WO2009152298A1 - Methods, compositions and and diagnostic kits for the detection and treatment for staphylococcal nasal decolonization using achromopeptidase - Google Patents

Abstract

This invention provides achromopeptidase and related compositions for intranasal use that can be administered to the anterior nares of the nose and applied by any means to the nasal carriage to those at risk for staphylococcal nasal colonization and subsequent infection, and methods for their use, as well as diagnostic kits useful for performing such methods.

Description

Title: Methods, Compositions and and Diagnostic Kits for the Detection and Treatment for Staphylococcal Nasal Decolonization Using Achromopeptidase.

Inventor: Shawn Mark O'Hara

Background:

• Field of the Invention

The present invention relates to the administration of achromopeptidase (ACP) for the treatment or diagnosis of Staphylococcus Aureus (SA) infections in mammals, including humans, as well as pharmaceutical preparations used in the same treatment or diagnosis. The invention embraces the use of achromopeptidase broadly, including all recombinant forms, ACP variants from genetic mutations, and wild type form.

• Background Art

Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) is now endemic in many United States hospitals. According to the Centers for Disease Control and Prevention about 30 percent (30%) of the population carries SA and about 1 percent (1 %) carries MRSA. The burden of SA infections on hospitals in the United States has recently been demonstrated in reports showing that SA infections were reported in patient discharge diagnosis for 0.8% of all hospital inpatients, or 292,045 stays per year. Inpatients with SA infection had, on average, 3 times the length of hospital stay than inpatients without this infection (14.3 vs 4.5 days; P=O.001 ), 3 times the total charges ($48 vs $14; P=0.001 ), and 5 times the risk of in-hospital death (11.2% vs 2.3%; P=0.001 ). Even when controlling for hospital-fixed effects and for patient differences in diagnosis-related groups, age, sex, race, and co-morbidities, the differences in mean length of stay, total charges, and mortality were significantly higher for hospitalizations associated with SA. The potential benefits to hospitals in terms of reduced use of resources and costs as well as improved outcomes from preventing SA, MRSA and similarinfections are significant

Lysostaphin, an enzyme that lyses the cell walls of S.A., is effective in the treatment of established staphylococcal infections. Its action is due to a peptidase that cleaves cell wall polyglycine bridges and is found exclusively in coagulase-positive staphylococci.

Persistent nasal carriers, especially in a hospital setting, are an important source of

S.A. Lysostaphin, and conventional antibacterial therapy, whether topical or systemic, has not consistently eliminated S.A. from nasal carriages in a safe, cost-effective manner. Staphylococcal infections are a significant cause of death in the US, particularly in settings such as hospitals, nursing homes, schools, and infirmaries.

Patients particularly at risk include infants, the elderly, the immuno-compromised, the immuno-suppressed, those convalescing, and those with chronic conditions, requiring frequent hospital stays. Further, multiple drug resistant strains of Staphylococcus

Aureus (SA) increase the concern and need for detection, blocking and treatment of such infections.

Among the areas applicable are infections in hospitalized patients. In the United

States alone, approximately 14,000 people are infected and die each year as a result of drug-resistant microbes acquired in hospitals. As many as 60% of hospital-acquired infections world-wide are caused by drug-resistant microbes.

In infections caused by S.A., the principal ecological niches for S.A. in humans is the anterior nares. Nasal carriage of staphylococci plays a key role in epidemiology and pathogenesis of infection. In healthy subjects, three patterns of S.A. nasal carriage can be distinguished over time: approximately 20% of people are persistent carriers, approximately 60% are intermittent carriers, and approximately 20% apparently never carry S.A.

Nasal carriage of staphylococci is an important risk factor for contracting S.A. infection. Patients at greatest risk are those undergoing impatient or outpatient surgery, in an Intensive Care Unit, on continuous hemodialysis, with HIV infection, with AIDS, burn victims, people with diminished natural immunity from treatments or disease, chronically ill or debilitated patients, geriatric populations, infants with immature immune systems, and people with intravascular devices or other foreign bodies.

In one study of ICU patients, it was found that on admission 166 of 752, or 22%, of patients were S.A. nasal carriers. The probability of developing a staphylococcal infection was significantly greater in these patients than in non-carriers. In 28 out of 30 cases that subsequently developed staphylococcal infection, researchers found the S.A. strain colonizing the nares to be identical to the strain isolated from the infection. In addition, Mest, et al. showed that 19 patients who were admitted to the ICU with positive, nasal cultures for S.A. (26%) subsequently developed staphylococcal infections as compared to only 6 S.A. infections in a group of patients (1.3%) negative for nasal carriage of staphylococci.

