HK1233167B - A therapeutic agent for use in the treatment of infections - Google Patents

Description

Therapeutic agents used in the treatment of infections

The present invention relates to a micro- or nano-scale therapeutic agent for use in the treatment of infections in humans or animals. The infection may be systemic or localized.

Antibiotic resistance, especially the emergence of widespread multidrug-resistant infections, poses a catastrophic risk to human health and involves substantial costs. Therefore, new approaches to combat infections are urgently needed.

The object of the present invention is to provide a therapeutic agent for treating infections in humans and animals, including multidrug-resistant infections, in a variety of infection settings and physiological environments.

It is also an object of the present invention to provide therapeutic agents that, at least in some embodiments, are capable of minimizing the potential for resistance in target infectious organisms.

According to the present invention, there is provided a nano- or micro-sized therapeutic agent comprising a micro- and/or nanoparticle carrier loaded alone or in combination with one or more inert precursor chemicals, wherein the carrier encapsulates the one or more precursor chemicals, which can be activated by the physiological environment after being released from the carrier in situ at the infection site to form an antimicrobial agent used in the treatment of human or animal infections.

Preferably, the one or more precursor chemicals form an oxidizing microbicide upon activation.

Because highly oxidizing molecules have high energy transfer capabilities, by imposing a large amount of and extensive disseminated damage to bacterial cells, these molecules produce a powerful microbicidal effect on microorganisms. Therefore, tolerance to these chemicals is rarely found. Examples of these molecules include: ozone, hypochlorous acid, sodium hypochlorite or sodium hypobromite, chlorine dioxide, peracids such as peracetic acid and hydrogen peroxide. Generally, it is not practical to use these powerful chemicals for therapeutic purposes, but when encapsulated precursor chemicals are directly delivered to the target infection organism in the form of micron and/or nanoparticles, these chemicals will not be produced until the precursor chemicals are activated in situ at the site of infection, for example, before being activated by contact with body fluids.

In particular, the precursor chemical preferably comprises one or more peroxygen donors that are activated upon release to form hydrogen peroxide.

Also preferably, release of the precursor chemical occurs when the carrier bursts, degrades, or changes porosity in situ within the body of a human or animal host, or in the case of topical application, on the body of a human or animal host. Advantageously, the carrier degrades by hydrolysis over time to provide controlled release of the precursor chemical.

The example of suitable micron and/or nanoparticle carrier used in the present invention comprises micelle, branched polymer, buckyball (buckyball), liposome, ethosome (ethosome), mesoporous silica, carbon nanotube, all of which can encapsulate other chemical substances. Beneficial but not necessarily, carrier is the form of micron and/or nanoparticle produced by thermal induced phase separation (TIPS) process. This method minimizes the residual of solvent used in the encapsulation process that may damage the safety and effectiveness of the therapeutic agent obtained. In addition, in some cases, preferably, the carrier is biodegradable in the host to produce harmless by-products. Therefore, preferably, the carrier comprises a biodegradable polymer, for example poly (lactic acid-co-glycolic acid) (PLGA), and micron and/or nanoparticles of encapsulated precursor chemical substances can be prepared by the TIPS method.

PLGA is a copolymer synthesized by the ring-opening copolymerization of two different monomers: glycolic acid and a cyclic dimer of lactic acid (1,4-dioxane-2,5-dione). It hydrolyzes in vivo to produce the original monomers, lactic acid and glycolic acid, which, under normal physiological conditions, are byproducts of various metabolic pathways in the body. Therefore, the systemic toxicity associated with the use of PLGA in the present invention is minimized.

Examples of the preparation of therapeutic agents of this type and according to the invention are described below.

Preferably, the precursor chemical further comprises one or more acetyl donors that react with the hydrogen peroxide produced by the peroxygen donor upon release to form a mixture of peracetic acid and hydrogen peroxide. The one or more acetyl donors may be encapsulated within the same carrier particles as the peroxygen donor. Thus, preferably, the therapeutic agent of the present invention comprises micro- and/or nanoparticle carriers, wherein at least a certain proportion of the carriers each encapsulate a peroxygen donor and an acetyl donor. Alternatively or in addition, the acetyl donor is encapsulated within its own micro- and/or nanoparticle carrier, and the therapeutic agent of the present invention comprises a combination of micro- and/or nanoparticle carriers that individually encapsulate one or more peroxygen donors or one or more acetyl donors.

