WO2023079335A1

WO2023079335A1 - Composition based on nystatin and vancomycin nanoparticles for the treatment of clostridioides difficile infection and prevention of recurrence

Info

Publication number: WO2023079335A1
Application number: PCT/IB2021/060174
Authority: WO
Inventors: Daniel PAREDES-SABJA; Pablo CASTRO CÓRDOVA; María Carolina OTERO ACUÑA
Original assignee: Universidad Andrés Bello
Priority date: 2021-11-03
Filing date: 2021-11-03
Publication date: 2023-05-11

Abstract

A pharmaceutical composition comprising nanoencapsulated nystatin and nanoencapsulated vancomycin, for use in the treatment of infections by C. difficile (CDI) and in the treatment of and for reducing the risk of developing recurrent infections by C. difficile (R-CDI) in a subject. A method for the treatment of CDI and for the treatment of R-CDI, and for reduction of the risk of developing R-CDI in a subject in need thereof, which comprises the administration of said pharmaceutical composition. A method for the treatment or prevention of a first CDI in a subject in need thereof, which comprises the administration of said pharmaceutical composition to a subject.

Description

COMPOSITION BASED ON NYSTATIN AND VANCOMYCIN

NANOPARTICLES FOR THE TREATMENT OF CLOSTRIDIOIDES DIFFICILE INFECTION AND PREVENTION OF RECURRENCE

DESCRIPTION

The present invention relates to a pharmaceutical composition comprising nystatin nanoparticles and vancomycin nanoparticles for use in a method for treating a first infection by Clostridioides difficile (CDI) and for use in a method for treating or preventing the risk of developing a recurrent infection by C. difficile (R-CDI). The present invention also relates to a method for treating a CDI and for treating or preventing the risk of developing a R-CDI which comprises the administration of a pharmaceutical composition comprising vancomycin nanoparticles and nystatin nanoparticles, where the nanoparticles comprise as an encapsulating agent, a polysaccharide polymer which allows to target the intestinal mucosa and cause extended release.

Description of the Background Art

C. difficile is a sporulating pathogen characterized by being the causative agent of ~20 to 30% of diarrheas associated to antibiotic consumption, with a recurrence rate of ~25% and an attributable mortality of ~6.9%. In addition, it is a primary infectious agent in hospital acquired diarrheas, exceeding in some health centers methicillin-resistant Staphylococcus aureus as the first cause of nosocomial infections. Whereby the composition of the present invention is designed to reduce or eliminate cases of recurrence and, consequently, reduce or eliminate mortality attributed to C. difficile.

The invention is directed to a pharmaceutical composition that can be administered in health facilities for reducing the risk of an epidemiologic outbreak. The pharmaceutical composition can also be used as a prophylactic, that is, it can be administered to patients who are under treatment with antibiotics and who are infected with C. difficile, in order to prevent colonization by C. difficile and the development of a CDI. Generally, CDI clinical pictures occur in patients who have been hospitalized, with a higher incidence in the population above 65 years of age. In the United States, hospital discharged patients under 18 years of age have 0.1 % of CDI, while between 65 and 84 years old said rate increases to 1 .5%, giving origin to an average rate adjusted by age group of 0.98% which is very similar to the Chilean rate, estimated at an average of 1 %. Applying these factors to the total population, it is estimated that the annual incidence of CDI is of 223,900 persons in the United States and 16,600 persons in Chile.

In general, manifestations of CDI vary from mild diarrhea conditions without systemic manifestations, to conditions characterized by fulminant colitis with toxic megacolon and perforations in the colon tract. The standard antibiotic treatment for CDI is orally administered vancomycin and/or metronidazole, with which favorable results and mortality rates varying between ~1 and ~5% are obtained. However, the main clinical challenge of CDI is that 30% of the patients with a first episode of CDI present a second episode of CDI or R-CDI. This is aggravated because the probabilities of presenting a third and a fourth episode of R-CDI increase by 40 and 65%. Likewise, mortality rates can also increase by over 30% after several recurrence episodes. This clinical challenge has directed efforts to developing new drugs and therapeutic strategies seeking to prevent R-CDI, with limited success or whose massive use has been difficult. Therefore, the high recurrence rate of CDI and the reduced spectrum of available therapies represent a great opportunity for the development of new formulations that can address key aspects of the pathogenesis of R-CDI.

The present invention is directed to the pharmaceutical industry for the treatment of patients who receive the first diagnosis of CDI, where, according to the results obtained, a composition of vancomycin nanoparticles and nystatin nanoparticles would allow to reduce recurrence rates to 44% in comparison to non-encapsulated vancomycin and nystatin. While the administration of vancomycin nanoparticles would allow to reduce recurrence rates to 66% in comparison to the administration of nonencapsulated vancomycin. The treatment of R-CDI is expensive to the health system since it requires extended hospitalization periods, whereby this pharmaceutical composition and method of treatment could contribute to reduce the cost for the health system in the treatment of R-CDI. Various drug patents were found which describe the treatment of CDI. Application WO 2001035983 mentions a pharmaceutical composition based on cysteine and cysteine derivatives for the treatment of diarrhea caused by C. difficile. Patent applications CA 2779413 A1 , US 20110280847 and WO 2010062369 A2, and Rineh et al., 2014, mention methods and bile salt formulations for inhibiting the germination of C. difficile spores and the growth of the vegetative form of C. difficile.

Within the prior art, mention can be made of document WO 2020035720 by the same inventors of the present document, which discloses a method and a pharmaceutical composition based on vancomycin, nystatin and taurocholate for preventing R-CDI.

On the other hand, document CN 106334184 relates to a method of preparation of sustained-release microspheres incorporating vascular endothelial growth factor (VEGF) and vancomycin for bone repair, where VEGF is incorporated in calcium alginate in the core and vancomycin is incorporated in chitosan in the outer layer. Document CN 107049988 discloses a vancomycin hydrochloride nanoparticle where the carrier material is a combination of carboxylation chitosan and chitosan quaternary ammonium salt that may be used for the treatment of osteomyelitis.

US 2016000924 provides methods for generating biodegradable chitosan compositions for delivering one or more therapeutic, antimicrobial and antiviral or antifungal agents. US 2016243243 discloses a composition that includes hydrophobized chitosan which may be used as an antiparasitic and/or antimycotic agent, or in the treatment of parasitic and/or fungal infections.

Cerchiara et al. (2015) demonstrated that encapsulation of vancomycin in chitosan nanoparticles contributes to protect vancomycin from degradation by digestive enzymes, and confers extended release of vancomycin. Consequently, encapsulation in chitosan nanoparticles permits to optimize the delivery of vancomycin to the intestinal mucosa, favoring the localized effect of the components in the colon, and would permit an extended release for the delivery of vancomycin in the colon for the treatment of Staphylococcus aureus.

None of the above cited documents mentions the treatment of a CDI or a R- CDI with a composition of vancomycin nanoparticles and nystatin nanoparticles. Additionally, bearing in mind that pharmaceutical nanotechnology focuses on the development of formulations of therapeutic agents that can be used for targeting drug delivery toward a specific type of cells or tissue, the effectiveness of an encapsulated formulation for treating CDI is evaluated.

The present invention discloses a pharmaceutical composition for the treatment of a first CDI through the use of an antibiotic such as vancomycin, and seeks to reduce the risk of manifesting a R-CDI through the administration of nystatin nanoparticles. The present invention discloses a method for treating a CDI and reducing the risk of a R-CDI by means of a solution that is different from those presently used. The present solutions are based on metronidazole and/or vancomycin. However, conventional therapies for treating CDI and R-CDI do not consider several biological aspects of the infection. First, conventional therapies do not inhibit the internalization of C. difficile spores by the intestinal epithelium. Second, existing treatments do not act on the (metabolically inactive) spores, but instead act on the vegetative form of C. difficile. Consequently, for antibiotics to act, the spores must be germinated by the action of primary bile salts such as taurocholate which induce the formation of a vegetative cell.