Chang et al. studied 84 patients with cirrhosis admitted to a liver transplant unit. Overall, 39 (46%) were nasal carriers of S.A. and 23% of these patients subsequently developed S.A. infections as compared to only 4% of non-carriers. A study of HIV patients showed that 49% of patients had at least one positive nasal culture of S.A.

34% of 201 patients were considered nasal carriers, with 38% of these being persistent carriers and 62% intermittent carriers. Interestingly, molecular strain typing indicated that the strain of S.A. isolated from the infected site was the same as that previously cultured from the nares. The nasal S.A. carrier patients were significantly more likely to develop S.A. infection. These findings suggest that the nasal carriage can be an important risk factor for S.A. infection.

Antibiotic resistance in staphylococci is a problem that extends even to nasal colonization by S.A., where many strains found in studies of nasal colonization are antibiotic resistant. For example, methicillin resistant staphylococcus aureus (MRSA) is a well documented public health problem. In one study of post operative intraabdominal infection, it was concluded that MRSA may be a causative pathogen in postoperative intra-abdominal infection and that this may be related to nasal colonization.

So while it may be clear that nasal colonization by antibiotic sensitive and antibiotic resistant S.A. is a major health risk that needs to be addressed, there is very little technology addressing this issue. One compound, Bactroban Nasal (2% mupirocin cream), is used to clear staphylococcal nasal colonization. While this compound may be effective for S.A. nasal colonization, mupirocin resistant strains of S.A. are continuing to emerge. US Patent Application Serial No. 10/323,907 provides for the use of lysostaphin intranasal compositions that can be administered to the anterior nares of those at risk for staphylococcal nasal colonization and subsequent infection. However, some questions have developed with the use of lysostaphin. In a study by Goldberg et al., a dog model for endocarditis reported high dosages of lysostaphin (at more than 50 mg/kg treatment) were only moderately effective (Goldberg, et al., ntimicrob. Ag. Chemother. 1967; 45-53 (1967). In limited human trials aimed at eradication of nasal carriage S.A. by topical application of lysostaphin to the nares, it was concluded that nasal carriage infection alone does not constitute a disease state, but does constitute a risk factor for infection of patients treated by colonized health care professionals or for self-infection in the case of a colonized patient, brining into question the role of nasally-applied lysostaphin in the disease state. US Patent No. 6,028,051 provides for a therapeutic regimen of a single or short course of lysostaphin administration using relatively high doses. This method provides for effective therapy. However, the use of high doses of lysostaphin as an intranasal application may involve higher than acceptable risks.

In considering these issues, it remains an objective for those in the art to develop the most tolerable and most effective means of addressing S.A. infections in the nasal carriage. In addressing these infections, a therapeutic regimen that is cost effective, efficacious and having minimum side affects would provide a useful tool in treating S.A.

Summary of the Invention This invention relates to compositions that comprise ACP for use in the intranasal treatment of disease, and methods for use of such compositions. Achromopeptidase is known to have potent bacteriolytic activity for most of the gram-positive aerobic bacteria, and is also used for the lysis of anaerobic cocci. It has also been used for the lysis of S.A. and streptococcus faecalis. The present invention takes advantage of such lysis ability, and provides an advantageous method of lysing S.A. in nasal carriages. The method provided by the invention offers a number of advantages, including that the compositions of the invention that it uses can be simply, quickly, and safely applied to the nasal carriage for effective treatment. Diagnostic kits comprising the compositions provided by the invention are also contemplated, and can be used for practicing the advantageous methods provided thereby. The process provided by the invention is particularly advantageous since only a single step or a few steps are involved. It has the advantage of being expedient compared to prior processes, and in its practice requires little instrumentation to achieve the advantages sought. Although there exist numerous enzymes and procedures for lysing S.A. and nasal infections, ACP, which can effectively lyse bacteria such as mycobacteria, provides an unexpected advantage of being effective and safe in the nasal carriage.

Figures: Figure 1 is a flow chart depicting a general outline of steps in obtaining decolonization using ACP.

Detailed Description of the Invention:

The present invention embodies a rapid, safe, and inexpens ive method for topical application of ACP. The practic e of this me thod can result in signi ficant decolonization and subsequent reduction and elimination of S.A. colonization.

