WO2022225394A1

WO2022225394A1 - Bulking agent for the treatment of stress urinary and fecal incontinence

Info

Publication number: WO2022225394A1
Application number: PCT/NL2022/050216
Authority: WO
Inventors: Jasper Johannes Franciscus VAN GOOL; Marcela Armine GARCIA MARTINEZ; Antonius Andreas Maria VOERMANS
Original assignee: Biomed Elements B.V.
Priority date: 2021-04-22
Filing date: 2022-04-22
Publication date: 2022-10-27
Also published as: NL2028044B1

Abstract

The invention relates to a bulking agent for use in the treatment of stress urinary incontinence and/or stress fecal incontinence, wherein the bulking agent is in the form of a gel, comprising a carrier fluid comprising water; cross- linked polysaccharide; and spherical microparticles of cross-linked polysaccharide having an average diameter in the range of 10-200 μm. When injected into periurethral or perianal tissue, the bulking agent allows the urethra or anus to close more effectively and so prevents urine and feces, respectively, from leaking.

Description

BULKING AGENT FOR THE TREATMENT OF STRESS URINARY AND FECAL

INCONTINENCE

FIELD OF THE INVENTION

The invention relates to a bulking agent for use in the treatment of stress urinary incontinence and/or stress fecal incontinence, to a container comprising such bulking agent and to a method for treating stress urinary incontinence or stress fecal incontinence.

BACKGROUND

Stress Urinary Incontinence (SUI) is when urine leaks out with sudden pressure on the bladder and urethra, causing the urinary sphincter muscles to open briefly. With mild SUI, pressure may be from sudden forceful activities, like exercise, sneezing, laughing or coughing. When SUI is more severe, leaking may occur with less forceful activities like standing up, walking or bending over. Urinary ‘accidents’ like this can range from a few drops of urine to enough urine to soak through clothing. Similar inconveniences may be experienced when the anal sphincter is prone to unintentional opening due to sudden pressure, resulting in the unwanted release of feces. This is named Stress Fecal Incontinence (SFI).

SUI is often treated by injection of a bulking agent in tissue at or near the urethra or sphincter, which adds bulk to the tissue, allowing for coaptation of the urethral wall and ultimately resulting in resistance to the passive outflow of urine. The treatment of SFI may be performed according to the same principle by injection at corresponding areas in tissue of the anus and anal sphincter. Although the injection of bulking agents is quite common, its clinical outcome is only limited positive. For example, most SUI therapies do not reach a higher clinical effectiveness than 60%, thereby leaving a large group of patients untreated.

Also, the injection of a bulking agent may give complications. The most common side effects are pain related to the site of injection and complications in the immediate post-procedure period such as urinary retention and voiding dysfunction. Surgical procedures are undesired as it is known that for example slings and tapes have adverse effects. Their application can result in permanent scarring of the surrounding tissue. Minimal invasive procedures such as injections are in that respect safer and preferable.

Other adverse effects of many bulking agents are related to their non-biological nature. They comprise synthetic materials such as polyacrylates ( e.g . polymethylmethacrylate), silicones or ethylene vinyl alcohol, which may trigger unwanted physical reactions in the body, such as an allergic reaction. Further, their poor biodegradability makes them very persistent in the body. When the presence of the material in the body is not desired anymore, or when the material has even become harmful due to e.g. undesired side effects, it is not an option to wait for its natural breakdown in the body. Instead, undesired invasive surgery is required to remove it from the body.

Therefore, there is a need for a safe bulking agent to treat stress urinary and fecal incontinence. It is also a desire to provide a bulking agent with a prolonged effectivity in the body.

SUMMARY OF THE INVENTION

It is therefore an objective of the present invention to provide a bulking agent that has prolonged effectiveness, a higher safety profile and causes less pain, irritation and inflammation upon injection at or near the urethra or the anus. It is in particular an objective that the bulking agent has an improved biodegradability compared to known bulking agents.

It has now been found that one or more of these objectives can be reached by applying a particular bulking agent.

Accordingly, the present invention relates to a bulking agent for use in the treatment of stress urinary incontinence and/or stress fecal incontinence, wherein the bulking agent is in the form of a gel and comprises

- a carrier fluid comprising water;

- a gel component of cross-linked polysaccharide;

- spherical microparticles of cross-linked polysaccharide having an average diameter in the range of 10-200 pm. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 displays a micrograph of the microparticles that are used in a bulking agent according to the invention.

Figure 2 displays hematoxylin and eosin stained tissues obtained at several intervals after the injection of different hyaluronic-acid based formulations.

Figure 3 displays the measured collagen density in tissues at twelve months after the injection of different hyaluronic-acid based formulations.

Figure 4 displays the progress of the biostimulation by the bulking agent of the invention over the 12-month period after injection.

Figure 5 displays the micrographs of the tissues involved in the biostimulation investigations referred to in Figure 4.

DETAILED DESCRIPTION OF THE INVENTION

Within the meaning of the present invention, the term “bulking agent” refers to a material or composition designed to directly and indirectly add volume to areas of soft tissue when injected in such tissue, in particular periurethral tissue or perianal tissue so that it helps to increase pressure at the opening of the urethra or anus and so prevents involuntary loss of urine or feces, respectively. When this is performed in a direct manner, the volume of the injected substance itself provides the added volume. When this is performed in an indirect manner, then the injected substance instigates or stimulates the body to produce additional tissue which on its turn provides the added volume. It usually takes some time before a significant contribution of the indirect volume addition is made. Therefore, it usually follows the direct volume addition.

Within the meaning of the present invention, the term “soft tissue” generally relates to tissues that connect, support, or surround other structures and organs of the body. In the present invention, soft tissues include, for example, periurethral tissue, perianal tissue pelvic floor muscles and submucosal tissue.

