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). Further membranes. Moreover, it has been shown that the ex more, pristane induces histiocytomas in BALB/c and 65 tent of the adsorption of surfactants at oil-water inter BALB/Mo mice (28) and plasmacytomas in BALB faces is largely determined by the nature of the hydro /cAnPt mice (29). Murine plasmacytomagenesis is by carbon oil itself (e.g., 87), indicating that the solubiliza far the most widely studied tumor system involving tion system must be optimized for each individual al 5,321,014 3 4. kane under consideration. This would necessitate rather the organic solvent dimethyl sulphoxide (DMSO). By extensive solubilization studies prior to any biological employing NMR techniques, the present inventors also testing. Despite the drawbacks of employing organic attempted to i) localize pristane within the membrane, solvents and surfactants to introduce alkanes into aque ii) investigate changed membrane properties such as ous media, they are still widely used. 5 altered mobilities and conformations of lipid head The preparation of lipid vesicles is another widely groups and fatty acids, and iii) study pristane induced and successfully used method to deliver highly lipo lamellar-hexagonal phase transitions. The results de philic compounds to mammalian cells. Nonetheless, this scribed below should allow better comparison of the approach also requires careful consideration of poten effects of pristane on lipid bilayers to the effects of other tial artifacts. Problems associated with using liposomes O membrane-targeted compounds. This should facilitate are dependent on the composition of the vesicles. For development of new approaches to studying the inter example, lipid exchange or fusion between vesicles and action of pristane, other alkanes, alkenes, and alkynes target cells can alter many properties of the target cell with mammalian cells vitro. membrane, such as its fluidity or the mobility and activ In applying knowledge from the area of clathrate ity of membrane bound proteins involved in signal 15 chemistry, which describes the interactions between transduction pathways Hence, specific effects of the so-called host and guest molecules (14), the present alkane of interest may be obscured by effects of the inventors devised a new, effective, and simple method liposome itself, thus complicating interpretation of the of solubilizing pristane by forming a monomolecular results. A number of practical disadvantages in employ inclusion complex with 3-cyclodextrin. Cyclodextrins ing lipsome vesicles may also be encountered such as 20 are ring-shaped oligomers of D-glucose which have a problems of reproducibility between different liposome hydrophobic cavity and a hydrophilic surface. They are preparations or stability and storage problems. Pristane presently used in numerous processes in the chemical can be dissolved in unilamellar liposomes, but the effec industry (reviewed in 15 and 16), and are increasingly tiveness of the liposomes is dependent upon the phos employed for pharmaceutical applications such as drug pholipids employed for their preparation. Hence, the 25 complexation and delivery (17, 18). The molecular en solubilization vehicle itself is a variable in the experi capsulation of pristane into the hydrophobic cavity of a ments. Thus there is a need to find an inert vehicle to (3-cyclodextrin molecule represents an innovative and deliver pristane and other alkanes and hydrophobic superior concept for delivering this alkane to living substances to cells. mammalian cells in vitro. Moreover, this approach 30 holds great promise for biological testing of a whole SUMMARY OF THE INVENTION range of other straight and branched chained alkanes, The induction of plasmacytomas by pristane has pro alkenes, and alkynes that are also of interest in cancer vided a useful model system in cancer research, genet research (19-21). Thus, the formation of cyclodextrin ics, and immunology, and has led to important biotech /alkane inclusion complexes should enable the develop nological applications such as hybridoma technology 35 ment of a number of new and important applications and monoclonal antibody production. both for basic research on other alkanes (e.g., cancer In spite of considerable progress in our understanding research) and for applied research, such as in risk assess of the pathogenesis of murine plasmacytomas, the pre ment for environmental and occupational health pur cise role of the plasmacytomagenic agent pristane is poses (e.g., genotoxicity testing). Finally, this approach poorly defined, partly because of a continuous failure to may ultimately lead to new product developments in find effective methods to test the effects of this hydro biotechnology, e.g., the manufacturing of new alkane phobic compound on mammalian cells in vitro. Pristane preparations with qualitatively different physiochemi and other lipophilic alkanes are immiscible with the cal properties compared to the parent compounds (e.g., aqueous environment required for mammalian cells to alkanes in the form of ready-to-use water-soluble pow function properly. Initially, attempts to overcome this 45 ders). problem employed either conventional solubilization The preparation of £3-cyclodextrin/pristane inclusion methods such as mixing with organic solvents and sur complexes may serve as a new and alternative system factants, or the use of liposomes of different composi for testing the effects of pristane and other hydrophobic tions. All attempts to design a compromise between compounds on mammalian cells in vitro. The same delivering sufficient amounts of pristane on the one 50 approach should also be well suited for improving bi hand and meeting the requirements of sensitive mamma oavailability of n-alkanes with chain lengths similar to lian tester cells on the other hand contained unaccept that of pristane such as n-dodecane, n-tetradecane, and able methodological limitations. n-hexadecane. These compounds are of interest in can One goal of the present work is to elucidate the plas cer research because of their promoter effects in skin macytomagenic activity of pristane at the cellular level. 55 carcinoma model systems. The preparation of inclusion It is likely that at least part of the effects caused by complexes with different natural, substituted, and poly pristane are connected with alterations of membrane merized cyclodextrins also holds future promise both properties. Yet, quantitative data on the uptake of pris for optimization of the delivery of pristane to living tane by lipid membranes are not available. As described cells and for new applications for studying other al below, the incorporation of pristane into model mem 60 kanes, alkenes, and alkynes in the field of biomedical branes was studied via three physico-chemical methods. research, Experiments were aimed at quantifying the uptake of Accordingly, it is an object of the present invention pristane into model membranes and determining the to provide a complex of a cyclodextrin and an alkane, influence of the mode of solubilization of pristane on its an alkene, or an alkyne. Said complex may further con incorporation into lipid bilayers. For the latter, two 65 tain a biologically active molecule other than said al methods were compared: a novel solubilization proce kane, alkene, or alkyne. dure for pristane in 6-cyclodextrin (A-Cy), and the Another object of the present invention is to provide conventionally used method of dissolving pristane in a method for preparing a cyclodextrin inclusion coin 5,321,014 5 6 plex containing an alkane, an alkene, or an alkyne, com FIG. 8: Time course of inclusion complex formation. prising introducing an alkane, alkene, or alkyne into an Pristane (final concentration 2 mM) was mixed with aqueous solution of a cyclodextrin, stirring said solu R-CyD (1, 2, 4 and 8 mM solutions) in water horizontal tion, collecting the crystalline inclusion complexes lines, open, dots, and cross-hatched bars, respectively) which precipitate out from said solution, and washing as described in Methods. In order to monitor the solubi said crystalline inclusion complexes. lization of pristane, the compound was mixed with 3H Yet another object of the present invention is to pro pristane. The portion of pristane incorporated into vide a method of delivering an alkane, an alkene, or an As-Cyd was determined by measuring radioactivity alkyne to a mammalian cell, either in vivo or in vitro, (liquid scintillation counting) in the inclusion complex comprising contacting said cell with a complex of a 10 precipitate at the given time points. cyclodextrin and an alkane, alkene, or alkyne, or with FIG. 9: Stability of the g-CyD/pristane inclusion such compounds plus a biologically active molecule complex. Suspensions of 3-CyD/pristane inclusion other than said alkane, alkene, or alkyne. complexes (10 uM-2 mM) were incubated either at A further object of the present invention is to provide ambient temperature (open symbols) or at 37 C. (closed a method for testing the effects of an alkane, an alkene, 15 symbols) for 40 min (circles), 70 min (squares) or 16 h or an alkyne on mammalian cells, comprising contact (triangles). The starting suspension (zero time) is shown ing said cells with a complex of a cyclodextrin and an as x-x. Light scatter from the suspension of crystalline alkane, alkene, or alkyne, or with a cyclodextrin and inclusion complex was measured in a diode array spec such compounds plus a biologically active molecule trophotometer as described in Methods. other than said alkane, alkene, or alkyne, and observing 20 FIG. 10: Estimation of the dissolution of the B the effects of said complex on said cells. CyD/pristane inclusion complex by organic solvents Further scope of the applicability of the present in and/or detergents. S-cyclodextrin/pristane inclusion vention will become apparent from the detailed descrip complex was suspended at the indicated concentrations tion and drawings provided below. However, it should in PBS containing either no additions (control, solid be understood that the detailed description and specific 25 squares) or the following additions: 2% DMSO-2% examples, while indicating preferred embodiments of ethanol (open squares); 5% DMSO--5% ethanol (solid the invention, are given by way of illustration only since circle); 1% Triton X-100 (solid triangle); 1% Triton various changes and modifications within the spirit and X-100+2% DMSO-2% ethanol (open triangle); 1% scope of the invention will become apparent to those Triton X-100+5% DMSO-4-5% ethanol (open circle). skilled in the art from this detailed description. 