In addition, nystatin is used for treating gastrointestinal infections caused by Candida yeast, since it binds to the ergosterol present in the fungal cell membrane. There is no disclosure of the use of nanoencapsulated nystatin. Therefore, it is not obvious that by using nanoencapsulated nystatin in a composition combined with nanoencapsulated vancomycin, a surprising combined effect could be obtained that would be effective for treating a CDI and treating or reducing the incidence of R-CDI.

Antibiotic treatments cause a dysbiosis of the intestinal microflora and, consequently, the metabolism of bile salts by the intestinal microbiota is reduced. In this sense, treatment with vancomycin generates an increase in the concentration of primary bile salts, such as cholate and taurocholate, and a decrease of secondary bile salts which inhibit the growth and germination of C. difficile spores. This results in that the dysbiosis produced by antibiotics such as vancomycin generates a colonic environment that favors spore germination, and vegetative cells are produced which die by the effect of vancomycin.

The use of nanoencapsulated nystatin in the present invention permits a targeted action to inhibit the internalization of the spores and keep them in extracellular form, leaving them exposed to primary bile salts such as taurocholate which induce the germination of C. difficile spores and subsequent inactivation with antibiotics, thus reducing the abundance of internalized spores and total spores in the colon tract, improving the effectiveness of the treatment as reflected in a lower incidence of R-CDI.

During a treatment with vancomycin nanoparticles and nystatin nanoparticles, the nystatin nanoparticles favor that the spores remain in extracellular form exposed to primary bile salts, whose concentration increases when administering antibiotics such as vancomycin, which permits germination of the spores and formation of vegetative cells which are susceptible to antibiotic treatment. Consistent with this, we have observed in a murine model that oral administration of nystatin nanoparticles and vancomycin nanoparticles to mice with CDI is able to treat CDI and surprisingly reduce the rates and severity of R-CDI. The vancomycin and nystatin nanoparticles of the present invention improve the bioavailability of the active principle for an extended effect in the specific colon tissue.

Unlike humans, mice in just a few days have a natural recovery from C. difficile infection. To evaluate CDI and R-CDI, mice need to be treated with antibiotics (i.e., vancomycin). Once the antibiotic treatment is suspended, the R-CDI symptoms are manifested after 2-5 days. For this reason, evaluating the effect of nystatin by itself is not possible according to the animal models described because the mice improve their symptoms even without treatment.

On the other hand, due to the mechanism of action of the molecules used for this formulation, it is not possible to evaluate a synergic effect between vancomycin and nystatin either. While vancomycin eliminates C. difficile vegetative cells, nystatin inhibits the internalization of the spore into intestinal epithelial cells. Nystatin by itself does not generate a measurable or visible effect on the infection. In consequence, we evaluated nanoparticles of vancomycin and nystatin, and nanoparticles of vancomycin as monotherapy but not nystatin as monotherapy, observing a meaningful and unexpected effect in the combined therapy, which is also unexpectedly better than the non-nanoencapsulated combined therapy and unexpectedly better than the prior art, where a mix of vancomycin and nystatin in combination with taurocholate, working as spore germinant, was used to treat R-CDI. In this disclosure, no spore germinant was required, and the results were unexpectedly better than the combined therapy that used a spore germ inant.

The present invention proposes a pharmaceutical composition comprising nanoencapsulated vancomycin and nanoencapsulated nystatin, additionally it proposes the use of nanoencapsulated vancomycin as monotherapy, wherein for nanoencapsuting these drugs, a nanoparticles are used as a carrier having as an encapsulating polymer, a polysaccharide polymer which is selected from the group comprising alginate, chitosan, maltodextrin, cyclodextrin, ethyl cellulose, ethyl methyl cellulose and carboxymethyl cellulose. Nanoencapsulation of vancomycin and nystatin, using a polysaccharide polymer such as chitosan, is used for improving the localized effect of the components in the colon and achieving extended release of the components. Chitosan is a cationic polysaccharide that exhibits mucoadhesive properties and it has been used for drug delivery to the gastrointestinal tract. The adhesion of chitosan loaded with drugs to the intestinal mucosa, increase the residence time, the concentration and bioavailability, prolongating the therapeutic effects, improving the patient compliance.

Likewise, the present invention provides a method of treatment for a CDI and/or treatment or prevention for a R-CDI by means of a composition of nanoencapsulated vancomycin and nanoencapsulated nystatin, obtaining as a result a surprising reduction in the incidence of the infection.

In the first place, the activity of nanoencapsulated vancomycin on CDI was assessed, for which purpose a composition of nanoencapsulated vancomycin, or free vancomycin, was administered to mice exhibiting symptoms of CDI. It was observed that the administration of nanoencapsulated vancomycin achieved an effective reduction of 34% of the R-CDI as compared to its non-encapsulated form.

The present invention discloses experimental evidence demonstrating the advantage of the use of a composition of nanoencapsulated vancomycin as monotherapy, since during the R-CDI, it was observed that 66% of the mice treated with nanoencapsulated vancomycin manifested diarrhea during the R-CDI, while 100% of the mice treated with free vancomycin manifested diarrhea during the R-CDI.

In addition, it was observed that the administration of a composition of nanoencapsulated vancomycin and nanoencapsulated nystatin is more effective for treating R-CDI than a composition of non-encapsulated vancomycin and nystatin and also than encapsulated vancomycin.

The present invention discloses experimental evidence demonstrating the advantage of the use of nanoencapsulated vancomycin and nystatin in a chitosan-based nanoparticles as combined therapy. It was observed that the administration of nanoencapsulated vancomycin and nystatin achieved an effective reduction of 56% of the R-CDI as compared to its non-encapsulated form. Since in the treatment of CDI with nanoencapsulated vancomycin and nystatin in mice, it was observed that 44% of the group of mice manifested diarrhea during the R-CDI, compared to the group of mice that was treated with vancomycin and nystatin where 100% manifested diarrhea during the R-CDI.

The results appear to be surprising when comparing the treatments with the same formulations but non-nanoencapsulated.

Description of the figures

Figure 1 shows the experimental design used for the production of vancomycin nanoparticles in chitosan-based nanoparticles.

Figure 2 shows the experimental design used for the production of nystatin nanoparticles in chitosan-based nanoparticles.

Figure 3. Diagram of the experimental design of a murine model of R-CDI. Mice were administered antibiotics to induce susceptibility to C. difficile (days -6, -3 before infection). Then they were challenged with C. difficile (day 0). Once CDI symptoms manifested (day 3), the mice were administered during 5 days with formulations of vancomycin (VAN), nanoencapsulated vancomycin (nanoVAN), vancomycin and nystatin (VAN + NYS) or nanoencapsulated vancomycin and nystatin (nanoVAN+nanoNYS). Once the treatment was suspended (day 7), the mice were evaluated for manifestation of R-CDI symptoms (days 8 to 13).

Figure 4. Relative weight during the R-CDI of the infection experiment described in Figure 3. It is observed that vancomycin nanoparticles (nanoVAN) and vancomycin nanoparticles + nystatin nanoparticles (nanoVAN+nanoNYS) protect the mice from weight loss during the R-CDI, in comparison to those treated with vancomycin (VAN) and vancomycin and nystatin (VAN+NYS). Shown as average ± SEM. Statistical analysis was performed by Kruskal Wallis post-Dunn's test; ns, p > 0.05.