The term "periurethral tissue" refers to the tissue in the vicinity of the urethra, in particular tissue surrounding the urethra. The term "perianal tissue" refers to the tissue in the vicinity of the anus, in particular tissue surrounding the anus.

A bulking agent of the invention is in the form of a gel, i.e. it is a gel.

The term “gel”, as used herein, generally refers to a material having a fluidity between that of a liquid and a solid at mammalian body temperature (typically 37 °C).

A bulking agent is for use in the treatment of stress urinary incontinence (SUI) and stress fecal incontinence (SFI). To this end, it is typically introduced into tissues surrounding the urethra or anus, respectively (periurethral tissue or perianal tissue). Usually, such introduction into the tissue is made via an injection. The bulking agent is for example introduced (injected) into the wall of the urethra or anus; in the vicinity of the urinary sphincter or anal sphincter; or into the urinary sphincter or anal sphincter. In particular, it is introduced (injected) into a tissue selected from the group of submucosal tissue of the urethra, urinary sphincter tissue, anal sphincter tissue or pelvis floor tissue. The increased "bulk" at the site of injection allows the urethra or anus to close more effectively and prevents urine or feces from leaking.

This increased bulk following injection of a bulking agent of the invention is initially due to the presence of the bulking agent as such, i.e. due to the volume of the introduced matter as a direct result of the injection. On the longer term, however, the matter that causes the bulk at the injection site gains an increasing level of collagen. This usually occurs when the level of the initially injected matter decreases, because this continues to degrade. The increasing level of collagen that causes the bulkiness is the result of the so-called biostimulative effect of the bulking agent. It has namely been found that the bulking agent of the invention incites the natural tissues at the injection site to an increased production of collagen.

When injected (or otherwise introduced into the relevant tissue), the amount of bulking agent that is typically injected during a medical intervention is 2 ml_ or more. It may also be 3 ml_ or more, 4 ml_ or more or 5 ml_ or more. The amount is usually in the range of 2-15 ml_, in particular in the range of 3-10 ml_ For a useful effect, usually a plurality of injections is performed, for example 2, 3,

4, 5, 6, 7 or 8 injections. These are typically symmetrically or equally distributed around the urethra or anus. The separate introductions or injections as such (as a part of a medical intervention) may each involve the introduction of at least 0.5 ml_, at least 1 .0 ml_ or at least 1 .5 ml_ of the bulking agent. The carrier fluid is the medium in which the active compounds ( e.g . active in the treatment of stress urinary or fecal incontinence) are present. The carrier fluid comprises water. Optionally, it comprises a polyalcohol. By a polyalcohol is meant an alcohol that contains more than one hydroxyl group, such as a diol or a triol. It is for example selected from the group of ethylene glycol, glycerol, 1 ,3-propanediol, 1 ,4-butanediol, mannitol, sorbitol and polyethylene glycol).

The carrier fluid is in principle designed to be a physiologically acceptable carrier fluid. When water is present in substantial amounts {e.g. constituting more than 50 wt.% of the carried fluid), then the carrier fluid is typically buffered at or around physiological pH, e.g. with a physiological saline solution such as phosphate buffered saline (PBS). Other suitable buffers are, for example, Ringer’s solution (typically comprising sodium chloride, potassium chloride, calcium chloride and sodium bicarbonate) or Tyrode’s solution (typically comprising sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium dihydrogen phosphate and sodium bicarbonate).

The pH of such aqueous gel of the invention is usually in the range of 5.2 to 7.8, in particular in the range of 6.0 to 7.4. Such pH may be reached by applying a buffer as provided above having an appropriate pH, or by setting the pH at a desired value by using appropriate amounts of acid and/or base.

The content of carrier fluid in a bulking agent of the invention is usually at least 50 wt.%, based on the total weight of the bulking agent as such. The content may also be at least 60 wt.%, at least 70 wt.%, at least 90 wt.%, at least 95 wt.%, at least 97.5 wt.%, at least 98 wt.%, at least 98.5 wt.%, or at least 99 wt.%. The content may also be 99 wt.% or less, 98 wt.% or less, 95 wt.% or less, 90 wt.% or less, 85 wt.% or less or 75 wt.% or less. Preferably, the content is in the range of 90-98 wt.%.

The gel properties of a bulking agent of the invention are mostly derived from the gel of cross-linked polysaccharide, which gel is a component of the bulking agent. The polysaccharide is cross-linked to such extent that it has the properties of a gel. The skilled person knows how to arrive at such gel without exerting inventive efforts and without undue experimentation. The polysaccharide in the gel component of a bulking agent of the invention may be selected from the group of cellulose, cellulose derivatives such as carboxymethyl cellulose, hemicellulose, starch, chitosan, chitosan derivatives, alginate, agar-agar (agarose), starch, dextran, xanthan, levan, pectin, pullulan, carrageenan, curdlan, konjac, and natural gums such as gellan gum, beta-mannan gum, carob gum, fenugreek gum, guar gum, tara gum, karaya gum, tragacanth gum, and arabinoxylan gum.

The polysaccharide in the gel component may also be a glycosaminoglycan or glycosaminoglycan derivative, for example a glycosaminoglycan selected from the group of hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and keratan sulfate.

Preferably, the polysaccharide in the gel component comprises a polysaccharide that is selected from the group of chitins and glycosaminoglycans, in particular from the group of chitosan, chitosan derivatives, hyaluronic acid, hyaluronic acid derivatives, chondroitin sulfate and chondroitin sulfate derivatives.