The present invention further encompasses the same method and topical application for other staphylococci infections such as, but not limited to, staphylococcal epidermidis. The method of the invention inv olves the purification of ACP to a sufficient purity level to be therapeutic ally effective for this purpose. Purification can be by any means known in the art. The purified ACP is us ed in a formulation that is optimized to determined an effectiv e concentration and v iscosity for nasal applic ations. F or topical delivery to the anterior nares, the present invention contemplates formulations that include saline, buffered saline, ointments, and creams. Delivery of the formulation c an be accomplishedby any means known in the art. However, a nasal spray or swab ointment or cream, applied through a coated finger and sp read in the nares are the preferred modes. (See Figure 1 ) The following examples demonstrate the ability of ACP to provide effective disruption of the cell wall of S.A. and potential decolonizati on in the n asal carriage, i n accordance with the present invention. Example 1

Achromopeptidase Disruption of the SA Cell Wall is Compatible with Direct- PCR & Nasal swab samples containing PCR inhibitors

Nasal samples were obtained from nasal swabs after elution with 200 micro liters of TE. Samples were then inc ubated with or without achromopeptidase (ACP) incubation at 1 Unit/ul 37C for 15 minutes followed by 99C for 5 minutes. Direct T aqMan PCR amplification of an exogenous spiked in control template DNA at a volume of up to 2. 5 micro liters of this ACP lysate in a 25 micro liter PCR r eaction confirmed compatibility . Further, transfer of volumes greater than 2.5ul into the 25ul PCR showed inhibition from both sample types suggesting that inhibition might start to negatively effect PCR above this volume proportion if not removed. T hese results confirm what has been previously demonstrated in the literature on ACP SA- PCR. Thus ACP Direct PCR from nasal swab sam ples can be improved by removal of PCR inhibitors using methods such as cell or DNA enrichment, activated charcoal etc. as described in the body of the text.

Example 2

ACP followed by Silica DNA Isolation When the above ACP disruptio n system was performed on TE buffer spiked with varying CF U numbers of SA strain ATCC- 29213 and t hen followed by Micr o kit DNA isolation (using comm ercially av ailable products from Qi agen) the reprodu cible lower limit measured by TaqMan nuc 137 real-time PCR was less than or equal to 10 colon y forming uni ts (C FU). These sampl e ampl ification resul ts are consi stent w ith and suggest that the vast majority of SA cells are also disrupted due to ACP treatment.

Example 3

Prevalence of Nasal SA by Culture & PCR

In a preliminary study using routine SA culture methods, 15 random subjects were tested for nasal swab SA and 4 s ubjects were shown to be positive by Culture resulting in a prevalence of SA at 27 %. This same n=15 sample set was also disrupted by ACP (1 u/ul), after being eluted in TE (1 OmM, 1 mM EDTA) by vo rtexing the nas al swab for 1 minuet. DNA was isolated us ing the above-referenced Qiagen Micro kit and SA specific T aqMan nuc 137 real-time PCR s howed 100% concordance with the culture results. P rocess blanks and controls indi cated no contamination present during this study. This SA prevalence num ber is in agreement with the expected percentage found in the literature.

Claims

What is claimed is:

1. A method for the prevention, protection from and treatment of disease in a subject or on an object, which method comprises topically administering to said subject or said object a composition that comprises a pharmaceutically-effective amount of achromopeptidase.

2. The method of claim I, wherein said prevention, protection and treatment in said subject is via intranasal decolonization of Staphylococcal reservoirs.

3. The method of claim 2, wherein said decolonization is to external body sites of said subject.

4. The method of claim 1 , wherein said composition comprises at least 4 separate Staphylococcal peptidoglycan digestion enzymes capable of digestion, lysing and killing gram positive Staphylococci.

5. The method of claim 4, wherein said gram positive Staphylococci comprise methicillin-resistant Staphylococcus aureus.

6. The method of claim 1 , wherein said prevention, protection and treatment in said subject is via human external microbiome decolonization of genus Staphylococcus.

7. The method of claim 6, wherein said gram positive Staphylococci comprise Staphylococcus epide rmidis prior to phlebotomy and installation of indwelling tubes.

8. A method for the diagnosis of gram positive Staphylococci, comprising obtaining a sample of said gram positive Staphylococci, and analyzing said sample using of a composition comprising a pharmaceutically-effective amount of achromopeptidase against said Staphylococci.

9. A composition for the prevention, protection from and treatment of disease in a subject or o n an object, said composition comprising a pharmaceutically-effective amount of achromopeptidase.

10. The composition of claim 7, wherein said composition is effective to provide intranasal decolonization of Staphylococcal reservoirs.

11. The composition of claim 8, wherein said composition comprises at least 4 separate Staphylococcal peptidoglycan digestion enzymes capable of digestion, lysing and killing gram positive Staphylococci.

12. The composition of claim 9, wherein said gram positive Staphylococci comprise methicillin-resistant Staphylococcus aureus.

13. The composition of claim 9, wherein said gram positive Staphylococci comprise Staphylococcus epidermidis biofilms.