30 The location of the turbidity curve over this concentra tion range serves again as a measure of dissolution of BRIEF DESCRIPTION OF THE DRAWINGS crystalline inclusion complex (see also FIG. 2). The above and other objects, features, and advan FIG. 11: Cytotoxicity of pristane to P388 cells as tages of the present invention will be better understood measured by the release of 51Cr. Pristane was solubi from the following detailed descriptions taken in con 35 lized either in DMSO, three different preparations of junction with the accompanying drawings, all of which liposomes prepared from heterogeneous PC, or by mo are given by way of illustration only, and are not limita lecular encapsulation in 3-CyD. The solutions were tive of the present invention, in which: added to P388 cells at 37 C. for 2 hours. Cytotoxicity FIG. 1(a-d): 2H-NMR spectra of perdeuterated pris was assayed as described in Methods. For comparison, tane in DPPC water (50 wt.% water) dispersions. The cytotoxicity is shown for 2% chloroform (v/v) and for concentration of pristane is given in mol % of the sum 2% Triton X-100 (v/v), which also serves as a standard of the molar concentrations of pristane and phospho for maximal 51Cr release. lipid: a, 1.0 mol %; b, 9.7 c, 16.3 mol %; d, 34.6 mol%. FIG. 12: Cytotoxic effects of pristane complexed by FIG. 2(a-d): 2H NMR spectra of (a) DPCC-d62 (50 different CyD preparations. Inclusion complexes wt.%) in water without pristane and (b) with pristane 45 formed between pristane and aqueous solutions of a-, (74.3 mol %) at 50° C. A-, and y-cyclodextrin cross-hatched, dote, and hori FIG. 3(a-b): 31P NMR spectra of DOPE (a, inverse zontal lines, respectively) were prepared by the copre hexagonal phase) and DPPC (b, lamellar phase) in the cipitation method as described in Methods. Cytotoxic presence of pristane (a, pristane 4.2 mol %, b, pristane ity in NSF-1 cells was measured with the 5Cr release 3.7 mol%). The anisotropy of the chemical shift was 50 test after a 2 h incubation with the indicated concentra +20.2 ppm (a) and - 46.8 ppm (b respectively. tions of inclusion complexes as described in Methods. FIG. 4-(a-d): Enhancement of the formation of an FIG. 13(A-B): Pristane (FIG. 13A) inhibits the LPS inverse hexagonal phase in DOPE/DOPC (3:1, mol/- induced transformation of splenic B lymphocytes to B mol) mixtures by pristane. The concentration of pris lymphoblasts. LPS blasts were generated from tane is given in mol % of the sum of molar concentra 55 BALB/c splenic cells that were cultured for 48 h in tions of pristane and phospholipid: a, 5.5 mol %; b, 8.5 serum free medium containing no LPS (horizontal mol %; c, 12.7 mol %; d, 13.3 mol %. lines), 18.5 ug/ml LPS (cross-hatched), or 37.5 g/ml FIG. 5: DSC thermograms of dispersions of DPPC in LPS (dots). Blast formation and cell number were mea water (a) without pristane and (b) with pristane (21.6 sured by Coulter Counter analysis as described in Meth mol %). ods. Addition of £3-CyD alone had no effect on LPS FIG. 6: Comparison of the uptake of pristane (final blast formation (FIG. 13B). concentration 50 uM) solubilized in g-CyD into uni FIG. 14: Effect of pristane on 3H-TdR incorporation. lamellar DOPC liposomes during 1, 3, and 12 hours at Pristane was added at time zero at the indicated concen T=37 C. Measurements were done in duplicates. trations to SJL-4 cells (circles), P388 cells (squares), or FIG. 7: Partitioning of 6-CyD/pristane (final con 65 an initiated keratinocyte cell line (308 cells, triangles. centration 50 uM; t = 12 h; T =37°C.) into the liposome H-TdR incorporation assay was performed as de phase (DOPC 250-4,000 uM), the D2O phase, and the scribed in Methods. Data are from three independent surface phase of the plastic vial. experiments run in triplicate. Standard deviations were 5,321,014 7 8 less than 10% and are not shown. Pristane was added as gen and then by vacuum (10 min). The samples were an inclusion complex with g-CyD, and 3-CyD served kept for a further 7 to 12 hours in a desiccator under as the solvent control. Values are presented as % of reduced pressure. The rate of evaporation of pristane control. 3-CyD alone had no effect on 3H-TdR incor was checked with a pure sample of pristane in the same poration (data not shown). 5 desiccator and a loss of only 1.5% pristane was de DETAILED DESCRIPTION OF THE tected. For measurements on samples containing deu INVENTION terated lipid or pristane, deuterium-depleted water was added. The glass tubes were sealed in a flame. Sample The following detailed description of the invention is contents were mixed and pelleted by centrifugation. provided to aid those skilled in the art in practicing the O present invention. Even so, the following detailed de Prior to the NMR measurements, the samples were scription should not be construed to unduly limit the stored for several days at room temperature, except for present invention, as modifications and variations in the DPPC-d62 samples that were kept at 50° C. embodiments herein discussed can be made by those of Measurements ordinary skill in the art without departing from the 15 NMR measurements were performed on a Bruker spirit or scope of the present inventive discovery. MSL 300 spectrometer using a high power probe with Physico-Chemical Studies an 8mm solenoidal sample coil which was doubly tuned for 31P and 1H at 121.513 MHz and 300.133 MHz, re Abbreviations spectively. Gated broadband decoupled 3P spectra (3-CyD, g-cyclodextrin; D2O, deuterium oxide; 20 were observed with a phase cycled Hahn echo sequence DMSO, dimethyl sulphoxide; DOPC, dioleoyl phos as described in (40). A delay time between the 90 and phatidylcholine; DOPE, dioleoyl phosphatidylethanol 180° pulse of 100 us was chosen. Accumulation was amine; DPPC, dipalmitoyl phosphatidylcholine; DSC, started at the top of the echo. Typically 1024 scans with differential scanning calorimetry; NMR, nuclear mag a recycle delay time of 1 s were accumulated. 2H NMR netic resonance; PBS, phosphate buffered saline. 25 spectra were observed using the same 8 mm solenoidal Chemicals sample coil as for 31P NMR but tuned for a resonance at Synthetic L-o-dioleoyl phosphatidylcholine 46.073 MHz. 2H NMR spectra were obtained using a (DOPC), L-a-dioleoyl phosphatidylethanolamine quadruple echo sequence (41). The delay time between (DOPE), and L-a-dipalmitoyl phosphatidylcholine-dg2 30 the two 90 pulses was 100 us and the recycle delay time (DPPC) were purchased from Avanti Polar Lipids 1 s. Sample temperatures were adjusted using a Bruker (Alabaster, AL, USA). The lipids were checked for temperature control unit. impurities by thin layer chromatography and were Calorimetry judged to be at least 98% pure. Pristane (2,6,10,14-tet ramethylpentadecane, 96% pure) was purchased from 35 A Perkin-Elmer-DSC-2 was used to perform differ Aldrich Chemical Company (Milwaukee, WI, USA). ential scanning calorimetry experiments. Dry DPPC Perdeuterated pristane was obtained from MSD Iso was filled into aluminum pans. The weight of the dry topes, Merck Division (Montreal, Canada). Deuterium lipid (= 5 mg) was determined with Cahn electrobal oxide (minimal isotopic purity 99.9 atom% deuterium) ances. Between 0.5 and 1 mg of pristane were added was from MSD Isotopes (Merck, Rahway, NJ, USA), 40 with a microsyringe to the dry lipid. Water was added deuterium depleted water was from Cambridge Isotope in excess. Samples were sealed and equilibrated for 24h Laboratories (Woburn, MA, USA), Tris (enzyme at room temperature prior to the measurements. The grade) was from BRL (Gaithersburg, MD, USA), so calorimeter was calibrated against the known tempera dium chloride was from Mallinckrodt (Paris, KY, ture (41.5 C.) of the gel-liquid crystalline phase transi USA), and (3-cyclodextrin was from Sigma (St. Louis, tion of DPPC (42). MO, USA). Solubilization of pristane NMR Pristane was solubilized by its molecular inclusion Sample preparation into (3-cyclodextrin (3-CyD) in a novel coprecipitation A. Lipid mixtures of DOPE/DOPC (molar ratio 3:1) SO procedure that was modified from a published method were prepared by dissolving both lipids in chloroform for complexing cinnarizine, a cerebral blood flow en /methanol (2:1, v/v). The solvent was first removed in hancer (43). Briefly, a bolus of pristane (final concentra a stream of nitrogen and then in vacuum. About 50 mg tion 2 mM) was pipetted into a 4 mM 6-CyD solution in of the dry lipids or lipid mixture were put into short distilled, deionized water and vigorously stirred on a Pyrex glass tubes with an outer diameter of 8 mm. Pris 55 simple benchtop mixer at ambient temperature for 4 tane was added to the dry lipid using a microsyringe. days. The crystalline inclusion complexes which precip The exact amount of lipid and pristane was deter itated out of solution were washed two times in PBS mined with microbalances. Pristane concentrations (pH 7.4, 2,800Xg, 20 min) in order to remove remaining given in mol % are calculated on the basis of the dry free g-CyD. Aliquots were stored at -20° C. Working weight of the lipid in the respective sample. After the portions were kept at 4 C. Alternatively, pristane was addition of pristane the tubes were closed with rubber dissolved in plain DMSO. This can be accomplished stoppers. On the next day, water was added to prepare without phase separation problems at an initial dilution a 50 wt.