In case the cross-linked polysaccharide in the gel component comprises hyaluronic acid, then the mass average molecular mass (M_w) of the hyaluronic acid is usually at least 50 kDa. Typically, it is in the range of 100-10,000 kDa. Preferably it is in the range of 200-5,000 kDa or in the range of 500-3,000 kDa.

In case the cross-linked polysaccharide in the gel component comprises chondroitin sulfate, then the mass average molecular mass (M_w) of the chondroitin sulfate is usually at least 1 kDa. Typically, it is in the range of 2-50 kDa. Preferably it is in the range of 3-45 kDa or in the range of 5-40 kDa.

In a bulking agent of the invention, the content of the cross-linked polysaccharide, in particular hyaluronic acid or chondroitin sulfate, that constitutes the gel component of cross-linked polysaccharide (in particular hyaluronic acid or chondroitin sulfate) is usually in the range of 0.1-10 wt.% of cross-linked polysaccharide (in particular hyaluronic acid or chondroitin sulfate), in particular in the range of 0.5-5.0 wt.%, more in particular in the range of 1.O^f.O wt.%, based on the total weight of the bulking agent as such.

The cross-links in the cross-linked polysaccharide (of the gel component) are usually chemical cross-links. These are formed by reaction of the polysaccharide with a chemical cross-linking agent. For example, such cross- linking agent is a diglycidyl ether ( e.g . 1 ,2-ethanediol diglycidyl ether or 1 ,4- butanediol diglycidyl ether) or a di-epoxyalkane {e.g. 1-(2,3-epoxypropyl)-2,3- epoxycyclohexane or 1 ,2,7,8-diepoxyoctane), Preferably, the cross-linking agent is divinyl sulfone or 1 ,4-butanediol diglycidyl ether.

The cross-links that result from these cross-linking agents are commonly said to be derived from the respective cross-linking agent. Accordingly, in a bulking agent of the invention, the chemical cross-links of the hyaluronic acid are typically derived from a cross-linking agent selected from the group of diglycidyl ethers and di-epoxyalkanes, preferably from 1 ,4-butanediol diglycidyl ether or divinyl sulfone.

The spherical microparticles in a bulking agent of the invention are of a cross-linked polysaccharide, which is usually prepared by the cross-linking of linear polysaccharide chains.

The polysaccharide of the spherical microparticles may be selected from the group of cellulose, cellulose derivatives such as carboxymethyl cellulose, hemicellulose, starch, chitosan, chitosan derivatives, alginate, agar-agar (agarose), starch, dextran, xanthan, levan, pectin, pullulan, carrageenan, curdlan, konjac, and natural gums such as gellan gum, beta-mannan gum, carob gum, fenugreek gum, guar gum, tara gum, karaya gum, tragacanth gum, and arabinoxylan gum.

The polysaccharide of the spherical microparticles may also be a glycosaminoglycan or glycosaminoglycan derivative, for example a glycosaminoglycan selected from the group of hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and keratan sulfate.

Preferably, the polysaccharide of the spherical microparticles comprises a polysaccharide that is selected from the group of chitins and glycosaminoglycans, in particular from the group of chitosan, chitosan derivatives, hyaluronic acid, hyaluronic acid derivatives, chondroitin sulfate and chondroitin sulfate derivatives.

In case the cross-linked polysaccharide in the spherical microparticles comprises hyaluronic acid, then the mass average molecular mass (M_w) of the cross-linked hyaluronic acid is usually at least 3 kDa. Typically, it is in the range of 1-5,000 kDa. Preferably it is in the range of 3-3,000 kDa or in the range of 5- 1,000 kDa.

In case the cross-linked polysaccharide in the spherical microparticles comprises chondroitin sulfate, then the mass average molecular mass (M_w) of the chondroitin sulfate is usually at least 1 kDa. Typically, it is in the range of 2-50 kDa. Preferably it is in the range of 3-45 kDa or in the range of 5-40 kDa.

The polysaccharide in the gel component and the polysaccharide of the cross-linked microparticles can be selected independently of one another.

The spherical microparticles in a bulking agent of the invention differ from microparticles that are used in known bulking agents in that they have a different shape. Known microparticles in such bulking agent applications are non- spherical, while those applied in the invention are spherical, as demonstrated in Figure 1. Moreover, the microparticles of the invention are also rather soft and may easily and reversibly deform when pressed against. They easily swell or shrink when they absorb or release water, respectively. These differences are the result of different processes of manufacturing the microparticles. In the art, this concerns the cross-linking of hyaluronic acid to form a gel, after which the gel is pulverized into the microparticles by e.g. grinding in a mortar (as in e.g. WO2018/159983A1). Microparticles thus obtained have an irregular shape, e.g. they have sharp edges and high aspect ratio’s.

Microparticles of the invention are prepared in a fundamentally different way. For example, the hyaluronic acid is dissolved in an aqueous medium. A water / ethyl acetate emulsion is made from this solution, yielding water droplets comprising the dissolved hyaluronic acid. Being slightly soluble in ethyl acetate, the water is drawn out of the droplets by the ethyl acetate, leaving behind globules of hyaluronic acid. Subsequently, the hyaluronic acid in the created globules is cross-linked. Further work-up then yields the spherical microparticles of cross- linked hyaluronic acid; the exposure to excess water in the work-up makes that they can swell substantially. These particles are thus highly regular and quite soft.

