US 2003O198664A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0198664 A1 Sullivan et al. (43) Pub. Date: Oct. 23, 2003 (54) LIPID MEDIATED SCREENING OF DRUG Publication Classification CANDDATES FOR IDENTIFICATION OF ACTIVE COMPOUNDS (51) Int. Cl." ......................... C12Q 1/68; G01N 33/574; A61K 9/127; C12N 15/88 (76) Inventors: Sean Michael Sullivan, Gainesville, FL (52) U.S. Cl. ............................. 424/450; 435/6; 435/723; (US); Robert A. Copeland, Hockessin, 435/.458 DE (US) Correspondence Address: (57) ABSTRACT SALIWANCHIK LLOYD & SALWANCHIK A PROFESSIONAL ASSOCATION The Subject invention provides lipoSome formulations that 2421 N.W. 41ST STREET are capable of Specifically targeting cell types. The Subject SUTE A-1 invention also provides for the encapsulation of new chemi GAINESVILLE FL 326066669 cal entities (NCE) or other drug candidate molecules (DCM) 9 within liposomes that Specifically target a particular cell (21) Appl. No.: 10/404,896 type. The Subject invention, advantageously, Solubilizes compounds, with low Solubility in aqueous environments, (22) Filed: Mar. 31, 2003 and permits Screening of these compounds against intact cells for biological activity in the absence of detergents that Related U.S. Application Data can damage cell membranes. Also provided are methods of preparing liposome formulations that target a specific cell (60) Provisional application No. 60/368,529, filed on Mar. type and methods of delivering therapeutic agents to target 29, 2002. cells. US 2003/O198664 A1 Oct. 23, 2003 LPD MEDIATED SCREENING OF DRUG (DCM) within liposomes that specifically target a particular CANDDATES FOR IDENTIFICATION OF ACTIVE cell type. The Subject invention, advantageously, Solubilizes COMPOUNDS compounds, with low Solubility in aqueous environments, and permits Screening of these compounds against intact CROSS-REFERENCE TO RELATED cells for biological activity in the absence of detergents that APPLICATION can damage cell membranes. Also provided are methods of 0001. The application claims priority to U.S. Provisional preparing liposome formulations that target a specific cell Application Serial No. 60/368,529, filed Mar. 29, 2002, type and methods of delivering therapeutic agents to target which is hereby incorporated by reference in its entirety cells. (including all figures, amino acid and polynucleotide Sequences, and formulae). DETAILED DISCLOSURE 0008. In one embodiment, the Subject invention provides BACKGROUND OF THE INVENTION a method of preparing liposomes that are cell-type specific 0002 Liposomes are microscopic vesicles having single (CTSL). One aspect of the invention utilizes targeting or multiple phospholipid bilayers that can be used to entrap agents, Such as antibodies, receptors, or ligands for the compounds. Liposomes with multiple bilayers are known as targeting of the liposome formulations. Other embodiments multilamellar vesicles (MLVs); liposomes with a single utilize the chemical composition of the liposome formula bilayer are known as unilamellar vesicles (UV). Liposomes tions for targeting to Specific cell types. In certain aspects of have been formed in sizes as Small as tens of Angstroms to the invention, liposomes contain compounds that are to be as large as a few microns. Most liposomes are non-toxic, Screened for biological activity in cell-based assays. Other non-antigenic and biodegradable in character Since they embodiments provide for uptake of the liposomes via have the molecular characteristics similar to mammalian endocytosis, endocytosis can be receptor-mediated or occur membranes. in the absence of receptorS. 0003. The advent of combinatorial chemistry, combina 0009. In one embodiment of the invention, liposomes of torial libraries, compound libraries, and automated Synthesis the invention are prepared from “standard liposomes” (SL) methods has significantly expanded the numbers of poten according to methods well known in the art. A "standard tially beneficial therapeutic compounds produced in the liposome” contains phosphatidylcholine (PC) and choles pharmaceutical industry. After these compounds have been terol (Chol). In certain preferred embodiments, the “standard Synthesized, they are, typically, Screened for their ability to liposome” contains dioleoylphosphatidylcholine (DOPC) impact a particular process, target molecule, or molecular and cholesterol. Ratios of PC: Chol or DOPC:Chol in “stan interaction. Compounds Successfully demonstrating a dard liposomes are, typically, about 2:1 (e.g., about 66.67 mole % PC or DOPC and about 33.33 mole % Chol). To desired effect are then tested for biological activity in cell provide liposomes that are taken up by target cells, via based Screening Systems. endocytosis, additional