Figure 5. Time to diarrhea during the course of R-CDI. It is observed that the administration of vancomycin nanoparticles (nanoVAN) or vancomycin nanoparticles + nystatin nanoparticles (nanoVAN+nanoNYS) reduces diarrhea episodes during the R-CDI, in comparison to those treated with vancomycin (VAN) or vancomycin + nystatin (VAN+NYS). Statistical analysis was performed with Log-rank (Mantel-Cox) test; ns, p > 0.05; *, p < 0.05.

Figure 6 shows diarrhea score during the course of R-CDI. Shown as average ± SEM. Statistical analysis was performed with Kruskal-Wallis, post-Dunn’s test, ns p > 0.05; *, p < 0.05.

Figure 7. Quantification of CFU/g of C. difficile spores in feces during the R- CDI. Shown as average ± SEM. Dotted line indicates limit of detection. Statistical analysis was performed with Kruskal-Wallis, post-Dunn’s test, ns, p > 0.05; *, p < 0.05; **, p < 0.01 .

Figure 8. Time to colonization, which was defined as the time when at least 1 x 10⁴ CFU/g of spores in feces were present during the R-CDI. Statistical analysis was performed with Log-rank (Mantel-Cox) test; ns, p > 0.05; *, p

< 0.05.

Figure 9. Adherence of C. difficile spores to different sections of the colon on day 13 post-infection. Dotted line indicates limit of detection. Statistical analysis was performed with two-tailed Mann-Whitney test, ns p > 0.05; *, p

< 0.05.

Figure 10 shows cytotoxicity of the cecal content on day 13 post-infection. Statistical analysis was performed with two-tailed Mann-Whitney test, ns p > 0.05.

Description of the Invention

The present invention relates to a pharmaceutical composition comprising nystatin and vancomycin, wherein the nystatin and vancomycin are encapsulated in the form of nanoparticles.

The present invention relates to a pharmaceutical composition wherein the nystatin and vancomycin are separately encapsulated in the form of nanoparticles. The present invention relates to a pharmaceutical composition wherein the nystatin and vancomycin are encapsulated together in the form of nanoparticles.

The present invention relates to a pharmaceutical composition wherein at least one encapsulating agent is a polysaccharide that improves targeting the intestinal mucosa and causes extended release, wherein at least one encapsulating agent is biocompatible or mucoadhesive, or both; wherein the polysaccharide is linear, without branching; wherein the polysaccharide is a polysalt or polyelectrolyte; wherein the polysaccharide is a polycation or a polyanion.

The present invention relates to a pharmaceutical composition comprising nystatin and vancomycin, wherein the nystatin and vancomycin are encapsulated in the form of nanoparticles, wherein the nanoparticles further comprise at least one encapsulating agent selected from the group consisting of alginate, chitosan, maltodextrin, cyclodextrin, ethyl cellulose, ethyl methyl cellulose or carboxymethyl cellulose, or any combination thereof; wherein the at least one encapsulating agent is preferably chitosan.

The present invention relates to a pharmaceutical composition comprising nystatin and vancomycin, wherein the nystatin and vancomycin are encapsulated in the form of nanoparticles, wherein the nanoparticles further comprise at least one encapsulating agent, and wherein the nanoparticles have one or more of the following properties: (i) an encapsulation efficiency between 70% and 100%; or

(ii) an average diameter or particle size between 1 nm and 500 nm; or

(iii) a polydispersion index between 0.100 and 0.300; or

(iv) an average zeta potential between +1 and +30 mV.

The present invention relates to a pharmaceutical composition comprising nystatin and vancomycin, wherein the nystatin and vancomycin are encapsulated in the form of nanoparticles, and wherein the

(a) the amount of vancomycin present in the composition is between 41 .3% and 62.96% by weight; and

(b) the amount of nystatin present in the composition is between 15.74% and 44.03% by weight.

The present invention relates to a pharmaceutical composition comprising nanoencapsulated nystatin and nanoencapsulated vancomycin, where the nanoencapsulated nystatin and nanoencapsulated vancomycin are nanoencapsulated in a chitosan matrix.

The present invention relates to a pharmaceutical composition for use in a method for treating an infection by C. difficile and for treating or preventing one or more recurrent infections by C. difficile in a subject. The present invention relates to the pharmaceutical composition of the invention for use in a method for treating or preventing one or more recurrent infections by C. difficile and for reducing the risk of developing recurrent infections by C. difficile in a subject, where the infection or the recurrent infection by C. difficile is colitis or pseudomembranous colitis.

The present invention relates to the use of a pharmaceutical composition of the invention for preparing a medicament useful for treating an infection by C. difficile. The invention also relates to the use of the pharmaceutical composition of the invention for preparing a medicament useful for treating or preventing one or more recurrent infections by C. difficile or for reducing the risk of developing recurrent infections by C. difficile in a subject, where the infection or the recurrent infection by C. difficile is colitis or pseudomembranous colitis.

The present invention relates to a method for treating an infection by C. difficile, or for treating or preventing one or more recurrent infections by C. difficile, or for reducing the risk of developing recurrent infections by C. difficile in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising nystatin and vancomycin, wherein the nystatin and vancomycin are encapsulated in the form of nanoparticles, wherein the nanoparticles further comprise at least one encapsulating agent; wherein the at least one encapsulating agent is a polysaccharide that improves targeting the intestinal mucosa and causes extended release, wherein at least one encapsulating agent is biocompatible or mucoadhesive, or both; wherein the polysaccharide is linear, without branching; wherein the polysaccharide is a polysalt or polyelectrolyte; wherein the polysaccharide is a polycation or a polyanion.

The method for treating an infection by C. difficile, or for treating or preventing one or more recurrent infections by C. difficile, or for reducing the risk of developing recurrent infections by C. difficile in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising vancomycin at a dosage of 100 mg to 4 g per day and nystatin at a dosage of 100,000 III to 3,000,000 IU per day.

The method for treating an infection by C. difficile, or for treating or preventing one or more recurrent infections by C. difficile, or for reducing the risk of developing recurrent infections by C. difficile in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising vancomycin at an amount of approximately 50 mg/kg/day and nystatin at an amount of approximately 5.66 mg/kg/day.

The present invention relates to a method for treating a CDI in a subject, which comprises the administration of a pharmaceutical composition comprising nanoencapsulated vancomycin and nanoencapsulated nystatin, where the CDI is colitis or pseudomembranous colitis; where the pharmaceutical composition includes as an encapsulating agent, a polysaccharide polymer which allows to target the intestinal mucosa and cause extended release; where the encapsulating agent is selected from the group comprising alginate, chitosan, maltodextrin, cyclodextrin, ethyl cellulose, ethyl methyl cellulose and carboxymethyl cellulose; where in the pharmaceutical composition the nanoencapsulated nystatin and nanoencapsulated vancomycin are nanoencapsulated in a chitosan matrix.

The present invention also relates to a method for treating CDI and for treating or reducing the risk of developing R-CDI in a subject, which comprises the administration of a pharmaceutical composition to the subject, where the pharmaceutical composition comprises nanoencapsulated vancomycin in a chitosan matrix, where the R-CDI is colitis or pseudomembranous colitis by C. difficile.

The pharmaceutical composition was manufactured at nanoscale and an encapsulating agent was included which allows to target the intestinal mucosa and cause an extended release. The particles of active principle nanoencapsulated in a polysaccharide polymer such as chitosan lead to significant advantages in respect of the prior treatment in which the drugs are administered free, that is without nanoencapsulation.

Nanoencapsulation represents a technical advantage over a previously described combined therapy. The nanoparticles formed are within the range between 1 nm and 500 nm, or 80 nm and 500 nm, preferably having an average diameter or particle size between 100 nm and 300 nm, or 150 nm and 250 nm or 170 nm and 220 nm, more preferably between 170 nm and 190 nm; more preferably an average diameter or particle size of approximately 200 nm. The pharmaceutical composition of the present invention comprises vancomycin that is administered within a range of 50 mg to 4 g per day, or within a range of 100 mg to 4 g per day, or in an amount of 500 mg per day. The pharmaceutical composition of the present invention comprises nystatin that is administered within a range of 100,000 III to 3,000,000 IU per day, preferably in an amount of 1 ,600,000 IU per day.

