AU2018285342A1

AU2018285342A1 - Antibiotic composition for the treatment of infections with resistent microorganism

Info

Publication number: AU2018285342A1
Application number: AU2018285342A
Authority: AU
Inventors: Per Bendix Jeppesen; Dan Mønster NIELSEN
Original assignee: Pulmopharma Ivs
Current assignee: Pulmopharma Aps
Priority date: 2017-06-13
Filing date: 2018-06-13
Publication date: 2020-01-02
Anticipated expiration: 2038-06-13
Also published as: WO2018229143A1; CA3066851A1; AU2018285342B2

Abstract

A composition is provided, which comprises a mixture of a polymeric biguanide polymer and an alkyl and/or dialkyl oxyethylene methyl ammonium salt, or pharmaceutical acceptable salts or tautomers thereof, for use in the treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as methicillin resistant Staphylococcus aureus (MRSA).

Description

ANTIBIOTIC COMPOSITION FOR THE TREATMENT OF INFECTIONS WITH RESISTENT MICROORGANISM

Technical field

The present invention relates to treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, in particular Staphylococcus aureus, such as methicillin resistant Staphylococcus aureus (MRSA).

Background

Resistant microbes are increasingly difficult to treat, requiring alternative medications or higher doses, both of which may be more expensive or more toxic. Microbes resistant to multiple antimicrobials are called multidrug resistant (MDR). Antimicrobial resistance is on the rise with millions of deaths every year. All classes of microbes develop resistance: fungi develop antifungal resistance, viruses develop antiviral resistance, protozoa develop antiprotozoal resistance, and bacteria develop antibiotic resistance.

Rising drug resistance is caused mainly by improper use of antimicrobials in humans as well as in animals, and spread of resistant strains between the two. Antibiotics increase selective pressure in bacterial populations, causing vulnerable bacteria to die; this increases the percentage of resistant bacteria which continue growing. With resistance to antibiotics becoming more common there is greater need for alternative treatments. Calls for new antibiotic therapies have been issued, but new drug development is becoming rarer. A World Health Organization (WHO) report released April 2014 stated, this serious threat is no longer a prediction for the future, it is happening right now in every region of the world and has the potential to affect anyone, of any age, in any country. Antibiotic resistance - when bacteria change so antibiotics no longer work in people who need them to treat infections - is now a major threat to public health. According to the Centers for Disease Control and Prevention: Each year in the United States, at least 2 million people become infected with bacteria that are resistant to antibiotics and at least 23,000 people die each year as a direct result of these infections. There are multiple national and international monitoring programs for drug-resistant threats, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant S. aureus (VRSA), extended spectrum beta-lactamase (ESBL), vancomycin-resistant Enterococcus (VRE), multidrug-resistant A. baumannii (MRAB).

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Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for several types of infections in humans, which are difficult-to-treat. MRSA comprise any strain of Staphylococcus aureus that has developed multiple drug resistance to beta-lactam antibiotics, β-lactam antibiotics are a broad spectrum group which includes some penams - penicillin derivatives such as methicillin and oxacillin, and cephems such as the cephalosporins. MRSA is prevalent in hospitals, prisons, and nursing homes, where people with open wounds, invasive devices such as catheters, and weakened immune systems are at greater risk of nosocomial infection (hospital-acquired infection). While MRSA began as a hospital-acquired infection, it has developed limited endemic status and is now community-acquired as well as livestock-acquired.

MRSA is currently very difficult to treat and there is a great demand for effective antibiotic drugs for MRSA treatment.

Summary

Methods and uses for treatment, prevention and/or amelioration of an infection with methicillin resistant Staphylococcus aureus (MRSA) is provided herein.

In one aspect, a composition is provided comprising a mixture of:

i. a polymeric biguanide polymer and ii. an alkyl and/or dialkyl oxyethylene methyl ammonium salt or pharmaceutical acceptable salts or tautomers thereof, for use in a therapeutic treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as methicillin resistant Staphylococcus aureus (MRSA).

The same composition is also provided for treatment of open wounds, both preoperative and post-operative wounds as well as in connection with surgery and dialysis.

In another aspect, a method is provided for the treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as methicillin resistant Staphylococcus aureus (MRSA), comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a mixture of:

i. a polymeric biguanide polymer and

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PCT/EP2018/065684 ιι. an alkyl and/or dialkyl oxyethylene methyl ammonium salt or pharmaceutical acceptable salts or tautomers thereof to a human or animal in need thereof.

In a third aspect, a use is provided of composition comprising a mixture of:

I. a polymeric biguanide polymer and ii. an alkyl and/or dialkyl oxyethylene methyl ammonium salt or salts or tautomers thereof for nontherapeutic treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as methicillin resistant Staphylococcus aureus (MRSA), of an object or subject.

A fourth aspect relates to a method for non-therapeutic treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as methicillin resistant Staphylococcus aureus (MRSA), said method comprising

a. providing a mixture of:

I. a polymeric biguanide polymer and ii. an alkyl and/or dialkyl oxyethylene methyl ammonium salt or pharmaceutical acceptable salts or tautomers thereof, and

b. exposing an object or subject to said mixture.

Detailed description

Methods, compositions and uses thereof for the treatment, prevention and/or amelioration of an infection with one or more microorganisms having acquired antimicrobial resistance, in particular MRSA infections, are provided herein. The methods and uses encompass both therapeutic and nontherapeutic applications. Therapeutic applications include treatment, prevention and/or amelioration of an infection with one or more microorganisms with antimicrobial resistance in humans and animals infected or at risk thereof. The non-therapeutic applications are directed to disinfection of objects which serve or could serve as agents of dissemination of resistant microorganisms, objects such as a devices, clothes, installations and/or premises.

Terms and definitions

To facilitate the understanding of the following description, a number of definitions are presented in the following paragraphs.

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The term treatment , as used anywhere herein comprises any type of therapy, which aims at terminating, preventing, ameliorating and/or reducing the susceptibility to a clinical condition as described herein. In a preferred embodiment, the term treatment relates to prophylactic treatment (i.e. a therapy to reduce the susceptibility of a clinical condition, a disorder or condition as defined herein).

Thus, treatment, treating, and the like, as used herein, refer to obtaining a desired effect, such as a biological, pharmacologic and/or physiologic effect, covering any treatment of a pathological and/or clinical condition or disorder in a mammal, including a human. The term is also used in the context of disinfecting objects from which disease may disseminate. The effect may be prophylactic in terms of completely or partially preventing a disorder or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder. That is, treatment includes (1) preventing the disorder or clinical condition from occurring or recurring in a subject, (2) inhibiting the disorder or clinical condition, such as arresting its development, (3) stopping, terminating or alleviating the disorder or clinical condition or at least symptoms associated therewith, so that the host no longer suffers from the disorder or clinical condition or its symptoms, such as causing regression of the disorder or clinical condition or its symptoms, for example, by restoring or repairing a lost, missing or defective function, or stimulating an inefficient process, or (4) relieving, alleviating, or ameliorating the disorder or clinical condition, or symptoms associated therewith, where ameliorating is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, such as inflammation, pain, and/or immune deficiency.

The terms prevent, preventing, and prevention, as used herein, refer to a decrease in the occurrence of symptoms or characteristics of a biological effect, a disorder or clinical condition. The term is also used in the context of preventing disease dissemination, for example from objects/equipment/facilities, from which disease may disseminate. The prevention may be complete. The prevention may also be partial, such that for example the occurrence of symptoms or characteristics of a disorder in a subject is less than that which would have occurred without the present invention. Prevention also refers to reduced susceptibility to a clinical condition.

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The terms ameliorate , ameliorating and amelioration , are also used separately herein to refer to a reduction of the severity of the occurrence of biological effects or symptoms or characteristics of a disorder or clinical condition.

The term “antimicrobial resistance” as used herein refers to microorganisms, which has acquired resistance to an antimicrobial drug that was once able to treat an infection by that microorganism. In particular, the term is meant to include “antibiotic resistance”, which applies to bacteria having acquired resistance to antibiotics.

