WO2013164652A2

WO2013164652A2 - Microspheres

Info

Publication number: WO2013164652A2
Application number: PCT/GB2013/051181
Authority: WO
Inventors: Cristina MARTINS; Inês GONÇALVES; Paula GOMES; José Ricardo OLIVEIRA; Celso REIS; Ana MAGALHÃES
Original assignee: Ineb-Instituto De Engenharia Biomédica; Ipatimup-Institute Of Molecular Pathology And Immunology Of The University Of Porto; Universidade Do Porto; WILSON, Justin
Priority date: 2012-05-04
Filing date: 2013-05-07
Publication date: 2013-11-07
Also published as: WO2013164652A3; EP2844301A2

Abstract

There is provided a microsphere for binding H. pylori, wherein the microsphere comprises receptors for H. pylori, and wherein the microspheres and the receptors can bind H. pylori. The microspheres can be used in binding H. pylori bacteria and in treating or preventing H. pylori infection. There is also provided a method of treating or preventing H. pylori infection of the gastric mucosa and/or mucus layer, the method comprising administering an effective amount of microspheres to a patient, wherein the microspheres can bind H. pylori. Pharmaceutical compositions comprising the microspheres are also described.

Description

Microspheres

Field of the Invention

The present invention relates to the use of microspheres for binding Helicobacter pylori before and after adhesion to the gastric mucosa and/or gastric mucous layer.

Background to the Invention

Helicobacter pylori (H. pylori), a gram-negative bacterium, is one of the most common infectious agents worldwide, colonizing the gastric mucosa of over 50% of the world's population [1]. Chronic infection by H. pylori has been associated with increased risk for development of gastritis, peptic ulcer and gastric cancer [2, 3, 4]. Accordingly, H. pylori is classified as a class I carcinogen by the International Agency for Research on Cancer (IARC) [5]. Gastric cancer is the second leading cause of cancer-related deaths in the world [6]. The adhesion of H. pylori to the gastric mucosa and gastric mucus layer is mediated by bacterial surface-bound proteins, named adhesins, which recognize glycans expressed on the surface of gastric cells and also present on the mucus layer lining the gastric mucosa [7, 8]. The mucus layer formed by high-molecular-weight and heavily glycosylated glycoproteins known as mucins, protects gastric epithelial cells against chemical, enzymatic, microbial and mechanical damage. Carbohydrate structures expressed on the surface of epithelial cells and mucins serve as main specific ligands for H. pylori adhesins. The carbohydrate structures specific for H. pylori (also referred as glycosylated receptors, glycan receptors or Gly-Rs) include: Lewis B (LeB; Fuca2Galp3[Fuca4]GlcNAc-R) and Η type 1 (Fuca2Galp3GlcNAc- R), among others which are recognized by the blood group antigen-binding adhesin (BabA) [7], and Sialyl-Lewis X (sLeX; NeuAca3Galp4[Fuca3]GlcNAc-R) and Sialyl-Lewis A (NeuAca3Gaip3[Fuca4]GlcNAc-R), among others, which are the ligands for the sialic acid binding adhesin (Sab A) [8]. These two adhesins constitute so far the only well characterized bacterial adhesins regarding its host ligands. H. pylori adhesion is a crucial step for gastric mucosa colonization and establishment of infection since it provides protection from clearance mechanisms. Moreover, infection with strains that express BabA is associated with increased risk for development of duodenal ulcer, intestinal metaplasia and gastric adenocarcinoma [9, 10]. The LeB glycan structure is naturally expressed on the gastric mucosa of secretor and Lewis-positive individuals which constitute the majority of the human population. Using an animal model that is knock out for an enzyme essential for LeB and H-type 1 antigens biosynthesis, an impairment of BabA- dependent adhesion of H. pylori to Fut2-null mice gastric mucosa has been demonstrated, whereas binding mediated by SabA was not affected. This inhibition in adhesion could be strictly attributed to decreased expression of BabA ligands [11]. In addition, the binding forces between BabA and LeB-albumin conjugated have been determined using optical tweezers. This technique measures the interactions between individual bacteria and polystyrene beads coated with the compound to be tested (Bjornham et al. Journal of biomedical Optics 10(4), 044024, 2005). However, this paper does not refer to treatment or prevention of H. pylori infection of the gastric mucosa.

Persistent gastric mucosa colonization by H. pylori results in inflammation that is accompanied by de novo expression of sialylated glycans, including sLeX. It has been demonstrated that H. pylori is able to induce the expression of a host glycosyltransferase that leads to sLeX overexpression by the host cells and therefore increases bacterial capacity to adhere to epithelial cells [12].

The interplay between bacterial adhesins and host epithelial cells constitutes an essential feature for achievement of successful long term colonization and disease progression.

Current H. pylori eradication treatment relies on a triple therapy approach that combines at least two antibiotics and one proton pump inhibitor [13,14]. However, this treatment raises concern due to possible recurrence of infection, high costs, side effects, poor compliance to therapy and, most importantly, the increasing acquired resistance to some antibiotics [15]. In fact, it has been estimated that eradication therapy is unsuccessful in nearly one in every five patients [16]. In addition, H. pylori vaccines are not established yet. WO 2008/105740 describes the preparation of antibodies against the H. pylori BabA adhesin and suggests their application in the development of H. pylori passive vaccination. However, this requires the use of micro-organisms expressing an antibody fragment which is a complex system to produce and maintain. Further, its action remains unproven and alternative therapies are needed. In order to improve the efficacy of the antibacterial drug therapy, namely to protect the drugs (usually antibiotics) from the very acidic gastric fluids and increase its retention time in the stomach, several strategies for drug encapsulation have been described [17,18] and patented (WO 98/52547). A local antibiotic delivery system using H. pylori antibody-conjugated polymer beads were also suggested (Marquette MJ et al. Targeting Helicobacter pylori with antibodies: Exploring drug delivery with polymer beads. Experimental Biology 2004). However, this approach was for delivering antibiotics to H. pylori rather than treatment or prevention of H. pylori infection in the absence of antibiotics. Chitosan has been used for drug encapsulation and gastric delivery due to its gastric retentive capacity [19, 20] and because it is a Food and Drug Administration (FDA) approved polymer [21]. Chitosan is a natural, biodegradable and non-toxic cationic polysaccharide obtained by N-deacetylation of chitin. The gastric mucoadhesive properties of chitosan result from electrostatic interactions between its positively charged free amines and gastric mucins, which are negatively charged at the acidic stomach pH. Furthermore, chitosan is known for its bacteriostatic properties. Due to electrostatic interactions of chitosan cationic amino groups with anions on the bacterial wall, chitosan has an intrinsic antibacterial activity, inhibiting bacterial proliferation [22]. The utilization of genipin as crosslinking agent for chitosan microspheres has been previously described [23, 24].

