Patent Application: US-95020710-A

Abstract:
this invention describes a method for delivery of a therapeutic agent to target cells . liposome delivery vesicles binds to a specific cell population via a targeting molecule attached to the liposome surface . after the liposomes bind to a target cell they are internalized into compartments within the cell called endosomes . it has been shown by prior art that by encapsulating a pore forming bacterial hemolysin into the lumen of a liposome , the endosome is broken down and drug is delivered into the cell cytoplasm . instead of encapsulating the hemolysin within the liposome , this invention improves on current techniques by associating the hemolysin with the lipid membrane of the liposome . this modification reduces development cost significantly , eases production methods and increases the effectiveness and versatility of the treatment . using this technique , we have demonstrated effective targeting and killing of her - 2 overexpressing tumor cells .

Description:
the invention involves the attachment of hemolysin to the membrane of a liposome that carries a therapeutic agent and is attached to a targeting molecule . lee et al . showed that when encapsulated in the lumen of ph - sensitive liposomes , a hemolysin formed pores in the endosomal membrane , increasing cytoplasmic delivery . this technique has been patented by lee et al . 1996 , u . s . pat . no . 5 , 643 , 599 . our technique improves on this prior art because of how the hemolysin is associated with the liposome . instead of being encapsulated within the lumen of the liposome as described by lee et al ., with this invention , the hemolysin is associated with the lipid membrane of the liposome either conjugated to the outside of the liposome or incorporated into the lipid membrane bilayer . this provides an advantage to the prior art in two regards . first , the liposomes are simpler and more cost effective to produce since they use a much smaller quantity of protein . second , since the hemolysin is associated with the membrane it can easily transfer from the liposome to the endosome , allowing for more effective delivery and versatile application . we will discuss these two advantages in more detail . in the current invention , a small aliquot of hemolysin is added to liposomes after the liposomes are formed and a targeting molecule is attached . with the prior art , hemolysin is added at a concentration of 250 μg / ml ( lee et al . 1995 ). a large quantity of hemolysin is needed because the liposomes are forming around the hemolysin protein . the number of protein molecules within each liposome is dependent on the concentration of hemolysin present . with this invention , we take advantage of the hemolysin &# 39 ; s ability to conjugate to the outside of a lipid membrane . the liposomes are exposed to a small amount of the hemolysin which incorporates into the membrane of the liposome . to achieve adequate concentrations of hemolysin it is only necessary to use 4 μg / ml , a 62 fold reduction in quantity of protein compared to the prior art ( kullberg et al . 2010 ). the predominate hemolysin used in both the prior art and this invention is listeriolysin o ( llo ) which is sold on the market at $ 192 for 10 μg ( bio - world incorporated ). with our invention one milliliter of liposomes would have a protein cost of $ 77 compared to $ 4 , 800 using methods described in the prior art . this is a considerable cost advantage which makes a treatment using hemolysin more realistic . in addition to cost , production methods of this invention are more simple than the prior art . in the prior art , liposomes are formed in the presence of hemolysin . most liposome production involves extruding liposomes down to nanometer sizes ( torchilin and weissig 2003 ). if hemolysin is present during the production , the temperatures necessary for this extrusion will damage the hemolysin and render the therapy ineffective . also , targeting antibodies are often attached through chemistry that binds the amine group of the antibody ( amiji 2007 , kullberg et al . 2010 ). this reaction occurs spontaneously after liposomes are formed and would be impossible to carry out in the presence of hemolysin which also has amine groups . the method we use for attaching antibody to our liposomes would not be possible using techniques from the prior art . after liposomes are internalized into a target cell , the hemolysin must transfer from the liposome to the membrane wall of the endosome . with the prior art , hemolysin is trapped within the lumen of the liposome . in order for the hemolysin to transfer to the endosome , the liposome must be broken down . the liposomes are eventually broken down in the lysosomes of the cells , but this environment will render the hemolysin ineffective . lysosomes are like the garbage disposal of the cell and are designed to chew up proteins , lipids and other molecules that enter them . to be effective the liposomes must release the hemolysin before this harsh environment . therefore , lee et al . designed their system using ph - sensitive liposomes ( lee et al . 