Microorganisms with antimicrobial resistance

The methods and uses provided herein can be applied to treatment, prevention and/or amelioration of an infection with any microorganism having acquired antimicrobial resistance. The microorganism is in a preferred embodiment selected from the group consisting of methicillin-resistant Staphylococcus aureus (MRSA), vancomycinresistant S. aureus (VRSA), extended spectrum beta-lactamase (ESBL), vancomycinresistant Enterococcus (VRE) and multidrug-resistant A. baumannii (MRAB). In one embodiment, the microorganism is Klebsiella pneumonia. In a preferred embodiment, the microorganism is MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA)

Methicillin-resistant Staphylococcus aureus (MRSA) is a gram-positive bacterium responsible for several types of infections in humans, which are difficuIt-to-treat. MRSA comprise any strain of Staphylococcus aureus that has developed multiple drug resistance to beta-lactam antibiotics. The strains may have gained drug resistance through horizontal gene transfer and natural selection, β-lactam antibiotics are a broad spectrum group which includes some penams - penicillin derivatives such as methicillin and oxacillin, and cephems such as the cephalosporins. MRSA have evolved from horizontal gene transfer of the mecA gene to at least five distinct S. aureus lineages.

The antibiotic resistance is believed to arise from alteration of the antibiotic's target site. Beta-lactam antibiotics permanently inactivate PBP enzymes, which are essential for bacterial life, by permanently binding to their active sites. Some forms of MRSA, however, express a PBP that will not allow the antibiotic into their active site.

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Acquisition of SCCmec in methicillin-sensitive staphylococcus aureus (MSSA) gives rise to a number of genetically different MRSA lineages. These genetic variations within different MRSA strains likely explain the variability in virulence and associated MRSA infections. The first MRSA strain, ST250 MRSA-1 originated from SCCmec and ST250MSSA integration. Historically, major MRSA clones: ST2470-MRSA-I, ST239-MRSA-III, ST5-MRSA-II, and ST5-MRSA-IV were responsible for causing hospital-acquired MRSA (HA-MRSA) infections. ST239-MRSA-III, known as the Brazilian clone, was highly transmissible compared to others and distributed in Argentina, Czech Republic, and Portugal. In the UK, the most common strains of MRSA are EMRSA15 and EMRSA16. EMRSA16 has been found to be identical to the ST36:USA200 strain, which circulates in the United States, and to carry the SCCmec type II, enterotoxin A and toxic shock syndrome toxin 1 genes. Under the new international typing system, this strain is now called MRSA252. EMRSA 15 is also found to be one of the common MRSA strains in Asia. Other common strains include ST5:USA100 and EMRSA 1. These strains are genetic characteristics of HA-MRSA.

Community-acquired MRSA (CA-MRSA) strains emerged in late 1990 to 2000, infecting healthy people who had not been in contact with health care facilities. It has been proposed that CA-MRSA did not evolve from the HA-MRSA. This is supported by molecular typing of CA-MRSA strains and genome comparison between CA-MRSA and HA-MRSA, which indicate that novel MRSA strains integrated SCCmec into MSSA separately on its own. By mid-2000, CA-MRSA was introduced into the health care systems and distinguishing CA-MRSA from HA-MRSA became a difficult process. Community-acquired MRSA (CA-MRSA) is more easily treated and more virulent than hospital-acquired MRSA (HA-MRSA). The genetic mechanism for the enhanced virulence in CA-MRSA remains an active area of research. Especially the PantonValentine leukocidin (PVL) genes are of interest because they are a unique feature of CA-MRSA.

In the United States, most cases of CA-MRSA are caused by a CC8 strain designated ST8:USA300, which carries SCCmec type IV, Panton-Valentine leukocidin, PSM-alpha and enterotoxins Q and K, and ST1:USA400. The ST8:USA300 strain results in skin infections, necrotizing fasciitis and toxic shock syndrome, whereas the ST1:USA400 strain results in necrotizing pneumonia and pulmonary sepsis. Other communityacquired strains of MRSA are ST8:USA500 and ST59:USA1000. In many nations of

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PCT/EP2018/065684 the world, MRSA strains with different predominant genetic background types have come to predominate among CA-MRSA strains; USA300 easily tops the list in the U.S. and is becoming more common in Canada after its first appearance there in 2004. For example, in Australia ST93 strains are common, while in continental Europe ST80 strains, which carry SCCmec type IV, predominate. In Taiwan, ST59 strains, some of which are resistant to many non-beta-lactam antibiotics, have arisen as common causes of skin and soft tissue infections in the community. In a remote region of Alaska, unlike most of the continental U.S., USA300 was found rarely in a study of MRSA strains from outbreaks in 1996 and 2000 as well as in surveillance from 200406.

An MRSA strain, CC398, is found in intensively reared production animals (primarily pigs, but also cattle and poultry), where it can be transmitted to humans as LA-MRSA (livestock-associated MRSA).

For CA-MRSA strains, it has been found in a study that 15 (63%) are associated with skin infections, 5 (21%) bloodstream, 3 (13%) respiratory, and 1 (4%) a catheter-site infection. One third of the patients were <18 years of age (3 were <2 years of age), and 50% were female. Nineteen (79%) patients were initially seen at hospitals, 3 (13%) at outpatient centers, and 2 (8%) at a prison. Three (13%) were considered to have healthcare-onset infections.

Twenty-two (92%) of 24 CA-MRSA isolates were of PFGE type USA1100, a community strain. These 22 isolates contained SCCmec type IVc and the PVL locus, and 19 of the 22 had indistinguishable Smal PFGE patterns. The other 3 USA1100 isolates were at least 91% related to the homogeneous group. The 3 healthcare-onset infections were of PFGE type USA1100. The 2 non-USA1100 isolates had microbiologic properties consistent with HA-MRSA belonging to PFGE type USA600 and USA800—one from the respiratory tract and the other from a catheter site. Both of these isolates were from adult patients who were considered to have community-onset infections.

The term “MRSA” as used herein includes any Methicillin-resistant strain of Staphylococcus aureus. The term also includes any strain regardless of how it is acquired and thus includes HA-MRSA (healthcare-associated MRSA), CA-MRSA (community-associated MRSA) and LA-MRSA (livestock-associated).Several newly

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PCT/EP2018/065684 discovered strains of MRSA show antibiotic resistance even to vancomycin and teicoplanin. These new evolutions of the MRSA bacterium have been coined vancomycin intermediate-resistant Staphylococcus aureus (VISA). Thus, in one specific embodiment, MRSA of the present invention also includes VISA strains.

Specifically, the methods and uses, whether therapeutic or non-therapeutic, is applied in specific embodiments for treatment, prevention and/or amelioration of an infection with at least one MRSA strain selected from the group consisting of CC1, CC5, CC8 (USA300), CC22, CC30, CC45, CC80 and CC398. In another embodiment, the strain 10 is selected from the group consisting of ST250 MRSA-1, EMRSA15 and EMRSA16/

USA200. In another embodiment, the strain is selected from the group consisting of

ST250 MRSA-1, ST2470-MRSA-I, ST239-MRSA-III, ST5-MRSA-II, ST5-MRSA-IV ST239-MRSA-III, EMRSA15, EMRSA16, ST5:USA100, EMRSA 1, ST8:USA300 (CC8), ST1:USA400, ST8:USA500, ST59:USA1000 and CC398

In another embodiment, the at least one MRSA strain is selected from the group set out in table 1 Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 15442, Enterococcus hirae ATCC 10541 and Escherichia coll ATCC 10536.