% lipid-in-water dispersion. factor of 1:200 (v/v) or greater. That corresponds to a B. Part of the samples (containing less than 10 mol % stock concentration of pristane of about 14.6 mM. Using pristane) were prepared using stock solutions of pris 65 tritiated pristane, this factor was determined in experi tane in chloroform. 50 mg of the matrix lipid were dis ments aimed at estimating the solubility limit of pristane solved in an appropriate volume of the stock solution. in plain DMSO and the requirements for its further The chloroform was removed first in a stream of nitro dilution in a DMSO/phosphate buffer with progres 5,321,014 10 sively decreasing concentrations of DMSO (data not groups of pristane are poorly resolved and overlapping. shown). While the largest of the overlapping splittings is in the Preparation of unilamellar liposomes order of 4 kHz, other quadrupolar interactions in the same molecule are only in the order of a few hundred 250 mg of DOPC were dispersed in 25 ml of 10 mM Hz or less, thus contributing to a broadened isotropic Tris, 10 mM. NaCl, 0.02% (w/v) sodium azide in D2O. signal. It is known that the broadening of doublets is The pD was adjusted to 7.4 using 1 NHCl. After initial caused by reorientation processes with a correlation vortexing the dispersion was sonicated with a Fisher times of the order of 10-4s. Slow reorientations of Sonic Dismembrator, Model 300 (Artek Systems Corp., pristane molecules during lateral diffusion along mem Farmingdale, NY, USA) with a tip sonicator at 60% 10 power output for about 45 minutes. During sonication branes with radii of curvature of 1 um and less is the the dispersion was cooled using a ice-cold water bath. most appropriate explanation of the broadening (44, 45). Prior to the experiments the dispersion was stored in a In agreement with this explanation is the fact that the refrigerator at a temperature of 4° C. shape of spectra depends to a certain extent on the sam ple preparation itself. We demonstrated previously that Uptake of pristane into liposomes 15 the preparation procedures decisively determine the The incorporation of pristane into model lipid bi fraction of liposomes with smaller radii of curvature layers was studied using 3H-pristane, DOPC liposomes, that form in aqueous dispersions (46, 47). and Tris/NaCl/D2O buffer. 3H-pristane (specific activ The shape of 2H NMR spectra is independent of the ity 30 mCi/ml) was prepared according to the catalytic amount of pristane at concentrations of up to 5 mol % hydrogen exchange method and was purified (final 20 in DOPC bilayers. Above this concentration, the quad radiochemical purity >95%) by silica gel chromatogra rupolar splittings of pristane decrease slightly, while the phy using n-hexane as solvent (American Radiolabeled intensity of the broadened isotropic component in Co., St. Louis, MO, USA). The experiments were car creases (FIG. 1). Unfortunately, it is impossible to dis ried out in 13 X51 mm polyallomer centrifuge tubes in a tinguish between the spectral contribution of pristane final volume of 3 ml. Liposome buffer (Tris 10 mM, 25 moving isotropically in a separate phase from the NaCl 10 mM, D2O, pH 7.0/pD 7.4), liposomes and methyl group signals of incorporated pristane that also 3H-pristane were pipetted in the given order into centri have very small quadrupolar splittings. For this reason fuge tubes that were carefully vortexed after addition of we are not able to give the exact concentration of pris each reactant and then adapted (20 min) to a tempera 30 tane at which a truly isotropic pristane signal can be ture of 37 C. In different experiments, 3H-pristane was seen for the first time. Nevertheless, all data indicate a added either solubilized in DMSO or as an inclusion change in the orientation and/or motion of pristane complex with (3-CyD. Samples were incubated between molecules at concentrations above 5 mol %. 1 and 12 hours at 37° C. with intermittent vortexing every 15–20 min. Then the tubes were centrifuged (2 h, It is noteworthy that we reproducibly observed dif 150,000Xg, 25 C.) in a Beckman L8-T70 Ultracentri 35 ferences between spectra obtained after direct addition fuge using a SW-Ti55 rotor. After centrifugation a of pristane to the lipid matrix (see NMR sample prepa lower D2O phase and an upper liposome phase were ration A., supra) and spectra recorded after prior simul clearly separated. The lower D2O phase was carefully taneous dissolution of pristane and lipids in chloroform aspirated in a plastic 3 ml syringe with a 20 gauge, 1.5 (see NMR sample preparation B., supra). Direct addi inch needle. The upper liposome phase was left in the tion results in a stronger isotropic component at con tube. Triplicates of both phases were counted in a Pack centrations as low as 3 mol%. On the other hand, the ard Tri-Carb 1500 liquid scintillation analyzer. The largest quadrupolar splittings, as reflected by the spec activities in both phases were compared to the total tral width close to the baseline, at pristane concentra activity, thus allowing estimation of the uptake of pris tions as high as 16 mol % are still close to the values 45 measured at 1 mol%. The 31P NMR spectra of DOPC tane into liposomes, the concentration of free pristane in in the presence of increasing concentrations of pristane the D2O phase, and the amount of pristane adsorbed to do not show any significant changes in the effective the wall of the centrifuge tube. anisotropy of chemical shift of the lipid phosphate Results groups upon pristane addition (cf. FIG. 3). Indications NMR 50 of medium speed exchange processes that are connected to the decrease of the radii of curvature of liposomes a) DOPC--pristane-dao after pristane addition can be found in both the 2H The 2H-NMR spectrum of free pristane appears as a NMR spectra of pristane-daoin DPPC water dispersions single resonance line with a linewidth of the order of and the 31P NMR spectra of lipids. 100 Hz that is indicative of isotropic motions. As a 55 consequence of the incorporation of pristane into the b) DPPC-d62+pristane lipid bilayers, the molecular motions of pristane acquire The influence of the incorporation of pristane on the a certain degree of anisotropy. This results in a splitting fatty acids of the phospholipids was investigated using of the 2H-NMR lines into doublets due to the angular fatty acid deuterated DPPC-d62 in the presence of a dependent quadrupolar interaction between the deute 60 large excess of pristane (74.3 mol%). The quadrupolar rium nucleus and the internal electric field gradient of splittings observed for the DPPC-d62, without added C-D (carbon-deuterium) bonds. The magnitude of split pristane were identical to values reported in (48). Pris ting depends on the average orientation of the particu tane addition resulted in a slight broadening of reso lar C-D bond to the magnetic field and fast motions of nance lines but had no influence on the position of reso that bond axis. The spectra in FIG. 1 represent so-called 65 nance peaks. As in the case of 3P NMR measurements, powder patterns of doublet splittings of pristane incor the broadening probably reflects the formation of parti porated into non-oriented bilayers. It can be seen that cles with a radius of curvature of membranes less than 1 the quadrupolar splittings of the methyl and methylene um (FIG. 2). 