However, several difficulties such as low retention time and difficulty to cross the mucus barrier have been observed when using chitosan microspheres for drug release in the stomach caused by a fast dissolution of chitosan upon arrival in the acidic gastric fluids.

Summary of the Invention

The present invention provides a microsphere for H. pylori binding, wherein the microsphere comprises receptors for H. pylori, wherein the microspheres and the receptors can bind H. pylori. These microspheres are used as a scavenging system, to remove H. pylori from the stomach of infected patients and not for a local drug delivery system. It has been found that the microspheres of the invention can bind H. pylori bacteria. When administered into the stomach, the microspheres can bind H. pylori bacteria which are present on the gastric mucosa and/or gastric mucus layer. This removes the H. pylori bacteria from the gastric mucosa and mucus layer. Further, H. pylori bacteria which are contained in the stomach but which are not adhered to the gastric mucosa or mucus layer can also be bound by the microspheres. This prevents the H. pylori bacteria from adhering to the gastric mucosa or mucus layer. The result of these effects is that H. pylori bacteria can be removed from the stomach. This reduces colonisation of the gastric mucosa and mucus layer by H. pylori bacteria and reduces or prevents re-colonisation. The microspheres with the bound H. pylori bacteria pass out of the stomach, after gastric mucus layer turnover, into the duodenum and are eventually excreted from the body, thereby removing the H. pylori bacteria from the body. By reducing, eliminating and/or preventing colonisation of the gastric mucosa and gastric cells by H. pylori bacteria, the risk of developing conditions associated with H. pylori infection can be reduced. For example, the risk of developing gastritis, peptic ulcer and gastric cancer can be reduced.

The microsphere can be made up of any suitable material which allows the microsphere to maintain its form at the range of pHs present in gastric and intestinal organs, i.e. between about 1.2 and about 9. The material should be such that the microsphere does not disintegrate at the range of pHs present in gastric and intestinal organs. The material is preferably a polymeric material. Suitable polymeric materials that could be used to make the microsphere include chitosan, polygalactosamine, polylysine, diethylaminoethyldextran (DEAE), DEAE- imine, etc. The microsphere itself (not including the receptors for H. pylori) should also be able to bind to H. pylori. The material from which the microsphere is made and, therefore, the microsphere itself, has mucoadhesive properties so that it binds gastric cells and mucins. This helps to increase the retention time of the microsphere in the stomach. The microsphere and the material from which it is made also binds to bacteria. This may be unspecific binding. Both of these types of bioadhesion can occur due to electrostatic forces. Therefore, the microsphere and the material from which it is made is positively charged so that it binds to the negative charges on the surface of bacteria, mucins and cells. The material used to make the microspheres may be a bioadhesive cationic polymer. Suitable materials include chitosan, polygalactosamine, polylysine, diethylaminoethyldextran (DEAE), DEAE-imine, etc. Polystyrene is not a suitable material so the microsphere should not be made from polystyrene.

The microsphere is preferably a chitosan microsphere. In other words, the microsphere is made up of chitosan molecules. Chitosan has advantageous bioadhesive properties. Chitosan is prepared by the deacetylation of chitin. The degree of deacetylation of chitosan should be preferably greater than 80%. The chitosan molecular weight should be preferably greater than 100,000 Da. The material which forms the microspheres may be modified to attach chemical and biological compounds for a desired purpose. This is in addition to the receptors for H. pylori. However, the attachment of these compounds should not modify the microspheres stability at the range of pHs present in gastric and intestinal organs. The microsphere is preferably crosslinked. In other words, the material forming the microsphere is preferably crosslinked. This helps to ensure that the microsphere is stable, retains its three dimensional structure and does not dissolve. A microsphere is stable if it does not disintegrate in simulated gastric fluid (SGF) with pepsin at a pH of 1.2. The degree of crosslinking is preferably at the minimum level necessary to avoid microsphere disintegration in simulated gastric fluid, particularly in acidic conditions, for example, at a pH of about 1.2. The crosslinking of the microsphere should not affect its required properties to allow it to be used to bind H. pylori. Preferably, the microsphere is partially crosslinked. For example, when the microsphere is made of chitosan, the chitosan molecules may be partially crosslinked rather than fully crosslinked. Complete crosslinking of chitosan microspheres can reduce the bioadhesive properties of the chitosan microsphere as the amine groups which are involved in bioadhesion can also be used to crosslink the chitosan. If the majority or all the amine groups are used for crosslinking, very few, if any, of the amine groups will remain to provide the advantageous bioadhesive properties. The degree of crosslinking to ensure that the microsphere is stable, but also that it retains its advantageous properties, can be determined by one skilled in the art.

The term "crosslinked" is intended to include various stages of crosslinking and includes partial crosslinking. The important aspect is that the microsphere is crosslinked to a sufficient degree to ensure that the microsphere is stable. The microsphere can be crosslinked with any suitable crosslinking agent. Suitable crosslinking agents are well known to those skilled in the art. For example, suitable crosslinking agents include glutaraldehyde, glyoxal, epichlorohydrin, succinaldehyde and genipin. Preferably, the crosslinking agent that is used is genipin due to its low toxicity. Genipin and the other crosslinking agents mentioned above are particularly suitable for crosslinking chitosan. A suitable concentration of crosslinking agent and an appropriate length of time for the crosslinking reaction can easily be determined to provide the desired amount of crosslinking.

In a particularly preferred embodiment, the microsphere is a chitosan microsphere which has been crosslinked with genipin. In this embodiment, partial crosslinking can be achieved, for example, by using lOmM of genipin for between 30 min and 90 min. Alternatively, ImM of genipin can be used for between 1 hr and 6 hr.

The microsphere should have a structure which allows it to bind H. pylori bacteria. The microsphere may have a porous structure. This means that the bacteria can be contained in the interior of the microsphere as well as on the surface in order to increase the maximum number of H. pylori bacteria that can be contained in/on the microsphere.

Microspheres used in this invention are microparticles were the longest measurable diameter is less than or equal to two times longer than the shortest measurable diameter.

The microsphere should have a size which allows it to effectively bind H. pylori bacteria. For example, the microsphere may have a diameter of between about 10 μιη and about 1000 μιη. Microspheres with these diameters have been shown to give good retention in the stomach. The microsphere may have a diameter of between about 10 μιη and about 750 μιη, or between about 10 μιη and about 500 μιη. When using the microsphere of the invention, a number of microspheres will be used. A mixture of sizes may be used. The mean diameter may be between about 10 μιη and about 1000 μιη at a pH of about 7.4. The mean diameter may be between about 10 μιη and about 750 μιη, or between about 10 μιη and about 500 μιη. The mean diameter of the microspheres may be between about 10 μιη and about 1000 μιη in simulated gastric fluid at a pH of about 1.2. The mean diameter may be between about 10 μιη and about 750 μηι, or between about 10 μηι and about 500 μηι in simulated gastric fluid at a pH of about 1.2.