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Table 1. List of MRSA strains (Khokhlova et al, 2015, PLoS ONE 10(6):e0128017)

Isolation year Name ofMRSA ST type (epidemiologicalclassificationof MRSA) Patients

	Disease	Outcome	Age	Sex
	ST239 (HA)	;+++7+/+/+/+777//+///////+++/++7/FfoMimM	Death	71Y :	//?/+/
////////)//+///·/ΐ//+ί/+/////++++^ΐ^Λ///ΐ7///+7/++ί/7	SiB'CA.			: ¥	..
/Oill	/l^;+iW:irtWiAlii+/i+	Pneumonia, sepsis (bacteremia?	Death	39Y	M
SiilliillliMllllii	ST8(CA)			41 V	/10+
//00237/+/7/:+	ST8 (CA)		Death	40Y	M
ιι·ιβ··®··ι	ST8(CA)	p_w>ww :			F
1^52·++·/	ST8 (HACA) ^:			34Y :	F++
OC^SO	ST239 (HA)		Ηο-:'ο·.·ο·γ	J4Y
2010 OC22B	ST239 (HA)	Osteomyelitis	Recovery	22Y :	M
	ST239 (HA)	Erysipelas-like necrotic cefkihits	Recovery	59Y .	io
:ΐΐί/^ι+^ΐΐι/1^ϊ·ΐ4/ϊΐΐ7	ST239 (HA)	Wound infection, cellulitis	Recovery	58Y :	M
»»111·····	ST239(HA)	Wound infection	Recovery	SOY	M
	/l:/:///Wt^S/71+777://++;	Skin abscesses	Recovery	50Y	Fll
liSie/lieil··	ST8(CA)	\|Ι·Ι\|··Μ^	ill··	19Y
:///7+_:/_::///++77+/://///:^^;/://:/i/i+7)	ST12(HA)	Surgical site infection, (sepsis)	Death	84Y	M
/<)” ocec	ST239(HA)	Pneumonia	1®·β·1	48r	M
/70014/+//77/7::	ST239 (HA) :	/7//777////+7+//	+:/://+//+/+++:		/+F++
	ST239(HA)	Pneumonia, sepsis (bacteremia)	Recovery	27V	M
1)0098::):)::)=	ST239 (HA)	:·/·/7+++7++++7+++^^^^	:<//:+///++/	31Y	M
ocw	ST239(HA)	Osteomyelitis	Recovery	30V	M
+OC111+//+/+/	ST239 (HA)	Osteomyelitis	Recovery	76Y	7/F+)
•.•A	ST239(HA)	ellliBIIlllIillllillllli	<+++ Recovery
//OC2+++7+/+:	ST239 (HA)	+////7+77++A///%+;/7:+7/F//77/+//+++/w+re+:Born^;inf^itort:+//+/+/+/7/+//:++::7+++++//+	Recovery	53Y	M
»··\|Β·	ST239(HA)	Wound infection	Recovery	ιόβ®	M
OC44	ST239 (HA) :	//+^:////:7//:7:/7+///+/++///+/++////////+/_:+_:/++/::+Y:///+:p^rft<mitia²/ /7+///7//+//+++7/+g/+://g	Recovery	30Y	M
OCt4C	ST239WCA)		//+0^^	43V	F
OC160	ST8 (CA)	Wound infection, cetitilius	Recovery	53Y	M
ock;	SW.CA,		irecmc.y	Iffl	-

“Y, years; M. mate; F. female; HA, healthcare-associated MRSA; CA. community· associated MRSA. ^s*Heatthy carrier (hospital worker) ^cHealfty carrier (stocfent) ^information not available.

da:10.13714ownal.paw.012^17S02

MRSA is generally obtained by touching the skin of another person who is colonized with MRSA, or by touching a contaminated surface (such as a countertop, door handle, or phone). You can develop an infection from MRSA if your skin is colonized and the bacteria enter an opening (e.g. a cut, scrape, or wound) in the skin. Infection with is most commonly seen under the nostrils. The rest of the respiratory tract, open wounds, 10 intravenous catheters, and the urinary tract are also potential sites for infection. Healthy individuals may carry MRSA asymptomatically for periods ranging from a few weeks to many years. People with compromised immune systems are at a significantly greater risk of symptomatic secondary infection. MRSA can usually be detected by swabbing the nostrils and isolating the bacteria found inside the nostrils. Combined with extra 15 sanitary measures for those in contact with infected people, swab screening people admitted to hospitals has been found to be effective in minimizing the spread of MRSA in hospitals in the United States, Denmark, Finland, and the Netherlands.

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MRSA may progress substantially within 24-48 hours of initial topical symptoms. After 72 hours, MRSA can take hold in human tissues and eventually become resistant to treatment. The initial presentation of MRSA is small red bumps that resemble pimples, spider bites, or boils; they may be accompanied by fever and, occasionally, rashes. Within a few days, the bumps become larger and more painful; they eventually open into deep, pus-filled boils.[5] About 75 percent of community-associated (CA-) MRSA infections are localized to skin and soft tissue. Infections can usually be treated effectively with conventional antibiotics. Some CA-MRSA strains display enhanced virulence, spreading more rapidly and causing illness much more severe than traditional HA-MRSA infections, and they can affect vital organs and lead to widespread infection (sepsis), toxic shock syndrome, and necrotizing pneumonia. This is thought to be due to toxins carried by CA-MRSA strains, such as PVL and PSM, though PVL was recently found not to be a factor in a study by the National Institute of Allergy and Infectious Diseases at the National Institutes of Health. It is not known why some healthy people develop CA-MRSA skin infections that are treatable while others infected with the same strain develop severe infections or die.

People are occasionally colonized with CA-MRSA and are completely asymptomatic. The most common manifestations of CA-MRSA are simple skin infections, such as impetigo, boils, abscesses, folliculitis, and cellulitis. Rarer, but more serious, manifestations can occur, such as necrotizing fasciitis and pyomyositis (most commonly found in the tropics), necrotizing pneumonia, and infective endocarditis (which affects the valves of the heart), and bone (osteomyelitis) and joint infections. CA-MRSA often results in abscess formation that requires incision and drainage. Before the spread of MRSA into the community, abscesses were not considered contagious, because infection was assumed to require violation of skin integrity and the introduction of staphylococci from normal skin colonization. However, newly emerging CA-MRSA is transmissible from HA-MRSA.

Diagnostic microbiology laboratories and reference laboratories use molecular techniques for identifying and characterizing MRSA have recently been developed. Normally, the bacterium must be cultured from blood, urine, sputum, or other body-fluid samples, and in sufficient quantities to perform confirmatory tests early-on. Still, because no quick and easy method exists to diagnose MRSA, initial treatment of the infection is often based upon 'strong suspicion' and techniques by the treating

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PCT/EP2018/065684 physician; these include quantitative PCR procedures, which are employed in clinical laboratories for quickly detecting and identifying MRSA strains.

Another common laboratory test is a rapid latex agglutination test that detects the PBP2a protein. PBP2a is a variant penicillin-binding protein that imparts the ability of S. aureus to be resistant to oxacillin.

MRSA infections can also be defined as illness compatible with staphylococcal disease in a patient from whom a strain of S. aureus resistant to oxacillin by disk diffusion was isolated from a clinically relevant site. Because it is suspected that community strains has entered the healthcare setting, epidemiologic risk factor data are not always useful in distinguishing community versus healthcare strains. Therefore, microbiologic definitions can be used. An MRSA isolate can be considered to be an HA-MRSA strain if it is resistant to at least 2 of the following antimicrobial agents: trimethoprim/ sulfamethoxazole (TMP/SMX), ciprofloxacin, gentamicin, rifampin, and tetracycline. A MRSA isolate is considered to be a CA-MRSA strain if 1) antimicrobial susceptibility results are available for at least 2 of the following agents: TMP/SMX, ciprofloxacin, gentamicin, rifampin, tetracycline, and 2) the isolate was resistant to no more than 1 of the agents and was confirmed to be susceptible to at least 2 of these agents.

An infection can be considered to be healthcare onset if the MRSA culture is obtained >48 hours after a patient was admitted to the hospital and the patient had no evidence of the infection at the time of admission. A MRSA culture obtained within 48 hours of hospital admission or evidence of infection on admission can be considered an indication of a community-onset infection.

Skin disease was defined as a primary skin infection such as abscess, cellulitis, folliculitis, or a skin infection spreading to contiguous tissues. Surgical site infections (SSIs) were not considered to be skin disease.