Hydrogen peroxide is widely used in endodontics, in the treatment of periodontitis, and as an antimicrobial antiseptic for wounds and mucous membranes. Dilute solutions of hypochlorous acid are advocated for topical disinfection, while peracetic acid is widely used in daily hygiene as a teat disinfectant during and after milking. However, conventional methods for delivering these highly active agents to more severe, deep sites of infection in a controlled, safe, and effective manner are largely impractical. The present invention overcomes this problem by encapsulating a precursor chemical in a carrier so that the antimicrobial agent is only produced when the carrier reaches the target and releases the precursor chemical, which is then activated by contact with the target in the physiological environment (e.g., by contact with body fluids).

The use of inert precursor chemicals that can be activated in situ overcomes the stability and safety issues of active antimicrobial agents.In the present invention, a peroxygen donor is preferably used in combination with acetyl peroxide to produce a dynamic equilibrium mixture of hydrogen peroxide and peracetic acid.

The peroxygen donor preferably comprises any one or a combination of the chemicals in List A below.

List A

Sodium perborate

Sodium percarbonate

sodium superphosphate

Urea peroxide

Peresters

Superoxide

Dioxide (Dioxygenyl)

ozone

hydrogen peroxide

lithium peroxide

Barium peroxide

Di-tert-butyl peroxide

Ammonium peroxydisulfate

Potassium persulfate

Any one or a combination of sodium percarbonate, potassium percarbonate, ammonium percarbonate, sodium perborate, ammonium perborate, ammonium persulfate and urea peroxide in List A is particularly preferred.

There are many acetyl donors that can react with the hydrogen peroxide released from the peroxygen donor upon addition of water. The acetyl donor preferably comprises any one or a combination of the chemicals in List B below.

List B

Tetraacetylethylenediamine (TAED)

Methylcellulose-encapsulated TAED or encapsulated donor

Acetylsalicylic acid

Diacetyldioxohexahydrotriazine (DADHT)

Tetraacetylglycoluril

Acetylurea

Diacetyl urea

Triacetyl urea

Pentaacetyl Glucose (PAG)

Tetraacetyl glycoluril (TAGU)

Acetyl phosphate

Acetylimidazole

Acetyl Coenzyme A

Acetic anhydride

Compounds containing hemiacetal groups

Acetic acid

Diacetylmorpholine

Pyruvate

Acetyl chloride

Acetyl-caprolactam

N'N'-diacetyl-N'N'-dimethylurea.

Particularly preferably, tetraacetylethylenediamine (TAED) is used as the acetyl donor.

The choice of peroxygen donor and acetyl donor depends on the physiological environment encountered during the use of the therapeutic agent. Those that degrade to produce substances that occur naturally or are well tolerated in the body are preferred for in vivo use, while others are suitable for external use, such as topical treatment of skin infections, or use within voids, fissures, or cavities.

As mentioned above, the carrier may comprise microparticles, nanoparticles, or a mixture thereof. According to the IUPAC (International Union of Pure and Applied Chemistry) definition, microparticles are particles of any shape with a size between 1× ^10⁻⁷ and 1× ^10⁻⁴ μm, while nanoparticles are particles of any shape with a size between 1× ^10⁻⁷ and 1× ^10⁻⁷ μm. Particle size and size distribution are the most important characteristics of micro- and nanoparticle systems. They determine the carrier's in vivo distribution, biological fate, toxicity, and targeting ability. Furthermore, they influence drug loading, drug release, and carrier stability. As drug carriers, submicron-sized nanoparticles offer many advantages over microparticles. Compared to microparticles, they have higher intracellular uptake and can reach more biological targets due to their smaller size and relative mobility. However, in certain circumstances, such as when particle packing into voids, crevices, or cavities is beneficial, the selection and use of microparticles is preferred over nanoparticles.

Poly(lactic-co-glycolic acid) (PLGA)-based particles can be prepared in sizes ranging from approximately 20×10 ⁻³ μm in diameter up to micrometer sizes. The preparation of such particles is known and is described, for example, in WO 2008/155558. The preparation methods of these particles allow for manipulation of properties such as porosity, payload efficiency, and drug release profile. This makes them particularly suitable as carriers for the present invention. Loading of these particles with precursor chemicals can be achieved by known techniques during or after the particle manufacturing process.