In some cases, the microparticles may deviate slightly from perfectly spherical. For example, the ratio of the shortest diameter to the longest diameter is at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, at least 0.97, at least 0.98, at least 0.99 or at least 0.995. In animal studies (see Example 6), it was found that the presence of spherical microparticles in the bulking agent of the invention resulted in an increase of natural tissue formation as compared to compositions comprising only the gel of polysaccharide (and no microparticles) over extended periods. This is a strong indication that spherical microparticles have a biostimulating effect that supports or even triggers the natural tissue formation. It was also observed that the presence of the spherical microparticles did not result in increased levels of inflammation. This is surprising, since compositions with the irregular microparticles are known for effecting a high level of inflammation in the first time after injection. Thus, the application of the spherical microparticles in the bulking agent provides an increased biostimulation that does not go hand in hand with an increased inflammation.

In particular, it appeared that the bulking agent of the invention has an initial volumizing effect which is in the course of 12 months taken over by a biostimulating effect. The combination of both effects is that the bulking is perceived more constant over time, as compared to a situation wherein the gel component of cross-linked polysaccharide is injected in the absence of microparticles.

Testing the effectivity of the bulking agent of the invention, as elaborated in Example 6, was not performed by injection into a periurethral tissue or a perianal tissue, but by injection into the skin. In this way, the bulking agent fulfills the function of a dermal filler. Given the similar nature of fascia on the one hand and periurethral tissue or perianal tissue on the other hand, the results of injection into the skin provide a strong indication of what the effects will be of injection into periurethral tissue or perianal tissue.

In the method of preparation as outlined above, it appeared that the higher the mass average molecular mass of the initial polysaccharide (in particular hyaluronic acid or chondroitin sulfate), the more difficult it is to obtain particles of a certain desired size. For example, when the polysaccharide had a mass average molecular mass well above 500 kDa, particles with a size in a desired range ( e.g . of 20-150 pm, 40-140 pm, or 60-125 pm) were difficult to prepare, which manifested e.g. as low particle yields and low process efficiency. It was initially not regarded as an option to lower the mass average molecular mass of the polysaccharide, since this is known to cause undesired inflammation side-effects, as is explained in the following.

Polysaccharides, and hyaluronic acid in particular, are known to have different effects on macrophage expression. The macrophage can undergo a phenotype change depending on the mass average molecular mass (M_w) of hyaluronic acid that the macrophage is in contact with. It is known that macrophages display a pro-inflammatory response when in contact with hyaluronic acid with lower mass average molecular mass (M_w), such as those as low as 500 kDa, 100 kDa, or 10 kDa. On the other hand, macrophages with a higher mass average molecular mass (M_w), typically higher than 500 kDa, are known to give a distinct anti-inflammatory response when in contact with hyaluronic acid.

In the context of the present invention, however, this appeared to be a prejudice. This is because it was surprisingly found in the animal studies that a bulking agent of the invention wherein the microparticles comprise hyaluronic acid with a mass average molecular mass (M_w) as low as 10 kDa does not result in a significant pro-inflammatory response.

It was also found that the decrease of the mass average molecular mass (Mw) did not cancel any of the other beneficial effects of the invention, in particular its effectivity as a biostimulator.

In conclusion, the decrease of the mass average molecular mass (M_w) of the polysaccharide (in particular of the hyaluronic acid) has opened the way to a more viable preparation of the microparticles, without giving in to the effectivity of the bulking agent and without attracting other undesired effects such as an inflammatory response.

Accordingly, in the spherical microparticles of cross-linked polysaccharide (in particular of hyaluronic acid), the mass average molecular mass (Mw) of the polysaccharide (in particular of the hyaluronic acid) is usually at least 1.0 kDa. Typically, it is in the range of 1-5,000 kDa. It may also be in the range of 1.2-3,000 kDa, in the range of 5-2,000 kDa, in the range of 2.4-500 kDa, in the range of 5-100 kDa or in the range of 10-1 ,000 kDa. Preferably, it is in the range of 5-500 kDa, more preferably in the range of 10-100 kDa.

The spherical microparticles in principle contain water. The water content in the microparticles is usually in the range of 50-99 wt.%, typically it is in the range of 75-95 wt.%. It may for example be 99 wt.% or less, 98 wt.% or less, 97 wt.% or less, 95 wt.% or less, 90 wt.% or less, 85 wt.% or less, 80 wt.% or less, or 75 wt.% or less. It may also be 60 wt.% or more, 75 wt.% or more, 80 wt.% or more, 85 wt.% or more, 90 wt.% or more, 93 wt.% or more or 95 wt.% or more.

The cross-links in the polysaccharide of the spherical microparticles are usually chemical cross-links, and are e.g. derived from a cross-linking agent selected from the group of diglycidyl ethers, di-epoxyalkanes and divinyl sulfone, in particular from 1 ,4-butanediol diglycidyl ether or divinyl sulfone.

The spherical microparticles in a bulking agent of the invention usually have an average diameter of 300 pm or less, 150 pm or less, 100 pm or less or 90 pm or less. It is usually 10 pm or more, 15 pm or more, 20 pm or more, 25 pm or more, 30 pm or more, 40 pm or more or 50 pm or more. Typically, it is in the range of 20-150 pm, in particular in the range of 40-140 pm, more in particular in the range of 60-125 pm.

The Dynamic Modulus (Storage Modulus) of the spherical microparticles is usually in the range of 10-3,000 Pa, in particular in the range of 100-2,500 Pa, more in particular in the range of 200-2,000 Pa and even more in particular in the range of 500-1 ,500 Pa.

The content of spherical microparticles in a bulking agent of the invention is usually in the range of 0.4-10 vol.%, in particular in the range of 0.5-8 vol.%, more in particular in the range of 1-6 vol.%. It may also be in the range of 4.0-30 vol.%, in the range of 8.0-24 vol.% or in the range of 12-18 vol.%. It may also be 30 vol.% or less, 25 vol.% or less, 20 vol.% or less, 15 vol.% or less or 10 vol.% or less. It may also be at least 1.0 vol.%, at least 2.0 vol.%, at least 3.0 vol.%, at least 5.0 vol.%, at least 10 vol.% or at least 15 vol.%.