Thus, the compositions, uses and methods, whether therapeutic or non-therapeutic are in one preferred embodiment applied to object or subjects for which S. aureus have been identified using one or more of the techniques described above, such as I particular qPCR.

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Composition

It is within the scope of the present invention to provide a composition for use as a disinfectant. In one aspect, the composition is provided for use in the therapeutic treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as methicillin resistant Staphylococcus aureus (MRSA). In another aspect a use the composition is provided for non-therapeutic treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as MRSA.

The inventors have shown that the combined use of polymeric biguanide polymer and alkyl and/or dialkyl oxyethylene methyl ammonium salt surprisingly results in an increased and synergistic biocidal effect against MRSA thus minimizing the concentration required to obtain the desired effect. Use of a 2 component biocidal composition may also reduce the risk that the targeted MRSA gain resistance towards the biocide composition. Further, the synergistic effect obtained when combining the two biocides reduces the amount of biocides needed to obtain a therapeutic effect thereby reducing the risk of side effects.

In one aspect, the invention relates to a composition comprising a mixture of:

I. a polymeric biguanide polymer and ii. an alkyl and/or dialkyl oxyethylene methyl ammonium salt or pharmaceutical acceptable salts or tautomers thereof, for use in a therapeutic treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as MRSA.

In another aspect, the invention relates to a composition comprising a mixture of:

I. a polymeric biguanide polymer and ii. an alkyl and/or dialkyl oxyethylene methyl ammonium salt or salts or tautomers thereof for nontherapeutic treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as methicillin resistant Staphylococcus aureus (MRSA) of an object or subject.

In a preferred embodiment of the present invention, the polymeric biguanide polymer is Poly-(hexamethylene-guanidium chloride).

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In another preferred embodiment the dialkyl oxyethylene methyl ammonium salt is N,Ndidecyl-N-methyl-poly(oxyethyl) ammonium propionate.

It is preferred that the composition according to the present invention comprises a mixture of Poly-(hexamethylene-guanidium chloride) and N,N-didecyl-N-methylpoly(oxyethyl) ammonium propionate.

In an embodiment of the invention, the one or more biocides is a mixture of poly(hexamethylene-guanidium chloride) and N,N-didecyl-N-methyl-poly(oxyethyl) ammonium propionate in a ratio of 0,5-1,5 : 1-5 by weight. In another embodiment of the invention, the one or more biocides is a mixture of poly-(hexamethylene-guanidium chloride) and N,N-didecyl-N-methyl-poly(oxyethyl) ammonium propionate in a ratio of 0,8-1,2 : 2-4 by weight. In yet another embodiment of the invention, the one or more biocides is a mixture of poly-(hexamethylene-guanidium chloride) and N,N-didecyl-Nmethyl-poly(oxyethyl) ammonium propionate in a ratio of about 1 : 2 by weight. In a preferred embodiment of the invention, the one or more biocides is a mixture of poly(hexamethylene-guanidium chloride) and N,N-didecyl-N-methyl-poly(oxyethyl) ammonium propionate in a ratio of about 1 : 1 by weight. In yet another preferred embodiment of the invention, the one or more biocides is a mixture of poly(hexamethylene-guanidium chloride) and N,N-didecyl-N-methyl-poly(oxyethyl) ammonium propionate in a ratio of about 1 : 3 by weight.

In a preferred embodiment of the present invention the one or more biocides is a mixture of Poly-(hexamethylene-guanidium chloride) (and N,N-didecyl-N-methylpoly(oxyethyl) ammonium propionate, such as e.g. in a ratio of about 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2, 1:3, 1:4, 1:5, 1.25:1, 1.5:1, 1.75:1,2:1,3:1, or 4:1; more preferably a mixture in a ratio of about 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2, 1:3, 1.25:1, 1.5:1, 1.75:1,2:1, or 3:1; even more preferably a mixture in a ratio of about 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2, 1.25:1, 1.5:1, 1.75:1, or 2:1; yet even more preferably essentially a one to one mixture of Poly-(hexamethylene-guanidium chloride) and N,N-didecyl-N-methyl-poly(oxyethyl) ammonium propionate. This specific combination of biocides has surprisingly been found to provide a synergistically effect against MRSA.

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Pharmaceutical formulation

Whilst it is possible for the composition of the present invention to be used as the raw mixture of biocides, it is preferred to present them in the form of a pharmaceutical formulation when used in therapeutic methods. Accordingly, the composition provided herein in a preferred embodiment is provided as a pharmaceutical formulation, which comprises the composition or a pharmaceutically acceptable salt or ester thereof, as herein defined, and a pharmaceutically acceptable carrier therefor. The pharmaceutical formulations may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 2005, Lippincott, Williams & Wilkins.

The pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more excipients which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents, or an encapsulating material.

Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

The composition may be formulated for parenteral administration and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers, optionally with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or non-aqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water. The

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PCT/EP2018/065684 term active ingredients as used herein refers to Poly-(hexamethylene-guanidium chloride) and N,N-didecyl-N-methyl-poly(oxyethyl) ammonium propionate.

The composition of the invention may also be formulated for topical delivery. The topical formulation may include a pharmaceutically acceptable carrier adapted for topical administration. Thus, the composition may take the form of a suspension, solution, ointment, lotion, sexual lubricant, cream, foam, aerosol, spray, suppository, implant, inhalant, tablet, capsule, dry powder, syrup, balm or lozenge, for example.

Preferably, the formulation will comprise about 0.5% to 75% by weight of the active ingredient(s) with the remainder consisting of suitable pharmaceutical excipients as described herein.

Pharmaceutically acceptable salts of the instant compounds, where they can be prepared, are also intended to be covered by this invention. These salts will be ones which are acceptable in their application to a pharmaceutical use. By that it is meant that the salt will retain the biological activity of the parent compound and the salt will not have untoward or deleterious effects in its application and use in treating diseases.

Pharmaceutically acceptable salts are prepared in a standard manner. If the parent compound is a base it is treated with an excess of an organic or inorganic acid in a suitable solvent. If the parent compound is an acid, it is treated with an inorganic or organic base in a suitable solvent.

The compounds of the invention may be administered in the form of an alkali metal or earth alkali metal salt thereof, concurrently, simultaneously, or together with a pharmaceutically acceptable carrier or diluent, especially and preferably in the form of a pharmaceutical composition thereof, whether by oral, rectal, or parenteral (including subcutaneous) route, in an effective amount.

Examples of pharmaceutically acceptable acid addition salts for use in the present inventive pharmaceutical composition include those derived from mineral acids, such as for example hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric,

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PCT/EP2018/065684 benzoic, glycolic, gluconic, succinic, p-toluenesulphomc acids, arylsulphomc, and antifoam products such as for example Struktol®.

The composition for use as described herein can for example be formulated for enteral, topical, oral or parenteral administration or as part of a sustained release implant.

Pharmaceutical formulations for oral administration

The composition of the present invention may be formulated in a wide variety of formulations for oral administration. Solid form preparations may include powders, tablets, drops, capsules, cachets, lozenges, and dispersible granules. Other forms suitable for oral administration may include liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, toothpaste, gel dentrifrice, chewing gum, or solid form preparations which are intended to be converted shortly before use to liquid form preparations, such as solutions, suspensions, and emulsions.

In powders, the carrier is a finely divided solid which is a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.

Drops according to the present invention may comprise sterile or non-sterile aqueous or oil solutions or suspensions, and may be prepared by dissolving the active ingredient in a suitable aqueous solution, optionally including a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.

Emulsions may be prepared in solutions in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents. Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous

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PCT/EP2018/065684 material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

In one aspect, a pharmaceutical composition is deliverable from a powder inhaler, a nebulizer and/or a metered dose inhaler, is provided, wherein the composition comprises: a suspension medium comprising

I. a polymeric biguanide polymer and ii. an alkyl and/or dialkyl oxyethylene methyl ammonium salt iii. a pharmaceutically acceptable propellant, and iv. respirable suspending particles, wherein polymeric biguanide polymer (i.) and alkyl and/or dialkyl oxyethylene methyl ammonium salt (ii.) associate with the suspending particles (iv.) to form a cosuspension.