phospholipid components (AC) are 0004. Many compounds exhibiting desirable characteris titrated into the “standard liposome” formulation. “Standard tics are lost to drug development because of failure in the liposomes” and/or CTSL can be unilamellar or multilamel cell-based Screening assayS. Failure can, often, be attributed lar. "Standard liposomes can also be drug/lipid complexes to a lack of Solubility in aqueous environments or lack of where the drugs are non-covalently bound to the lipid. cellular uptake. Failed compounds are often discarded in the drug discovery process because the time and expense 0010 Another aspect of the invention provides for the required to increase drug Solubility via medicinal chemistry preparation of CTSL by combining components of the or because the time and expense of identifying properties Standard liposome formulation, additional phospholipid that reduce or abrogate cellular uptake is prohibitive. components, water, ethanol, and marker Substances into a Suspension. The Suspension is dried under vacuum to 0005. Other compounds are discarded in drug develop remove the ethanol and the Suspension can be re-hydrated ment programs because of a failure to meet acceptance with isotonic Solutions and assayed for uptake by a specific criteria for new chemical entity (NCE) development. Accep cell type. Another embodiment provides for Spontaneous tance criteria that eliminate an NCE include undesirable liposome formation using lipids with asymmetric acyl physiochemical properties (e.g., poor activity in a variety of chains. delivery means), poor water Solubility, manufacturing issues, regulatory issues, and/or marketing issues. 0011 Additional phospholipid components (AC) that are suitable for titration into the “standard liposome” include, 0006 The subject invention addresses issues in the drug but are not limited to, phosphatidylglycerol, phosphatidyle discovery proceSS and provides liposome formulations and thanolamine, phosphatidic acid, phosphatidylinositol, methods that facilitate the Screening of compounds in cell mono-, di-, and triglycerides, gangliosides, Sphingomyelin, based Screening Systems. The invention has applicability to and cerebrosides. In Some embodiments, a single additional Virtually all compounds and can be used to specifically target component is added into the SL formulation. Other embodi a variety of cell types. ments provide for the addition of more than one additional component into the SL formulation. BRIEF SUMMARY 0012 Another embodiment allows for the formulation of 0007. The subject invention provides liposome formula CTSL encapsulating (containing) agents selected from the tions that are capable of Specifically targeting cell types. The group consisting of therapeutic agents, marker Substances, Subject invention also provides for the encapsulation of new NCE, and drug candidates by combining these agents with chemical entities (NCE) or other drug candidate molecules an ethanol/water/lipid Suspension. The lipid component of US 2003/O198664 A1 Oct. 23, 2003 the Suspension is provided in ratioS Specific for uptake by a Specific target cell. Ethanol facilitates freezing of the SuS TABLE 1. pension and can be removed under Vacuum. The resultant lipid/agent film can, then, be re-hydrated in isotonic Solu Titration of a single AC into an SL formulation tions, diluted to varying concentrations, and assayed for Mole % Mole % Mole % biological activity or cell uptake. Optionally, therapeutic Chol PC or DOPC AC1 agents, NCE, drug candidates, or marker Substances can be 33.33 61.66 5 33.33 56.66 1O added to the CTSL at the time the dried CTSL are rehy 33.33 51.66 15 drated. Drugs containing weak acids or weak bases can be 33.33 46.66 2O loaded into preformed liposomes using gradients of coun 33.33 41.66 25 terions. Another method of introducing drugs into liposomes is to use preformed liposomes comprising lipids that undergo a phase transition from gel to liquid State. Incuba 0016 tion of the drug with the liposomes at the transition tem perature allows the drug to diffuse into the liposomes. TABLE 2 Subsequent return to the liquid or gel State temperature traps Titration of multiple AC into an SL formulation the drug inside the liposome. Mole % Mole % Mole % Mole % 0013 Phospholipids used in the formulation of the lipo Chol PC or DOPC AC1 AC2 33.33 56.67 5 5 Somes of the invention can contain short chain (C. to Cs) or 33.33 46.67 1O 1O long chain (Co to Cs) fatty acids. Short or long chain 33.33 36.67 15 15 phospholipids can also contain Saturated or unsaturated fatty acid tails. In various embodiments, the phospholipids con tain Zero, one, two, or three Saturated bonds. Preferred 0017) embodiments utilize phospholipids containing Zero or one Saturated