A bulking agent of the invention may have undergone a sterilization process to provide the bulking agent as a sterile bulking agent. For example, it is sterilized by exposure to high temperature {e.g. by steam sterilization) or to high energy radiation {e.g. gamma irradiation).

The viscosity of a bulking agent of the invention may be tuned by varying certain characteristics of the bulking agent, such as the degree of cross- linking of the polysaccharide used in the gel component, the amount and size of the microparticles, and the relative abundancy of the different components, in particular of 1) the gel of cross-linked polysaccharide; 2) the microparticles; and 3) an eventual additive with an influence on the viscosity, such as linear polysaccharide ( vide infra). Depending on the specific application of the bulking agent, a higher or a lower viscosity can be set.

The dynamic viscosity of a bulking agent of the invention is usually in the range of 10-1100 Pa.s. It may also be in the range of 20-800 Pa.s or in the range of 30-700 Pa.s. A person skilled in the art will be able to find the conditions that are required for reaching a certain viscosity by routine experimentation and without exerting an inventive effort.

The Dynamic Modulus (Storage Modulus) of a bulking agent of the invention is usually in the range of 1-3,000 Pa, in particular in the range of 5- 2,500 Pa, more in particular in the range of 15-2,000 Pa and even more in particular in the range of 20-1 ,500 Pa.

The tan d of a bulking agent of the invention is usually 1.0 or less, in particular in the range of 0.05-0.90, more in particular in the range of 0.10-0.90 and even more particular in the range of 0.20-0.80. It can also be in the range of 0.20-0.70 or in the range of 0.30-0.60. The tan d (tangent delta) of a viscoelastic material is also known as the damping factor and is the ratio of the Loss Modulus to the Storage Modulus of the material.

A bulking agent of the invention may comprise a linear polysaccharide such as linear hyaluronic acid. The main purpose of this additive is that it may be used to tune the viscosity and so optimize the injectability of the bulking agent.

When a linear polysaccharide is present in a bulking agent of the invention, it usually has a mass average molecular mass (M_w) of at least 10 kDa. Typically, it is in the range of 12-5,000 kDa. Preferably it is in the range of 15- 3,000 kDa or in the range of 20-2,000 kDa.

When a linear polysaccharide is present in a bulking agent of the invention, it is usually present in the range of 0.05-5.0 wt.%, in particular in the range of 0.1 -4.0 wt.%, based on the total weight of the bulking agent as such.

The linear polysaccharide, when present, and the cross-linked polysaccharide are usually present in a mass ratio in the range of 1.0 : 0.25 to 1.0 : 15.0, in particular in the range of 1.0 : 1.0 to 1.0 : 10.0, based on their dry matter content. It is an advantage of the bulking agent of the invention that the bulking agent comprises polysaccharides, which make the bulking agent biodegradable.

It is also an advantage that a patient who is injected with a bulking agent of the invention experiences less side-effects than when a conventional bulking agent is injected, in particular less pain and/or less inflammation. It is all the more an advantage that this decrease of side-effects does not go at the expense of the effectivity of biostimulation. In other words, a bulking agent of the invention provides an increased biostimulation that does not go hand in hand with an increased inflammation and/or with a continuous inflammation.

A bulking agent of the invention may comprise other ingredients, in particular active pharmaceutical ingredients. For example, it may comprise a local anesthetic such as lidocaine or vitamins ( e.g . vitamin B, C, or E).

The invention further relates to a method for preparing a bulking agent in the form of a gel, comprising preparing a gel of a cross-linked polysaccharide; preparing spherical microparticles of a cross-linked polysaccharide, wherein the spherical microparticles have an average diameter in the range of 10-200 pm when they are present in the gel; mixing the cross-linked polysaccharide and the spherical microparticles with a carrier fluid comprising water to form the gel (/.e. the bulking agent).

In a method of the invention, the gel component of cross-linked polysaccharide and the spherical microparticles are usually prepared separately, after which they are mixed with the carrier fluid to form the gel of the invention. Thus, there are then at least three components that are combined in a method of the invention (in addition optionally also e.g. the linear polysaccharide).

There are multiple modes of combining these components. The three polymers may be void of water when combined, but one or more of them may also contain water upon combining the four components.

For example, the cross-linked polysaccharide may be contained in the carrier fluid to form gel, while the linear polysaccharide and the microparticles may be added to this gel as a dry solid. The linear polysaccharide is often applied as a dry solid, since it is commonly purchased as a dry powder that is ready for use in the method of the invention. The microparticles are typically prepared in an aqueous environment, which makes it convenient to apply them in wet form in a method of the invention. They may however also be dried prior to combining them with the other components.

The cross-linked polysaccharide is usually prepared by treating linear polysaccharide with a chemical cross-linking agent, e.g. divinyl sulfone or 1 ,4-butanediol diglycidyl ether. Analogously, the spherical microparticles of polysaccharide are usually also prepared by cross-linking the linear polysaccharide. The process for preparing the spherical microparticles is performed in such manner that the microparticles have an average diameter in the range of 10-200 pm when they are present in the final product of the process, which is the bulking agent. Their average diameter, when measured directly after their preparation in an aqueous environment, may be different from the average diameter of the microparticles in the final product, especially when the aqueous environment of their preparation is different from the carrier fluid in the final product (the latter may e.g. comprise a buffer, while the former may lack it).

