wetted by Mesna is not an option.

Water is selected as a pharmaceutically acceptable solvent for Examples 1-15 and 31-45, a 10 mM Na ₂ HP0 ₄ aqueous solution is selected as a pharmaceutically5 acceptable buffer for Examples 16 to 31 and Examples 46 to 60 and a solution of 0.9 mass/volume percent of NaCI in water is selected as a pharmaceutically acceptable solvent for Examples 61-90. A 10 mM Na ₂ HPC> ₄ solution is a desired pharmaceutically acceptable buffer in the present context, owing to its solubilizing properties for solubilizing Mesna and the selected dyes and its buffering properties0 for stabilizing the pH of the composition at a pH from 6 to 8. The buffer, taken on its own, has a pH of at least 9.

Examples 1 -90 disclose compositions comprising Mesna and a dye selected from the group consisting of indocyanine green, riboflavin, riboflavin 5’-phosphate sodium,5 methylene blue and fluorescein. All these dyes are accepted for parenteral use in humans. Indocyanine green is a fluorescent cyanine dye with a peak spectral absorption at about 800 nm, i.e. it is a green-colored dye. For use in combination with Mesna, indocyanine green shows the advantage of acting as a reductant, thus not reacting with Mesna which is prone to oxidation. Besides, the green color has the advantage of being especially visible on tissues.

As a fluorescent dye, riboflavin provides a yellow to orange-yellow coloring to objects stained with it. For use in combination with Mesna, riboflavin shows the advantage of acting as a reductant, thus not reacting with Mesna which is prone to oxidation.

Riboflavin 5’-phosphate sodium is the phosphate sodium salt of riboflavin. Like riboflavin, riboflavin 5’-phosphate sodium provides a yellow to orange-yellow coloring to objects stained with it. Like riboflavin, for use in combination with Mesna, riboflavin 5’-phosphate shows the advantage of acting as a reductant, thus not reacting with Mesna which is prone to oxidation.

Methylene blue is a fluorescent thiazine dye with a blue color. Methylene blue acts as an oxidant, yet in the relative amounts of methylene blue in respect of Mesna in Examples 10-12, Examples 25-27, Examples 40-42, Examples 55-57, Examples 70- 72 and Examples 85-87 below, oxidation of Mesna by methylene blue is negligible.

Fluorescein is a red-colored fluorophore which appears purple under UV light. When applying UV light, fluorescein is thus well-suited for visualizing a composition comprising Mesna on tissues.

In Examples 1-30 and 61-75, the selected dyes are comprised in the compositions from 0.01 weight per volume percent to 0.03 weight per volume percent and in Examples 31-60 and 76-90, selected dyes are comprised in the compositions from 0.04 weight per volume percent to 0.06 weight per volume percent. Said concentrations of the one or more dyes are large enough to allow sufficient staining and clear visualization of tissues on which a composition comprising Mesna and one or more dyes is applied while not too large so that excessive use or wastage of the one or more dyes occurs or that undesired damage to the tissues would take place because of the one or more dyes. The staining of tissues with the one or more dyes functions to visualize parts of the tissues on which the Mesna composition is applied. This entails the advantage that a medical practitioner can see the parts where the composition is applied, allowing intuitive and quick ascertaining of a desired application of Mesna to the tissues, or if the application of Mesna should be adjusted, which adjustments can also be directly seen by the visualization brought about by the one or more dyes. The use of a dye allows to visualize whether the mesna solution remains in place for a sufficient duration for effective chemical action. It also helps visualize the cleavage plane, which is very useful to the surgeon. This is of great importance for allowing efficient dissection of tissues facilitated by Mesna, and certainly in view of the knowledge that flooding a surgical cavity in order to make sure that all tissues to be dissected are wetted by Mesna is not an option, because the amount of Mesna which can be used in a single surgical procedure is limited by toxicity considerations.

Table 1 : Compositions of Examples (Ex.) 1-15 with specific concentrations of Mesna and selected dyes solubilized in a volume of water, the concentrations expressed in weight per volume percent (w / v% ) with respect to the volume of water, according to embodiments of the first aspect of the invention

Table 2 : Com positions of Exam pies ( Ex.) 16-30 w ith specific concent rations of Mesna and selected dyes solubilized in 10 mM Na2HP04 aqueous solution, the concentrations expressed in weight per volume percent ( w/ V% ) w ith respect to the volume of the 10 mM Na2HP04 aqueous solution, according to embodiments of the first aspect of the invention

Table 3 : Com positions of Exam pies ( Ex.) 31-45 w ith specific concent rations of Mesna and selected dyes solubilized in water, the concentrations expressed in weight per volume percent (w/ V%) with respect to the volume of water, according to embodiments of the first aspect of the invention

Table 4: Compositions of Examples (Ex.) 46-60 with specific concentrations of Mesna and selected dyes in a 10 mM Na2HP04 aqueous solution, the concentrations expressed in weight per volume percent (w/ v% ) with respect to the volume of the 10 mM Na2HP04 aqueous solution, according to embodiments of the first aspect of the invention

Example 91: Stability study of solutions comprising Mesna and riboflavin 5’- phosphate sodium

A stability study was conducted for the purpose of evaluating a possible interaction between riboflavin 5’-phosphate sodium, a dye accepted for parenteral use in humans, and Mesna.

