III US005321 014A United States Patent 19 (11) Patent Number: 5,321,014 Janz et al. (45. Date of Patent: Jun. 14, 1994 (54) MOLECULAR ENCAPSULATION AND 4,834,985 5/1989 Elger et al...................... 424/488 DELVERY OF ALKENES ALKYNES AND 4,956,351 9/1990 Mesens et al. ........................ 514/58 LONG CHAINALKANES, TO LIVING 5,007,966 4/1991 Hedges et al. ..... ... 514/58 5,019,563 5/1991 Hunter et al. .. 536/03 MAMMALIAN CELLS 5,070,081 12/1991 Majid et al......... ... 514/58 (75) Inventors: Siegfried Janz, Bethesda; Emily 5,079,000 1/1992 Takahashi et al., ... 514/58 Shacter, Kensington, both of Md. FOREIGN PATENT DOCUMENTS 73) Assignee: The United States of America as represented by the Department of 53-10153158-21602 2/19829/1978 JapanE. Health and Human Services, 62-120344 6/1987 Japan. Washington, D.C. 1-287094; 11/1989 Japan . (21) Appl. No.:v Vy 723,2409 2090738 7/1982 United Kingdom . ila. Primary Examiner-Ronald W. Griffin (22 Filed: Jun. 28, 1991 Attorney, Agent, or Firm-Susan S. Rucker 51 Int. Cl. ...................... C12Q 1/02; A61K 31/715 (52 U.S.C. ...................................... 5/S8,536/103. 57 ABSTRACT 435/29 A complex of a cyclodextrin and an alkane, alkene, 58) Field of Search .......................... 514/58; 536/103; alkyne, aromatic compound, etc. can be prepared ac 435/29 cording to the method of the present invention. These (56) References Cited complexes can be delivered to prokaryotic and eukary otic cells, tissues, and organs in vitro and in vivo. In this U.S. PATENT DOCUMENTS manner, the toxic, genotoxic, and mitogenic effects of 2,827,452 3/1958 Schlenk et al. ....................... 514/58 these compounds can be determined. Ternary com 2,959,580 1 1/1960 Schlenk et al. ..................... 536/103 plexes further including one or more biologically active E. : 93. s al - - - - - - - - - - - - - - - - - - - - - : molecules in addition to an alkane, etc. can be employed 3,846,551s 1 1/1974 Mifuneelin et et al. al. .............................................. ... 514/58 to determine the modulatory effects of such biologically 4,024,223 5/1977 Noda et al. ... 514/770 active molecules on these alkanes, etc. 4,228, 160 10/1980 Szejtli et al. .514/5s 4,228, 160 10/1980 Szejili et al. ........................ 536/103 27 Claims, 11 Drawing Sheets U.S. Patent June 14, 1994 Sheet 1 of 11 5,321,014 U.S. Patent June 14, 1994 Sheet 2 of 11 5,321,014 U.S. Patent June 14, 1994 Sheet 3 of 11 5,321,014 U.S. Patent June 14, 1994 Sheet 4 of 11 5,321,014 U.S. Patent June 14, 1994 Sheet 5 of 11 5,321,014 5. s : CH Oueuoxe U.S. Patent June 14, 1994 Sheet 6 of 11 5,321,014 FIG.6 3H-pristone uptoke (%) 4O h 3O 3h 2h 2O O O O OOO 200O 3OOO 4OOO 5OOO DOPC (M) FG.7 3H-pristone (%) OO 8O Odsorption 4O liposome phose 2O deuterium oxide phose O OOO 2OOO 3OOO 4OOO 5OOO DOPC (M) U.S. Patent June 14, 1994 Sheet 7 of 11 5,321,014 FG. 8 Pristone solubilized (%) 72 time (h) FG. 9 Average OD (500-6OOnm) In pristone, uM U.S. Patent June 14, 1994 sheet 8 of 11 5,321,014 FG.O Average OD (500-600 nm) 4. in pristone, uM FG. 4 3H-TdR uptoke (%) 8O 6 O 4 O 2O OO 8O O 5 2.5 3.5 4.5 5.5 In pristone), uM U.S. Patent June 14, 1994 Sheet 9 of 11 5,321,014 (%)9S0919),JOIG U.S. Patent June 14, 1994 Sheet 10 of 11 5,321,014 F. G. 2 5 Cr releose (%) 6O 3O 2O 6 3 63 25 25O 5OO. KOOO 20OO pristone (JM) U.S. Patent 5,321,014 B lymphoblasts (%) 25 2O ø.» O O.39 56 6.25 25 <^^^^^^^^^^^^^^^^OO pristone (M) F. G. 3B B lymphoblasts (%) 25 O39 56 6.25 25 OO B-CyD (M) 5,321,014 1. 2 pristane (reviewed in 10, 11). Hence, much of the cur MOLECULAR ENCAPSULATION AND DELVERY rent interest in this simple compound, an isoprenoid OF ALKENES ALKYNES AND LONG CHAIN C19-isoalkane, is derived from the desire to better un ALKANES, TO LIVING MAMMALIAN CELLS derstand plasmacytomagenesis in mice. The tumor inducing activity of pristane, thought to BACKGROUND OF THE INVENTION be a non-genotoxic, "complete' carcinogen, is still 1. Field of the Invention poorly understood. This is reflected by several diverse The present invention relates to the interaction be hypotheses on its putative effects in vivo. Changes in tween hydrophobic compounds and mammalian cells. It the conformation of DNA in lymphocytes (30) and includes the areas of in vitro testing of the effects of 10 hybridoma cells (31) after incorporation of pristane hydrophobic compounds on cells for biomedical re have been observed fluorometrically with propidium search purposes, including cancer research and geno iodide. A subsequent fluorescence polarization study toxicity assessment, and for the production and applica revealed pristane-induced changes in the membrane tion of new pharmaceutical compositions containing fluidity of rat lymphocytes (32). The generation of oxi hydrophobic substances for administration in liquid or 15 dation products of pristane by reactive oxygen interme powder form. diates (33) had been proposed as a possible additional 2. Description