Polymeric biguanide polymer is preferably Poly-(hexamethylene-guanidium chloride) and dialkyl oxyethylene methyl ammonium salt is preferably N,N-didecyl-N-methylpoly(oxyethyl) ammonium propionate. These active compounds are preferably present in equimolar amounts or substantially equimolar amounts.

Pharmaceutical formulations for parenteral administration

The composition of the present invention may be formulated in a wide variety of formulations for parenteral administration. For example, the parenteral administration can be intravenous, subcutaneous, intramuscular, intracranial or intraperitoneal.

Injections and infusions

For injections and infusions the formulations may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Alternatively, the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules, vials, pre-filled syringes, infusion bags, or can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection

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PCT/EP2018/065684 solutions and suspensions can be prepared from sterile powders, granules, and tablets.

Examples of oily or non-aqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents.

The formulations for injection will typically contain from about 0.5 to about 25% by weight of the active ingredients in solution.

Topical delivery

The composition may also be administered topically. Regions for topical administration include the skin surface and also mucous membrane tissues of the vagina, rectum, nose, mouth, and throat. For example, the topical administration can be dermal, epicutaneous, vaginal, intravesical, pulmonary, intranasal, intratracheal or as eye drops. The composition may in particular be administered to the skin or hair. For animals, the composition may be administered to the fur. When administered to the fur or skin, the composition is preferably formulated as a shampoo or skin soap.

The topical composition will typically include a pharmaceutically acceptable carrier adapted for topical administration. Thus, the composition may take the form of a suspension, solution, ointment, lotion, sexual lubricant, cream, foam, aerosol, spray, suppository, implant, inhalant, tablet, capsule, dry powder, syrup, balm or lozenge, for example. In a preferred embodiment the composition takes the form of a shampoo. Methods for preparing such compositions are well known in the pharmaceutical industry.

The composition of the present invention may be formulated for topical administration to the epidermis as ointments, creams or lotions, or as a transdermal patch. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy base. The base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin or a fatty acid. The formulation may incorporate any suitable surface

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PCT/EP2018/065684 active agent such as an anionic, cationic or non-iomc surfactant such as a sorbitan ester or a polyoxyethylene derivative thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

Lotions according to the present invention also include those suitable for application to the eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide.

Nasal, pulmonary and bronchial administration

Formulations for use in nasal, pulmonary and/or bronchial administration are normally administered as aerosols in order to ensure that the aerosolized dose actually reaches the mucous membranes of the nasal passages, bronchial tract or the lung. The term aerosol particle is used herein to describe the liquid or solid particle suitable for nasal, bronchial or pulmonary administration, i.e., that will reach the mucous membranes.

In a preferred embodiment of the present invention the composition for use is formulated for nasal, pulmonary and/or bronchial administration. Preferably, said composition is formulated for inhalation. It is further preferred that the composition for use or the composition for use formulated for nasal, pulmonary and/or bronchial administration is administered as aerosols. Such administration is particularly useful for treatment of infections in the respiratory system, such as lung infections.

Typically aerosols are administered by use of a mechanical devices designed for pulmonary and/or bronchial delivery, including but not limited to nebulizers, metered dose inhalers, and powder inhalers. With regard to construction of the delivery device, any form of aerosolization known in the art, including but not limited to spray bottles, nebulization, atomization or pump aerosolization of a liquid formulation, and aerosolization of a dry powder formulation, can be used.

Liquid Aerosol Formulations in general contain a composition of the present invention in a pharmaceutically acceptable diluent. Pharmaceutically acceptable diluents include but are not limited to sterile water, saline, buffered saline, dextrose solution, and the like.

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Formulations for dispensing from a powder inhaler device will normally comprise a finely divided dry powder containing pharmaceutical composition of the present invention (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device. Dry powder formulations for inhalation may also be formulated using powderfilled capsules, in particularly capsules the material of which is selected from among the synthetic plastics.

The formulation is formulated to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in therapy and known to the person skilled in the art. The propellant may be any propellant generally used in the art. Specific non-limiting examples of such useful propellants are a chlorofluorocarbon, a hydrofluorocarbon, a hydrochlorofluorocarbon, ora hydrocarbon.

The formulations of the present embodiment may also include other agents useful for pH maintenance, solution stabilization, or for the regulation of osmotic pressure.

Transdermal Delivery

The pharmaceutical agent-chemical modifier complexes described herein can be administered transdermally. Transdermal administration typically involves the delivery of a pharmaceutical agent for percutaneous passage of the drug into the systemic circulation of the patient. The skin sites include anatomic regions for transdermally administering the drug and include the forearm, abdomen, chest, back, buttock, mastoidal area, and the like.

Transdermal delivery is accomplished by exposing a source of the complex to a patient's skin for an extended period of time. Transdermal patches have the added advantage of providing controlled delivery of a pharmaceutical agent-chemical modifier complex to the body. Such dosage forms can be made by dissolving, dispersing, or otherwise incorporating the pharmaceutical agent-chemical modifier complex in a proper medium, such as an elastomeric matrix material. Absorption enhancers can also

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PCT/EP2018/065684 be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel. For example, a simple adhesive patch can be prepared from a backing material and an acrylate adhesive.

Vaginal administration

The composition of the present invention may be formulated for vaginal administration. Pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Nasal administration

The composition of the present invention may be formulated for nasal administration. The solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The formulations may be provided in a single or multidose form. In the latter case of a dropper or pipette this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray this may be achieved for example by means of a metering atomizing spray pump.

Enteric coating

When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient.

Dosages and dosing regimes

The dosage requirements will vary with the particular biocide composition, the route of administration and the particular subject or object being treated. It will also be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of active ingredients or a pharmaceutically acceptable salt thereof will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a compound or a pharmaceutically acceptable salt thereof given per day for a defined number of days, can be ascertained using conventional

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It is appreciated that the composition of the present invention comprises at least 50 % active ingredients, such as at least 30 wt. % active ingredients, such as at least 25 wt. % active ingredients, such as for example at least 20 wt. % active ingredients, at least 15 wt. % active ingredients, such as at least 25 wt. % active ingredients, such as for example at least 20 wt. % active ingredients, at least 15 wt. % active ingredients, such as at least 10 wt. % active ingredients, such as for example at least 8 wt. % active ingredients, at least 5 wt. % active ingredients, such as at least 4 wt. % active ingredients, such as for example at least 3 wt. % active ingredients, at least 2 wt. % active ingredients, such as at least 1 wt. % active ingredients, such as for example at least 0,5 wt. % active ingredients or at least 0,5 wt. % active ingredients.

Wt. % is an abbreviation for weight percent.

The active ingredients are polymeric biguanide polymer an alkyl and/or dialkyl oxyethylene methyl ammonium salt or pharmaceutical acceptable salts or tautomers thereof. Preferred embodiments of polymeric biguanide polymer an alkyl and/or dialkyl oxyethylene methyl ammonium salt present in the composition are as described elsewhere herein. It is preferred that the active ingredients is a mixture of poly(hexamethylene-guanidium chloride) (e.g. Akacid Forte) and N,N-didecyl-N-methylpoly(oxyethyl) ammonium propionate (e.g. Bardap 26). Preferred rations between the active ingredients are as described herein above.

The daily oral dosage regimen of the active ingredients or the composition is preferably from about 0.01 to about 80 mg/kg of total body weight. The daily parenteral dosage regimen about 0.001 to about 80 mg/kg of total body weight. The daily topical dosage regimen will preferably be from 0.1 mg to 150 mg, administered one to four, preferably two or three times daily. The daily inhalation dosage regimen will preferably be from about 0.01 mg/kg to about 1 mg/kg per day.

In one embodiment the composition or active ingredients is to be administered in a dosage of from 1 pg/kg -10,000 pg/kg body weight, such as 1 pg/kg - 7,500 pg/kg, such as 1 pg/kg - 5,000 pg/kg, such as 1 pg/kg - 2,000 pg/kg, such as 1 pg/kg -1,000 pg/kg, such as 1 pg/kg - 700 pg/kg, such as 5 pg/kg - 500 pg/kg, such as 10 pg/kg to 100 pg/kg bodyweight.

