Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
  • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents

Definitions

• 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.
• Photodynamic therapy 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' Js 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.
• 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.
• U.S. Patent 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.
• 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.
• Anti-microbial PDT is effective mostly against Gram-positive bacteria when compared to Gram-negative bacteria.
• a molecular conjugate which can actively target both Gram-positive and Gram- negative bacteria.
• Objectives and Brief Summary of the Invention It is an objective of the present invention to provide a molecular conjugate for targeting pathogenic microorganism causing infectious diseases. It is another objective of the present invention to develop photodynamic method for inactivation/reduction of bacteria (both Gram-positive and Gram-negative) in complex environment like blood, serum and saliva.
• the present invention provides antimicrobial molecular conjugates for the treatment and prevention of infectious diseases caused by pathogenic microorganisms in human and animals.
• the key to these conjugates is a special spacer connecting at least one photosensitizer to a microorganism receptor (vector) which in turn binds selectively to the surface of a microorganism bringing about photo-destruction upon irradiation.
• Spacers having hydrophilic structure such as ethylene glycol units and amino carboxyl end capped ethylene glycol units must be used for linking the vector to the photosensitizer.
• a spacer would have at least 3 ethylene glycol units and be end capped with a carboxyl group on one end and a amino group at the other end.
• the present invention effectively works to combat bacterial infection in the real patient-related environments where blood, serum and other body fluids are always present or at least nearby, of selected length and structure, in preferred embodiments, are used for linking the vector to the photosensitizer.
• conjugate are found to be very effective in combating bacterial infection in the real patient-related environments where blood, serum and other body fluids are always present or a least nearby.
• a method of use is also provided.
• Fig 1 Graph showing photodynamic inactivation of Staphylococcus aureus DSMl 104, Pseudomonas aeruginose DSMl 777 and Escherichia coli DSM8698 by using Safranin O as a photosensitizer.
• Fig 2 Graph showing photodynamic inactivation of Staphylococcus aureus DSMl 104, Pseudomonas aeruginose DSMl 117 and Escherichia coli DSM8698Staphylococcus aureus DSMl 104 by using of compounds 1, 3a, 3b, 3c, 3d and 3e as photosensitizers.
• a photodynamic method for inactivation/reduction of bacteria in complex environment is disclosed.
• combating bacterial infection in the complex media present in vivo, like serum, plasma or blood is most difficult part as seen in the prior art.
• antimicrobial photodynamic therapy is used to target pathogenic microorganisms using conjugated photosensitizers to treat various infectious diseases and also to induce photodestruction in the complex media normally found in vivo for real patients.
• a molecular conjugate disclosed is designed to target both Gram-positive and Gram-negative bacteria even their resistant strains.
• the molecular conjugate comprises of one photosensitizer linked to a vector (antimicrobial peptide) via a specially selected spacer molecule whose presence in the structure of a conjugate between vector and photosensitizer plays critical influence on conjugate activity as well as the structure of the spacer.
• antimicrobial peptides of varying length and structure are used as vector which can improve the targeting ability of the photosensitizer and also facilitate the membrane permeability in the bacterial cell wall because of the ability of such peptide sequences to associate with microbial membranes.
• the peptide vectors used in present invention include the oligopeptide fragments of eel 1- permeabilizing peptides or derived from lipopolysaccharide binding proteins but are not limited thereby.
• the selectivity of targeting moiety permits increased targeting of photosensitizer by using the conjugate of the present invention thus minimizing the dosage and adverse side-effect.