The microsphere comprises receptors for H. pylori. These are receptors which can bind to H. pylori bacteria. More specifically, these receptors bind to molecules which are displayed on the surface of H. pylori bacteria. This allows the microspheres to bind to the H. pylori bacteria, thereby adsorbing and/or absorbing them so that they can be removed from the gastric mucosa and/or mucus layer or prevented from binding to the gastric mucosa and/or mucus layer. The receptors can be throughout the microsphere and not just on the surface. The receptors can be any suitable receptors which are able to bind to the surface of the H. pylori bacterium. Preferably, the receptors should be specific for the H. pylori bacterium so that the receptors do not bind to other molecules. In some embodiments, the receptors may be antibodies which can bind to H. pylori bacteria. Preferably, the glycan receptors bind to adhesins on the surface of the H. pylori bacterium. Adhesins are bacterial surface components that facilitate bacterial adhesion, in particular, to the gastric mucosa and/or mucus layer in the case of H. pylori. Preferably, the receptors are glycan receptors specific for H. pylori. These can also be called glycosylated receptors or Gly-R. Glycan receptors bind to adhesins on the surface of the H. pylori bacterium. The glycan receptors of H. pylori include fucosylated ABO blood group antigens, glycans with charged groups such as sialic acid or sulfate, and glycans which expose fucose, sialic acid, GlcNac and Gal. The blood group antigen-binding adhesin (Bab A) binds the Lewis b (Fuca2Galp3[Fuca4]GlcNAc-R) and H type 1 (Fuca2Gaip3GlcNAc-R) histo-blood group carbohydrate structures. The sialic-acid binding adhesin (SabA) mediates binding to carbohydrate structures such as Sialyl-Lewis X (NeuAca3Galp4[Fuca3]GlcNAc-R) and Sialyl-Lewis A (NeuAca3Galp3[Fuca4]GlcNAc-R). Further glycan receptors include Lewis A, Lewis Y, Lewis X, B-type and A-type receptors. Particular glycan receptors that can be used in the invention include: Lewis B (LeB) and H type 1 which are recognized by the blood group antigen-binding adhesin (BabA); and Sialyl- Lewis X (sLeX) and Sialyl-Lewis A (sLeA) which are ligands for the sialic acid binding adhesin (SabA). In some embodiments, the glycan receptors may be selected from Lewis B and Sialyl-Lewis X receptors.

The microsphere may comprise a single type of receptor. For example, the microsphere may only comprise Lewis B (LeB) receptors or Sialyl-Lewis X (sLeX) receptors. Alternatively, the microspheres may comprise a plurality of receptor types. For example, the microsphere may comprise Lewis B (LeB) receptors and Sialyl-Lewis X (sLeX) receptors.

The receptors may be attached to the microspheres in any suitable way so that the receptors can bind to H. pylori bacteria. When the microsphere is made of chitosan, the receptors are preferably attached to the chitosan via the primary alcohol group rather than the more reactive primary amine group. This preserves the primary amine groups of chitosan unchanged since these groups are responsible for chitosan' s mucoadhesive properties. An example of this can be seen in Figure 1.

Microspheres have been used previously to deliver active ingredients to the stomach. Current H. pylori treatment relies on a triple therapy approach that combines at least two antibiotics and one proton pump inhibitor. In some embodiments, the microsphere of the invention may further comprise an active pharmaceutical ingredient for the treatment or prevention of H. pylori infection. The microsphere may comprise an antibiotic against H. pylori or other drugs with antimicrobial activity such as amoxicillin, clarithromycin, metronidazole, ampicillin, tetracycline, doxycycline, oxytetracycline, bismuth, etc. The microsphere may comprise more than one antibiotic. The microsphere may comprise a proton pump inhibitor such as omeprazole, lansoprazole, rabeprazole, esomeprazole, cimetidine, etc. The microspheres may comprise one or more antibiotics and a proton pump inhibitor.

In other embodiments, the microsphere comprises a sub-therapeutic level of an active pharmaceutical ingredient for the treatment or prevention of H. pylori infection. In a particularly preferred embodiment, the microsphere does not contain an active pharmaceutical ingredient for the treatment or prevention of H. pylori infection. The microsphere may contain no active pharmaceutical ingredient whatsoever. In some embodiments, the microsphere may be in a lyophilised form, i.e. it is a lyophilised microsphere. It has been found that lyophilised microspheres retain their three dimensional structure and their H. pylori binding characteristics. In one aspect, there is provided a pharmaceutical composition comprising microspheres as described above and optionally one or more pharmaceutically acceptable excipients. Suitable pharmaceutically acceptable excipients are well known to those skilled in the art. In another aspect, there is provided a microsphere as described above for use in binding H. pylori bacteria. Further, there is provided a microsphere as described above for use in treating or preventing H. pylori infection, in particular, of the gastric mucosa and/or mucus layer.

In a related aspect, there is provided the use of a microsphere as described above in the manufacture of a medicament for binding H. pylori bacteria. Further, there is provided the use of a microsphere as described above in the manufacture of a medicament for treating or preventing H. pylori infection, in particular, of the gastric mucosa and/or mucus layer.

In a further aspect, there is provided a method of treating or preventing H. pylori infection of the gastric mucosa and/or mucus layer, the method comprising administering an effective amount of the microspheres described above to a patient.

The term "treating H. pylori infection" means the level of infection is reduced. In other words, the total number of H. pylori bacteria is reduced. Although it is preferred that all H. pylori bacteria should be removed/eliminated from the gastric mucosa and/or mucus layer, in reality this may not be possible. Therefore, the term treating is intended to have a broader meaning of reducing infection and not just eliminating infection.

Preferably, the microspheres are administered orally so that the microspheres are delivered to the stomach of the patient. The microspheres may be contained in a capsule (e.g. a gelatin capsule) which can dissolve in the stomach to release the microspheres.

Preferably, the patient is human. Preferably, the patient has H. pylori colonisation of the gastric mucosa and/or mucus layer.

In the pharmaceutical composition described above comprising microspheres of the invention and the method of treatment comprising administering microspheres, the microspheres can be a mixture of different microspheres. For example, some may have one receptor and others may have a different receptor. In an additional aspect, there is provided a method of manufacturing a microsphere for binding H. pylori, the method comprising:

forming a microsphere; and

attaching receptors for H. pylori to the microsphere.

The skilled person will appreciate that the features of the microsphere described above are equally applicable to the method of manufacturing the microsphere. For example, the microsphere is preferably a chitosan microsphere.

The method may further comprise the step of crosslinking the microsphere. Preferably, the crosslinking is carried out with genipin, especially when the microsphere is a chitosan microsphere.

Preferably, the receptors are glycan receptors specific for H. pylori such as Lewis B and/or Sialyl-Lewis X receptors.

When the microsphere is made of chitosan, the receptors are preferably attached to the chitosan via the primary alcohol group.