5,32,014 12 +5% we were not able to determine the exact value. In c) DOPE/DOPC (3:1 molar)+pristane-dao: contrast, solubilization of pristane in 6-CyD led to sub Rand et. al. (49) reported that addition of tetradecane stantially higher incorporation rates into liposomes. In to a DOPE/DOPC (3:1 molar) mixture transformed the FIG. 7, the partition of 6-CyD/pristane into the three lipid mixture from a lamellar to an inverse hexagonal compartments of this simple system, i.e., the liposome phase. As shown in FIG. 4, pristane also caused the phase, the D2O phase, and the plastic surface of the transformation of part of the lipid into a hexagonal centrifuge tube, is given. The partition proved to be phase in a dose-dependent fashion. Measurements of the quite different from that of DMSO/pristane where no anisotropy of chemical shift of the 31P NMR signal of significant adsorption to the plastic surface and a much DOPE in the hexagonal phase in the presence of pris 10 lower uptake into the liposome phase were observed. tane did not show any significant changes of lipid order parameters in comparison with samples not containing Significance of the Experimental Results pristane. The 2H NMR spectrum of deuterated pristane Incorporation of alkanes into lipid bilayers in the hexagonal phase consists of poorly resolved over Because of the wide use of alkanes in the formation of lapping quadrupolar splittings with a maximum splitting 15 Black Lipid Membranes (BLM), numerous studies of around 1 kHz (data not shown). Pristane-dao was incor n-alkane incorporation into lipid bilayers have been porated into both lamellar and hexagonal phases in the performed in the last 25 years. Pope et. al. (50) found same sample. Due to overlapping resonance signals it that n-alkanes with chain lengths of 12 carbon atoms or was not possible to determine the exact amount of pris less could be dissolved in amounts above 10 mol % in all tane in both phases. Therefore, we are not able to pre 20 bilayers examined. The same authors showed that the dict whether pristane exhibits a preference for a particu solubility of the longer chain alkanes at concentrations lar phase, whether bilayer or non-bilayer. around 10 mol % alkane increased with increasing acyl Calorimetry chain length of the matrix lipid. Cholesterol in the mem branes reduced the solubility for the longer chain al Addition of an excess of pristane of up to 25 mol % to 25 kanes, whereas the solubility increased with increasing DPPC bilayers resulted in no measurable shift (i-1" C.) temperature. Using methylated alkanes, White (51) of the phase transition temperature of DPPC from a gel showed that the molecular volume of alkanes has only a to a liquid crystalline lamellar phase. However, the second order effect on alkane incorporation. pretransition from the L8 to the Pe phase as well as the Alkanes in general do not change the area per mole PB/La phase transitions were significantly broadened cule in the liquid crystalline phase. Short chain alkanes, (FIG. 5). however, increase the thickness of the hydrophobic Uptake of pristane by liposomes portion of the bilayer. Neutron diffraction measure The uptake of pristane into model bilayer membranes ments (52) show that n-hexane forms a layer in the was determined using tritiated pristane and small uni 35 middle between the two bilayers. The mode of incorpo lamellar DOPC liposomes with diameters of about 40 ration and the amount of incorporated material seems to nm. The diameter of liposomes was determined by 31P derive from a complicated interplay between the en NMR measurements after addition of the shift reagent thalpy and entropy difference of alkanes in a bulk phase Pr03. At ambient temperature, DOPC is in a liquid and in ordered membranes. The enthalpy of interaction crystalline, lamellar phase that is equivalent to the phase increases if the alkanes line up with the lipid molecule, state of membrane phospholipids in vivo. Deuterium while a less ordered state of alkane molecules in the oxide was used instead of water since previous me middle of the bilayers is preferred entropically. With thodologic experiments showed a better separation be increasing chain length the presence of alkanes in the tween the (upper) liposome phase and the (lower) aque center of bilayers is restricted to lower concentrations. ous phase after ultracentrifugation (data not shown). 45 The longer molecules are more likely to line up with the Pristane was added as an aqueous suspension of (3- lipid fatty acid chains. Pope et, al. (50) relate the solubil CyD/pristane inclusion complexes. First, the reproduc ity of alkanes to an increased motional freedom of lipid ibility of the uptake of pristane by three different DOPC carbohydrate chains in the middle of the bilayer. They liposome preparations was measured. The uptake conclude that alkanes dissolve in the disordered region proved to be reproducible with variations of about 10% 50 in the middle of the membrane. The disordered region is in the maximal amount of pristane taken up (data not larger for lipids with larger acyl chains and, therefore, shown). In order to estimate the kinetics of the uptake these lipids incorporate more of the longer chain al of pristane, the incubation time at 37° C. was varied kanes. from 1 to 12 hours. FIG. 6 illustrates that the incorpora Incorporation of pristane into lipid bilayers tion of pristane is complete after 1 hour and that prolon 55 gation of the incubation time up to 12 hours does not Addition of up to 5 mol % pristane to DOPC via a further increase the amount of pristane incorporated. chloroform solution results in the formation of a homo The difference between using two different modes of geneous lipid pristane mixture in which all pristane solubilizing pristane on its subsequent incorporation molecules occupy identical binding sites. The apparent into DOPC liposomes was analyzed. Pristane was 60 decrease of pristane order parameters at higher pristane added either solubilized in DMSO or as a molecular concentrations is either connected with the occupation inclusion complex with g-CyD. The latter method dis of binding sites of lower order in the same phase or the perses pristane at the molecular level by individually formation of other lipid structures containing increasing encapsulating each hydrophobic pristane molecule with amounts of pristane. Possible candidates of other lipid a hydrophilic (3-CyD coat. The amount of pristane in 65 structures containing pristane are pristane lenses cov DOPC bilayers after incorporation via DMSO solubili ered by lipid monolayers and lipid micelles encapsulat zation was smaller than 5% of the total pristane added ing pristiane. How much of these structures is formed (data not shown). Because of an experimental error of depends on the way pristane is added. Addition of pris 5,321,014 13 14 tane to the dry matrix lipid prior to sonication results in in the hydrocarbon region, making the formation of a formation of the above mentioned structures at pristane hexagonal lipid phase more likely. The formation of concentrations as low as 3 mol %. The remaining por particles with radii of curvature less than um after pris tion of pristane is built into regular bilayers and main tane addition could be caused by a similar effect. That tains the quadrupolar splitting of pristane that is charac behavior might suggest that the rate of pristane incorpo teristic of low concentrations in bilayers. In other ration depends strongly on membrane curvature (56), words, samples with plain pristane added directly to the but until now we have no direct evidence for this, 2H dry lipid are nonhomogeneous and are characterized by NMR of deuterated pristane in coexisting lamellar and high local concentrations of the compound. The less hexagonal phases showed that pristane is located in ordered or isotropic spectral components in these sam 10 both phases. Nonetheless, we can not rule out a slightly ples resemble the structures obtained in chloroform different uptake of pristane by one of the phases. preparations at higher concentrations of pristane. The In the liposome experiments a maximal uptake of differences in free energy of pristane between these about 2 mol % pristane, relative to the dry weight of structures are small. As a result, different forms of the DOPC in the preparations, was observed. This amount incorporation of pristane coexist with low exchange 15 rates of pristane molecules between them. is considerably lower than the 5 mol % of pristane The maximum quadrupolar splittings of pristane are homogeneously incorporated after its prior mixing with only 25 to 30 % of the corresponding splittings of n matrix lipid in chloroform. The lower uptake of pristane alkanes (53), indicating a higher motional freedom and in the liposome experiments is not surprising consider /or different orientations of the individual methyl and 20 ing that at least four different phases that compete for methylene segments of pristane. This is supposedly pristane need to be taken into account; i) the watery caused by the influence of the bulky methyl groups of phase, ii) the g-CyD phase, iii) the plastic surface, and pristane. In contrast, the presence of pristane in a phos iv) the lipid phase. The amount of pristane in these pholipid bilayer does not seem to have any influence on phases depends on the concentration of binding sites the lipid order parameters in the headgroup and fatty 25 and on the differences between them in free energy of acid regions. The mobility and conformation of phos pristane. Moreover, in the NMR experiments the lipid phatidylcholine as measured by NMR are unchanged. concentration was considerably higher (625 mM) than In good agreement with that result is the fact that the in the liposome experiments (4 mM). It is also important quadrupolar splitting of pristane itself is not concentra to realize that increased solubility of pristane in the