The pharmaceutical composition of the present invention comprises 500 mg per day of vancomycin and 1 ,600,000 mg per day of nystatin.

The pharmaceutical composition of the present invention comprises vancomycin within a range of 40% to 70% by weight, nystatin within a range of 7% to 44 % by weight and chitosan within a range of 14% to 24 % by weight.

The pharmaceutical composition of the present invention comprises vancomycin within a range of 41 % to 63% by weight, nystatin within a range of 15% to 44 % by weight and chitosan within a range of 14% to 21 % by weight.

The concentrations of vancomycin, nystatin and chitosan in the pharmaceutical composition of the present invention used in animal tests, contain the following: 69.09% by weight (at a dose of 50 mg/kg) of vancomycin, 7.82% by weight (at a dose of 5.66 mg/kg) of nystatin, and 23.09% by weight (16.71 mg/kg) of chitosan. The pharmaceutical composition used in animal tests comprises about 69% by weight vancomycin, about 8 % by weight of nystatin and about 23 % by weight of chitosan.

The maximum concentration of vancomycin and nystatin would be the maximum recommended concentrations in humans, that would be 4000 mg/day of vancomycin and 3,000,000 lll/day of nystatin. Then maximum concentrations of vancomycin, nystatin and chitosan in the pharmaceutical composition of the present invention used in human comprise 62.96% by weight of vancomycin (4000 mg/day of vancomycin), 15.74% by weight of nystatin (3,000,000 lll/day of nystatin) and 21.30% by weight of chitosan. The pharmaceutical composition comprises an amount of about 63% by weight vancomycin, about 16% by weight of nystatin and about 21 % by weight of chitosan.

The concentration of vancomycin and nystatin for human use corresponds to 500 mg/day of vancomycin and 1 ,600,000 lll/day of nystatin. Then preferably, the pharmaceutical composition comprises an amount of about 41 % by weight vancomycin, about 44 % by weight of nystatin and about 15 % by weight of chitosan. Preferably, the pharmaceutical composition comprises about 42% by weight vancomycin, about 44 % by weight of nystatin and about 14 % by weight of chitosan.

The pharmaceutical composition of the present invention comprises the preparation of nystatin and vancomycin nanoparticles separately in a chitosan-based nanoparticles which targets the intestinal mucosa, allowing an extended release of the components in a specific zone. In addition, chitosan has properties which allow it to bind to the intestinal mucosa, which allows to improve the localized effect of the components in the colon and the extended release, relevantly improving the therapeutic effect.

The pharmaceutical composition comprising nanoencapsulated vancomycin and nanoencapsulated nystatin is formulated in injectable, water soluble form for medical use. Methods of nanoencapsulation in a chitosan polymeric matrix are described.

In order to assess the effectiveness of the nanoencapsulated pharmaceutical composition, protocols were designed for the generation of chitosan nanoparticles with nystatin and vancomycin. Then, the encapsulation efficiency for nanoparticles with nystatin and vancomycin was evaluated. Once the protocols were validated, scale production was conducted and the effectiveness of the nanoencapsulated composition was assessed in comparison to the non-encapsulated composition in a murine model of R-CDI, administering both the treatment of vancomycin nanoparticles in comparison to free vancomycin, and the treatment of vancomycin and nystatin nanoparticles in comparison to free vancomycin and nystatin.

Examples of preparation of solutions for the synthesis of chitosan nanoparticles The synthesis of chitosan nanoparticles with vancomycin and nystatin in a chitosan-nanoparticles was conducted as follows:

A stock solution of chitosan was prepared: for which a solution was prepared of 1 % low molecular weight chitosan with 75-85% deacetylation grade (Sigma-Aldrich) in 1 % acetic acid (HPLC grade, Merck) with constant stirring for 24-48 hours on a heating plate at 60°C (MRC-HSCD-7). The solution was covered to prevent solvent evaporation.

Then, a chitosan-acetic acid solution was prepared, with a stock solution of 1 % chitosan, ultra-pure water and 1 % acetic acid which were mixed in a proportion of 1 :5:1 , respectively, the solution was stirred for 5 minutes at 400 rpm and 60°C, and then the solution was stirred for 10 min at 400 rpm at room temperature. The solution was covered to prevent solvent evaporation.

The vancomycin solution was prepared by dilution of vancomycin (Sigma- Aldrich) in ultra-pure water to a concentration of 0.755 mg/mL, according to Figure 1 .

A stock solution of nystatin was prepared by dilution of 4 mg nystatin (Sigma-Aldrich) in 1 mL DMSO at a concentration of 5 mg/mL. Then, 0.5 mg of the stock solution of nystatin was diluted in 25 mL ultra-pure water to form a diluted nystatin solution at a final concentration of 0.0755 mg/mL, according to Figure 2. The TPP (pentasodium tripolyphosphate) solution was prepared by dilution of 3.4 mL pentasodium tripolyphosphate (TPP; Sigma-Aldrich) in 18.6 mL ultra-pure water to a concentration of 1 .55 mg/mL. It was stirred at 400 rpm.

Example of preparation of chitosan nanoparticles

The protocol for the synthesis of vancomycin nanoparticles in chitosan nanoparticles is shown in Figure 1. 10 mL of the chitosan-acetic acid solution were mixed with 26.5 mL of the vancomycin solution and stirred at 400 rpm for 15 min to form a vancomycin-chitosan solution. Then 36.5 mL of the vancomycin-chitosan solution were added with a syringe pump (Tong Li Tech-TL-F6) at a flow rate of 0.7 mL/min downward to 22 mL of the TPP solution with stirring at 400 rpm. The vancomycin-chitosan-TPP solution was stirred for 15-30 minutes at 400 rpm to remove excess solvents in the solution. The change in the appearance of the solution from transparent to opaque indicates formation of nanoparticles. The vancomycin-chitosan- TPP solution was separated into 8 tubes of ~8 mL each, which were sonicated twice for 1 min with a 60% amplitude. Then they were centrifuged at 9,000 rpm for 30 min at 14 °C. The supernatant was collected to measure the encapsulation efficiency as is indicated below. The pellet was resuspended in 4 mL ultra-pure water and sonicated for 1 min with a 30% or 60% amplitude, then 4 mL ultra-pure water were added and it was sonicated again for 1 min with a 30% or 60% amplitude. The protocol for the synthesis of nystatin nanoparticles in chitosan nanoparticles is shown in Figure 2. 10 mL of the chitosan-acetic acid solution were mixed with 26.5 mL of the nystatin diluted solution and stirred at 400 rpm for 15 min to form a nystatin-chitosan solution. Then 36.5 mL of the nystatin-chitosan solution were added with a syringe pump (Tong Li Tech-TL-F6) at a flow rate of 0.7 mL/min downward to 22 mL of the TPP solution with stirring at 400 rpm. The nystatin-chitosan-TPP solution was stirred for 15-30 minutes at 400 rpm to remove excess solvents in the solution. The change in the appearance of the solution from transparent to opaque indicated the formation of nanoparticles. The nystatin-chitosan-TPP solution was separated into 8 tubes of ~8 mL each, which were sonicated twice for 1 min with a 60% amplitude. Then they were centrifuged at 9,000 rpm for 30 min at 14°C. The supernatant was discarded. The pellet was resuspended in 4 mL ultra-pure water and sonicated for 1 min with a 30% or 60% amplitude, then 4 mL ultra-pure water were added and it was sonicated again for 1 min with a 30% or 60% amplitude.