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In another embodiment the composition as described herein is to be administered in a dosage of from 1 pg/kg -1,000 pg/kg body weight, such as 1 pg/kg - 500 pg/kg, such as 1 pg/kg - 250 pg/kg, such as 1 pg/kg -100 pg/kg, such as 1 pg/kg - 50 pg/kg, such as 1 pg/kg to 10 pg/kg bodyweight.

In yet another embodiment the compound as described herein is to be administered in a dosage of from 10 pg/kg - 30,000 pg/kg body weight, such as 10 pg/kg - 7,500 pg/kg, such as 10 pg/kg - 5,000 pg/kg, such as 10 pg/kg - 2,000 pg/kg, such as 10 pg/kg 1,000 pg/kg, such as 10 pg/kg - 700 pg/kg, such as 10 pg/kg - 500 pg/kg, such as 10 pg/kg to 100 pg/kg bodyweight.

The term unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a compound, alone or in combination with other agents, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular compound or compounds employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host. The dose administered should be an effective amount or an amount necessary to achieve an effective level in the individual patient.

When the effective level is used as the preferred endpoint for dosing, the actual dose and schedule can vary, depending on inter-individual differences in pharmacokinetics, drug distribution, and metabolism. The effective level can be defined, for example, as the blood or tissue level desired in the patient that corresponds to a concentration of one or more compounds according to the invention.

In one embodiment the administration of the composition as described herein is repeated at least 1,2, 3, 4, 5 or 6 times weekly.

In one embodiment the administration of the composition according to the present invention is repeated daily. In another embodiment said administration is repeated at least 1-3 times weekly, such as 2-5 times weekly, such as 3-6 times weekly.

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In a further embodiment the administration is repeated daily. The administration of the composition may for example be repeated 1,2, 3, 4, 5, 6, 7 or 8 times daily. In one embodiment the administration is repeated 1 to 8 times daily, such as 2 to 5 times daily.

When the composition provided herein is used for nontherapeutic treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as MRSA, (e.g. for disinfection of objects such as a devices, clothes, installations and/or premises) the working range could vary from 1 - 100.000 ppm. However, a concentrate may be provided, which is to be diluted by the used. In one embodiment, the working concentration is at least 1 ppm, such as at least 5, such as at least 10, 20, 30, 40, 50, 60, 70, 80, 90, such as at least 100 ppm, for example at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, such as at least 20000 ppm. It is preferred that the concentration used is at low as possible while maintaining a desired and acceptable biological effect against a microorganism with antimicrobial resistance, such as MRSA. Thus in another embodiment, the working concentration is less than 20000 ppm, such as below 15000, such as below 10000, such as below 5000, such as below 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 ppm. In one preferred embodiment, the working concentration is between 25 and 1000 ppm, such as between 25 and 500 ppm or preferably between 25 and 100 ppm.

Therapeutic methods

In one aspect, a method is provided for the treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as MRSA, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a mixture of:

I. a polymeric biguanide polymer and

II. an alkyl and/or dialkyl oxyethylene methyl ammonium salt or pharmaceutical acceptable salts or tautomers thereof to human or animal in need thereof.

The composition is as defined herein above, as either a general composition or a pharmaceutical formulation.

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A method is also provided for the treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as MRSA, the method comprising:

i. providing a powder inhaler, a nebulizer or a metered dose inhaler comprising a pharmaceutically acceptable co- suspension, the co-suspension comprising:

a. a polymeric biguanide polymer and

b. an alkyl and/or dialkyl oxyethylene methyl ammonium salt

c. a pharmaceutically acceptable propellant, and

d. respirable suspending particles, wherein polymeric biguanide polymer (a.) and alkyl and/or dialkyl oxyethylene methyl ammonium salt (b.) associate with the suspending particles (d.) to form a co-suspension, and

II. administering the co-suspension to the patient by actuating the powder inhaler, nebulizer or metered dose inhaler, wherein said administering of the cosuspension composition comprises delivering a therapeutically effective amount of biguanide polymer (a.) and alkyl and/or dialkyl oxyethylene methyl ammonium salt (b.) to the patient.

Non-therapeutic methods

Generally, methods and uses are also provided herein for nontherapeutic treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as MRSA. Accordingly, in one aspect a use is provided of a composition comprising a mixture of:

i. a polymeric biguanide polymer and

II. an alkyl and/or dialkyl oxyethylene methyl ammonium salt or salts or tautomers thereof for nontherapeutic treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as methicillin resistant Staphylococcus aureus (MRSA), of an object or subject.

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A method is also provided for non-therapeutic treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as methicillin resistant Staphylococcus aureus (MRSA), said method comprising

a. providing a mixture of:

b. exposing an object or subject to said mixture.

This use or method is preferably used for disinfecting objects or subjects with the mixture.

Microorganism with antimicrobial resistance, such as MRSA, are prevalent in hospitals, prisons, and nursing homes, where people with open wounds, invasive devices such as catheters, and weakened immune systems are at greater risk of hospital-acquired infection. It is therefore preferred that the methods and uses is applied to objects such as hospitals, prisons, and nursing homes. In general, the object for non-therapeutic treatment, prevention and/or amelioration is a device, clothe, installation and/or premise infected or at risk of being infected with microorganism with antimicrobial resistance, such as MRSA.

Specifically, the object may be a building set apart and adapted for keeping domesticated animals (a stable), in particular for keeping pigs. The object may also be a slaughterhouse. Further, the clothe may be work wear, which is used in stables, including foot wear, such as boots, in particular rubber boots, which can be treated/disinfected by stepping into a small washbasin between the stable and the outside.

The object can also be an installation, such as any installation present in hospitals, prisons, nursing homes and stables, from where resistant microorganisms may spread. For example, the installation is a stable installation, such as cages, apparatuses used for handling of domesticated animals.

In another preferred embodiment, the object is a medical device or equipment, generally those, which are subject to skin contact. Such devices and equipment include

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MR scanners, x-ray devices and the like. Such devices and equipment can be sterilized/disinfected using the mixtures provided herein.

A number of subjects are of increased risk of acquiring infection from resistant microorganisms. Broadly, methods and uses of the composition provided herein can be applied to human and animal subjects, in particular mammalian subjects. The methods and uses are particularly preferred for treatment, prevention and/or amelioration of MRSA infection in domesticated animals, in particular pigs.

The following human subjects are of particular risk of acquiring infection with resistant microorganisms, such as MRSA, and the methods and uses provided herein are therefore in preferred embodiments applied to one or more of the following subjects:

• People who are frequently in crowded places, especially with shared equipment and skin-to-skin contact • People with weak immune systems (HIV/AIDS, lupus, or cancer sufferers; transplant recipients, severe asthmatics) • Diabetics • Intravenous drug users • Users of quinolone antibiotics • The elderly • School children sharing sports and other equipment • College students living in dormitories • Women with frequent urinary tract or kidney infections due to infections in the bladder • People staying or working in a health care facility for an extended period of time • People with tattoos or body piercing • People who spend time in coastal waters where MRSA is present, such as some eaches in Florida and the west coast of the United States • People who spend time in confined spaces with other people, including occupants of homeless shelters and warming centers, prison inmates, military recruits in basic training, and individuals who spend considerable time in changing rooms or gyms • Elite athletes • Veterinarians, livestock handlers, and pet owners

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Additional active agents

The compositions, methods, uses and kits provided herein may involve or comprise at least one further active agent. The further active agent is preferably suitable the treatment, prevention and/or amelioration of a microbial infection, in particular for use in therapeutic and/or non-therapeutic treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as MRSA.

