Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing
Reexamination Certificate
1998-03-16
2002-10-29
Clark, Deborah J. R. (Department: 1633)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
C514S002600, C424S130100, C424S134100, C424S141100, C424S152100
Reexamination Certificate
active
06472365
ABSTRACT:
The present invention relates to methods for releasing an agent under pre-determined conditions, for example at a pre-determined site or in the presence of a pre-determined material, and in particular for releasing an agent for therapeutic, diagnostic or investigative purposes. The invention further relates to pharmaceutical compositions incorporating such methods, materials and kits for use in such methods. It is frequently desirable in the bioscience field to be able to deposit or release a particular agent under, pre-determined conditions, for instance at a specific site within an organism or to mark the presence or absence of an analyte during an assay. At present such specificity is frequently achieved by use of antibodies bound directly to active agents. For instance tumour associated monoclonal antibodies (MABS) have been used to selectively carry chemotherapeutic drugs to tumour cells. Clinical studies have investigated the delivery of methotrexate in patients colorectal carcinoma (Ballantyne et al. 1988. Int. J. Cancer, 42: 103-108) and also the use of adriamycin (see “Principles of Cancer Biotherapy” Ed. Oldham, R. K., Pub. Raven Press, New York, 1987). Similarly. MABS conjugated to toxins such as ricin, abrin, Pseudomonas toxin, Diptheria toxin and other have also been used as anti-cancer agents. Studies in vitro and in vivo have indicated that such conjugates can be extremely toxic to tumour cells ( Roffler et al. 1991. Cancer Res. 51:4001-4007; Embleton et al 1991; Bri. J. Cancer 63:670-674).
The use of MABS to provide selectivity avoids the side-effect problems associated with traditional chemotherapeutic treatment of cancer either in metastatic disease or in an adjuvant or primary setting. However, a major problem arises because many agents require internalisation before killing the target cell. Additionally immunotoxins usually give rise to unacceptable toxicity due to interaction with non-target cells during passage to the site.
A potential alternative delivery system for selected agents is based around the use of synthetic liposomes. Liposomes were originally described in 1974 (Bangham e al. Methods Membr. Biol. 1: 1-68). Liposomes consist of one or more phospholipid bilayers arranged in concentric rings of alternate aqueous spaces. Many compounds (both lipid and Water soluble) including cancer chemotherapeutics, antimicrobial drugs, enzymes, hormones and nucleic acids have been incorporated into either the aqueous or lipid phase of liposomes. The behaviour of drug-containing liposomes in animal and human subjects has formed the subject of several studies (Gregoriadis 1990, Immunol. Today 11: 89-97).
Thus liposomes offer considerable promise as vehicles for delivery of agents for use in a variety of applications including biochemical and immunological assays, diagnosis, and also pharmaceutical delivery systems for eternal and parenteral use. Unfortunately their application is undermined by the difficulty associated with selectively releasing their contents at a specific time or location.
The present invention has now provided methods for releasing a selected agent at a specific disease site or at a specific time or location and pharmaceutical compositions incorporating such methods, kits and materials for use in such methods, which seek to address some, and in preferred forms all, of the aforementioned problems.
According to a first aspect of the present invention there is provided a method of releasing an agent under predetermined conditions comprising the steps of protecting the agent within a lipid structure, causing lipase activity to be constituted in response to the predetermined conditions, and exposing the lipid structure to the constituted lipase activity such as to release the agent.
By lipase is meant any enzyme which hydrolyses lipids and includes, but is not limited to. enzymes which hydrolyse complex lipids such as phospholipids and grycolipids.
The term constituted as used herein is intended to denote localised, created or significantly increased i.e. a significant achievement or increase in lipase activity is initiated when the predetermined conditions are met.
A large number of naturally occurring lipases are known. For instance many gram-positive and negative bacteria produce enzymes having phospholipase C (PLC) activity. These enzymes hydrolyse phospholipids with varying efficiencies and posses a variety of haemolytic and lethal properties which generally makes them unsuitable for administration to living subjects.
One characterised enzyme is
Clostridium perfringens
alpha-toxin (CPAT). CPAT promotes direct lysis of certain mammalian cells and is the most toxic PLC described to date (see McDonel, J.L. (1986) pp 617-655 “Pharmacology of Bacterial Toxins” Eds. Dorner & Drews, Pub. Pergamon Press, Oxford). CPAT is a peptide containing 370 amino acids.
Preferably the lipid structure employed by the present invention comprises a phospholipid membrane defining a core. More preferably the lipid structure is a liposome. The agent to be released is chosen in accordance with the precise application in which the invention is being employed, however the nature of the agent must be such that it is protectable by a lipid structure.
Preferably the lipase activity employed in the present invention comprises a PLC activity, and more preferably is derived from CPAT. CPAT activity has not previously been demonstrated against liposomes; however the inventors of the present invention have shown that CPAT has significant activity against liposomes.
Preferabiy the lipase activity employed in the present invention is constituted by combining two or more components whereby the lipase activity of the product formed by the components is greater than the sum of the individual components, or alternatively the lipase activity is constituted at a specific location normally with much less or no lipase activity by virtue of the localisation of the lipase either as a holoenzyme or a combination of two or more components in either case in combination with a targeting molecule.
More preferably the components correspond to, or are derived from, an active lipase holoenzyme such that their recombination recovers all or part of the activity of the holoenzyme lipase preferably at a specific location. These components may both be proteins—however the invention embraces all systems wherein lipase activity is enhanced. localised or recovered by the combination of two or more components, including non-protein components such as co-factors.
Most preferably the components are derived from or include CPAT.
The N-terminal two-thirds of CPAT shares sequence homology with the phosphatidylcholine-PLC from
Bacillus cereus
. It has been demonstrated that N-terminal recombinant truncated CPAT (aa 1-249) retains phosphatidylcholine hydrolysing activity but has reduced sphingomyelinase activity and is neither haemolytic nor lethal. (Titball et al 1991. Infect. Immun. 59:1872-1874). Recombinant protein comprising the C-terminal third of CPAT (aa 247-370) is devoid of sphingomyelinase and haemolytic activity and is not toxic for murine lymphocytes (Titball et al. 1993, FEMS Microbiology Letters 110: 4550). It has been demonstrated that haemolytic activity (as assessed by an in vitro murine erythrocyte lysis assay) can be restored when the N-terminal and C-terminal recombinant proteins are added together (reconstituted CPAT).
The inventors of the present invention have shown that reconstituted CPAT has significant activity against liposomes.
The pre-determined conditions of the present invention may require that the agent be released only in the vicinity of a tumour or pathogen, or in the presence of an anaiyte or DNA sequence, or under any other suitable detectable condition.
Preferably the achievement of predetermined conditions is causally related to the constitution of lipase activity at a specific location by conjugating at least one of the lipase components or the holoenzyme to a targeting molecule capable of specific binding to a predetermined target under the pre-determined conditions. Su
Carr Francis J
Titball Richard W
Biovation Limited
Chen Shin-Lin
Clark Deborah J. R.
Nixon & Vanderhye P.C.
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