• conjugate between vector and photosensitizer of present invention and the importance of spacer moiety is demonstrated by us on the examples of conjugates 3a-e where the compound 3a has no spacer in its structure, and other compounds (3b-e) have the spacer with varied length and hydrophobicity.
• Preparation of products 3a-e from meso pyropheophorbide-a (1) is demonstrated in the Examples 1-6.
• Preparation of conjugate 3a (no spacer in the structure) comprises direct attachment of vector moiety to the compound 1 by conventional solid and liquid phase methods know in the art and exemplified below.
• Synthesis of spacer-armed conjugates 3b-e includes first attachment of the appropriate spacer to the compound 1 and further coupling with the vector by conventional solid and liquid phase methods know in the art and exemplified below.
• the antibacterial treatment includes the administration of photosensitizers to an environment containing bacteria and is allowed to incubate for a sufficient period of time thus providing accumulation of the photosensitizer into the bacterial cells.
• the incubation time varies depending on many factors. In this experiment it is incubated for 30 min before being irradiated with 665 nm laser at 100 J/cm 2 to initiate photodestruction of bacterial cells.
• the photosensitizer can be administered either by systemic application, or local injection in the affected area. For infection on or near the skin it can be administered topically.
• Amide 2a was prepared in a 92% yield by, coupling of compound 1 and aminocapronic acid according to the typical procedure as described in the Example 1.
• Amide 2b was prepared in a 79% yield, by coupling of 2a and aminocapronic acid according to the typical procedure as described in the Example 1.
• Amide 2d was prepared in an 86% yield by coupling of compound 1 and amino acid 5 (product of GlycoSense AG, Jena, Germany) according to the typical procedure as described in the Example 1.
• Example 5 and 6 illustrates the preparation of photosensitizer — spacer— vector conjugate for one of the example compound.
• Acid derivative 2c (40 mg, 0.055 mrnol, 2.97 eq.) was dissolved in 5 mL of dichloromethane, then 0.1 mL (excess) of triethylamine was added followed by addition of 0.05 mL (excess) of pentafluorophenyl trifluoroacetate. The mixture was stirred at room temperature for 20 min, washed with water, concentrated, and dissolved in 2 mL of pyridine.
• This derivative was treated with 2 mL of HCl cone, at room temperature for 25 min, evaporated to dryness, and purified via MPLC on Lobar-RPl 8 (size B) column and gradient elution with acetonitrile-water (+0.1% TFA) (0-»50%) to give 29mg (56%) of pure conjugate 3d.
• the organisms used in our studies were three members of the microflora of wounds: Staphylococcus aureus DSMJ 104, Gram-positive; Escherichia coli DSM8698, Gram-negative; Pseudomonas aeruginosa DSMl 117, Gram-negative.
• Gram-positive bacteria e.g. Staphylococcus aureus
• Gram-negative bacteria e.g. Escherichia coli, Pseudomonas aeruginosa
• complex media e.g. blood, plasma, blood serum, saliva
• Cultured cells are suspended in sterile phosphate-buffered saline (PBS) or sterile PBS supplemented with 10% sterile horse blood serum, 10% human plasma or 10% human blood respectively.
• the final OD (Optical Density) at 600 nm, 1 cm in all cases was 0.03.
• the bacterial suspensions are placed into sterile black well plates with clear bottoms.
• Concentrations of photosensitizer 1 and photosensitizer conjugate 3a (without spacer), and 3b, 3c, 3d, 3e (with spacer) used in the study is as follows: lOO ⁇ M, l ⁇ M, 10 ⁇ M, and 100 ⁇ M . After incubation the samples are exposed to laser light of 665 nm, power set to

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Epidemiology (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Organic Chemistry (AREA)
• Biochemistry (AREA)
• Oncology (AREA)
• Communicable Diseases (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Radiation-Therapy Devices (AREA)
• Apparatus For Disinfection Or Sterilisation (AREA)

Priority Applications (4)

Applications Claiming Priority (4)

Publications (2)

Family

ID=40282008

Family Applications (1)

Country Status (4)

Cited By (2)

Family Cites Families (7)

• 2007
  • 2007-07-27 EP EP07797049.9A patent/EP2049105A4/de not_active Withdrawn
  • 2007-07-27 WO PCT/US2007/016951 patent/WO2009014524A2/en not_active Ceased
  • 2007-07-27 BR BRPI0714667A patent/BRPI0714667A8/pt not_active Application Discontinuation
  • 2007-07-27 MX MX2009001074A patent/MX2009001074A/es active IP Right Grant

Also Published As

Similar Documents

Legal Events