An example of a method for preparing the therapeutic agent according to the present invention will be described below with reference to the accompanying drawings.

Figure 1 shows a photograph of a scanning electron microscope image of PLGA micro- and nanoparticles encapsulating sodium percarbonate.

The photograph in FIG2 shows a scanning electron microscope image of one of the microparticles shown in FIG1 , but at a higher magnification.

Figure 3 shows photographs of scanning electron microscopy images of PLGA micro- and nanoparticles encapsulating urea hydrogen peroxide.

The photograph in FIG4 shows a scanning electron microscope image of one of the microparticles shown in FIG3, but at a higher magnification.

FIG5 shows a graph of the average pH over time of a sample of ultrapure water containing 20 mg of the particles shown in FIG1 and 2 per 1 ml of water at 23°C.

FIG6 shows a graph of the average pH over time of a sample of ultrapure water containing 20 mg of the particles shown in FIG3 and 4 per 1 ml of water at 23°C.

In one manufacturing method, the therapeutic agent of the present invention is prepared by a thermally induced phase separation (TIPS) process using a PLGA solution containing active precursor chemicals. However, one skilled in the art will appreciate that other manufacturing methods are possible.

The TIPS process begins by preparing a PLGA solution at high temperature to produce a homogeneous solution. Precursor chemicals are dissolved in a suitable solvent and then blended into the PLGA solution. Using another immiscible cooling liquid to remove heat by rapidly cooling to below the biomodel solubility curve can induce phase separation of the homogeneous PLGA solution to form a multiphase system comprising a PLGA-rich phase and a PLGA-poor phase. The phase-separated PLGA solution is then freeze-dried to remove the solvent, producing the micro- and/or nanoparticles of the present invention.

Specifically, to prepare samples of micro- and nanoparticles of encapsulated sodium percarbonate, poly(DL-lactic-co-glycolic acid), a product sold under the trade name PDLG IV 0.68 dl/g by the Corbion Group Netherlands BV, was dissolved in dimethyl carbonate to prepare a 4 wt% solution, which was stirred magnetically for 24 hours before use. Sodium percarbonate was dissolved in ultrapure water at a concentration of 100 mg/ml by vortexing and then left standing for 24 hours. Then, before vortexing, 833 μl of the sodium percarbonate solution was added to 7.5 ml of the PLGA solution in a 10 ml covered glass tube.

TIPS micro- and nanoparticles are then prepared using a conventional microencapsulator, such as the VAR-D encapsulator manufactured by Nisco Engineering AG. The prepared particles are collected in liquid nitrogen and then transferred to a freeze dryer for freeze drying. The resulting particles are shown in Figures 1 and 2.

A similar method can also be used to prepare micro- and nanoparticles encapsulating urea hydrogen peroxide, except that urea hydrogen peroxide is used instead of sodium percarbonate and dissolved in methanol at a concentration of 100 mg/ml. The resulting particles are shown in Figures 3 and 4.

The above method prepared micro- and nanoparticles with a drug loading concentration of 278 mg of the relevant precursor chemical per gram of PLGA. It is expected that higher concentrations can be achieved if necessary, up to approximately double the dosage.

As shown in Figures 1 and 3, the prepared micro- and nanoparticles exhibited the typical wrinkled and porous surfaces of TIPS particles. The surfaces of both groups of particles showed deposits of amorphous material, likely containing the loaded precursor chemical active ingredient. Macropores were visible on the surface of many of the microparticles, a characteristic feature of TIPS micro- and nanoparticles. Similarly, the precursor chemical was encapsulated in a PLGA carrier and contained within the particles in dry form, with the solvent removed by freeze-drying. Ideally, solvents used in the manufacturing process should be eliminated or present in minimal amounts, as they can increase the toxicity of the particles. Furthermore, if the precursor chemical is encapsulated in solution, its stability over time is reduced. The TIPS preparation method tends to minimize or even eliminate residual solvent and offers the advantage of better control over the porosity of the micro- and nanoparticles, which determines the controlled release time of the precursor chemical from the particles. Micro- and nanoparticles encapsulating TAED acetyl donors using acetonitrile as the solvent for TAED can also be prepared using a similar TIPS method.