tion dependent up to 5 mol%. At low concentrations, 30 watery phase caused by the addition of £3-CyD im the binding sites for pristane between the lipid fatty acid proves the establishment of an equilibrium distribution chains are independent of each other. of pristane between all binding sites, while at the same Even high concentrations of added pristane (21.6 mol time decreasing the amount of pristane in different loca %, FIG. 5) in DPPC bilayers did not result in a measur tions at thermodynamic equilibrium. The complexity of able change in the gel-liquid crystalline phase transition 35 the thermodynamic picture is further illustrated by the temperature in the DSC measurements. Therefore, pris effects of the vehicle on delivery of pristane to the tane molecules are unlikely to perturb intermolecular liposome matrix. Solubilization of pristane in 6-CyD, as lipid interactions and membrane hydration to any signif compared to DMSO, led to significantly higher incor icant extent. This is in contrast to n-alkanes, in which poration rates. This result is intriguing since these solu chain lengths of about 12 carbon atoms or less decrease 40 bilization methods differ in principal. Whereas DMSO the phase transition temperature of DPPC, while longer is an organic solvent that simply dissolves pristane, n-alkanes cause an increase (54). The chain length of g-CyD disperses the compound at the molecular level pristane corresponds to that of n-pentadecane. If even by wrapping individual molecules with ring-shaped odd effects of the number of carbon atoms can be ne oligomers of glucose that have a hydrophobic molecu glected, n-pentadecane can be expected to increase the 45 lar core and a hydrophilic surface. Thus, the 6-CyD phase transition temperature by about 6' C. But such an method solubilizes pristane in an aqueous phase in the increase was not observed for pristane. We ascribe the complete absence of organic solvents deviation from the behavior of n-alkanes to the presence Biological significance of the incorporation of pristance of bulky methyl groups along the chain in the pristane into biomembranes molecule. However, the DSC measurement did reveal 50 broadened L62, P62, and PB/Lo, phase transitions which During the uptake of pristane by a cell, this hydro eflect an apparent loss of cooperativity after the incor phobic compound should partition into the membrane poration of pristane into the DPPC bilayer. Such broad interior, where it may act as a perturbant. Using a enings (and even a splitting) of the gel to liquid crystal >51Cr release short-term cytotoxicity assay, we deter line transition has also been described for n-alkanes (54). 55 mined that the critical concentration of pristane (solubi The reason for the observed loss of cooperativity might lized in (3-CyD) capable of disrupting several cell lines be a changed arrangement of the packing of the lipid during an 120 min incubation is about 200 uM, depend bilayers. But we consider also the possibility that differ ing on the cell type used. Yet, the exact nature of this ent structural isomers of pristane or impurities that are membrane perturbing/lytic activity has not been clari always present in commercial pristane preparations in fied. The only significant perturbation observed with low concentrations (55) may obscure true pristane ef NMR techniques was a tendency to form an inverse fects. The above-mentioned inhomogeneities in ar hexagonal phase after addition of pristane. At present it rangement of pristane in the lipid bilayer might also is unclear to which extent membrane stress caused by contribute to this effect. such a mechanism may be related to physiological men Pristane, like other alkanes, increases the hydropho 65 brane activity such as cell fusion, lysis, pore formation, bic volume of bilayers. In lipid mixtures like DOPE/- vesiculation, and the activity of integral membrane DOPC which are capable of transforming into nonlam proteins; any of these might contribute to the tumor ellar phases, pristane helps to reduce the packing stress inducing/promoting activity of pristane. 5,3 2 O 4. 15 6 The generalization has been made that non-genotoxic carcinogens are primarily hydrophobic. It has further Ceil lines and cell culture been proposed that in the absence of specific membrane The B-lineage lymphoma cell lines SJL-4 (subclone receptor mediated effects, some compounds derive their SJL-4/2) and NSF-1 (subclone NSF-1/12080–7) repre activity solely from their hydrophobicity (57). This may sent immunoblastic and follicular center cells, respec be the case for pristane. Along these lines, C10 to C18 tively (65). P388 is defined as an early B lymphoblastic n-alkanes are tumor promoting in murine skin carcino lymphoma (66). All B cells were grown continuously in genesis (58). Moreover, a strong correlation between suspension culture in RPMI 1640 medium containing the length of the alkyl chains and their promotional 10% (v/v) fetal calf serum (FCS), 200 mM L-glutamine, activity in murine skin carcinogenesis has been ob 10 and 50 uM 2-mercaptoethanol at 37°C. in a humidified served for organic peroxides and hydroperoxides (59). atmosphere containing 5% CO2 in air. An initiated mu The subsequent problem then becomes, how does the rine keratinocyte cell line, 308 (67), was cultured in hydrophobicity alter cell functions? In this respect, it is EMEM medium supplemented with 8% (v/v) fetal calf recognized that the incorporation of external lipids into 15 serum and 1.2 mM CaCl2. The 308 cells were plated in the cell membrane can cause diverse phenomena such as 60 mm dishes at a density of 2x10 cells/plate. The triggering of homeostatic processes aimed at preserving culture medium was changed 3 times/week. physico-chemical properties of the membrane (60), lipid exchanges near integral membrane proteins with poten Liposomes tial alterations in conformation and activity (61), or 20 Lipid vesicles were prepared from heterogeneous modulation of enzyme activity through hydrophobic L-a-phosphatidylcholine (PC) and honogeneous dis effects (62). Biochemical studies are warranted to see teacoryl L-a-phosphatidylcholine (DSPC) with a sonica whether such processes might be brought about by tion technique modified from von Hofeet. al. (68). pristane. Briefly, the lipids were dissolved in chloroform/me Finally, the finding of an enhancement of the uptake 25 thanol (9:1, v/v) in a sterile glass centrifuge tube and the of pristane into unilamellar liposomes after its solubiliza solvents were evaporated under a stream of nitrogen. tion with 6-CyD seems particularly important from a Pristane and PBS, pH 7.4, were added to the bottoms of practical point of view, i.e., for devising experiments to the tubes and the mixtures were sonicated with a steril test effects of pristane on cells in aqueous tissue culture ized sonicator probe at 60W for 180 min on ice in order media. In the past, such experiments have been frus 30 to form unilamellar vesicles. Sonicated preparations trated by insufficient bioavailability of pristane after its were centrifuged for 20 min at 20,000Xg. The optically dissolution in DMSO. This situation is very similar to clear center portion of the preparation, containing the other systems where the solubilization of alkanes unilamellar lipid vesicles, was separated from the coal proved to be the limiting step for studying their biologi escing micelles on the top of the tube and from the cal effect (63, 64). Using g-CyD-solubilization, it is now 35 multilamellar lipid vesicles and debris on the bottom. possible to obtain reproducible results when assaying Unilamellar lipid vesicles were divided into aliquots and toxicity, mitogenicity, and genotoxicity of pristane on stored at 4 C. For some experiments, aliquots were various B lymphocyte cell lines, as described infra. filtered through 0.80 um, 0.47 um, and 0.27 um poly Biological Studies carbonate filters prior to use. The incorporation of pris tane into PC liposomes proved to be much more effec Abbreviations tive than into DSPC liposomes (data not shown). Con a-CyD, a -cyclodextrin; (3-CyD, S-cyclodextrin; y sequently, data on cytotoxicity experiments using lipo CyD, y-cyclodextrin; DMSO, dimethyl sulphoxide; some-solubilized pristane described here will only be EMEM, Eagle's minimal essential medium; HBSS, 45 given for PC liposomes. Hanks' balanced salt solution; LPS, lipopolysaccharide; PBS, Dulbecco's phosphate buffered saline (without Methods of preparing the g-CyD/pristane inclusion Ca2+ and Mg2+); PC, L-a-phosphatidylcholine; complex DSPC, disteadryl L-a-phosphatidylcholine; 3H-TdR, The inclusion complex between g-CyD and pristane tritiated-methyl- thymidine. SO was prepared by modifying a published procedure for complexing cinnarizine, a cerebral blood flow en Chemicals hancer, with g-CyD according to the coprecipitation a-Cyd, g-CyD, y-Cy), L-a-phosphatidylcholine and the neutralization methods (69). In the neutraliza (PC, type II-S, approx. 