Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
1999-06-30
2001-10-16
Prouty, Rebecca E. (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C435S209000, C536S023200
Reexamination Certificate
active
06303118
ABSTRACT:
A human chitinase, its recombinant production, its use for decomposing chitin, its use in therapy or prophylaxis against infection diseases.
1. Field of the Invention
The subject invention is in the fields of therapeutical and prophylactic treatment of human individuals against infections, especially by chitin-containing organisms, and recombinant DNA technology for the production of a substance useful in the above treatment. The invention furthermore has implications on some other fields, such as diagnostics, gene therapy, controlled drug release from chitin-containing drug carriers or implants, and cosmetic, dental and even food products.
2. Background of the Invention
Infectious Diseases and Natural Defence
Man is constantly at risk for chronic infections by a variety of agents such as viruses, bacteria, fungi, protozoa and multicellular parasites. Some of the associated infectious diseases are seriously disabilitating or even life-threatening. In response to the threat exerted by these pathogens a variety of defence mechanisms, the so called immune responses, have evolved in mammalians. For an excellent overview on the topic see chapters 1, 2, 15 and 16 in ref.
1
.
A distinction can be made between innate (or non-adaptive) immune responses and adaptive immune responses. The latter type of response is highly specific for a particular pathogen and improves with each successive encounter with the infectious agent. The adaptive immune responses are mediated by various types of lymphocytes. The innate immune responses are primarily produced by the phagocytic cells. These more primitive responses are not based on a highly specific recognition and act as a first line of defence against infection. An important group of phagocytes are long-lived cells (monocyte/macrophages) that belong to the mononuclear phagocyte lineage. Monocytes are formed from bone marrow stem cells and enter the blood stream. These cells can migrate out into the tissue where they develop into various types of tissue macrophages. Examples are the microglial cells in brain, the alveolar macrophages in the lung, the Kupffer cells in the liver, the mesanglial phagocytes in the kidney, the splenic macrophages, the lymph node resident and recirculating macrophages and the synovial cells. The second category of phagocytes is formed by the polymorphonuclear neutrophils that are short-lived cells and constitute the majority of the blood leukocytes.
Role of Phagocytes in Defence
Well documented is the key role played by phagocytes in immunity to bacterial infections. Phagocytes are attracted chemotactically to a bacterial infection. Attachment to the bacterium can occur via numerous interactions, e.g. complement-mediated, antibody-mediated or mannose-binding protein-mediated or via lectin-oligosaccharide interaction. Subsequently, the organism is exposed to a sequence of killing mechanisms in phagosomes and lysosomes. Of great importance are the oxygen-dependent killing mechanisms that generate the superoxide anion and subsequently other reactive oxygen intermediates that are toxic. More recently the importance of killing via the nitric oxide pathway in neutrophils has become evident. Oxygen-independent killing is mediated by defensins, small cationic polypeptides, lysosomal enzymes, lysozyme and lactoferrin. The precise roles of phagocytes in immunity to fungal and parasite infections are less well understood, but it is thought that they are similar to those involved in resistance to bacterial infections.
Besides their direct role in killing of organisms, macro-phages play other important roles in the immunity to foreign organisms. Firstly, these cells are very effective at presenting antigens to T lymphocytes followed by further responses of the immune system. Secondly, exposure of macrophages to microbial products can be accompanied by release of cytokines that affect other components of the immune system. Thirdly, macrophages respond to cytokines released by T lymphocytes. For example, in some parasite infections the body reduces damage by walling off the parasite behind a capsule of inflammatory cells. This T-lymphocyte dependent process results in local accumulation of macrophages that release fibrogenic factors which stimulate the formation of granulomatous tissue and ultimately fibrosis.
Intervention of Infectious Diseases
The natural defence mechanisms against pathogens are not always sufficiently effective to prevent clinical complications and (preventive) intervention is therefore required.
One preventive approach is immunization, i.e. stimulation of defence mechanisms by prior vaccination of the host with (components of) pathogens. To be effective, a vaccine must induce a long-lived response from the right kind of T-lympho-cytes that produce a strong cell-mediated immunity. Although vaccination has proved to be effective for some pathogens, a number of intrinsic problems are associated with this approach. Most importantly, it has to be avoided that antigens used for vaccination induce the wrong kind of immune response, such as suppression or even autoimmunity. With some infections there is a need to achieve immunity in specific body locations that can be only obtained by local or oral immunization. Due to the complexity of the immune system, the heterogeneity in pathogens and the ability of some pathogens to escape from the specific immune responses, a generalized approach for effective immunization against pathogens is not available. Various strategies for specific infectious diseases remain under investigation by trial and error.
Another approach in the intervention of infectious diseases is the use of pharmacological agents that prevent further proliferation or survival of pathogens. In this connection use is made preferably of compounds that specifically act at the level of the pathogen and do not affect the host. Such specificity is based on differences in the composition and needs of mammalian cells and their pathogenic invaders. Some illustrative examples are the following. Penicillins and cephalosporins are specific inhibitors of bacterial cell wall synthesis. Aminoglycosides, chloramphenicol, tetracyclines, macrolides are inhibitors of bacterial protein synthesis; rifampicin, 4-quinolones are specific inhibitors of bacterial DNA replication. Amphotericin B and nystatin are antibiotics that are fungicidal due to binding specific sterols in the fungal cell membrane, thus causing leakage of cell components.
The pharmacological approach to intervene with pathogens at the level of a specific target that is absent in the host cells is also used in nature. A good example is the hydrolase lysozyme that is present in vertebrates as well as many invertebrates. Cell walls of many bacteria contain interlinked polymers of muramic acid and N-acetylglucosamine. The hydrolase lysozyme is capable of cleaving the glucosidic bond between muramic acid and N-acetylglucosamine moieties and consequently the integrity of the cell wall. The presence of lysozyme is without harm for the host since a similar structure is absent in non-bacterial cells.
Chitin
Chitin is a glycopolymer that is absent in mammalian cells but is present in a variety of organisms that cause infectious diseases in man. Chitin therefore forms an attractive target for selectively attacking these type of pathogens.
Chitin is a polymer of &bgr;(1-4) linked N-acetyl-D-glucosamine units. It may also contain glucosamine units in different proportions. Mainly deacetylated chitin is called chitosan. For overviews on the topic see refs.2-5.
Chitin and its derivatives are one of the most abundant macromolecular biological products on earth. The estimated annual production is 10,000-100,000 million tons. Chitin is a structural component of cell walls of fungi and of the exoskeleton of almost all invertebrates (except sponges, most anthozoa, scyphozoa, and echinoderms), but is absent in vertebrates and autotrophic organisms. Chitin fulfils important functions: it protects cells and organisms against mechanical and chemical stress from the environment and it also
Hoffman & Baron LLP
Prouty Rebecca E.
Universiteit Van Amsterdam
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