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Anti-microbial photodynamic therapy

Anti-microbial photodynamic therapy description/claims

1. Field of the Invention

The present invention relates generally to photodynamic therapy, and particularly to molecular conjugates for the treatment and prevention of microbial infectious diseases in human and animals. A molecular conjugate of the present invention comprises a special spacer to connect at least one photosensitizer, with a vector.

2. Invention Disclosure Statement

Photodynamic therapy (PDT) is a relatively new treating modality for cancers and other diseases. Photosensitizers are administered systemically, locally or topically and accumulate in the tumor or other lesion; illuminating the area with light energy to excite the sensitizer, which, in the presence of oxygen, produces cytotoxic effects in the cells. Another important application of PDT is the treatment of infectious diseases caused by pathogenic microorganisms.

Antimicrobial photodynamic therapy is very promising method for combating bacterial infection even for resistant strains. Fortunately, no resistance to photodynamic destruction has been reported to be acquired by bacteria nor is it likely since the ‘killing species’ is oxygen. Bacterial cells treated with photosensitizers were shown to be successfully killed by photo illumination. None of the known photosensitizers and photosensitizer conjugates is effective against all bacteria, as activity mainly depends on their chemical structure. Effectiveness of the photosensitizer also depends on the bacterial cell wall as it becomes the limiting factor for the sensitizer penetration. In the case of Gram-negative bacteria their double-layer outer membrane structure is the main obstacle.

Cell structures of Gram-positive and Gram-negative bacteria are different and it is differentiated by their Gram staining characteristics. The Gram-positive cell wall is characterized by the presence of a very thick layer of peptidoglycan. Embedded in the Gram-positive cell wall are polyalcohols called teichoic acids, some of which are lipid linked to form lipoteichoic acids. Teichoic acids give the Gram-positive cell wall an overall negative charge due to the presence of phosphodiester bonds between teichoic acid monomers. While Gram-negative bacterial cell wall contains a thin peptidoglycan layer adjacent to cytoplasmic membrane, in addition to this it has another outer membrane composed by phospholipids and lipopolysaccharides. The highly charged nature of lipopolysaccharides confers an overall negative charge to Gram-negative bacterial cell wall. The chemical structure of the outer membrane lipopolysaccharides is often unique to specific bacterial strains (i.e. sub-species) and is responsible for many of the antigenic properties of these strains.

One of the major problems for the use of anti-microbial PDT is a blocking action of the components of the blood whose presence decreases the activity of photosensitizers. This effect is exemplified on FIG. 1 demonstrating photodynamic inactivation of Staphylococcus aureus (Gram-positive bacterium), Pseudomonas aeruginosa and Escherichia coli with known photosensitiser (Gram-negative bacterium) Safranin O which exists in two tautomeric forms.

As seeing from FIG. 1 Safranin O exhibits high bactericidal photodynamic activity in PBS buffer that decreased remarkably when of the blood serum or blood is added. Particularly, the addition of even 10% of horse serum or human plasma or even whole blood could practically block the photodynamic activity of this sensitizer against Pseudomonas aeruginosa. The antibacterial effect strongly depends on the kind of bacterial cells. While Gram-positive cells of Staphylococcus aureus could be killed sufficiently, Gram-negative cells of Pseudomonas aeruginosa or Escherichia coli are more resistant to killing by PDT.

One of the prospective approaches to increase the specificity of photosensitizers and the effectiveness of PDT against bacterial infection is to conjugate a photosensitizer with a ligand-vector, which specifically binds to receptors on the surface of a target cell. In the prior art different methods have been used to effectively target the pathogen or the infected cells.

In U.S. Pat. No. 6,977,075 by Hasan et al., discloses a method of killing intracellular pathogen using antibiotics and PDT. The intracellular pathogens are targeted using conjugated photosensitizers. Targeting moiety used are molecules or a macromolecular structure that target macrophages or that interacts with a pathogen. Effectiveness of the conjugate against Gram-negative, and, in complex environment is not disclosed.

In U.S. Pat. No. 6,573,258 by Bommer, et al., he describes positively charged porphyrins which can effectively target both Gram-positive and Gram-negative bacteria when present at much lower concentrations and at much shorter irradiation times. The novel porphyrins have one hydrophobic tail consisting of at least one hydrocarbon chain of between 6 and 22 carbon in length. Bacterial targeting depends upon the carbon chain length and is not very effective.

In U.S. Pat. No. 6,462,070 by Hasan, et al., discloses a photosensitizer conjugated to polylysine which is linked to a histatin targeting moiety to treat disorder of the oral cavity infected by microorganism. Different types of targeting moiety disclosed here include non-pair member polypeptide, small anti-microbial peptide, low density lipoprotein etc. Effectiveness of the conjugates on the complex environment like blood, serum etc. is not disclosed in here.

U.S. Pat. No. 5,466,681 describes a variety of conjugates useful for the treatment of infectious diseases due to pathogenic microorganisms. The conjugates comprise at least one agent coupled to a microorganism receptor—a carbohydrate vector; said vector is able to bind selectively to a microorganism. The agent is a penicillin antibiotic and said vector is an asialoganglioside or another carbohydrate chain. The conjugates are administered for the treatment of bacterial infections, particularly, caused by Streptococcus pneumoniae and by Helicobacter pylori.

A wide variety of natural and synthetic molecules recognized by target cells could be used as vectors. The use of oligopeptides and big protein molecules, including lectins, growth factors and especially antibodies to specific tumor cell antigens are known in the art. The '681 patent discloses a conjugate comprising at least one agent that is an anti-infective coupled to a microorganism receptor. Agents such as antibiotics, synthetic drugs and steroids are mentioned. Since photosensitizers do not themselves interact with microbes, they are not considered agents as described in the '681 patent and were not disclosed therein.

“Polycationic photosensitizer conjugates: effects of chain length and Gram classification on the photodynamic inactivation of bacteria”, Michael R. Hamblin, David A. O'Donnell, Naveen Murthy, Krishanan Rajagopalan, Norman Michaud, Margaret E. Sherwood and Tayyaba Hasan, Journal of Antimicrobial Chemotherapy 49 (2002) pp. 941-951; In this publication the relationship between the size of the polylysine chain and its effectiveness for mediating the killing of Gram-negative and Gram-positive bacteria. The result of the present study implies that, in the case of polycationic photosensitizer conjugates, it is necessary for the photosensitizer to gain access through the outer membrane permeability barrier. The efficiency with which this occurs depends on the size of the polycationic chain. Conjugates with 8, 37 lysines and free chlorin e6 used in the study were found to be effective against bacterial infection but only 37-lysine conjugate killed the bacteria.

Anti-microbial PDT is effective mostly against Gram-positive bacteria when compared to Gram-negative bacteria. Hence there is an urgent requirement to develop a molecular conjugate which can actively target both Gram-positive and Gram-negative bacteria. Also needs to work in in vivo condition where typically or often blood and other body fluids are present, to use with patients directly to help protect them from deleterious microorganisms.

It is an objective of the present invention to provide a molecular conjugate for targeting pathogenic microorganism causing infectious diseases.

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