Likewise, the release of precursor chemicals from micro- and nanoparticles is observed to simulate potential efficacy when used in the treatment of human or animal infections.

The particles encapsulated with sodium percarbonate and urea hydrogen peroxide described above were tested for their ability to induce pH changes in ultrapure water with an initial pH of 6.01 over a 7-day test period. Particle samples were placed in 2 ml polypropylene screw-cap microtubes, and ultrapure water was added to produce a final concentration of 20 mg of micro- and nanoparticles per 1 ml of water. The particles were mixed by vortexing for 10 seconds and then incubated at 23°C. The pH of the samples was measured at predetermined time intervals. A pH electrode was inserted into the sample for 2 minutes before each measurement was recorded. The results are shown in Figures 5 and 6, with measurements from four replicates plotted as the mean and standard deviation of the mean.

The pH increased due to sodium percarbonate and decreased due to urea hydrogen peroxide throughout the incubation period, even when the water in which the particles were immersed was replaced at 24-hour intervals. The pH changes produced by the particles corresponded to the pH of a solution containing only the active ingredient dissolved in ultrapure water, as shown in Table 1 below.

Table 1

280mg/ml 140mg/ml 70mg/ml 35mg/ml 17.5mg/ml weight% 28% 14% 7% 3.5% 1.75% Sodium percarbonate 10.74 10.83 10.87 10.89 10.83 Urea hydrogen peroxide 4.97 6.38 6.9 6.92 7.90

It should be understood that the change in pH observed after the particle incubation shows that the precursor chemical substance can maintain the release from the particle to produce the therapeutic agent of the present invention in an extended time range as the particle degrades. Release occurs when the encapsulating polymer degrades, which is by hydrolysis for the PLGA polymer. It is expected that the release kinetics (rate and duration) can be adjusted by adjusting the composition of the polymer used to prepare micron and nanoparticles. Therefore, the present invention is not limited to the use of micron and nanoparticles based on poly (lactic acid-co-glycolic acid) (PLGA). The micron and nanoparticles using various synthetic and sudden polymers can be adopted. The example of this polymer includes: polyhydrochloric acid (allylamine), poly (diallylmethylammonium chloride), polyethyleneimine (PEI), polyvinylpyrrolidone, poly-L ornithine, poly-L arginine, protamine, chitosan, alginate, polystyrene sulfonate/ester, poly (acrylic acid), poly (methacrylic acid), polyvinyl sulfonate/ester, polyphosphoric acid, poly-L glutamic acid, alginate and dextran sulfate. Nanomicelle carriers can also be made from, for example, polyethylene oxide/polypropylene oxide diblock and triblock copolymers, phospholipids, or precursor surfactants. These carriers can be used to replace or supplement other micro- or nanoparticle carriers.

The carrier can also be treated with auxiliary processes, such as treatment with polyethylene glycol, typically designed for PEGylation, to protect the particles in physiological environments and, for example, to achieve extended circulation time in the bloodstream. Alternatively or in addition, the carrier can be treated with a targeting ligand to increase targeting specificity. For example, the carrier can be targeted by a biosensor such as a monoclonal antibody.

Some additional examples of therapeutic agents of the invention are described below.

1. Poly(lactic-co-glycolic acid) (PLGA)-based nanoparticle carrier with a diameter of 200×10 ⁻³ μm and loaded with 10% sodium percarbonate.

2. Poly(lactic-co-glycolic acid) (PLGA)-based nanoparticle carriers with a diameter of 20×10 ⁻³ μm to 100×10 ⁻³ μm and loaded with sodium percarbonate to a loading efficiency of 80%.

3. Poly(lactic-co-glycolic acid) (PLGA)-based nanoparticle carriers loaded with sodium percarbonate and tetraacetylethylenediamine alone or in combination to a loading efficiency of 0.1% to 50%.

4. A polyethyleneimine (PEI) carrier loaded with sodium persulfate and acetylsalicylic acid alone or in combination to a combined loading efficiency of 65%.

5. Poly(lactic-co-glycolic acid) (PLGA)-based nanoparticle carriers with a diameter of 100 to 1000 μm loaded with urea peroxide to a loading efficiency of 75%.