17% phosphatidylcholine), dis tion method, pristane (final concentration 2 mM) was teacoryl L-a-phosphatidylcholine (DSPC, synthetic, 55 added as a bolus to a g-CyD solution (4 mM in 0.1 N purity as 99%), and Triton X-100 were from Sigma (St. HCl). The mixture was adjusted to pH 11 with 1 N Louis, MO). Pristane (2,6,10,14-tetramethylpentadec NaOH and subsequently neutralized to pH 7 with 1 N ane, 96% pure) and 3,5-diaminobenzoic acid (DABA, HCl. The crystalline precipitate which formed was 99% pure) were purchased from Aldrich (Milwaukee, washed twice in distilled, deionized water and resus WI).3H-pristane (specific activity 10.3 mCi/mmol) was pended in PBS, pH 7.4. In the coprecipitation method, prepared according to the catalytic hydrogen exchange a bolus of pristane (final concentration 2 mM) was pi method and was purified (final radiochemical purity petted into a 4 mM (3-CyD solution in distilled, deion >95%) by silica gel chromatography using n-hexane as ized water and stirred at room temperature for 4 days. solvent (American Radiolabeled Co., St. Louis, MO). 65 The crystalline inclusion complexes which precipitated 3H-TdR (20 Ci/mmol) and 51Cr (400-12,000 Ci/g) were out of solution were washed two times in PBS and obtained from NEN (Boston, MA). LPS (Escherichia stored at 4°C. The coprecipitation method will be the coli 055:B5) was from Difco (Detroit, MI). only method further described. 5,321,014 17 18 Cr release cytotoxicity assay Results Target cells (2x 107/ml) were labeled in HBSS with Characterization of the g-CyD/pristane inclusion Cr (100 uCi/ml) for 1 hour at 37° C. and then washed complex three times in PBS, pH 7.4, with 7.5 mM glucose at 5 a) Time course of inclusion complex formation 5'-10 C. Labeled cells (1.2 x 106/ml) were incubated in In order to estimate the rate of formation of the g a final volume of 0.5 ml with different concentrations of CyD/pristane inclusion complex, the time course of the pristane for 2 hours in a 37 C. shaking water bath. The molecular dispersion of pristane (2 mM final concentra assay was stopped by addition of 3 ml ice cold HBSS. O tion) was measured using 4 different concentrations of Cells were centrifuged at 400Xg for 10 min at 5-10 C. R-CyD (1, 2, 4, and 8 mM) in water. This was accom and 1 ml aliquots of the supernatant containing 51Cr plished by measuring the incorporation of H-pristane released were transferred to plastic tubes and counted in into the crystalline (3-CyD/pristane inclusion complex. a Gamma 4000 Beckman counter for 2 min. Positive FIG. 8 shows that the time required to complex pristane controls representing 100% 5Cr release were deter 15 was dependent upon the concentration of £3-CyD used. mined by complete cell lysis with Triton X-100 (2%, A 2 or 4 mM (3-CyD solution was sufficient to complex the pristane within 4 days and higher concentrations of v/v). Cytotoxicity is defined as (Activity sample-Ac A-CyD did not improve the solubilization process. The tivity Background)/(Activity Positive contro-Activity Back total recovery of complexed pristane after repeated ground). Background activity represents 51Cr release 20 washing and resuspension in an appropriate buffer was from negative (buffer only) control samples. approximately 80%. Considering unavoidable losses of LPS-induced splenic B lymphocyte transformation pristane during sample preparation (washing, centrifu gation, etc.) and due to adsorption on the walls of the Normal proliferating splenic B lymphocytes were reaction vials, it may be assumed that the compound is prepared from 8-12 week old BALB/cAnPt mice ac 25 solubilized quantitatively by the described method. cording to standard techniques. Briefly, splenic perfu b) Stability and dissolution of R-CyD/pristane inclusion sates were freed of erythrocytes in hypotonic lysis complexes buffer (0.004% NH4C1, 0.85% KHCO3, 0.1% EDTA, Preliminary data were collected to allow prediction pH 7.4) and seeded at a starting density of 1 x 106 30 cells/nl. The cells were cultured for 48-72 hours in of the general behavior of the complex in a biological assay system. Because it forms a turbid suspension in serum-free medium (70) containing 0, 18.5, or 37.5 aqueous solutions, whereas (3-CyD and pristane are ug/ml Escherichia coli 055:B5 LPS. Cell counts and size both optically clear solutions, the concentration of in distribution were determined with a model ZM Coulter clusion complex can be followed by measuring light counter. Blast cells were defined by the relatively strin 35 scattering in a spectrophotometer. To this end, the aver gent criterion as cells having a diameter larger than 14 age optical densities of suspensions of g-CyD/pristane Ln. at 500-600 nm were measured on a Hewlett-Packard 8452A diode array spectrophotometer. FIG. 9 illus 3H-TdR incorporation assay trates the high stability of the inclusion complex in Cells (1 X 10.5/ml, 100 ul aliquots) suspended in dou water. The concentration of 6-CyD/pristane required ble-supplemented medium (RPMI 1640 with 20% FCS, to give half-maximal turbidity is higher at 37° C. than at 400 mM L-glutamine, 100 uM 2-mercaptoethanol) were ambient temperature. Nonetheless, at either tempera pipetted into 96-well culture plates containing 100 ul ture, the complex is stable for at least 16 h. FIG. 10 shows preliminary findings on the dissolution of the aliquots of serial dilutions (2-fold) of test sample (pris 45 R-CyD/pristane inclusion complex. It is evident that tane or controls) diluted in RPMI/1% FCS. In each the addition of organic solvents such as ethanol or series, a well containing only cells and buffer was in DMSO, as well as detergents such as Triton X-100, cluded to determine background counts. In order to enhance the dissolution rate of the inclusion complex as minimize evaporation and temperature gradient effects reflected by the loss of turbidity. In addition, organic the outer wells contained water only and were not used 50 solvents increase the effect of Triton. As expected, the to assay samples. The plates were incubated at 37 C. in dissolution of inclusion complex in the presence of other a humidified atmosphere of 5% CO2. After 24-36 hours, solubilizers is enhanced by increased temperature (data 50 pil of pre-warmed 3H-TdR in culture medium were not shown). added to each well to give a final activity of 0.5-1.0 55 Biological testing of the g-CyD/pristane inclusion uCi/ml and the plates were further incubated for 4 complex hours. Cells were harvested in a Cambridge Scientific cell harvester and radioactivity was determined in a a) Cr release (cytotoxicity) Packard LSC 1050 counter. The assay was carried out In order to evaluate the efficiency with which differ in a modified version for studying 308 cells. Briefly, the ent solubilization principles allow pristane to interact with mammalian cells, a well-established short-term cells were plated in 60 mm dishes at a density of 2x 105 cytotoxicity assay was employed, i.e., the 51Cr release cells/ml. After pulse-labeling the cells (1 uCi/ml, 60 test. The rationale behind this approach was the as min), cellular DNA from the entire plate was precipi sumption that the most likely primary target site of tated, hydrolyzed, and measured fluorometrically with 65 pristane at the cellular level is the cell membrane. It was 3,5-diaminobenzoic acid (71). H-TdR uptake was de anticipated that the potential for pristane to act as a termined by liquid scintillation counting. The results are nonspecific membrane perturbant subsequent to its par given as dpm/ug DNA. titioning into the lipid bilayer would be proportional the 5,321,014 19 20 amount of pristane delivered to the target cell. Conse its direct effects on cells could be investigated. The quently, the percentage of 51Cr released per time unit problem of delivering pristane to mammalian cells in should reflect the effectiveness of the solubilization vitro is not trivial. In its pure form, pristane is not bi vehicle employed. Moreover, equitoxic concentrations oavailable. It is almost totally immiscible in water and of pristane delivered by different carriers should allow 5 hence cannot come into contact with a cell surrounded a direct comparison of their respective efficiencies. by an aqueous environment. In other systems where In a standardized procedure, cytotoxicity in P388 biological effects of alkanes have been studied, proper cells was compared for pristane solubilized in DMSO, solubilization has been the limiting precondition for PC liposomes, and (3-CyD (FIG. 11). It is shown that obtaining meaningful data (68, 72,73). It is interesting to the different solubilization methods differed widely in O note that certain alkane-utilizing microorganisms (bac their capacity to introduce pristane to the cells, and that teria and fungi) release biosurfactants in order to facili g-CyD was the most efficient vehicle. The relative tate uptake of hydrocarbon substrates (74). In order to efficacies of a-, 3-and y-CyD to deliver pristane to determine whether a particular solubilization protocol NSF-1 cells were also compared. These compounds was effective in delivering pristane to cells, effects on contain rings of 6, 7 and 8 glucose moieties, respec 15 cell function were assayed. This was accomplished tively, and hence differ in the size of the hydrophobic using well-established tests for cytotoxicity (Cr core. The results indicate that y- and a-CyD were effec release), proliferation (H-TdR incorporation), and tive in delivering pristane to the cells (FIG. 12), but less mitogen-driven B cell development (LPS-blast forma so than y-Cyd. When (3-CyD was employed, pristane tion). With these assays, it was assumed that a cellular concentrations as low as 50-100 uM were clearly