1996 ). their liposomes open up in the low ph of the endosome before reaching the lysosomes and the released hemolysin transfers to the endosome membrane . this method will only be effective with ph - sensitive liposomes . however , the vast majority of potential targeted therapies are not made with ph - sensitive liposomes and therefore cannot benefit from the prior art which encapsulates hemolysin in the liposome lumen . a problem with using ph - sensitive liposomes is that they are quickly taken up by the immune system unless they are coated with a polymer such as polyethylene glycol ( peg ) which helps disguise the liposomes ( torchilin and weissig 2003 ). peg removes the ph - sensitivity of ph - sensitive liposomes which has been a great hindrance to advancing the field ( momekova et al . 2010 ). with our invention , hemolysin is attached to the membrane of the liposome and transfers freely from the liposome surface to the endosome membrane once the liposome is internalized . any targeted liposome system , with or without peg , can be easily modified to incorporate the hemolysin in the liposome membrane . well before reaching the damaging lysosomes , the hemolysin transfers from the liposome to the endosome where it forms pores in the membrane . it is not necessary for the liposome to break down since hemolysin is associated with the liposome membrane . after the hemolysin transfers to the endosome and forms pores in the membrane , therapeutic molecules can transfer through the pores to the cytoplasm . both our system and most other targeted liposomes would not benefit from hemolysin being incorporated within the lumen of the liposome as is done in the prior art ( park et al . 2001 , kullberg et al . 2010 ). lee et al . produced an exciting system when they incorporated hemolysin within ph - sensitive liposomes and demonstrated increased delivery to cells . however , this invention demonstrates a clear improvement that leads to lower cost , ease of production and a more versatile delivery system that can be incorporated into almost any targeted liposome therapy . the term “ liposome ” refers to a vesicular membrane structure comprised of a natural or synthetic phospholipid membrane or membranes , and optionally other membrane components such as cholesterol and protein . the vesicle can have a unilamellar , oligolamellar , or multilamellar membrane . the term “ targeting molecule ” refers to an agent that binds to a defined target cell population , such as tumor cells . preferred targeting moieties useful in this regard include antibody and antibody fragments , peptides , cytokines , peptidomimetics and hormones . proteins corresponding to known cell surface - receptors ( including low density lipoproteins , transferrin , egf and insulin ), fibrinolytic enzymes , anti - her2 , platelet binding proteins such as annexins , and biological response modifiers ( including interleukin , interferon , erythropoietin and colony - stimulating factor ) are also preferred targeting moieties . ligands suitable for use within the present invention include biotin , haptens , lectins , epitopes , dsdna fragments , enzyme inhibitors and analogs and derivatives thereof . oligonucleotides binding to cell surfaces are also included . analogs of the above - listed targeting moieties that retain the capacity to bind to a defined target cell population may also be used within the claimed invention . in addition , synthetic targeting moieties may be designed . the term “ therapeutic agent ” refers to toxins , anti - tumor agents , drugs and radionuclides . preferred toxins include russell &# 39 ; s viper venom , activated factor ix , activated factor x , thrombin , phospholipase c , holotoxins , pertussis toxin , dodecandrin , shiga toxin , cobra venom factor , ricin , ricin a chain , pseudomonas exotoxin , diphtheria toxin , bovine pancreatic ribonuclease , ribonucleases , angiogenin , trichosanthin , pokeweed antiviral protein , abrin , abrin a chain , gelonin , saporin , modeccin , modeccin a chain , viscumin , volkensin , tritin , barley toxin , or any portion of these toxins . ribosomal inactivating proteins ( rips ), naturally occurring protein synthesis inhibitors that lack translocating and cell - binding ability , are also suitable for use herein . extremely highly toxic toxins , such as palytoxin and the like , are also contemplated for use in the practice of the present invention . preferred drugs suitable for use herein include conventional chemotherapeutics , such as vinblastine , doxorubicin , bleomycin , methotrexate , 5 - fluorouracil , 6 - thioguanine , cytarabine , cyclophosphamide and cisplatinum , as well as other conventional chemotherapeutics as described in cancer : principles and practice of oncology , 2d ed ., v . t . devita , jr ., s . hellman , s . a . rosenberg , j . b . lippincott co ., philadelphia , pa ., 1985 , chapter 14 . the term hemolysin refers to any pore forming protein expressed by gram - positive bacteria . some examples of hemolysins include lysteriolysinu o ( llo ), streptolysin o ( slo ) and perfringolysin o with this delivery system , liposomes are attached to a targeting molecule and carry a hemolysin that is associated with the lipid membrane . liposomes may also carry a therapeutic molecule that is delivered to the target cells . as described below a therapeutic molecule may also be administered separately and have its entry into the cell cytoplasm enabled by the liposomes . the liposomes will circulate through the body and bind to the target cells via the targeting molecule . after binding , the liposomes will be internalized into the endosomes of the target cells . the hemolysin which is located on the membrane of the liposomes transfers to the endosome membrane and forms pores in the endosome ( kullberg et al . 