Lyophilization of nanoparticles

To lyophilize the samples, 40 mg/mL of sucrose (Merck) was added to each tube, they were stored at -20°C overnight and lyophilized for 48 hours at - 20°C. Lyophilized samples are stored at -20°C until use.

Encapsulation efficiency Encapsulation efficiency was performed by measuring vancomycin or nystatin concentration in supernatants of the nanoprecipitation process, for this purpose, the supernatant of 3 independent batches for each composition was collected. Supernatants were stored at -20°C overnight and lyophilized for 48-72 hours. The lyophilized samples were stored at - 20°C until use. Quantification of vancomycin or nystatin in supernatants was carried out by high performance liquid chromatography (HPLC) and each antibiotic was evaluated with a calibration curve. The calibration curve for vancomycin and nystatin exhibits 9 points: 2.5, 5, 10, 15, 20, 25, 30, 35 and 40 mg/mL and 0.25, 0.5, 1 , 1.5, 2, 2.5, 3, 3.5 and 4 mg/mL, respectively. The mobile phase used was methanol : 0.05 M ammonium acetate buffer (pH 5.0) 70:30 v/v. Encapsulation efficiency of vancomycin or nystatin nanoparticles are between 70 and 100%. Specifically, an encapsulation efficiency of nanoparticles was determined as 75% for vancomycin and 98% for nystatin.

Table 1. Determination of encapsulation efficiency and characterization of nanoparticles.

Encapsulation Size (nm) Polydispersion Zeta

Efficiencies Index Potential

Vancomycin 75% 173.4±0.40 0.155±0.01 +16.3±0.2

HPLC Nystatin 98% 181.3±1.5 0.149±0.003 +15.8±0.03

HPLC

Once the encapsulation efficiency for nanoparticles with nystatin and vancomycin was known, the scale production of several batches of vancomycin and nystatin nanoparticles was carried out, in order to evaluate their efficiency in a murine model of CDI.

For nanoparticles with vancomycin, 8 batches were produced, obtaining a final mass of nanoparticles with vancomycin of 30.93 mg. For nanoparticles with nystatin, 16 batches were produced; obtaining a final mass of nanoparticles with nystatin of 73.44 mg.

Clostridioides difficile strains and spore purification

Suspensions of C. difficile R20291 spores were prepared by plating a 1 :750 dilution from a culture incubated overnight in a Bactron II-2 anaerobic chamber (Shellab, OR, USA) onto agar plates with 70:30 sporulation medium and incubated for 5 days at 37°C under anaerobic conditions. Spores were harvested with ice-cold sterile distilled water and purified with 50% Nicodenz as described in Castro-Cordova et al. 2021. Spore suspensions were purified until, by phase-contrast microscopy, they were > 99% free of vegetative cells, sporulating cells and cell debris. Spore concentration was quantified with a Neubauer chamber and they were stored at -80°C. Animals

Mice of strain C57BL/6 (age 8-12 weeks) were obtained from the bioterium of the Facultad de Ciencias de la Vida of Universidad Andres Bello (Santiago, Chile).

All mice used in the experiments were housed individually under conventional conditions at the animal infection facility of the Microbiota-Host Interactions and Clostridia Research Group of Universidad Andres Bello (Santiago, Chile), for one week before initiation of the study. The mice were housed individually in sterile cages with ad libitum access to food and water. Food (Chow; Prolab RMH 3000 rodent diet, St. Louis, MO, USA), water, bedding and cages were sterilized before use. All experimental protocols were conducted in strict accordance with, and under the formal approval of, the Institutional Animal Ethics Committee of Universidad Andres Bello (protocol #022/2016).

Application Examples

Animal infection model

To induce susceptibility to C. difficile in mice, an antibiotic cocktail (ATB cocktail) was used of 4.2 mg/kg kanamycin (Sigma-Aldrich), 3.5 mg/kg gentamicin (Sigma-Aldrich), 4.2 mg/kg colistin (Sigma-Aldrich), 21.5 mg/kg metronidazole (Sigma-Aldrich) and 4.5 mg/kg vancomycin (Sigma-Aldrich). The antibiotic cocktail was administered to C57BL/6 mice (n = 34) via gavage during three days, followed by two days of no antibiotic administration. One day before the infection, an intraperitoneal (i. p.) injection of 10 mg/kg clindamycin (Sigma-Aldrich) was administered as described in Castro-Cordova et al. 2021.

Figure 3 shows a design of an experimental model for evaluating the effectiveness of the nanoencapsulated composition in comparison to a nonnanoencapsulated composition in a murine model of R-CDI.

Mice were infected by gavage with 6 x 10⁷ C. difficile R20291 spores.

To evaluate the effectiveness of vancomycin nanoparticles and nystatin nanoparticles in reducing the incidence of R-CDI, once CDI symptoms manifested (day 3), the animals were treated for 5 days orally by gavage with: i) 50 mg/kg vancomycin (n = 9); ii) 50 mg/kg vancomycin in chitosan-based nanoparticles (n = 6); iii) 50 mg/kg vancomycin + 5.66 mg/kg nystatin (n = 9); iv) 50 mg/kg vancomycin in chitosan-based nanoparticles + 5.66 mg/kg nystatin in chitosan-based nanoparticles (n = 10).

To avoid reinfection caused by the reingestion of spores released by the same animals on days 3, 5 and 7, the bedding was changed after the infection and the cages were cleaned with bleach 5%. A non-blind experiment was conducted for better control of the groups and to avoid cross-contamination and transmission between the different groups during handling. Procedures and processing were performed aseptically in a biosafety cabinet. Before handling a new group, the biosafety cabinet was cleaned with a 5% bleach solution and with a sporicidal solution of 1 % potassium peroxymonosulfate (Virkon S), as described in Castro-Cordova et al. 2021.

Mice were monitored daily for weight loss, physical appearance (i.e., abnormal/hunched gait, piloerection), spontaneous behavior (i.e., lethargy, inactivity or lack of mobility) and emaciation, using a scoring system previously employed for CDI to determine the humanity endpoint, as described in Castro-Cordova et al. 2021. Clinical signs were monitored daily, and fecal samples, cecum content and colon tissue were collected. Diarrhea is the main clinical symptom of CDI in mice, whereby it is used as an indicator of disease during CDI and R-CDI. The presence of diarrhea was classified as positive if feces exhibited a color/consistency change, presence of wet tail, mucosa or liquid feces, and negative if they remained healthy as described in Castro-Cordova et al. 2021 .

Quantification of C. difficile spores in feces and intestinal tissue samples

C. difficile spore quantification in feces and in intestinal tissue was performed as previously described in Castro-Cordova et al. 2021. Briefly, fecal samples (~35 mg) were collected every day during the assay. Feces were diluted to 50 mg/mL with sterile MilliQ water for 16 hours at 4°C, homogenized and diluted to 100 mg/mL with absolute ethanol (Sigma- Aldrich) and incubated for 30 min. Each sample was serially diluted and plated onto selective medium supplemented with 0.1 % (w/v) taurocholate, 16 pg/mL cefoxitin, 250 pg/mL L-cycloserine (TCCFA plates (Taurocholate, Cycloserine, Cefoxitin, Fructose and Agar)) and incubated anaerobically at 37°C for 48 h. Colony forming units (CFU) were counted, and results were expressed as [Log-io (CFU/g of feces)].

At the end of the assay, the colon tissue and the cecum were extracted, washed with a syringe with phosphate-buffered saline (PBS) and divided into four sections: proximal colon tissue, middle colon tissue, distal colon tissue and cecum tissue. One centimeter (cm) of the cecum (from the cecum base) and 1 cm of each colon section (from the cecum as a reference) were collected and stored in sterile tubes at -20°C until quantification of spore CFU. Each sample was weighed and then the tissues were adjusted to 100 mg/mL with PBS: absolute ethanol (1 :1 ), samples were homogenized and incubated for 1 hour at room temperature. Then, samples were serially diluted, placed on TCCFA plates and incubated anaerobically at 37°C for 48 hours. The colonies obtained were counted and the results were expressed as [Log-io (CFU/g of tissue)].