Preferably, the method does not involve the step of inserting an active pharmaceutical ingredient into the microsphere. The method may further comprise the step of lyophilising the microsphere.

In a further aspect, there is provided a kit for forming microspheres for binding H. pylori, the kit comprising a material for forming microspheres and receptors for H. pylori. The kit may further comprise a crosslinking agent.

Surprisingly, it has also been found that microspheres which have bioadhesive properties can be used on their own to bind H. pylori bacteria in a non-specific way. This is thought to be through electrostatic interactions. Therefore, receptors for H. pylori attached to the microspheres are not essential. Therefore, there is provided a method of treating or preventing H. pylori infection of the gastric mucosa and/or mucus layer, the method comprising administering an effective amount of microspheres to a patient, wherein the microspheres can bind H. pylori.

The features described above for the microsphere comprising receptors are equally applicable to this aspect except for the fact that the receptors for H. pylori are not required.

In particular, the microspheres can be made up of any suitable material, preferably polymeric material, which can maintain microspheres intact at the range of pHs present in human gastrointestinal tract (the stomach and intestine) and which can bind to H. pylori bacteria. The material should be able to avoid microspheres degradation at a pH of between about 1.2 and about 9. The material used to make the microspheres may be a bioadhesive cationic polymer. Suitable materials include chitosan, polygalactosamine, polylysine, diethylaminoethyldextran (DEAE), DEAE-imine, etc. The microspheres are preferably chitosan microspheres. In other words, the microspheres are made up of chitosan molecules.

The material which forms the microspheres may be modified to attach functional groups for a desired purpose. Modified material should still be able to maintain microspheres without disintegration at the range of pHs present in human gastrointestinal tract and bind H. pylori.

In this aspect, the microspheres are preferably crosslinked as described above in detail. Preferably, the microspheres are crosslinked with genipin as described in detail above. In this aspect, the microspheres preferably comprise a sub-therapeutic level of an active pharmaceutical ingredient for the treatment or prevention of H. pylori infection. In a particularly preferred embodiment, the microspheres do not contain an active pharmaceutical ingredient for the treatment or prevention of H. pylori infection. The microspheres may contain no active pharmaceutical ingredient whatsoever.

The microspheres may be lyophilised microspheres.

As above, the term "treating H. pylori infection" means the level of infection is reduced. In other words, the total number of H. pylori bacteria is reduced. Preferably, the microspheres are administered orally.

Preferably, the patient is human. Preferably, the patient has H. pylori colonisation of the gastric mucosa and/or gastric cells.

The present invention provides a pharmaceutical composition for treating or preventing H. pylori infection, the composition comprising microspheres which can bind H. pylori and optionally one or more pharmaceutically acceptable excipients.

The present invention also provides a pharmaceutical composition for treating or preventing H. pylori infection, the composition consisting of microspheres which can bind H. pylori and optionally one or more pharmaceutically acceptable excipients. As will be appreciated by a skilled person, microspheres with receptors can be used in combination with microspheres without receptors.

Therefore, there is also provided a pharmaceutical composition for treating or preventing H. pylori infection, the composition comprising:

1) microspheres which can bind H. pylori; and

2) microspheres comprising receptors for H. pylori that can bind Η. pylori.

Further, there is provided a method of treating or preventing H. pylori infection of the gastric mucosa and/or mucus layer, the method comprising administering an effective amount of the above composition comprising microspheres with and without receptors to a patient.

Additionally, there is provided the above composition comprising microspheres with and without receptors for use in treating or preventing H. pylori infection of the gastric mucosa and/or mucus layer.

Also provided is use of the above composition comprising microspheres with and without receptors in the manufacture of a medicament for treating or preventing H. pylori infection of the gastric mucosa and/or mucus layer. As indicated above, the microspheres comprising receptors for H. pylori can be a mixture of different microspheres, for example, some having one receptor and others having a different receptor. The invention will now be described in detail, by way of example only, with reference to the figures which are as follows:

Brief Description of the Figures

Figure 1 shows a scheme for the chemical immobilization of glycan receptors (GlyRs) on chitosan microspheres: (a) Protection of chitosan reactive primary amine groups with phthalic anhydride; (b) Chitosan O-alkynylation; (c) Immobilization of N3-GlyR using the azide- alkyne conjugation "click reaction"; (d) Deprotection of chitosan primary amine groups.

Figure 2. Chitosan microspheres 2A) without glycosylated receptors (GlyRs) (Ch- Microsphere), 2B) with alkyne group (Alkyne-Microsphere) and with two different types of GlyRs: 2C) Lewis B (LeB-Microsphere) and 2D) sialyl-Lewis X (sLeX-Microsphere) with adherent H. pylori strain 17.1 (17875/Leb), which express functional BabA, but not SabA adhesins (Figures 2A-D, respectively). As can be observed, 17.1 strain, that express BabA (specific for LeB antigens), binds strongly and in a non-specific way to Ch-Microspheres, strongly and specifically to LeB-Microspheres and in small amounts to Alkyl- or sLeX- Microspheres. 2A) Ch-Microsphere + H. pylori strain 17.1 (BabA+/SabA-); 2B) Alkyne- Microsphere + H. pylori strain 17.1 (BabA+/SabA-); 2C) LeB-Microsphere + H. pylori strain 17.1 (BabA+/SabA-); and 2D) sLeX-Microsphere + H. pylori strain 17.1 (BabA+/SabA-). Figure 3. Chitosan microsphere image obtained by scanning electron microscopy (scale bar = 100 μπι).

Figure 4. Fluorescence kinetic of chitosan microspheres in the presence of 1 mM and 10 mM genipin.

Figure 5. [35S]-H. pylori adhesion to chitosan microspheres after incubation in pH 6 and pH 2.6. Four H. pylori strains differing in the functional expression of adhesins were used: J99 (BabA+/SabA+), 17875/Leb (17.1 ; BabA+/SabA-), 17875 isogenic babAlA2 mutant (DM; BabA-/SabA+) and 097UK (BabA-/SabA-).

Figure 6. [35S]-H. pylori adhesion (17.1 strain on the left and DM strain on the right) to MKN45 cells (per coverslip) without contact with microspheres (Cell+H. pylori) and after contact with chitosan microspheres before and after H. pylori infection (Cell+H./r /on+Mic and Cell+Mic+H./r /on, respectively).

Detailed Description of the Invention

Example 1

Use of Chitosan Microspheres to Bind H. pylori and to Prevent/Remove Adhesion of H. pylori to the Gastric Mucosa and Mucus Layer The present invention uses a different strategy to avoid and remove H. pylori gastric colonization. It exploits the bioadhesive properties of chitosan to create three dimensional porous microspheres that can be used to bind H. pylori before and after bacterial binding to gastric mucosa and mucus layer. Although chitosan microspheres have already been described as a good vehicle for gastric drug delivery (WO 98/52547), they have never been described as being used to impair H. pylori adhesion and therefore for infection treatment purposes. The inventors have found that chitosan microspheres can bind different strains of H. pylori and prevent H. pylori from adhering to the gastric cells. The inventors have also found that chitosan microspheres can bind to H. pylori which are already adhered to the gastric cells, thereby removing the H. pylori from the gastric cells.