In one embodiment, the further active agent is a sulfa drugs (like co-trimoxazole (trimethoprim/sulfamethoxazole)), tetracyclines (like doxycycline and minocycline) and clindamycin (for osteomyelitis). Generally, CA-MRSA has a greater spectrum of antimicrobial susceptibility to sulfa drugs. In one preferred embodiment, the further active agent is vancomycin, which is the current drug of choice for treating CA-MRSA. HA-MRSA is also often susceptible to vancomycin, but to lesser extent to other drugs. The further active agent could also be linezolid (belonging to the newer oxazolidinones class of drugs) and daptomycin, which are effective against both CA-MRSA and HAMRSA.

In another preferred embodiment, the at least one further active agent is one or more of vancomycin, linezolid, or clindamycin, which are recommended by The Infectious Disease Society of America for treating people with MRSA pneumonia.

The further agent may also be selected from Ceftaroline (fifth-generation cephalosporin), Vancomycin and teicoplanin. Teicoplanin is a structural congener of vancomycin that has a similar activity spectrum but a longer half-life. Because the oral absorption of vancomycin and teicoplanin may be low, these agents are preferably administered intravenously to control systemic infections.

In one embodiment, the further agent is selected from the group consisting of Linezolid, quinupristin/dalfopristin, daptomycin, ceftaroline, and tigecycline, which are currently used to treat more severe infections that do not respond to glycopeptides such as vancomycin. In particular, daptomycin is preferred for VISA bloodstream infections and endocarditis, and is therefore a preferred further agent.

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Examples

Example 1

In vitro susceptibility testing of a disinfectant with possible effect on methicillin resistant Staphylococcus aureus (MRSA).

Aim

To test the effect of a disinfectant composition comprising a 1:1 mixture of poly(hexamethylene-guanidium chloride) and N,N-didecyl-N-methyl-poly(oxyethyl) ammonium propionate on a small collection of Danish MRSA representing both Danish and international clonal lineages (CC) predominating in human infections.

Methods and Results

The disinfectant received was diluted 15X in MH broth to a 51200 ppm suspension followed by two - fold dilutions in a range from 25600 ppm -50 ppm.

MRSAs belonging to CC1, CC5, CC8 (USA300), CC22, CC30, CC45, CC80, CC398 were tested for purity on 5% blood agar plates, diluted to 10e6 CFU/mL and inoculated to the dilution series in a microwell plate (table 2) that was incubated overnight in 35 C, in a microwell plate reader with readings every 15 min after shaking.

Table 2

A

B C D

E F G H

112722

112725

112696

112741

112728

112688

112650

112742

2 3 4 56789 10 11 12 /no bacteria

25600	12800	6400	3200	1600	800	400	200	100	50	25	ΜΗ II
25600	12800	6400	3200	1600	800	400	200	100	50	25	ΜΗ II
25600	12800	6400	3200	1600	800	400	200	100	50	25	ΜΗ II
25600	12800	6400	3200	1600	800	400	200	100	50	25	ΜΗ II
25600	12800	6400	3200	1600	800	400	200	100	50	25	Desinfectant X
25600	12800	6400	3200	1600	800	400	200	100	50	25	Desinfectant X
25600	12800	6400	3200	1600	800	400	200	100	50	25	Desinfectant X
25600	12800	6400	3200	1600	800	400	200	100	50	25	Desinfectant X

Growth was inferred from visual inspections of the plates..

It was decided to check growth in the wells by CFU determination of 4 of the strains (CC1, CC8, CC30 and CC80) from wells grown with 3 different concentrations of the disinfectant (high: 25600 ppm; medium: 3200 ppm and low : 50 ppm).

After overnight incubation no visible growth was detected.

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An additional attempts to test the activity of the disinfectant was done by preparing paper filter discs with dilutions of the disinfectant and place them on blood agar plates with a semi confluent layer of the various MRSA strains tested.

Inhibition zones were visible at all concentrations tested (20 pl_ of 50, 800 and 25600 ppm/ disk) thus a concentration dependent inhibition of growth was observed.

Table 2. Results paper disc diffusion

Isolat	MH	II	Technician
	51200 ppm	25600 ppm	800 ppm	50 ppm
A	112722	CC1	17	16	9	6*	LRY
B	112725	CC5	17	16	9	6	LRY
C	112696	CC8	17	16	9	6	LRY
D	112741	CC22	16	15	9	6	LRY
E	112728	CC30	16	15	9	6	LRY
F	112688	CC45	17	16	9	6	LRY
G	112650	CC80	16	15	9	6	LRY
H	112742	CC398	16	15	9	6	LRY

*Growth to the edge of the disc

Conclusions

The tests performed show that the disinfectant has a profound effect on viability of the tested MRSA isolates. A concentration dependent killing was shown but no MIC since the bacteria were all killed even at the lowest concentrations tested. Thus, a disinfectant composition comprising a 1:1 mixture of poly-(hexamethylene-guanidium chloride) and N,N-didecyl-N-methyl-poly(oxyethyl) ammonium propionate is functional and extremely effective at even very low concentrations.

Example 2

In vitro susceptibility testing of disinfectant with possible effect on methicillin resistant Staphylococcus aureus (MRSA).

The disinfectant was tested on an MRSA strain represented by international clonal lineage (CC): CC8(USA300), which is relevant in human infections. The tested disinfectant was a liquid composition comprising equimolar amounts of Poly(hexamethylene-guanidium chloride) and N,N-didecyl-N-methyl-poly(oxyethyl) ammonium propionate. The disinfectant was diluted MH broth to a 51200 ppm

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PCT/EP2018/065684 suspension and thereof, two-fold dilutions were prepared ranging from 25600 to 25 pmm. CC8 (USA300) was tested by direct plating on 5% blood agar plates. After overnight growth at 35°C, only slight growth was visible on the plate containing 50ppm disinfectant, where 3 colonies were observed, while no growth was observed on the remaining plates; cf. table 3. Thus, it is seen that the disinfectant seems to have a potent effect on S aureus (MRSA-CC8-USA300).

Table 3. Dilution series of disinfectant tested for effect on growth of MRSA-CC8USA300 on blood agar plates.

Concentration of disinfectant/ppm	Colony forming units
25,600	0
12,800	0
6,400	0
3,200	0
1,600	0
800	0
400	0
200	0
100	0
50	0
25	3

Example 3

Testing of disinfectant by agar-dilution

Materials:

Staphylococcus aureus MSSA ATCC 6538

Staphylococcus aureus MRSA ATCC 33591 Pseudomonas aeruginosa ATCC 15442 Enterococcus hirae ATCC 10541

Escherichia co//ATCC 10536

Klebsiella pneumoniae ATCC 700603

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PCT/EP2018/065684 (BD) ΜΗ II bouillon (BD) ΜΗ II agar

Reagent A (equimolar mixture of Akacid Forte and Bardap 26)

Disinfectant:

The product (reagent A) is a concentrated aqueous solution, which is an equimolar mixture of Akacid Forte and Bardap 26. The product precipitates at temperatures below 6 °C, incompatible with metals

Do not expose the solution to direct sunlight and heat. Avoid addition of strong acids and bases.

Store in a ventilated area.

Shake well before use.

Hazard and Precautionary statements: H302 (harmful if swallowed), H314 (Causes severe skin burns and eye damage), H310 (Causes serious eye damage), P273 (Avoid release to the environment), P280 (Wear protective gloves/ protective clothing/ eye protection/face protection).

The usage of nitrile cloves and eye protection is required when working with the concentrated solution.

Methods:

Reagent A should be stored at room temperature. Shake well before usage.

Two experiments with agar-embedment of the product were conducted.

1) After an initial 50-fold dilution to 2 %, 10-fold dilutions of the product were produced and embedded in ΜΗ II agar with an end-concentration ranging from 10’³ to 10’⁷.

2) After an initial dilution to 10'⁴, 2-fold dilutions of the product were produced and embedded in ΜΗ II agar with end-concentrations ranging from 10'⁴ to 1.5625 x 10’⁶.

The six bacterial strains selected for investigation were adjusted to a concentration of 10⁷ CFU/ml and 1 μΙ of this suspension was transferred in triplicate to an agar plate. The plates were incubated for 16-20 hours at 35 °C before read-out.

The bacterial growth was registered as either confluent growth, single colonies or no growth.