Cytotoxicity assay of the cecum content Vero cell cytotoxicity was assayed as described in Castro-Cordova et al. 2021. Mice cecum content was suspended at a final concentration of 100 mg/mL with PBS, vortexed and centrifuged (18,400xg for 5 min). Filter- sterilized supernatant was serially diluted in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 LI/mL penicillin, 100 pg/mL streptomycin; 100 pl of each dilution were added to wells containing Vero cells cultured in 96-well flat-bottomed plates at a density of 10⁵ cells/well. Cell rounding caused by toxins was evaluated 16 hours after incubation at 37°C. Cytotoxic titer was defined as the reciprocal of the highest dilution that caused rounding in at least 80% of the Vero cells per gram.

Statistical analysis

Prism 8 (GraphPad Software, Inc.) was used for statistical analysis. Statistical significance between groups evaluated for measurements of relative weight, diarrhea score, CFU/g of feces, was assessed by Kruskal Wallis post-Dunn's test, and for time to diarrhea and time to colonization, it was assessed with the Log-rank Mantel-Cox test. While for CFU/g of tissue and cytotoxicity of cecal content, it was assessed with a two-tailed Mann Whitney test.

A p-value of < 0.05 was accepted as the level of statistical significance.

Characterization of chitosan nanoparticles containing vancomycin or nystatin The biophysicochemical properties of the nanoparticles influence the interface between the nanoparticles and the biological system, and also determine their in vivo biocompatibility. The main variables that determine the biocompatibility are the nanoparticle size, zeta potential, and dispersibility (measured as polydispersion index).

Nanoparticle Size

It has been reported that the size of nanoparticles influences pharmacokinetics, tissue distribution, and clearance. It has been reported that 50-100 nm nanoparticles have higher permeability across the mucus in vitro, and 200 nm nanoparticles have an importantly reduced permeability in the mucus membrane.

The nanoparticles of vancomycin and nystatin have an average diameter or particle size between 1 nm and 500 nm, or 80 nm and 500 nm, preferably having an average diameter or particle size between 100 nm and 300 nm, or 150 nm and 250 nm or 170 nm and 220 nm. Therefore, the nanoparticles of the invention having an average diameter or particle size of approximately 200nm, have a reduced permeability across the intestinal mucus; so that the nanoparticles with vancomycin and nystatin of the invention have an increased bioavailability in the intestinal lumen, place where C. difficile colonize and cause the infection.

Zeta potential Zeta potential is an indication of the nanoparticle surface charge. Meanwhile, positive values indicate a positive charge, negative values indicate a negative charge. Values close to 0 indicated neutral nanoparticles. The nanoparticles of vancomycin and nystatin have a zeta potential between +1 and +30 mV, preferably having a zeta potential between +10 and +20 mV; more preferably having an average zeta potential between +15.8 ± 0.03 and +16.3 ± 0.2 mV. The nanoparticles of the invention having an average zeta potential of approximately +15, in consequence, have a reduced permeability across the intestinal mucus, increasing the bioavailability of the nanoparticles with vancomycin and nystatin in the intestinal lumen, place where C. difficile colonize and cause the infection.

Polydispersion index (PDI)

PDI is an indicator of nanoparticle quality with respect to the size distribution. The size affects the physicochemical properties of the nanoparticles, and drug delivery is important to form a homogenous (monodisperse) population of particles of determined size. The polydispersion index describes the degree of non-uniform ity size distribution. PDI values smaller than 0.05 are seen as monodisperse, and PDI values over 0.7 indicate a very broad particle size distribution and are not recommended to use. PDI of the nanoparticles of vancomycin and nystatin are between 0.100 and 0.300, preferably between 0.130 and 0.190; or more preferably between 0.136 and 0.180. PDI of the nanoparticles of the present invention are around approximately 0.155. This value, being between acceptable values, ensures that almost all the nanoparticles have the same biophysicochemical properties.

Chitosan nanoparticles were synthesized: nanoparticles containing vancomycin hydrochloride and nanoparticles containing nystatin. Three batches of each type of nanoparticles were characterized physico- chemically. Using a Zetasizer Nano ZS, there were determined the hydrodynamic diameter of the nanoparticles indicating average diameter, the Zeta potential or the surface charge, useful parameters for evaluating the stability of any colloidal system, and the width of nanoparticle size distribution by the polydispersion index.

Three independent batches of chitosan nanoparticles containing vancomycin or nystatin were analyzed by dynamic light scattering. In the case of chitosan nanoparticles containing vancomycin, sizes were in average 173.4 ± 0.4 nm and a polydispersion index (PDI) of 0.155 ± 0.013. See Table 2. An electrostatic magnitude (Zeta potential) of +16.3 ± 0.2 mV with a conductivity of 0.0803 mS/cm was observed.

Table 2. Mean size (nm), polydispersion index (PDI) and Zeta potential (mV) of three independent batches of chitosan nanoparticles loaded with vancomycin hydrochloride (nanoVAN). nanoVAN (batch

Size (nm) PDI Zeta Potential (mV)

No.)

1 173.7 0.180 +16.1

2 172.7 0.136 +16.7

3 173.8 0.150 +16.2

Average 173.4 ± 0.4 0.155 ± 0.013 +16.3 ± 0.2

In the case of chitosan nanoparticles containing nystatin, sizes were in average 181.3 ± 1.5 nm and a PDI of 0.149 ± 0.003. See Table 3. An electrostatic magnitude (Zeta potential) of +15.8 ± 0.03 mV and a conductivity of 0.0804 mS/cm were observed. Encapsulation efficiency was determined as 75% for vancomycin and 98% for nystatin.

Table 3. Mean size (nm), polydispersion index (PDI) and Zeta potential (mV) of three independent batches of chitosan nanoparticles loaded with nystatin (nanoNYS). nanoNYS (batch

Size (nm) PDI Zeta Potential (mV)

No.)

1 184.2 0.149 +15.7 2 180.3 0.144 +15.8

3 179.5 0.153 +15.8

Average 181.3 ± 1.5 0.149 ± 0.003 +15.8 ± 0.03

Preparation of a composition of vancomycin-loaded nanoparticles and nystatin- loaded nanoparticles to be administered in a murine model

Based on calculations of encapsulation efficiency, for administering 50 mg/kg of vancomycin loaded in nanoparticles, a solution containing 66.66 mg/kg of vancomycin nanoparticles in saline (NaCI 0,9%) was administered. While for 50 mg/kg of vancomycin and 5.66 mg/kg of nystatin loaded in nanoparticles, a solution containing 66.66 mg/kg of vancomycin nanoparticles and 5.78 mg/kg of nystatin nanoparticles in saline was administered.

Administration of (i) vancomycin-loaded nanoparticles and (ii) vancomycin-loaded nanoparticles and nystatin-loaded nanoparticles to C. difficile-infected mice reduces weight loss and reduces diarrhea

Figure 3 shows the murine model experimental design of R-CDI for evaluating the effectiveness of the nanoencapsulated composition in comparison to a non-nanoencapsulated composition in a murine model of R-CDI, who were administered an antibiotic cocktail and then were infected with C. difficile R20291 spores. When measured relative weight post-infection from day 1 to day 4 of CDI no statistical differences are observed between the groups. Up to this point, all animals followed the same treatment. 100% of the mice manifested diarrhea between days 1 and 3 (data not shown).