In this example, the inventors studied the effect of chitosan microspheres crosslinked with genipin in preventing/removing H. pylori gastric cell adhesion, under different pH conditions.

EXPERIMENTAL METHODS

Preparation of chitosan microspheres Chitosan powder, obtained from France-Chitine (France), was purified by the reprecipitation method as previously described [25].

Chitosan microspheres were produced in a high voltage electrostatic system by extruding chitosan droplets (1% w/v in acetic acid) into a 5% w/v sodium triphosphate pentabasic (TPP) solution. Chitosan microspheres were crosslinked in lmM or lOmM genipin solutions (in PBS 0.01M) over different reaction times at 25°C and 120rpm.

Characterization of chitosan microspheres

Microsphere size and morphology was visualized by scanning electron microscopy (SEM) and optical microscopy. Genipin crosslinking kinetics was assessed with a time lapse using a fluorescence microscope (FM), since genipin fluoresces dark blue when crosslinked, and its chemical structural changes were monitored by Fourier transform infrared (FT-IR) spectroscopy. Microspheres stability and swelling in simulated gastric fluid (SGF) with pepsin was evaluated for 7 days by optical microscopy.

Mucoadhesiveness of microspheres were determined by measuring the capacity of the microspheres to bind porcine stomach mucin type III (M1778, Sigma). Quantification of the adsorbed mucins onto chitosan microspheres was performed applying a colorimetric Periodic Acid-Schiff (PAS) Kit (395B, Sigma).

H. pylori adhesion to gastric cells and chitosan microspheres

Four H. pylori strains differing on the adhesins expression were used: J99 (BabA+/SabA+), 17875/Leb (17.1; BabA+/SabA-), 17875 isogenic babAlA2 mutant (DM; BabA-/SabA+) and 097UK (BabA-/SabA-). The strains J99, 17.1 and DM were obtained from the Department of Medical Biochemistry and Biophysics, Umea University, Sweden. The strain 097UK was obtained from the Department of Molecular Biology, Max-Planck Institut fur Infektionsbiology, Berlin, Germany. Bacteria were cultured in microaerophily at 37 °C on trypticase soy agar with 5% sheep blood for 48h and afterwards on pylori gelose for 48h. For quantification assays bacteria were labelled with radioactive sulphur (S-35), for visualization, bacteria were labelled with fluorescein isothiocyanate (FITC). A gastric carcinoma cell line MKN45 (mainly expressing sLeX, which binds SabA) was used. Cells were grown in RPMI 1640 with glutamax, supplemented with 10% inactivated (30 min, 56°C) fetal bovine serum, lOU/ml penicillin and 10μg/ml streptomycin at 37°C in a humidified 5% C0₂ atmosphere.

H. pylori adhesion tests were performed in citrate-phosphate buffer under pH=2.6 and pH=6 for 2h, at 37°C, 120 rpm using an inoculum of 1x10 colony forming units/ml (CFU/ml). Quantification of [35S]-H. pylori adherent to gastric cells or chitosan microspheres was performed using a luminescence counter (Microbeta). Observation of microspheres with adherent FITC-H. pylori was performed by confocal laser scanning microscopy (CLSM).

Efficiency of chitosan microspheres in removing/preventing H. pylori adhesion from/to gastric cells.

Three different conditions were tested. In the first condition (control of bacteria adhesion to cells), MKN45 cells were incubated for 2h with [35S]-H. pylori (Cells+H. pylori); in the second condition, MKN45 cells were incubated for 2h with [35S]-H. pylori, rinsed with buffer to remove non-adherent bacteria and afterwards incubated with chitosan microspheres for another 2h (Cells+H. pylori +Mic); and finally, in the third condition, MKN45 cells were incubated for 2h with chitosan microspheres and afterwards incubated with [35S]-H. pylori for another 2h (Cells+Mic+H. pylori). The second condition will enable to infer on the capacity of the microspheres to remove H. pylori already adhered to gastric cells, while the third condition will allow concluding on the ability of chitosan microspheres to prevent H. pylori from adhering to gastric cells. The measurement of the radioactivity of [35S]-H. pylori adherent

Quantification of [35S]-H. pylori adhesion to gastric cells or chitosan microspheres were detected using a luminescence counter (Microbeta).

RESULTS AND DISCUSSION SEM (Figure 3) and optical microscopy revealed that microspheres have a mean diameter of

Genipin crosslinking kinetics revealed that microspheres with lOmM and ImM of genipin are completely crosslinked after 2hr and 7hr, respectively (Figure 4) Genipin crosslinking occurs through the amine groups that are also necessary for maintaining chitosan mucoadhesion. Therefore, optimal crosslinking time and concentration of genipin was determined taking into account that full crosslinking is not desirable, only the minimum to avoid microsphere disintegration in acidic conditions. FT-IR of microspheres with lOmM genipin showed a decrease of the characteristic peaks of the amine groups of chitosan at 3375 and 1598 cm^"1 with the increase of genipin crosslinking time, meaning that the amine groups are being replaced by amide binding from genipin reaction that appears at 1655 cm-1. The increase of the absorption band at 1420 cm^"1 with increase of genipin crosslinking time was assigned to the N-H stretching of amide band and ring stretching of genipin molecule (region at 1500-1300 cm-1) [26].

Microspheres crosslinked with lOmM of genipin for lhr were stable for seven days in simulated gastric fluid (SGF) and were used in all bacteria adhesion tests.

The mean diameter of these microspheres, after crosslinking (genipin lOmM/lh) and lyophilisation, is pH dependent and change between 186+29μιη (pH=7.4) to 345+71 μιη (SGF; pH=1.2). The mucoadhesiveness of chitosan microspheres decreased with increase of crosslinking time from 0.053+0.008 (0.5 hour) to 0.048+0.008 mg of mucins per mg of microspheres (2 hour).

Quantitative studies of bacterial adhesion using [35S]-H. pylori revealed that approximately 100 bacteria adhere to each microsphere at pH 6, while roughly 50 bacteria adhered to each chitosan microsphere at pH 2. These results confirmed that H. pylori adhesion to chitosan microspheres is higher at pH 6, but it also takes place in a more acidic pH (pH 2.6) (figure 5) and that chitosan microspheres are able to bind all the tested strains, regardless of the adhesin expression profile. The addition of chitosan microspheres to a culture that was previously infected with H. pylori (Cell + H. pylori + Mic) removed more than 50% of adherent H. pylori, independently of the bacteria strain and pH used. When 17.1 strain was used, the reduction in its binding to gastric cells was higher at pH=2.6 (70%) and when DM strain was used, this effect was higher at pH =6 (76%) (Figure 6). Regarding the effect of adding chitosan microspheres to a cell culture before infection with H. pylori (Cell + Mic + H. pylori), it was demonstrated that a reduction around 50% of bacterial adhesion was observed comparing to when no microspheres were added, despite the bacteria strain and pH at where incubations occurred (Figure 6).