The following protocol was followed:

• 30 ml of phosphate buffer was added to Mesna powder for reconstitution, resulting in a solution comprising Mesna;

• riboflavin 5’-phosphate sodium was then added at 7 and 15 mg to obtain 2 different concentrations, namely 0.23 mg/mL and 0.5 mg/mL;

• each solution was then placed at 4 °C and 20° C during 1 hour and 24 hours before analysis;

• HPLC analyses according to the recommendations of the European Pharmacopoeia 9th edition were performed (“R” refers to Pharmacopoeia reagents) :

o Standards of Mesna: 10.0 - 5.0 - 2.0 - 1.0 mg/mL

o Volume taken: 1 mLdiluted in 10 mLof mobile phase o Mobile phase:

^■ 2.94 g of potassium dihydrogenphosphate

2.94 g of dipotassium hydrogen phosphate R

2.6 g hydrogen sulfate tetrabutylam monium R

^■ Ad 600 mL of water R

^■ Adjust pH to 2.3 with phosphoric acid

^■ Add 335 mLof R-methanol

^■ Ad 1000 m L of water R

o Flow rate: 1 mL/min

o Detection: 235 nm

o Injection volume: 20 pL

o Run time: 18 minutes

o Number of analyzes per sample: 1

The first step was to verify the absence of interference between the peak corresponding to the Mesna and that corresponding to riboflavin 5’-phosphate sodium using the assay method / Mesna analysis conditions described in European Pharmacopoeia 9th edition and included in the protocol above. Using the assay / assay method of assaying Mesna and related substances described in European Pharmacopoeia 9th edition, detection of riboflavin 5’-phosphate sodium did not interfere with that of Mesna. The latter can therefore be quantified without risk of interference with the selected dye.

Staining of the Mesna solution after addition of riboflavin 5’-phosphate sodium, whatever the concentration of riboflavin 5’-phosphate sodium, turned the solution from colorless to translucent yellow. In order to validate the obtained data, a reference corresponding to a reconstituted solution of Mesna without riboflavin 5’-phosphate sodium was analyzed at the same time as the samples comprising riboflavin 5’-phosphate sodium (Table 5).

Table 5 Concentrations of Mesna in a phosphate buffer, without and with (0.23 and 0.50 mg/ mL) riboflavin 5’-phosphate sodium (RF5’PNa), immediately after reconstitution and after 1 h and 24 h, at 4 °C and 20°C.

^* The percentage variation is calculated relative to the reference under the same storage conditions Under the analytical conditions described by the European Pharmacopoeia 9th edition for the quantification of Mesna and its related substances, the variations in Mesna content do not increase by more than 1 % after addition of riboflavin 5’- phosphate sodium , whatever either its concentration in the device (0.23 mg/m L and 0.50 mg/m L) and the storage temperature (4 °C and 20 °C) . The increases evaluated are in line with those observed at the reference level.

After 24 hours, the percentages of variation increase significantly. However, relative to the reference not com prising riboflavin 5’-phosphate sodium , the increases in levels did not seem significant, except for the sam ple comprising 0.23 mg/m L riboflavin 5’-phosphate sodium placed at 20 °C.

I n conclusion for Exam ple 91 , it can be concluded that the riboflavin 5’-phosphate sodium does not interfere with or degrade Mesna under the storage conditions described .

Example 92 : Stability study of Mesna powder in the presence of riboflavin 5’- phosphate sodium About 1 .5 g of Mesna powder was accurately weighed, without or with 7 mg or 1 5 mg of riboflavin 5’- phosphate sodium , and placed in a dry oven at 20 °C for 1 month to test its stability (Table 6) .

Table 6 Resu lts of stability tests on Mesna pow der in t he absence and presence of ribof lavin 5’- phosphate sodiu m ( RF5’PNa) at 20 ° C

^* standard deviation

^{* *} W0 = weight before placem ent in dry oven at 20 °C

* * * W1 = weight after 1 month in dry oven at 20 °C Given the standard deviations, it can concluded that Mesna is stable in the presence of riboflavin 5’-phosphate sodium , in dry form , for 1 month at 20 °C.