At the third day after the infection, the animals were treated for 5 days with: i) Group A: vancomycin (shown as VAN); ii) Group B: vancomycin in chitosan-based nanoparticles (shown as nanoVAN); iii) Group C: vancomycin and nystatin (shown as VAN+NYS); iv) Group D: vancomycin and nystatin in chitosan-based nanoparticles (shown as nanoVAN+nanoNYS).

Once the treatment was suspended, the animals were monitored for R-CDI symptoms and weight loss, and diarrhea was evaluated (days 8 to 13 postinfection).

Figure 4 shows a graph of relative weight post-infection from day 8 to day 13 after the infection during the R-CDI. It is observed that the administration of vancomycin nanoparticles protects mice from weight loss by ~5% during days 10-12, in respect of mice treated with vancomycin during the R-CDI.

Groups treated with vancomycin show significant weight loss as compared to animals treated with vancomycin nanoparticles or vancomycin and nystatin nanoparticles, exhibiting a difference of 9% or 7%, respectively, on day 10, and of 8% or 7%, respectively, on day 11 post-infection.

Mice treated with vancomycin nanoparticles and nystatin nanoparticles (101 %) exhibit significantly lower weight loss than mice treated with vancomycin and nystatin (7% difference between groups), and on day 11 , mice administered with vancomycin nanoparticles and nystatin nanoparticles exhibit significantly lower weight loss in comparison to those administered with vancomycin (7% difference between groups).

Figure 5 shows the time to manifestation of diarrhea during the R-CDI days, after administration of the treatments. It was observed that 44% of the mice that were treated with vancomycin nanoparticles and nystatin nanoparticles manifested diarrhea during the R-CDI, in comparison to 100% of the mice treated with non-encapsulated vancomycin or vancomycin and nystatin which manifested diarrhea. Similarly, it was observed that 66% of the mice that were treated with vancomycin nanoparticles manifested diarrhea during the R-CDI, in comparison to 100% of the mice treated with nonencapsulated vancomycin which manifested diarrhea. This being consistent with the observations made regarding weight loss.

Administration of vancomycin nanoparticles and nystatin nanoparticles to C. cf/777c/7e-infected mice reduces weight loss and diarrhea during the R-CDI.

Figure 6 shows diarrhea scores during the R-CDI. It was observed that diarrhea scores on days 10 and 11 post-infection were significantly lower for the group of mice treated with vancomycin nanoparticles (average score for day 10: 0.16) and those treated with vancomycin nanoparticles and nystatin nanoparticles (average score for day 10: 0.5; average score for day 11 : 0.2), in comparison to the group treated with non-encapsulated vancomycin (average score for day 10: 1 .66; average score for day 11 : 1 .22) or non-encapsulated vancomycin and nystatin (average score for day 10: 1.66).

These results show that treatments for CDI with vancomycin nanoparticles, and vancomycin nanoparticles and nystatin nanoparticles, are more effective than non-encapsulated vancomycin in diminishing the severity of diarrhea during the R-CDI. In addition, the results show that administration of vancomycin nanoparticles and nystatin nanoparticles is more effective than non-encapsulated vancomycin and nystatin in diminishing the severity of diarrhea during the R-CDI.

Vancomycin- and nystatin-loaded nanoparticles decrease spore shedding in recurrence and cytotoxicity of the cecal content, but not spore adherence to the colon tract

To evaluate C. difficile spore load in feces, samples were collected daily during the treatment, plated in serial dilutions in TCCFA medium and incubated for 2 days in anaerobiosis, and CFU counting was performed.

Figure 7 shows a graph of spore shedding in feces from day 8 to day 13 during the R-CDI, expressed in base-10 logarithmic units of CFU per gram of fecal matter. On days 10, 11 , 12 and 13 after infection, a statistically significant decrease was observed in C. difficile spore CFU counts. The decrease in C. difficile spore counts was of 1 .89, 2.67 and 2.52 Log-io CFU/g of feces on days 10, 11 and 12, respectively, between the groups administered with formulations of vancomycin nanoparticles compared to vancomycin, demonstrating their effectiveness in the reduction of the severity of R-CDI. Likewise, a decrease was observed in C. difficile spore counts by 2.57, 1.85, 2.05 Log-io CFU/g of feces on days 10, 11 and 12, respectively, between the groups administered with formulations of vancomycin nanoparticles and nystatin nanoparticles as compared with vancomycin and nystatin, being statistically significant on days 10 and 12. These results demonstrate the effectiveness of nanoparticle formulations in decreasing spore load in feces during the R-CDI. On days 8 and 9, no differences were observed in C. difficile spore shedding between the groups.

Figure 8 shows time to colonization, which was defined as the time when spore load in feces reaches 1 x 10⁴ CFU/g. We observed that 100% of the mice treated with vancomycin were colonized on day 10 post-infection, while only 33% of the mice treated with vancomycin nanoparticles were colonized at the end of the experiment, this difference being statistically significant. Likewise, we observed that 100% of the animals treated with vancomycin and nystatin were colonized on day 10, while 44% of the mice treated with vancomycin nanoparticles and with nystatin nanoparticles were colonized on day 13 post-infection, this difference being statistically significant. These results demonstrate that nanoencapsulated formulations are more effective than non-encapsulated drugs in diminishing colonization by C. difficile.

Figure 9 shows C. difficile spore adherence to different segments of intestinal tissue, which were collected at the end of the experiment. A statistically significant decrease in spore adherence was observed in the distal colon between the groups of mice which were treated with vancomycin nanoparticles and nystatin nanoparticles and those which were treated with vancomycin. While in the other groups, the decrease was not significant. Additionally, no significant decrease was observed in spore adherence in the cecum, proximal colon or middle colon in the groups treated with the evaluated formulations.

Finally, in Figure 10, the cytotoxicity of the cecal content on Vero cells was evaluated on the last day of the experiment. A significantly lower toxin title, by 3.9 logarithmic units, was observed in the cecum of animals treated with formulations of vancomycin nanoparticles, in comparison to animals treated with formulations of non-encapsulated vancomycin. Similarly, a lower toxin title, by 2.54 logarithmic units, was observed in the group of animals treated with vancomycin nanoparticles and nystatin nanoparticles, in comparison to animals treated with vancomycin and nystatin. Consequently, for the group treated with vancomycin nanoparticles and nystatin nanoparticles, a significant reduction in cytotoxicity, by 2.87 logarithmic units, was observed in comparison to animals treated with vancomycin.

Data show that the administration of a composition of vancomycin nanoparticles or the administration of a composition of vancomycin nanoparticles and nystatin nanoparticles during the CDI, is effective in the treatment of CDI and reduces weight loss, incidence of diarrhea and severity during the R-CDI.

Together, these data show that the administration of a composition of vancomycin nanoparticles and the administration of a composition of vancomycin nanoparticles and nystatin nanoparticles have a higher effectiveness in the inhibition of R-CDI than non-encapsulated forms thereof.

Additionally, it is shown that the administration of vancomycin nanoparticles significantly reduces spore load in the colon, in comparison to the nonencapsulated form thereof during the R-CDI.

It was also observed that the administration of vancomycin nanoparticles and nystatin nanoparticles to mice with CDI significantly reduces spore load in the colon, in comparison to the non-encapsulated form thereof during the

R-CDI. It was observed that vancomycin nanoparticles as monotherapy would allow to reduce recurrent infection in a murine model by 34%, in comparison to the administration of non-encapsulated vancomycin.

Combined therapy, using vancomycin nanoparticles and nystatin nanoparticles in a chitosan-based matrix, allows to reduce recurrent infection in a murine model by 56%, in comparison to non-encapsulated vancomycin or non-encapsulated vancomycin and nystatin. Both results represent a considerable technical advantage in respect of those previously achieved in the treatment of R-CDI.