CONCLUSION

Microspheres crosslinked with lOmM of genipin for lhr proved to be stable under acidic conditions for at least 7 days. These microspheres are able to bind H. pylori regardless of bacteria adhesin expression and of the pH. Furthermore, they were able to both remove H. pylori adherent to gastric cells (50-76%) and prevent H. pylori from adhering to gastric cells (47-56%).

FURTHER RESULTS AND CONCLUSIONS

For the success of this application, microspheres should be porous and should not degrade so that they are removed intact from the stomach (through the intestinal tract) after gastric mucosal turnover.

The microspheres should be stable in the range of pHs present in gastric and intestinal organs. In order to avoid dissolution of the microspheres in acidic pH, they were crosslinked with genipin. Genipin crosslinking of the microspheres was optimized to maintain its three dimensional structure in acidic conditions without losing the mucoadhesive properties.

Chitosan microspheres were produced, crosslinked and lyophilized. The degree of crosslinking, size and porosity controls chitosan degradation in the gastric acidic conditions and bacteria binding. Crosslinking enables the microspheres to be removed through the gastrointestinal tract without dissolving.

Genipin was used as crosslinking agent to avoid degradation in simulated gastric fluid (SGF) for at least 1 week. Two different genipin concentrations (1 and 10 mM) were used over different reaction times. The degree of crosslinking was used to control the swelling and mucoadhesive properties. Lyophilisation was used to maintain the three dimensional porous structure that is essential for bacterial absorption. This process also allows the storage of the microspheres for a long period of time without chitosan degradation. The mean diameter of these microspheres, after crosslinking (genipin lOmM/lh) and lyophilisation, is pH dependent and change between 186+29 μιη (pH=7.4) to 345+71 μιη (SGF; pH=1.2).

Chitosan microspheres bind bacteria, namely H. pylori, in a range of pHs (2.6-7.4) when bacteria are in solution or adherent to cells. These microspheres were also able to prevent bacteria adhesion to gastric cells.

In conclusion, three dimensional chitosan porous structures, stable in simulated gastric fluid, with the capacity to bind mucins, swell and bind H. pylori over a range of different pHs.

Applications

Chitosan microspheres, crosslinked with genipin and lyophilized, can be used for gastric applications to bind H. pylori and remove it through the gastrointestinal tract. Chitosan microspheres, crosslinked with genipin and lyophilized, can be decorated with receptors for bacteria to increase its binding specificity.

The crosslinked chitosan microspheres can be used for the prevention or treatment of H. pylori infection and associated diseases, and the prevention of H. pylori re-infection on antibiotic treated patients.

Example 2

Chitosan Microspheres Decorated with Specific Glycan-receptors (GlyRs) for Helicobacter pylori Adhesins

In this example, mucoadhesive chitosan microspheres were used which had an additional chemical modification. The microspheres are decorated with glycosylated receptors (GlyRs) with affinity to H. pylori adhesins, to compete with gastric mucins and attract, bind and remove H. pylori from the stomach. These microspheres (bound to and carrying H. pylori) are eliminated from the stomach (through the intestinal tract) after gastric mucosal turnover.

During this work, it was demonstrated that H. pylori expressing the Bab A adhesin, bind specifically to chitosan microspheres (GlyR-Mic) decorated with LeB.

Crosslinked chitosan microspheres (e.g. those from Example 1) can attract and bind H. pylori, but unlike the GlyR-Mic that are H. pylori adhesin specific, they bind in a non-specific way. The presence of GlyRs on chitosan microspheres improves the attraction and binding of H. pylori since the GlyRs can compete directly with host glycans expressed on mucins and cell surfaces for H. pylori adhesins. A combination of chitosan microspheres with different GlyRs can be used in order to improve the biding of H. pylori that express different adhesins. A combination of chitosan microspheres with different GlyRs and microspheres without Gly-Rs (e.g. those from Example 1) can also be used to improve the biding of H. pylori by the combination of non-specific H. pylori binding with specific H. pylori binding.

Chemical modification of chitosan microspheres with GlyRs has never been described. Additionally, biomaterials designed to selectively attract and bind H. pylori have also never been described.

EXPERIMENTAL METHODS

Chitosan microspheres crosslinked with genipin and decorated with glycosylated receptors (GlyR), such as LeB (Fuca2Galp3[Fuca4]GlcNAc-R) and sLeX (NeuAca3Gaip4[Fuca3]GlcNAc-R) were produced.

Chitosan microspheres with a mean diameter between 186+29μιη (pH=7.4) to 345+71 μιη (SGF; pH=1.2) and stable for at least 7 days in simulated gastric fluid (SGF) with pepsin were produced as described above. GlyR immobilization onto chitosan microspheres was carried out through the primary alcohol group of chitosan using the "click reaction" (corresponding to an azide-alkyne coupling) instead of the more reactive primary amine groups. The possibility to modify the primary alcohol group of chitosan powder using the "click reaction" was previously described by the inventors [27] using an azide terminated-PEG but not with carbohydrates such as glycan receptors. Moreover, the methodology used for coupling compounds to powders and 3 dimensional structures (microspheres) are different. This strategy was chosen to preserve the primary amine groups of chitosan unchanged since these groups are responsible for chitosan's mucoadhesive properties. This covalent immobilization is briefly described in Figure 1.

Protection of chitosan reactive primary amine groups with phthalic anhydride ((N-phthaloyl- chitosan microspheres) - Figure la)

Chitosan microspheres were suspended in phthalic anhydride solution in 15 ml dimethylformamide (DMF) containing 5% (v/v) water and incubated for 15h at 75 °C under 200 rpm. The microspheres were afterwards rinsed with DMF and after with tetrahydrofuran (THF).

Preparation of N-phthaloyl-chitosan Q-prop-2-ynyl carbamate microspheres ((activation of the hydroxyl groups of chitosan and addition of alkyne groups) - Figure lb)

N-phthaloyl-chitosan microspheres were suspended in 30 mg/ml carbonyldiimidazole (CDI) solution in THF and incubated for 6h at 40°C under 200 rpm. After reaction, microspheres were rinsed with THF and then incubated in a propargylamine solution in THF for 15h at 25°C under 200 rpm. Finally the microspheres were rinsed with THF and dried. Preparation of N-phthaloyl-chitosan Q-Gly-R microspheres ((Addition of N3-G -R to chitosan using the azide-alkvne conjugation "click reaction") - Figure lc)

Three different solutions (5:5:4) were added to N-phthaloyl-chitosan Oprop-2-ynyl microspheres: (1) lOmg/ml aqueous sodium ascorbate solution; (2) 10 mg/ml aqueous copper acetate solution in DMF and (3) aqueous solution of glycosylated receptor Lewis B (LeB) or sialyl-Lewis X (sLeX) . The mixture was incubated for 15h at 40°C under 200 rpm.