The spherical microparticles are usually prepared in such manner that their average diameter in the final product is in the range of 20-150 pm, in particular in the range of 40-140 pm, more in particular in the range of 60-125 pm. This means that during the particle preparation, there is already accounted for a change in size of the particles that occurs later on when the particles are used to prepare the bulking agent of the invention, in particular when they are mixed with the cross-linked polysaccharide and the carrier fluid. Such change typically occurs when the amount of water that is contained by the microparticle changes, for example due to different concentrations of the components present, including salt concentration and pH. The skilled person knows which particle size should be initially present in order to arrive at the desired particle size in the final bulking agent.

The preparation of the microparticles may include the use of a sieve. The spherical microparticles are then sieved over a plurality of sieves to yield particles with an appropriate average diameter. When a sieving step is performed, the spherical microparticles are usually in a wet state, i.e. they comprise water.

A method of the invention typically includes a sterilization step, yielding the bulking agent of the invention as a sterile bulking agent. For example, a bulking agent formed according to a method of the invention may be exposed to an elevated temperature, e.g. to a temperature in the range of 80-140 °C, in particular in the range of 100-135 °C. The temperature and the period of exposure are then chosen such that any micro-organisms are destroyed to a desired extent, whilst not degrading the bulking agent too much. For example, the bulking agent is exposed during 15-20 minutes {e.g. at a temperature in the range of 115-125 °C), or it is exposed during 2-10 minutes {e.g. at a temperature in the range of 130— 140 °C).

Sterilization may also be achieved by exposing the gel to high energy radiation, in particular ionizing radiation such as gamma rays, electron rays,

X-rays, and the higher ultraviolet part of the electromagnetic spectrum. The dosage to which a gel may be exposed is e.g. 15, 25 or 50 kGy.

The invention further relates to a bulking agent obtainable by the method as described hereabove.

The invention further relates to a container, in particular a syringe, comprising at least 1 .5 ml_ of a bulking agent for use in the treatment of stress urinary incontinence and/or stress fecal incontinence, wherein the bulking agent is in the form of a gel and comprises

- a carrier fluid comprising water;

- a gel component of cross-linked polysaccharide;

- spherical microparticles of cross-linked polysaccharide having an average diameter in the range of 10-200 pm.

Such container or syringe may also comprise 2 ml_ or more, 3 ml_ or more, 4 ml_ or more or 5 ml_ or more of the bulking agent. Typically, the amount in the container or syringe in the range of 2-25 ml_, in particular in the range of 3-15 ml_, more in particular in the range of 4-10 ml_.

Typically, one container (or syringe) is used for one medical intervention. When such intervention comprises multiple subsequent injections at different sites, these injections may all be performed using the same syringe. It is of course also possible to use multiple syringes in one intervention.

A bulking agent of the invention is usually applied in the field of urology or gynecology. The invention further relates to a method for treating stress urinary incontinence or stress fecal incontinence, comprising administering to a human an effective amount of a bulking agent for use in the treatment of stress urinary incontinence and/or stress fecal incontinence, wherein the bulking agent is in the form of a gel and comprises

- a carrier fluid comprising water;

- a gel component of cross-linked polysaccharide;

The administration is typically performed by introducing the bulking agent into tissue in the vicinity of an urethra or anus, such as periurethral tissue or perianal tissue, in particular a tissue selected from the group of submucosal tissue of the urethra, urinary sphincter tissue, anal sphincter tissue or pelvis floor tissue. The introduction is usually performed by injection with a syringe via a needle.

The invention further relates to the use of a bulking agent as described hereabove for treating a tissue in an individual in need thereof.

The invention further relates to the use of a bulking agent for use in the treatment of stress urinary incontinence and/or stress fecal incontinence, wherein the bulking agent is in the form of a gel and comprises

- a carrier fluid comprising water;

- a gel component of cross-linked polysaccharide;

- spherical microparticles of cross-linked polysaccharide having an average diameter in the range of 10-200 pm; for the manufacture of a medicament for treating stress urinary incontinence and/or stress fecal incontinence in an individual in need thereof.

EXAMPLES

Example 1. Preparation of hyaluronic acid gel

Hyaluronic acid gels were prepared containing 1.5 g of 2,600 kDa hyaluronic acid (HA) in 13.5 g of 0.25 M sodium hydroxide (NaOH). After all HA was dissolved, 165 mg of 1 ,4-butanediol diglycidyl ether (BDDE) was added to the solution and mixed for 5 minutes with a spatula. The solution was put in a plastic cup which was closed off and transferred to an oven at 50 °C for 2 hours. The gel was then placed in an excess amount of PBS and left to hydrate till it reached a HA percentage of 1.5 wt.%.

Example 2. Preparation of cross-linked microparticles

50 mg of 10 kDa hyaluronic acid was dissolved in 2 ml_ 0.005 NaOH together with 7.5 mg of divinyl sulfone and left at room temperature for 2 hours. Then, 400 ml_ of ethyl acetate in an 800 ml_ beaker was stirred at 2,000 rpm with an overhead stirrer and the hyaluronic acid solution was added through a 30 G needle over a course of 2 minutes. The solution was left to stir for 1 hour. Afterwards the solution was left at room temperature for 24 hours. After purification (removal of ethyl acetate in vacuo) the particles were filtrated and washed. During the washing the particles swelled substantially, yielding soft hyaluronic acid microparticles.

Example 3. Combining all three components

22 g of the hydrated gel prepared in example 1 was mixed with 50 mg of 1 ,600 kDa hyaluronic acid powder for 5 minutes with a spatula. Afterwards,

90 mg of the particles prepared in example 2 were added and the resulting mixture was stirred for 5 minutes.