2010 ). as the therapeutic agent leaks from the liposome it will travel through these pores and exert its cytotoxic effect on the target cells . using her - 2 targeting liposomes that incorporate gelonin and are attached to llo we have shown specific killing of her - 2 overexpressing breast tumor cells ( fig6 ). in addition , we have recently used the liposomes to facilitate the entry of a chemotherapeutic agent that is administered seperately from the liposomes . the chemotherapeutic agent is taken up independently into the endosomes of the cells . the chemotherapeutic agent becomes much more effective if its entry into the cell is enabled by a hemolysin carrying liposome . when the chemotherapeutic agent collocalizes with the liposomes inside of target cell endosomes , the hemolysin breaks down the endosome membrane and the therapeutic agent kills the cell . as described in the claims , this invention includes systems where the therapeutic agent is carried by the liposomes and also systems where a therapeutic agent is adminstered seperately and its entry into the cellular cytoplasm is facillitated by the hemolysin carrying liposomes . hemolysin protein — hemolysins are proteins expressed by gram - positive bacteria . some examples of hemolysins include lysteriolysin o ( llo ), streptolysin o ( slo ) and perfringolysin o ( pfo ). the hemolysin used in this example is llo , preparation of liposomes — liposomes can be formed by a variety of methods by people familiar with the art . in this example liposomes were prepared by the film hydration - extrusion method ( 10 ). there are many different lipids and an infinite variety of combinations that will form liposomes . in the present invention liposomes can be made up of any combination of lipids , cholesterol or additives . in this example , liposomes are made with lipids dppc : mppc : dppg : dspe - peg ( 3400 )- nhs at a molar ratio of 82 : 10 : 3 . 5 : 4 . the lipids in chloroform are mixed and a thin lipid film was formed by drying the lipids under nitrogen at 46 ° c . for 2 hours . lipids were hydrated in 1 ml of distilled water at 46 ° c . for 4 minutes and filtered through a 220 nm filter system using a gas - tight sample - luer lock syringe from hamilton ( reno , usa ). cell targeting — liposomes can be targeted to a particular type of cell by conjugating the liposome with a targeting molecule that can bind to a structure or marker on the cell surface . the targeting molecule can be an antibody , antibody fragment , oligonucleotide , peptide , hormone , ligand , cytokine , peptidomimetic , protein carbohydrate , chemically modified protein , chemically modified nucleic acids , chemically modified carbohydrates or any other molecule that allows liposomes to be taken up by cells . in this preferred embodiment , a 0 . 5 ml aliquot of the antibody trastuzumab ( her - 2 antibody ) in water at a concentration of 1 . 5 mg / ml was added to the filtered lipid . the suspension was incubated at 46 ° c . for 10 minutes and left at 4 ° c . overnight to allow for conjugation of the trastuzumab through amine binding . liposomes were then extruded 10 times through a 200 nm filter at 46 ° c . using an extruder from eastern scientific ( new york , usa ). conjugation of hemolysin to liposomes — liposomes are coupled with a hemolysin or a hemolysin - cholesterol complex by exposing the liposomes to hemolysin or a hemolysin - cholesterol complex . in this preferred embodiment an 8 μl aliquot of llo ( 2 . 0 mg / ml storage buffer ) was added to 0 . 4 ml of liposomes at a lipid concentration of 0 . 33 mg / ml . the mixture was left at room temperature for 10 min and then 8 μl of cholesterol ( 25 mg / ml in 200 - proof ethanol ) was added to the solution . in this step , the llo or llo - cholesterol complexes associate with the lipid bilayer of the liposome . the liposomes were shaken slowly for 30 min and then spun at 3800 g for 3 min to pellet the cholesterol . the supernatant containing liposomes was run over a cl - 4b column one more time to remove any unconjugated llo or cholesterol that remained . therapeutic or diagnostic agent — a therapeutic agent or diagnostic agent is included either in the lumen of the liposome , associated with the liposome membrane or administered separately from the liposomes . in this preferred embodiment liposomes are filled with the therapeutic agent , gelonin . liposomes are mixed with gelonin at a concentration of 1 mg / ml and then heated 46 ° c . for 3 minutes to allow the gelonin to flow into the interior of the liposomes . loaded liposomes are separated from unencapsulated gelonin by size exclusion chromatography using a cl - 4b sepharose . after this column purification , liposomes are ready to be administered . examples of other therapeutic agents include diagnostic agent , peptide , an oligonucleotide , a nucleic acid , an antibiotic , an antimicotic , an anti - viral agent , an anti - cancer agent , an enzyme , a chemotherapeutic drug or toxin . this research was conducted at the university of alaska . following the recommendation of the university &# 39 ; s office of technology transfer , the rights to this invention have been signed over to max kullberg . 1 . amiji m m . 2007 nanotechnology for cancer therapy , crc press , london . 2 . azambuja , e ., durbecq , v ., rosa , d d , colozza m , larsimont , d ., piccart - 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