cyto 20 response could only be achieved if pristane was incor toxic depending upon the cell line studied and the porated into the cell, thus reflecting successful delivery. length of the incubation time (data on the kinetics of It was found that encapsulation in g-CyD was the most pristane-induced 51Cr release not shown). effective method for rendering pristane bioavailable. Hence, pristane was found to be cytotoxic at concentra b) Inhibition of LPS-induced solenic B lymphocyte 25 tions greater than or equal to 50 LM, mitogenic at sub transformation toxic concentrations (e.g., 25-50 uM), and inhibitory to In a second set of experiments, the effect of pristane LPS-blast formation at even lower concentrations on a physiologic function of murine B lymphocytes was (6.25-25 uM). The concentration of all of these effects assessed. FIG. 13 shows that in comparison to cytotox was dependent upon the cell line and type being exam icity (51Cr release), relatively low concentrations of 30 ined. pristane inhibited LPS-stimulated splenic B cell trans formation. Although a thorough dose response relation Limitations of using conventional solubilization ship between pristane and LPS was not determined, it methods to deliver pristane to mammalian cells may be noted that approximately 25 uM pristane was Conventional methods of solubilizing alkanes in aque required to inhibit the B cell transformation induced by 35 ous media rely either on organic solvents (with/without a low dose of LPS (18.5 ug/ml), whereas 6.25 uM surfactants) or on lipid vesicles such as uni- or multi pristane was sufficient to inhibit the transformation lamellar liposomes. Employment of organic solvents induced by 37.5 g/ml LPS, thus pointing to a differen imposes severe biological drawbacks because many of tial effect of pristane dependent upon the degree of them are highly toxic to cells. They can produce arti stimulation of the B cells (FIG. 13A). Addition of 40 facts even at very low concentrations (72). Moreover, R-CyD alone had no effect on LPS-blast formation initial solubilization in an organic solvent does not guar (FIG. 13B). antee that the lipophilic compound will remain dis solved after further dilution into an aqueous medium; c) Growth-modulating effects of pristane complexed the initial macroemulsion that results from addition of a with (3-CyD. 45 hydrophobic compound in a water-miscible organic In a third approach, potential growth-modulating solvent (e.g., DMSO) is not stable. This problem is effects of pristane on several B cell lines were examined circumvented somewhat by adding surface active com by measuring HJ-TdR incorporation. FIG. 14 illus pounds (biological detergents, tensids and co-tensids) to trates a modest increase in 3H-TdR uptake in SJL-4 keep the hydrocarbon in solution. However, surfactants cells (~30%) at concentrations around 25 uM. In con SO themselves alter and eventually disrupt cell membranes. trast, this effect was not observed in P388 cells. A cell They may also alter equilibria by forming micellar pha line completely unrelated to the lymphocytic lineage ses (75). The extent of adsorption of surfactants at the has also been employed, initiated mouse keratinocytes oil-water interface is largely determined by the nature (308 cells), to test the effects of pristane on cell growth. of the hydrocarbon oil involved (87). Hence any solubi Pristane was found to be maximally mitogenic at a sub 55 lization system that involves the use of detergents has to toxic concentration of 50 uM as reflected by a signifi be optimized for each individual alkane under consider cant (-60%) increase in 3H-TdR incorporation in com ation. This necessitates rather extensive solubilization parison to the solvent control. These 3H-TdR labeling studies prior to biological testing. experiments were repeated several times over a wide Lipid vesicles are also employed to deliver lipophilic range of concentrations of pristane, but only the narrow compounds to mammalian cells. This method, too, con concentration range around 5-50 uM pristane proved tains potential artifacts since removal of lipophilic com reproducibly to be growth-promoting. Concentrations pounds from liposomes occurs only under participation of pristane higher than 50 uM were cytotoxic depend of other membrane events such as fusion between the ing upon the cell line investigated. vesicles and the target cells (76) or lipid exchange (77, 65 78). Furthermore, retention of lipophilic materials Significance of the Experimental Results within liposomes can be considerable (79). Hence, spe The purpose of this work was to develop a reliable cific effects of the alkane of interest may be obscured by and effective solubilization method for pristane so that undesirable effects of the liposome itself. Some practical 5,321,014 21 22 disadvantages are also inherent in using liposomes, such two different compounds simultaneously, it is possible as poor reproducibility between different liposome to deliver the test compound together with a potential preparations, as well as stability and storage problems modulator of its function (85). For example, the prepa (data not shown). The absence of a suitable protocol for ration of a ternary y-CyD/pristane/indomethacin in delivering pristane to cells focused our attention on the 5 clusion complex may make it possible to study the effect need to develop an alternative method. of pristane on cells in the presence of a drug known to Advantages of using a 3-CyD/pristane inclusion inhibit murine plasmacytomagenesis (86). complex to deliver pristane to mammalian cells Complexes of cyclodextrins With Other Alkanes, The advantages of solubilizing pristane (or a variety 10 Alkenes, and Alkynes of other lipophilic guest compounds) by molecular The present method of complexing an alkane with complexation with 6-CyD may be found by considering cyclodextrins can be applied to any alkane, alkene, or the unique properties of the host compound and the alkyne, including aliphatic (open chain and cyclic) and behavior of the complex in an aqueous environment. aromatic compounds, for which there is a suitably sized A-CyD is a relatively innocuous molecule which is 15 cyclodextrin. This is defined by the stability constant of neither metabolized nor absorbed by mammalian cells. the complex, and is mainly determined by the hydro Furthermore, it has practically no disadvantageous phobicity, relative size, and geometry of the alkane, solvent effects (80). It is completely non-toxic to cells alkene, or alkyne. In general, complex formation will even at a concentration of 1 mM, which is the highest not take place when the guest compound is either too concentration tested (data not shown) Complexation 20 large or too small to fit in the host cavity. Guest com with g-CyD improves the bioavailability of pristane by pounds do not, however, have to fit completely into the dispersing it at the molecular level, thereby increasing CyD cavity, but the efficiency of solubilization is im the solubility of the alkane in water and allowing facili proved for compounds that do. With respect to the tated partitioning into the cell membrane (81). The upper size limit, longer or branched guests can be en A-CyD/pristane complex forms a stable suspension in 25 capsulated by "capping' the ends of the compound tissue culture media. When mixed with cells, pristane with two G-CyD molecules that are oriented in head-to partitions from the less hydrophobic cyclodextrin cav head fashion. Cyclodextrin complexation is a versatile ity to more hydrophobic absorption sites in the lipid method that is applicable to many alkanes, alkenes, and bilayer of the cell membrane. alkynes, and can be achieved via the routine method Another positive feature of delivering pristane by 30 described supra. In general, the upper size limit for means of an inclusion complex with g-CyD is high branched chain and cyclic compounds, including lighted by the concept of content-uniformity (82). It is arenes, will be higher (about C36) than for straight chain extremely difficult to control or define the actual con compounds, for which the upper limit is about C22. centration of a compound that has both low solubility in There is no lower size limit, and therefore compounds water and a low effective concentration range. For 35 such as methane and methanol can be employed. example, pristane is practically insoluble in water and is Representative aliphatic and aromatic compounds effective in the present biological tests at micromolar which can be employed in the present invention in concentrations. In most cases, conventional methods clude, but are not limited to, the following: that employ watermiscible solvents to dissolve an al kane are insufficient to reliably and reproducibly dis- 40 perse small amounts of the alkanes in aqueous media. Alkanes Alkenes Alkynes This is because of the tendency of the alkanes to co n-pentane pentene pentyne n-hexane n-hexene n-hexyne alesce and form a separate phase with a trend toward an n-heptane n-heptene n-heptyne increase in particle size (83). In contrast, encapsulation ri-octane -Octete n-octyne of the compound in a suitable host molecule keeps the 45 wOae -Onene n-nonyne alkane dispersed at the molecular level. The finding that n-decane n-decene n-decyne R-CyD proved to be the best cyclodextrin for this