Example 4. Microparticles size analysis

The microparticles prepared in example 2 were analyzed using a Leica upright DM2500 light microscope. With a bright field 200x magnification the particles were examined. The morphology as well as the size was analyzed using Image J. An average spherical particle size of 30-50 pm was obtained. Figure 1 displays a micrograph of the obtained microparticles. Example 5. Rheometric analysis of the gel

The product made in example 3 was analyzed with a Discovery Hybrid Rheometer (TA Instruments). With a 1 ,200 pm gap height at 25 °C, the storage and loss modulus were measured. The plate had a 25 mm diameter and a 1% strain was applied. A frequency sweep was performed from 0.1 Hz to 5.0 Hz. At 5.0 Hz the gel had a storage modulus of 372 Pa and a loss modulus of 52 Pa.

Example 6. Biological Response - Animal Study

6.1. Set-up

An animal study was conducted to test the safety and effectiveness of the bulking agent of the invention. The in vivo study was conducted from June 2^nd, 2019 to June 2^nd, 2020 at Hangzhou Huibo Science and Technology Co., Ltd in Hangzhou China. The bulking agent was tested on New Zealand white rabbits and progress of the study was monitored at three different timepoints: 3, 6, and 12 months. Three different gel formulations (A, B and C) were tested:

A) cross-linked hyaluronic acid gel with linear hyaluronic acid;

B) hyaluronic acid microparticles with linear hyaluronic acid; and

C) cross-linked hyaluronic acid gel with linear hyaluronic acid and hyaluronic acid microparticles (i.e. the bulking agent of the invention).

The three formulations were prepared according to the procedures described in Examples 1-3 above (formulation B was prepared according to the procedure of Example 3, with the exception that cross-linked hyaluronic acid was left out of the procedure).

The primary aim of testing these formulations was to investigate the effect of the microparticles on the biostimulating effect of the bulking agent. At the same time, any side effects such as inflammation, rejection and encapsulation were monitored.

A total of 21 New Zealand white rabbits were used in this study. The rabbits were allocated as follows. A total of one rabbit per timepoint was used for formulation A; three rabbits per timepoint were used for formulations B and C. A volume of 0.1 to 0.2 ml_ was injected in the dorsum of each rabbit, approximately 5 cm away from the spinal column, between the front and back extremities of the animal. The injection occurred through the skin and in the fascia.

The two staining used to verify the safety and effectiveness of the hyaluronic acid gel were hematoxylin and eosin (H&E) along with Pricosirius red (PSR), respectively. The H&E staining provided a very detailed status of the tissue structure after the infiltration of the gel, and the PSR staining highlighted the presence of collagen fibers at each specific region of interest.

All images were processed using the software Image J. All images were analyzed at the same scale and magnification of 10x. The images were processed through Colour deconvolution, using H&E staining within the scroll down option. The region of interest (ROI) and thresholding parameters were fixed and used across all pictures. The quantity data collected was then processed in Excel and plotted as seen in the Figures.

In the following, the term ‘hyaluronic acid’ is sometimes abbreviated as ΉA’; the term ‘microparticles’ is sometimes abbreviated as MP.

6.2. Results

First, the obtained data were analyzed on the volumizing effectivity of the bulking agent. This is the increase of volume caused by the injection of cross-linked hyaluronic acid. A volumizing effect is generally known to be the highest directly after injection. Thereafter, cross-linked hyaluronic acid degrades, which manifests as a decrease of the volumizing. The data were also analyzed on the occurrence of side-effects occurred such as inflammation, rejection, encapsulation.

Formulation A or formulation C was injected and the skin was monitored over time. Tissues of the rabbits were analyzed at certain time intervals (3, 6 and 12 months) after the injection.

In the first weeks after the injection, any irritation/inflammation that has been observed was equally divided over the tissues that were treated with each formulation. Figure 2 displays H&E stained dermal layer tissues obtained at 3, 6 and 12 months after the injection of formulation A or formulation C (the scale bars all run from 0-2.5 mm over five sections of 0.5 mm). The gel is identified as a darker area, of which the contours are indicated with a black line. After three months, much of the gel is still present. No rejection from the skin has occurred at the injection site, nor at surrounding areas. The dermal layer has fully embraced the surface area of the gel, not only from the perimeter but also within/across it. The pictures after six months show a fainter appearance of the gel. After one year, no gel was left at the injection site, indicating complete degradation of the gel. Such degradation profile is in line with that of conventional volumizing compositions. Encapsulation of the gel through fibrotic tissue was not observed at any time, indicating that no immunologic response has been triggered. In conclusion, these tests indicate that a bulking agent of the invention is an effective volumizer and that the presence of the microparticles herein do not give undesired side-effects upon and after injection. This is all the more surprising in view of the mass average molecular mass (M_w) of hyaluronic acid in the particles. The M_w is only 10 kDa, a value that is normally regarded as unsuitable since it triggers a pro- inflammatory response in the body. It appears that this low value may be key to the effectivity of the biostimulation as well as to the low inflammatory response of a bulking agent of the invention.

Second, the obtained data were analyzed on the biostimulating effectivity of the bulking agent. This is the extent to which the injected bulking agent stimulates the formation of collagen. To this end, the amount of collagen fibers within each tissue sample is quantified by measuring the collagen density at the injection site, which is based on PSR polarized images of the tissues.

Figure 3 displays the measured collagen density in tissues at twelve months after the injection with formulation A, formulation B or formulation C. With formulation A providing a reference value corresponding to the absence of biostimulation (lower bar), it follows from Figure 3 that formulations B and C indeed initiate the formation of collagen (middle bar and upper bar, respectively). This is supported by visual inspection of the tissues themselves, as tissues injected with formulation B or C have a more dense appearance and contain less voids than tissues injected with formulation A.