of Related Art component of the action of pristane under the condi The study of aliphatic hydrocarbons (alkanes) has tions of a prolonged inflammation in vivo (34). Immu become increasingly important in biomedical research nosuppression (35, 36, 37) and formation of a specific (1-3). The alkane components of mineral paraffin oils 20 histologic matrix for tumor origin and development (38) are implicated in a number of human pathologic condi after the intraperitoneal administration of pristane may tions such as mineral oil pneumonia, folliculitis of the contribute to tumorigenesis through different pathways. skin, oleogranuloma (paraffinoma), and systemic focal Whether or not pristane metabolites that may be gener lipidosis (4). Human exposure to mineral paraffin oils ated in vivo (39) are involved in the tumor inducing comes from workplace contaminants (5), environmental 25 activity of pristane is presently completely unclear. pollutants (6), food (7), and medications (8). Particular interest in the biological actions of alkanes derives from In early experiments, attempts were made to solubi the fact that in genetically susceptible inbred strains of lize and deliver pristane to cells by using conventional mice (BALB/c and NZB), intraperitoneal injection of solubilization methods such as mixing with organic mineral oils such as the isoalkane pristane (2,6,10,14-tet- 30 solvents and surfactants or by using liposomes of differ ramethylpentadecane) induces a malignant B cell lym ent compositions (13). Both methods were found to be phoma (plasmacytoma, 9). Over the years, pristane unacceptable. The key constraint derived from the need induced plasmacytomagenesis has become the most to deliver sufficient amounts of pristane without expos intensively studied prototype for plasma cell tumorigen ing the mammalian tester cells to toxic or “bioactive' esis (reviewed in 10 and 11). 35 solvents. Pristane and other lipophilic alkanes are practically There is no evidence that the complexation of pris immiscible with aqueous media (12) required for cell tane, or any other alkane, with cyclodextrins has ever growth and viability. However, when pristane is in been used for delivering these compounds in biological jected and then withdrawn from the peritoneal cavity assay systems. The predominant examples of the em of a mouse it appears as an emulsion even after only a ployment of cyclodextrins as solubilizers in biomedicine few days. Emulsification of pristane in vivo is probably are in pharmaceutical applications of drug complex facilitated by amphiphilic molecules (e.g., fatty acids, ation and delivery by cyclodextrins, including non fatty alcohols, phospholipids, and proteins) that are steroidal anti-inflammatory drugs, steroids, fat-soluble present in abundance in the inflammatory environment vitamins, prostaglandins, barbiturates, cardiac glyco that results from injection of the oil. In a conventional 45 sides, etc. (reviewed in 18). In addition, there are nu plasmacytoma induction protocol, 1.5 ml of pure pris merous applications of cyclodextrins in the chemical tane (as 4.4 mmol) are injected. The oil is gradually industry such as for separation and resolution of enan incorporated into a fixed inflammatory tissue (oil granu tiomers as well as for chromatographic separations on loma) but can still be found in large quantities in the cyclodextrin polymers (reviewed in 15 and 16). peritoneal fluid for months following its injection. 50 Conventional methods of solubilizing alkanes in wa Given that some sort of emulsification and hence solubi tery media rely either on the use of organic solvents lization of pristane takes place in vivo, it is clear that with or without surfactants, or on use of vesicles such as cells are exposed to pristane over a prolonged period of unilamellar or multilamellar liposomes. The employ time. Although pristane is thought to act indirectly by ment of organic solvents imposes severe biological inducing chronic inflammatory conditions which some- 55 drawbacks because many of them are highly toxic and how permit plasmacytomagenesis (11), direct biological may produce artifacts even at very low concentrations. effects of the alkane on cells may also contribute to the Moreover, organic solvents do not guarantee that lipo tumorigenic process. Some direct biological effects of philic compounds will remain dissolved after further pristane have been described (7). dilution into aqueous media. Although the solubilizing Pristane (2,6,10,14-tetramethylpentadecane) causes 60 properties of organic solvents may potentially be im numerous detrimental effects in animals (22, 23, 24, 25). proved by combining them with surface active com It promotes skin carcinogenesis in C3H mice (26) and pounds (biological detergents, tensids and co-tensids), the development of 3-methylcholanthrene induced lym surfactants frequently alter and eventually disrupt cell phoid malignancies in Copenhagen rats (27).