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Results:

This table shows the results from the first experiment with the 10-fold dilutions:

Cone.	S. aureus ATCC 6538	S. aureus ATCC 33591	P. aeruginosa ATCC 15442	E. hirae ATCC 10541	E. coll ATCC 10536	K. pneumoniae ATCC 700603
10’³	-r	-r	-r	-r	-r	-r
10’⁴	-r	-r	-r	-r	-r	-r
10’⁵	-r	-r	+	-r	(+)	+
10’⁶	+	+	+	+	+	+
10’⁷	+	+	+	+	+	+
+: Coni	luent growth, (+): growth o	’single colonies, +: No growth

All the bacteria were inhibited at concentrations down to 10’⁴, at 10'⁵ there was no inhibition of P. aeruginosa and only partial inhibition of E. coll, while at 10'⁶ there was no inhibition of any of the 6 species.

To determine the MIC-values, 2-fold dilutions in the concentrations ranging from 10'⁴ to 10'⁶ were produced in a follow-up study.

This table shows the results from the second experiment with the 2-fold dilutions from 10 10’⁴CFU/ml:

Cone.	S. aureus ATCC 6538	S. aureus ATCC 33591	P. aeruginosa ATCC 15442	E. hirae ATCC 10541	E. coll ATCC 10536	K. pneumon iae ATCC 700603
10’⁴	-r	-r	+	-r	-r	-r
5x 10’⁵	-r	-r	+	-r	-r	-r
2,5x 10’⁵	-r	-r	+	-r	-r	-r
1,25x 10’⁵	-r	-r	+	-r	+	-r
6,25x 10’⁶	-r	+	+	-r	+	-r
3,125x 10’⁶	-r	+	+	+	+	-r
1,5625x 10’ 6	+	+	+	+	+	+

+: Confluent growth, +: No growth

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It is normal that the MIC-value can vary for a 2-fold dilution step between experiments. This is the case for P. aeruginosa, where we detected a difference in growth for the 10 ⁴ dilution between the two experiments.

Conclusions:

We could determine the products bacteriostatic effect by embedding different concentrations of the product into ΜΗ II agar. Thus, we avoided the problems with precipitation of the product that we have previously observed with bouillon-dilution in microtiter plates.

The MIC-values for the product for the six reference strains based on these experiments are:

P. aeruginosa ATCC 15442	10’⁴
S. aureus MRSA ATCC 33591	1,25x 10’⁵
E. coll ATCC 10536	2,5x 10’⁵
K. pneumoniae ATCC 700603	3,125x 10’⁶
S. aureus MSSA ATCC 6538	3,125x 10’⁶
E. hirae ATCC 10541	6,25x 10’⁶

The MIC-values are assumed to vary between strains for the same bacterial species.

Claims

1. A composition comprising a mixture of:

2. The composition for use according to claim 1, wherein said polymeric biguanide polymer is Poly-(hexamethylene-guanidium chloride).

3. The composition for use according to any of the preceding claims, wherein said dialkyl oxyethylene methyl ammonium salt is N,N-didecyl-N-methyl-poly(oxyethyl) ammonium propionate.

4. The composition for use according to any of the preceding claims, wherein said composition comprises a mixture of Poly-(hexamethylene-guanidium chloride) and N,N-didecyl-N-methyl-poly(oxyethyl) ammonium propionate.

5. The composition for use according to claim 4, wherein said composition comprises a mixture of Poly-(hexamethylene-guanidium chloride) and N,Ndidecyl-N-methyl-poly(oxyethyl) ammonium propionate in a ratio of about 1:1.

6. The composition for use according to any of the preceding claims, further comprising at least one further active agent.

7. The composition for use according to any of the preceding claims wherein said further active agent is suitable the treatment, prevention and/or amelioration of a microbial infection, in particular for use in the treatment, prevention and/or amelioration of an infection with methicillin resistant Staphylococcus aureus (MRSA).

8. The composition for use according to claim 1, wherein MRSA is vancomycin intermediate-resistant Staphylococcus aureus (VISA).

9. The composition for use according to any of the preceding claims, wherein the composition is formulated for enteral, topical, oral or parenteral administration.

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10. The composition for use according to claim 8, wherein the parenteral administration is intravenous, subcutaneous, intramuscular, intracranial or intraperitoneal.

11. The composition for use according to claim 8, wherein the topical administration is dermal, epicutaneous, vaginal, intravesical, pulmonary, intranasal, intratracheal or as eye drops.

12. The composition for use according to any of the preceding claims, wherein the composition is formulated for nasal, pulmonary and/or bronchial administration.

13. The composition for use according to any of the preceding claims, wherein the composition is formulated for inhalation.

14. The composition for use according to any of the preceding claims, wherein the composition is administered as aerosols.

15. The composition for use according to any of the preceding claims, wherein said composition is to be administered in a dosage of from 1 pg/kg -10,000 pg/kg body weight, such as 1 pg/kg - 7,500 pg/kg, such as 1 pg/kg - 5,000 pg/kg, such as 1 pg/kg - 2,000 pg/kg, such as 1 pg/kg - 1,000 pg/kg, such as 1 pg/kg - 700 pg/kg, such as 5 pg/kg - 500 pg/kg, such as 10 pg/kg to 100 pg/kg bodyweight. The composition for use according to any of the preceding claims, wherein said administration is repeated daily.

16. The composition for use according to any of the preceding claims, wherein said administration is repeated at least 1-3 times weekly, such as 2-5 times weekly, such as 3-6 times weekly.

17. The composition for use according to any of the preceding claims, wherein said administration is repeated 1 to 8 times daily, such as 2 to 5 times daily.

18. A method for the treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as methicillin resistant Staphylococcus aureus (MRSA), comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a mixture of:

I. a polymeric biguanide polymer and

II. an alkyl and/or dialkyl oxyethylene methyl ammonium salt or pharmaceutical acceptable salts or tautomers thereof to a human or animal in need thereof.

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19. The method according to claim 18, wherein said subject is a human or mammalian subject with open wounds, invasive devices such as catheters, and weakened immune systems.

20. The method according to any of claim 18, wherein said composition is as described in any of claims 1 to 17.

21. Use of a composition comprising a mixture of:

i. a polymeric biguanide polymer and

II. an alkyl and/or dialkyl oxyethylene methyl ammonium salt or salts or tautomers thereof for nontherapeutic treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as methicillin resistant Staphylococcus aureus (MRSA) of an object or subject.

22. A method for non-therapeutic treatment, prevention and/or amelioration of an infection with a microorganism with antimicrobial resistance, such as methicillin resistant Staphylococcus aureus (MRSA), said method comprising

a. providing a mixture of:

i. a polymeric biguanide polymer and

II. an alkyl and/or dialkyl oxyethylene methyl ammonium salt or pharmaceutical acceptable salts or tautomers thereof, and

b. exposing an object or subject to said mixture.

23. The use or method of anyone of claims 21 and 22, wherein said object or subject is disinfected by said mixture

24. The use or method of anyone of claims 21 and 23, wherein said object is a device, clothe, installation and/or premise infected or at risk of being infected with methicillin resistant Staphylococcus aureus (MRSA)

25. The use or method of anyone of claims 21 and 24, wherein said subject is a human being or a domesticated animal, such as a pig.

26. The use or method of anyone of claims 21 and 25, wherein said clothe is footwear, such as rubber boots.

27. The use or method of anyone of claims 21 and 26, wherein said premise is a hospital, a nursing home, a prison or a building set apart and adapted for keeping domesticated animals, such as a pigs.

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28. The use or method of anyone of claims 21 and 27, wherein said installation is a stable installation, such as cages, apparatuses used for handling of domesticated animals.

29. The use or method of anyone of claims 21 and 28, comprising the use of at least

5 one further active agent, such as an agent suitable the treatment, prevention and/or amelioration of a microbial infection, in particular for use in the treatment, prevention and/or amelioration of an infection with methicillin resistant Staphylococcus aureus (MRSA).

30. The use or method of anyone of claims 21 and 29, comprising the concentration

10 of said mixture is between 25 and 100 ppm