Additionally, it was observed that mice treated with vancomycin nanoparticles showed a lower spore CFU load during the R-CDI of between ~1.6 and 2.7 logarithmic cycles, in comparison to the non-encapsulated form thereof on days 10, 11 , 12 and 13 post-infection. It was also observed that combined therapy of nanoencapsulated vancomycin and nanoencapsulated nystatin reduced spore CFU load from 2.5 to 1.5 logarithmic cycles, in comparison to the non-encapsulated form thereof on days 10, 11 , 12 and 13 post-infection.

Likewise, consistent with the foregoing, it was observed that the cytotoxicity of the cecal content of m ice that were treated with vancomycin nanoparticles was 3.9 logarithmic cycles (~8100-fold) lower than that of the group of mice treated with non-encapsulated vancomycin, and a ~350-fold reduction when comparing between the combined therapy of vancomycin nanoparticles and nystatin nanoparticles and non-encapsulated vancomycin and nystatin, thus representing considerable technical advantages in respect of known therapies.

Among the surprising results found in the present invention, it stands out that the administration of vancomycin nanoparticles and nystatin nanoparticles in a chitosan-based matrix to mice presenting CDI, reduces the manifestation of a R-CDI by 56% compared with the non-encapsulated therapy, and that the administration of vancomycin nanoparticles reduces the manifestation of the R-CDI by 34% in comparison to the nonencapsulated form thereof.

REFERENCES

Rineh A, Kelso MJ, Vatansever F, Tegos GP, Hamblin MR. Clostridium difficile infection: molecular pathogenesis and novel therapeutics. Expert Rev Anti Infect Then 2014; 12(1 ): 131 -150. doi: 10.1586/14787210.2014.866515

P. Castro-Cordova, P. Mora-Uribe, R. Reyes-Ramirez, G. Cofre-Araneda, J. Orozco-Aguilar, C. Brito-Silva, M. J. Mendoza-Leon, S. A Kuehne, N. Minton, M. Pizarro-Guajardo, D. Paredes-Sabja. Entry of spores into intestinal epithelial cells contributes to recurrence of Clostridioides difficile infection. Nature Communications 2021 12:1 , 12(1 ), 1-18. doi.org/10.1038/s41467-021 -21355-5. Bandi, S.P., Kumbhar, Y.S. & Venuganti, V.V.K. Effect of particle size and surface charge of nanoparticles in penetration through intestinal mucus barrier. J Nanopart Res 22, 62 (2020).

Cerchiara T, Abruzzo A, di Cagno M, et al. Chitosan based micro- and nanoparticles for colon-targeted delivery of vancomycin prepared by alternative processing methods. Eur J Pharm Biopharm. 2015;92:112-119. doi:10.1016/j.ejpb.2015.03.004

Claims

46 CLAIMS

1 . A pharmaceutical composition comprising nystatin and vancomycin, wherein the nystatin and vancomycin are encapsulated in the form of nanoparticles, wherein the nanoparticles further comprise at least one encapsulating agent.

2. The composition of claim 1 , wherein the nystatin and vancomycin are separately encapsulated in the form of nanoparticles.

3. The composition of claim 1 , wherein the nystatin and vancomycin are encapsulated together in the form of nanoparticles.

4. The composition of any one of claims 1 to 3, wherein at least one encapsulating agent is a polysaccharide that improves targeting the intestinal mucosa and causes extended release.

5. The composition of any one of claims 1 to 4, wherein at least one encapsulating agent is biocompatible or mucoadhesive, or both.

6. The composition of claim 4, wherein the polysaccharide is linear, without branching.

7. The composition of claim 4, wherein the polysaccharide is a polysalt or polyelectrolyte.

8. The composition of claim 4, wherein the polysaccharide is a polycation. 47

9. The composition of claim 4, wherein the polysaccharide is a polyanion.

10. The composition of any one of claims 1 to 5, wherein at least one encapsulating agent is alginate, chitosan, maltodextrin, cyclodextrin, ethyl cellulose, ethyl methyl cellulose or carboxymethyl cellulose, or any combination thereof.

11. The composition of any one of claims 1 to 5, wherein at least one encapsulating agent is chitosan.

12. The composition of claim 1 , wherein the nanoparticles have one or more of the following properties:

(i) an encapsulation efficiency between 70% and 100%;

(ii) an average diameter or particle size between 1 nm and 500 nm;

(iii) a polydispersion index between 0.100 and 0.300; or

(iv) an average zeta potential between +1 and +30 mV.

13. The composition of any one of claims 1 to 12, wherein:

(a) the amount of vancomycin present in the composition is within the range of 40% to 70% by weight, and

(b) the amount of nystatin present in the composition, nystatin within the range of 7% to 44 % by weight. 48

14. The composition of claim 13, wherein:

(a) the amount of vancomycin present in the composition is about 69% by weight; and

(b) the amount of nystatin present in the composition is about 8 % by weight.

15. The composition of any one of claims 1 to 12, wherein:

(a) the amount of vancomycin present in the composition is within the range of 41 % to 63% by weight; and

(b) the amount of nystatin present in the composition is within the range of between 15% to 44% by weight.

16. The composition of claim 15, wherein:

(a) the amount of vancomycin present in the composition is about 42% by weight; and b) the amount of nystatin present in the composition is about 44 % by weight.

17. A method for treating an infection by C. difficile, or for treating or preventing one or more recurrent infections by C. difficile, or for reducing the risk of developing recurrent infections by C. difficile in a subject in need thereof, comprising: administering to the subject a pharmaceutical composition of any one of claims 1 to 16.

18. The method of claim 17, wherein the method comprises treating an infection by C. difficile in the subject.

19. The method of claim 17, wherein the method comprises treating or preventing one or more recurrent infections by C. difficile in the subject.

20. The method of claim 17, wherein the method comprises reducing the risk of developing recurrent infections by C. difficile in the subject.

21 . The method of any one of claims 17 to 20, wherein the vancomycin in the pharmaceutical composition is administered to the subject at a dosage of 100 mg to 4 g per day.

22. The method of any one of claims 17 to 20, wherein the nystatin in the pharmaceutical composition is administered to the subject at a dosage of 100,000 IU to 3,000,000 IU per day.

23. The method of any one of claims 17 to 20, wherein:

(a) the vancomycin in the pharmaceutical composition is administered to the subject at a dosage of 100 mg to 4 g per day; and

(b) the nystatin in the pharmaceutical composition is administered to the subject at a dosage of 100,000 III to 3,000,000 IU per day.

24. The method of any one of claims 17 to 20, wherein vancomycin in the pharmaceutical composition is administered to the subject at an amount of approximately 50 mg/kg/day.

25. The method of any one of claims 17 to 20, wherein nystatin in the pharmaceutical composition is administered to the subject at an amount of approximately 5.66 mg/kg/day.

26. The method of any one of claims 17 to 20, wherein:

(a) the vancomycin in the pharmaceutical composition is administered to the subject at an amount of approximately 50 mg/kg/day; and

(b) the nystatin in the pharmaceutical composition is administered to the subject at an amount of approximately 5.66 mg/kg/day.

27. The method of any one of claims 17 to 20, wherein administration of the pharmaceutical composition reduces manifestation of recurrent infections by C. difficile by at least 56% as compared to administering a pharmaceutical composition comprising nystatin and vancomycin in nonencapsulated form.

28. Use of the pharmaceutical composition of any one of claims 1 to 16, wherein it serves for preparing a medicament useful for treating an infection by C. difficile.

29. Use of the pharmaceutical composition of any one of claims 1 to 16, wherein that it serves for preparing a medicament useful for treating or preventing one or more recurrent infections by C. difficile, and for reducing the risk of developing recurrent infections by C. difficile.

30. Use of the pharmaceutical composition of claims 28 and 29, wherein the infection or recurrent infection by C. difficile is colitis, including pseudomembranous colitis.