Moreover, formulation C (of the invention) appears to perform even better than formulation B as regards the biostimulation. This is an indication of a synergistic effect of the microparticles and the cross-linked hyaluronic acid gel. Possibly, the gel inhibits the bio-degradation of the microparticles so that there is a prolonged activity of the microparticles.

Third, the obtained data were analyzed on the progress of the biostimulation by the bulking agent of the invention (formulation C) over the 12-month period after injection. It is generally known that it takes some time before biostimulation has a measurable effect, e.g. one or more months.

Figure 4 demonstrates such time profile for formulation C, as the effect is moderate after three months and has almost tripled in the nine months thereafter.

Figure 5 displays the micrographs of the tissues that correspond to the bars of Figure 4, revealing more homogeneity and less voids as time progresses (the scale bars all run from 0-250 pm over five sections of 50 pm). This also evidences the biostimulating effect of the bulking agent of the invention.

6.3. Conclusions

The above data yield the following conclusions. . The bulking agent of the invention has an initial volumizing effect which is in the course of 12 months taken over by a biostimulating effect. The combination of both effects is that the bulking is perceived constant over time. . The microparticles are responsible for the biostimulating effect and act herein synergistically with the cross-linked hyaluronic acid gel. . The bulking agent of the invention exhibits the properties under 1) and 2) without giving undesired side-effects upon and after injection. This is in contrast to the known compositions that comprise the irregularly-shaped hyaluronic particles. It is contemplated that the smoothness of the spherical microparticles in the bulking agent of the invention is responsible for the absence of the side- effects.

Claims

1. Bulking agent for use in the treatment of stress urinary incontinence and/or stress fecal incontinence, wherein the bulking agent is in the form of a gel and comprises

- a carrier fluid comprising water;

- a gel component of cross-linked polysaccharide;

2. Bulking agent for use according to claim 1 , wherein the bulking agent is introduced or injected into periurethral tissue or perianal tissue, in particular into a tissue selected from the group of submucosal tissue of the urethra, urinary sphincter tissue, anal sphincter tissue and pelvis floor tissue.

3. Bulking agent for use according to claim 1 or 2, wherein at least 2 mL of the bulking agent is introduced, in particular injected.

4. Bulking agent for use according to claim 3, wherein the polysaccharide of the gel component and/or of the spherical microparticles is selected from the group of chitins and glycosaminoglycans, in particular from the group of chitosan, chitosan derivatives, hyaluronic acid, hyaluronic acid derivatives, chondroitin sulfate and chondroitin sulfate derivatives.

5. Bulking agent for use according to any one of claims 1-4, wherein the bulking agent has a tangent delta in the range of 0.1-1.0, in particular in the range of 0.2-0.9, more in particular in the range of 0.3-0.8.

6. Bulking agent for use according to any one of claims 1-5, wherein the content of the carrier fluid is at least 90 wt.%, based on the total weight of the bulking agent as such, preferably at least 95 wt.%.

7. Bulking agent for use according to any one of claims 1-6, wherein the content of the cross-linked polysaccharide, in particular of cross-linked hyaluronic acid, that constitutes the gel of cross-linked hyaluronic acid is in the range of 0.1-10 wt.%, in particular in the range of 0.5-5.0 wt.%, more in particular in the range of 1.0-3.5 wt.%, based on the total weight of the bulking agent as such.

8. Bulking agent for use according to any one of claims 1-7, wherein the cross- linked polysaccharide in the gel component comprises a substance selected from the group of hyaluronic acid, hyaluronic acid derivatives, chondroitin sulfate, chondroitin sulfate derivatives, chitosan and chitosan derivatives.

9. Bulking agent for use according to claim 8, wherein

- the hyaluronic acid or hyaluronic acid derivatives have a mass average molecular mass in the range of 100-5,000 kDa or in the range of 500- 3,000 kDa; or

- the chondroitin sulfate or chondroitin sulfate derivatives have a mass average molecular mass in the range of 2-50 kDa or in the range of 5-40 kDa; or

- the chitosan or chitosan derivatives have a mass average molecular mass in the range of 10-2,000 kDa or in the range of 50-1 ,000 kDa.

10. Bulking agent for use according to any one of claims 1-9, wherein the cross- linked polysaccharide in the spherical microparticles has a mass average molecular mass in the range of 3-500 kDa, in particular in the range of 5-100 kDa.

11. Bulking agent for use according to any one of claims 1-10, wherein the cross- linked polysaccharide in the gel component and/or in the microparticles comprises cross-links derived from a cross-linking agent selected from the group of diglycidyl ethers, di-epoxyalkanes and divinyl sulfone, in particular from 1 ,4-butanediol diglycidyl ether or divinyl sulfone.

12. Bulking agent for use according to any one of claims 1-11 , wherein the spherical microparticles have an average diameter in the range of 20-150 pm, in particular in the range of 40-140 pm, more in particular in the range of 60-

13. Bulking agent for use according to any one of claims 1-12, wherein the content of spherical microparticles in a bulking agent of the invention is in the range of 0.4-30 vol.%, in particular in the range of 2.0-20 vol.%, more in particular in the range of 5.0-15 vol.%.

14. Bulking agent for use according to any one of claims 1-13, further comprising a linear polysaccharide, in particular linear hyaluronic acid or linear chondroitin sulfate.

15. Bulking agent for use according to claim 14, wherein the mass average molecular mass of the linear hyaluronic acid is in the range of 10-3,000 kDa, in particular in the range of 50-2,000 kDa.

16. Container or syringe comprising at least 2 ml_ of a bulking agent as defined in any one of claims 1-15.

17. Method for treating stress urinary incontinence or stress fecal incontinence, comprising administering to a human an effective amount of a bulking agent as defined in any one of claims 1-15.