Control chitosan microspheres (Alkyne-Microspheres) were prepared by undergoing all the processes but in this last step, instead of being incubated in a solution of GlyR, they were incubated in the solvent. The microspheres were rinsed with DMF and dried with ethanol.

Removal of the N-phthaloyl protecting group. (Unblockage of the free amines of chitosan) - Figure Id) Chitosan microspheres with and without GlyR were rinsed with ethanol and incubated in hydrazine monohydrate in ethanol (1 : 1) for 6h at 40°C under 200 rpm. The microspheres were finally rinsed with ethanol and dried. Characterization of Gly-R microspheres

Immobilization of the GlyRs, LeB and sLeX into chitosan microspheres was confirmed by FT-IR spectroscopy and through immunogold labelling using monoclonal antibodies recognizing each GlyR and visualized by transmission electron microscopy (TEM). H. pylori adhesion to the Gly-R microspheres was performed using FITC-labelled bacteria and visualized using fluorescence microscopy. A strain expressing BabA adhesin and not the SabA adhesin (H. pylori strain 17.1 (BabA+/SabA-)) was tested against chitosan microspheres without (Alkyne-Microspheres) and with different GlyRs (LeB-Microspheres and sLeX-Microspheres).

RESULTS AND DISCUSSION

Gly-R microspheres were stable for at least 7 days in simulated gastric fluid (SGF) with pepsin. All the steps described at figure 1 were confirmed by FT-IR spectroscopy by the presence and disappearance of the typical absorption bands of the groups that were coupled onto chitosan [28], namely: la) the presence of the phthaloyl group was detected at 1776 and 1714 cm^"1 (represented C=0 stretching bands), imide C=C stretching at 1580 cm^"1 and =C-H out of plane deformation at 723 cm^"1; lb) incorporation of the alkyne group (C≡C) onto chitosan microspheres was detected by the presence of≡C-H stretching vibration at 3291 cm^"1 and C≡C stretching vibration at 2122 cm^"1 that typical of monosubstituted alkynes; lc) attachment of azide-Gly-R by the azide-alkyne coupling was provided by the higher decrease of the typical monosubstituted alkyne vibration modes at 3291 cm^"1 (≡C-H stretching) and 2122 cm^"1 (C≡C stretching). Id) final unblocking of the primary amines by dephthaloylation, was detected by the disappearance of the phthaloyl-related bands described above (la), such as C=0 stretching above 1700 cm^"1, imide C=C stretching at 1580 cm^"1; =C-H out of plane deformation at 723 cm^"1.

TEM with immunogold labelling using monoclonal antibodies recognizing each GlyR confirmed the presence of only LeB at LeB-Microspheres and only sLeX at sLeX- Microspheres.

Microspheres were able to attract and bind H. pylori specifically through its adhesins.

LeB-Microspheres were able to attract and bind H. pylori through its specific adhesin BabA. Specific H. pylori adhesion was achieved since this H. pylori strain 17.1, that express BabA (specific to LeB receptors) adhered in much higher amounts to LeB-Microspheres than to sLeX-Microspheres and to microspheres without receptors (Alkyne-Microspheres) (see Figure 2).

References

1. Wroblewski LE, Peek RM Jr, Wilson KT. Helicobacter pylori and gastric cancer: factors that modulate disease risk. Clin Microbiol Rev. 2010 Oct;23(4):713-39;

2. Correa P, Houghton J. Carcinogenesis of Helicobacter pylori. Gastroenterology 2007;133(2):659-72.;

3. Matysiak-Budnik T, Megraud F. Helicobacter pylori infection and gastric cancer. Eur J Cancer. 2006, 42(6):708-716;

4. Polk DB, Peek RM. Helicobacter pylori: gastric cancer and beyond. Nat Rev Cancer.

2010, 10:403-14

5. International Agency for Research on Cancer. 2003 World cancer report. IARC Press.

Lyon, France. 177-240;

6. Ferlay J, Shin HR, Bray F, Forman D, Mathers C and Parkin DM. GLOBOCAN 2008.

Cancer Incidence and Mortality worldwide: IARC CancerBase No. 10;

7. liver D, Arnqvist A, Ogren J, Frick IM, Kersulyte D, Incecik ET, Berg DE, Covacci A, Engstrand L, Boren T, Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging. Science. 1998, 279: 373-77; 8. Mahdavi, J., Sonden, B., Hurtig, M., Olfat, F.O., Forsberg, L., Roche, N., Angstrom, J., Larsson, T., Teneberg, S., Karlsson, K.-A., et al. (2002). Helicobacter pylori SabA Adhesin in Persistent Infection and Chronic Inflammation. Science 297, 573-578;

9. Gerhard M, Lehn N, Neumayer N, Boren T, Rad R, Schepp W, Miehlkei S, Classen M, Prinz C. Clinical relevance of the Helicobacter pylori gene for blood-group antigen- binding adhesion. PNAS 1999 96 (26) 12778-12783.

10. Prinz, C. et al. Key importance of the Helicobacter pylori adherence factor blood group antigen binding adhesion during gastric inflammation. Cancer Res., 61: 1174-80, 2001.

11. Magalhaes A, Gomes J, Ismail MN, Haslam SM, Mendes N, Osorio H, David L, LePendu J, Haas R, Dell A, Boren T, Reis CA. Fut2-null mice display an altered glycosylation profile and impaired BabA-mediated Helicobacter pylori adhesion to gastric mucosa. Glycobiology. 2009 Dec;19(12): 1525-36

12. Marcos NT, Magalhaes A, Ferreira B, Oliveira MJ, Carvalho AS, Mendes N, Gilmartin T, Head SR, Figueiredo C, David L, Santos-Silva F, Reis CA. Helicobacter pylori induces β3ΰηΤ5 in human gastric cell lines, modulating expression of the SabA ligand sialyl—

Lewis x. J Clin Invest. 2008;118(6):2325-2336.

13. Malfertheiner P, Magraud F, CTMorain C, Bazzoli F, El-Omar E, Graham D, Hunt R, Rokkas T, Vakil N, Kuipers EJ. The European Helicobacter Study Group (EHSG). Current concepts in the management of Helicobacter pylori infection: the Maastricht III consensus report. GUT. 2007, 56:772-781.

14. Malfertheiner P, Bazzoli F, Delchier JC, et al for the Pylera Study Group. Helicobacter pylori eradication with a capsule containing bismuth subcitrate potassium, metronidazole, and tetracycline given with omeprazole versus clarithromycin-based triple therapy: a randomised, open-label, non-inferiority, phase 3 trial. Lancet 2011; 377: 905-913.