6. A PEGylated nanoparticle carrier loaded with sodium percarbonate, with a particle diameter of 20×10 ^-3 μm to 300×10 ^-3 μm (including the end values) and a loading efficiency of 40%.

7. Poly(lactic-co-glycolic acid) (PLGA)-based microparticle carriers with a diameter of approximately 300 μm loaded with sodium percarbonate and a loading efficiency of 50% to 80% (inclusive).

8. A mixture of chitosan-based nanoparticles loaded with sodium percarbonate and PLGA particles loaded with tetraacetylethylenediamine, wherein the ratio of percarbonate to TAED is approximately 2:1.

Thus, the present invention combines a well-studied and widely used high-level environmental antimicrobial agent with its bioinert precursor encapsulated in a targeted micro- or nanoscale carrier. The other chemicals are protected from the host's immune response and do not cause unacceptable damage or side effects to host tissues. Thus, the present invention provides a therapeutically safe and effective method for targeting and killing infectious microorganisms (including multidrug-resistant microorganisms) that can be targeted to various body sites, including the bloodstream, lungs, liver, kidneys, intestines, urinary tract, and cortex.

Claims (11)

1. A nano- or micron-sized therapeutic agent for use in the treatment of human or animal infections, comprising a micron- and/or nanoparticle carrier loaded alone or in combination with one or more inert precursor chemicals comprising a peroxy donor and an acetyl donor. The carrier encapsulates the precursor chemical substance, which is activated by the physiological environment upon exposure at the site of infection, causing the peroxide donor to form hydrogen peroxide and the acetyl donor to react with the hydrogen peroxide to produce a mixture of peracetic acid and hydrogen peroxide. The particulate carrier comprises a biodegradable polymer and is prepared and incorporated with precursor chemicals through a thermally induced phase separation and freeze-drying process, wherein the thermally induced phase separation uses acetonitrile or dimethyl carbonate as a solvent. Wherein, the peroxide donor is any one or a combination of sodium percarbonate, potassium percarbonate, ammonium percarbonate, sodium perborate, ammonium perborate, ammonium persulfate, and urea peroxide, and The carrier comprises poly(lactic acid-co-glycolic acid) (PLGA). 2. The therapeutic agent of claim 1, wherein the acetyl donor is any one or a combination of any chemical substance from the following list: tetraacetylethylenediamine (TAED), methylcellulose-encapsulated TAED, acetylsalicylic acid, diacetyldioxohexahydrotriazine (DADHT), tetraacetylglycourea, acetylurea, diacetylurea, triacetylurea, pentaacetylglucose (PAG), tetraacetylglycourea (TAGU), acetylphosphate, acetimidazole, acetyl-CoA, acetic anhydride, compounds containing a hemiacetal group, acetic acid, diacetylmorpholine, pyruvate/ester, acetyl chloride, acetyl-caprolactam, N'N'-diacetyl-N'N'-dimethylurea. 3. The therapeutic agent of claim 1, wherein the acetyl donor comprises one of tetraacetylethylenediamine (TAED), acetylsalicylic acid, and pentaacetylglucose (PAG). 4. The therapeutic agent of claim 1, wherein at least a portion of the micron and/or nanoparticle carriers each encapsulates a peroxy donor and an acetyl donor. 5. The therapeutic agent of claim 1, comprising a combination of micron and/or nanoparticle carriers individually encapsulating one or more peroxy donors or one or more acetyl donors. 6. The therapeutic agent of claim 1, wherein the release of one or more precursor chemicals occurs when the carrier ruptures, degrades, or alters its porosity in situ at the site of infection. 7. The therapeutic agent of claim 6, wherein the carrier is hydrolyzed and degraded over time to provide controlled release of the one or more precursor chemicals. 8. The therapeutic agent of claim 1, wherein the micron and/or nanocarrier is any one or a mixture of micelles, branched polymers, briquette spheres, liposomes, liposomes, mesoporous silica, and carbon nanotubes. 9. The therapeutic agent of claim 1, wherein the carrier is PEGylated. 10. The therapeutic agent of claim 1, wherein the carrier has been treated with a targeting ligand. 11. The therapeutic agent of claim 1, wherein the carrier is targeted via a biosensor.