15. Parsons HK, Carter MJ, Sanders DS, Winstanley T, Lobo AJ. Helicobacter pylori antimicrobial resistance in the United Kingdom: the effect of age, sex and socioeconomic status. Aliment Pharmacol Ther. 2001, 15: 1473-1478;

16. Vakil N. Helicobacter pylori treatment: a practical approach [Editorial]. Am J Gastroenterol. 2006, 101:497-9;

17. Conway BR. Drug delivery strategies for the treatment of Helicobacter pylori infections.

Current Pharmaceutical Design. 2005, 11, 775-790;

18. Hejazi R, Amiji M. Stomach- specific anti-H. pylori therapy; part III: effect of chitosan microspheres crosslinking on the gastric residence and local tetracycline concentrations in fasted gerbils. Int J Pharm. 2004, 272(l-2):99-108;

19. Dhawan S, Singla AK, Sinha VR. Evaluation of mucoadhesive properties of chitosan microspheres prepared by different methods. AAPS PharmSciTech. 2004, 5(4): 1-7;

20. Hejazi R, Amiji M. Chitosan-based gastrointestinal delivery systems. Journal of controlled release. 2003, 89: 151-165.

21. McCurdy JD. FDA and the use of chitin and chitosan derivatives. In Advances in chitin and chitosan (Brine CJ, Sandford PA, Zikakis JP, eds.), Elsevier Applied Science, London and New York, 1992, pp659-662.

22. Strand SP, Vandvik MS, Varum KM, 0stgaard, K. Screening of chitosans and conditions for bacterial flocculation. Biomacromolecules. 2001 , 2(1): 126- 133.

Mi FL, Sung HW and Shyu SS, Release of Indomethacin from a Novel Chitosan Microsphere Prepared by a Naturally Occurring Crosslinker: Examination of Crosslinking and Polycation- Anionic Drug Interaction. (2001) Journal of Applied Polymer Science, Vol. 81 : 1700-1711.

Mi FL, Sung HW, Shyu SS, Su CC and Peng CK. Synthesis and characterization of biodegradable TPP/genipin cocrosslinked chitosan gel beads. (2003). Polymer 44: 6521- 6530.

Amaral IF, Granja PL and Barbosa MA. Chemical modification of chitosan by phosphorylation: an XPS, FT-IR and SEM study. J. Biomater. Sci. Polymer. 2005; 16(12): 1575-1593;

Mi FL, Sung HW and Shyu SS. Synthesis and Characterization of a Novel Chitosan- Based Network Prepared Using Naturally Occurring Crosslinker. J. Appl. Polym.2000;38:2804-2814.

Oliveira JR, Martins MCL, Mafra L and Gomes P. Synthesis of an O-alkynyl-chitosan and its chemo selective conjugation with a PEG-like amino-azide through click chemistry. Carbohydrate Polymers. 2012;87:240- 249.

Socrates, G. (2001). Infrared and Raman Characteristic Group Frequencies Tables and Charts (3rd edition). New York (USA): John Wiley & Sons, Ltd.

Claims

1. A microsphere for binding H. pylori, wherein the microsphere comprises receptors for H. pylori, and wherein the microspheres and the receptors can bind H. pylori.

2. The microsphere of claim 1, wherein the microsphere is a chitosan microsphere.

3. The microsphere of claim 1 or claim 2, wherein the microsphere is partially crosslinked.

4. The microsphere of claim 3, wherein the microsphere is crosslinked with genipin.

5. The microsphere of any preceding claim, wherein the microsphere has a porous structure.

6. The microsphere of any preceding claim, wherein the microsphere has a diameter of between about 10 μιη and about 1000 μιη.

7. The microsphere of any preceding claim, wherein the receptors bind to adhesins on the surface of the H. pylori bacterium.

8. The microsphere of any preceding claim, wherein the receptors are glycan receptors for H. pylori.

9. The microsphere of claim 8, wherein the glycan receptors are selected from Η type 1, Lewis B (LeB), Sialyl-Lewis X (sLeX) and Sialyl-Lewis A (sLeA) receptors..

10. The microsphere of claim 2, wherein the receptors are attached to the chitosan via the primary alcohol group.

11. The microsphere of any preceding claim, wherein the microsphere does not contain an active pharmaceutical ingredient for the treatment or prevention of H. pylori infection.

12. The microsphere of any preceding claim, wherein the microsphere is a lyophilised microsphere.

13. The microsphere of claim 1, wherein the microsphere is a chitosan microsphere which is partially crosslinked with genipin, wherein the receptors are glycan receptors which can bind to adhesins on the surface of H. pylori bacteria, and wherein the microsphere does not contain an active pharmaceutical ingredient for the treatment or prevention of H. pylori infection.

14. A microsphere according to any one of claims 1 to 13 for use in binding H. pylori bacteria.

15. The microsphere of claim 14 for use in treating or preventing H. pylori infection.

16. A method of manufacturing a microsphere for absorbing H. pylori, the method comprising:

forming a microsphere which can bind H. pylori; and

attaching receptors for H. pylori to the microsphere.

17. A kit for forming microspheres for absorbing H. pylori, the kit comprising a material for forming microspheres which can bind H. pylori and receptors for H. pylori.

18. The kit of claim 17, further comprising a crosslinking agent.

19. A method of treating or preventing H. pylori infection of the gastric mucosa and/or mucus layer, the method comprising administering an effective amount of microspheres to a patient, wherein the microspheres can bind H. pylori.

20. The method of claim 19, wherein the microspheres are chitosan microspheres.

21. The method of claim 20, wherein the microspheres are partially crosslinked with genipin.

22. The method of any one of claims 19 to 21, wherein the microspheres do not contain an active pharmaceutical ingredient for the treatment or prevention of H. pylori infection.

23. The method of claim 19, wherein the microspheres are the microspheres of any one of claims 1 to 13.

24. The method of claim 19, further comprising administering microspheres according to any one of claims 1 to 13.

25. A pharmaceutical composition for treating or preventing H. pylori infection, the composition comprising microspheres which can bind H. pylori and optionally one or more pharmaceutically acceptable excipients.

26. The pharmaceutical composition of claim 25, wherein the microspheres are chitosan microspheres which are partially crosslinked with genipin.

27. The pharmaceutical composition of claim 25 or claim 26, wherein the microspheres do not contain an active pharmaceutical ingredient for the treatment or prevention of H. pylori infection.

28. The pharmaceutical composition of any one of claims 25 to 27, wherein the pharmaceutical composition consists of microspheres which can bind H. pylori and optionally one or more pharmaceutically acceptable excipients.

29. The pharmaceutical composition of claim 25, wherein the microspheres are the microspheres of any one of claims 1 to 13.

30. The pharmaceutical composition of claim 25, further comprising the microspheres of any one of claims 1 to 13.