pur n-undecane n-undecene n-undecyne n-dodecane n-dodecene n-dodecyne pose was not unexpected because the ability to form an n-tridecane n-tridecene n-tridecyne inclusion complex in solution is mainly determined by n-tetradecane n-tetradecene n-tetradecyne the cavity size of cyclodextrins. Thus, a-CyD is most 50 n-pentadecane n-pentadecene n-pentadecyne useful for molecules smaller than benzene, 3-CyD is n-hexadecane n-hexadecene n-hexadecyne used for compounds larger than benzene rings but etc., through C22 etc., through C22 etc., through C22 smaller than benzo(a)pyrene, and y-CyD is used for larger molecules (84). Cyclized alkanes, alkenes, and alkynes useful in the The preparation of 3-CyD/pristane inclusion com- 55 present invention include, but are not limited to, for plexes establishes a new system for testing the effects of example, cyclopentane, cyclohexane, cycloheptane, pristane and other alkanes on mammalian cells in vitro. cyclooctane, etc. Further physicochemical studies are warranted in order Branched chain (i-) alkanes useful in the present in to define the stability constant, equilibrium phase solu vention include, for example, i-pentane, i-hexane, i-hep bility diagram, crystal structure, etc. of the inclusion 60 tane, etc. complex. It should be possible to optimize the delivery Also useful in the present invention are singly or system by using substituted cyclodextrins which have a multiply oxygenated, halogenated, nitrogenated, Sul higher aqueous solubility than 3-CyD. In addition, the fated, etc. derivatives of n-, c-, or i-alkanes substituted preparation of an inclusion complex between 6-CyD on any carbon atom, such as alcohols, alkylhalides, and pristane may allow for the conversion of pristane 65 haloalkenes, haloalkynes, ethers, epoxides, aldehydes, from an oily liquid into a microcrystalline solid which ketones, carboxylic acids, etc. may be dried, stored and packaged as a powder or a Further useful compounds include functional deriva tablet. Since cyclodextrins bear the potential to harbor tives of the aforementioned, such as acid chlorides, acid 5,32,014 23 24 anhydrides, amines, chloramines, amides, esters, phe nite characterization of the g-CyID/pristane complex, nols, etc. spectroscopic methods such as circular dichroism, UV Specific, non-limiting examples of such compounds absorption, and NMR, as well as X-ray and neutron include chloroform, ethyl chloride, ethylbromide, 1- or diffraction analysis of crystallized complexes, will be 2-chloropropane, 1- or 2-chlorobutane, 1-, 2-, or 3 5 required. In spite of an expected molar ratio of g-CyD chloropentane, chlorohexane, chloroheptane, etc.; to pristane different from 1:1, it is still correct to use the methanol, ethanol, propanol, butanol, pentanol, etc.; term "monomolecular inclusion complex' to describe methylamine, ethylamine, ethylamide, etc.; saturated such complexes. and unsaturated fatty acids such as octanoic acid, no In summary, £3-CyD primarily forms channel-type noic acid, decanoic acid, undecanoic acid, dodecanoic O acid, myristic acid, palmitic acid, stearic acid, oleic acid, complexes, with the cyclodextrins arranged head-to linoleic acid, linolenic acid, and arachidonic acid; iso head. A rare exception is the cage type complex that prenoid compounds and derivatives thereof such as forms only with small guests (e.g., methanol.6H2O, pristane, pristanol, pristanic acid, phytane, phytol, phy ethanol.8H2O). Secondarily, these channel type com tanic acid, etc.; carotenoid derivatives of isoprenoids 15 plexes form cage type superstructures. such as £3-carotene and limonene; mineral oils; fish oils; Ternary Complexes phytol; squalene and derivatives thereof, such as choles terol; triglycerides; and complex mixtures of hydrocar Ternary or three-component systems consisting of bons including, but not limited to, petroleum fractions CyD, an alkane, etc., and a second guest could provide such as natural gasoline (C5-C10 and cycloalkanes), 20 interesting tools to elucidate mechanistic aspects of the kerosene (C12-C18 and aromatics), and gas oil (C12 and plasmacytomagenic activity of such compounds. The higher), including alicyclic and aromatic hydrocarbons, second guest may modulate the biological activity of aromatic-aliphatic hydrocarbons (arenes), etc. the alkane. For example, indomethacin, and possibly The type of CyD used is dictated by the compound other non-steroidal anti-inflammatory drugs, inhibit being studied and the intended application. The unsub 25 pristane-induced plasmacytomagenesis. By treating stituted CyDs that are currently available are a-, y-, and cells with a ternary complex containing both pristane or y-CyD. These show differences in cavity size, solubility any other alkane, etc. and indomethacin, it is possible to in water, etc. By substituting some of the hydroxyl test whether the drug inhibits the effect of the hydro groups in the CyDs with different functional groups, carbon directly. Similarly, inclusion of a second guest properties such as solubility and encapsulating capacity 30 can be altered significantly. As an example, the main compound that can interact either with the hydrocar disadvantage of £3-CyD, its low aqueous solubility, can bon or with the target cell provides a method to en be markedly improved by substituting a hydroxyl group hance, inhibit, or modulate the biological activity of on the sugars with an alkyl moiety (e.g., 2-hydrox either compound. With respect to the hypothesis con ymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxy 35 cerning the generation of genotoxic oxidation products propyl, 2-hydroxyisobutyl derivatives of 6-CyD). The of pristane by reactive oxygen intermediates, ternary inclusion complexes of methylated (3-CyDs are highly complexes that include radical producing (e.g., ED water soluble, and are more stable. Alkylation also TA/Fe chelates) or radical scavenging (e.g., low mo makes (3-CyD surface active. Further modifications of lecular weight antioxidants) compounds could be use CyDs include tosylation, mesitylation, perbenzoylation, 40 ful. Other feasible ternary systems may include certain amination, esterification, and etherification. Many ap cofactors for catalysis of site-specific reactions, or phar plications for these substituted CyDs have already been macologically active compounds such as prostaglan described, e.g., as catalysts, pharmaceuticals (serum dins, retinoids, and sex hormones. cholesterol depressants), plasticizers, etc. Substituted In theory, there is no reason to limit the number of CyDs hold much promise for further improvements of 45 potential guest compounds to only two. Depending on the solubilization of alkanes in watery phases. CyID the size, net hydrophobicity, and geometry of the guest polymers, another class of modified CyDs, exist as molecules, quaternary and larger complexes are possi cross-linked and matrix-coupled CyDs. These polymers ble, and are encompassed within the scope of the pres are mainly used in chemical and biotechnological sepa ent invention. Examples of such complexes include, for ration techniques; however, their application for deliv 50 example, pristane/indomethacin/copper complexed in ering alkanes, alkenes, and alkynes to target cells ap gamma-CyD; EDTA or an amino acid/iron or copper pears readily feasible. This could be accomplished, for /an alkane such as n-dodecane or a fatty acid, fatty example, by coating tissue culture plates with a station alcohol, or other lipid complexed in gamma-CyD; etc. ary Cyid polymer phase. Non-limiting examples of second guest compounds Stoichiometry 55 which can be used to form ternary complexes with the The molar ratio of £8-CyD and pristane in the present alkanes, alkenes, alkynes, aromatics, etc. described invention has not yet been determined. On the basis of Supra include indomethacin; other non-steroidal antiin published results on the crystal structures of various flammatory drugs such as aspirin, naproxen, ibuprofen, f3-CyD complexes, a molar ratio of 6-CyD:pristane of 60 phenylbutazone, etc.; free radical-producing com about 2: 1 is predicted. This prediction is mainly based pounds such as transition metals, (iron, copper, nickel, on published results that £3-CyD inclusion complexes etc.); chelates of EDTA (ethylenediamine tetraacetic form dimers in aqueous solution, followed by subse acid), EGTA, amino acids (e.g., histidine), and nucleo quent crystallization in, commonly, channel-type, head tides (e.g., ATP); paraquat; bleomycin; adriamycin; free to-head aggregates. The prediction of the molar ratio is 65 radical scavenging compounds such as BHT, ethanol, indirectly confirmed by the experimental finding that DMSO, mannitol, cysteine, and derivatives thereof the 2:1 ratio proved to be optimal for complexing pris (e.g., N-acetyl-cysteine); and nitroxides (e.g., DMPO, tane by the coprecipitation method. However, for defi TEMPO, TEMPOL, PBN, etc.). 5,321,014 25 26 8. Moloney, S. J. & Teal, J. J. (1988). Alkane-induced Delivery of Inclusion Complexes to Cells edema formation and cutaneous barrier dysfunction. The cyclodextrin inclusion complexes of the present Archives of Dermatology Research 280, 375–379. invention can be delivered to cells in vivo or in vitro. 9. Anderson, P. N. & Potter, M. (1969). Induction of Methods of delivering in vivo include, for example, 5 plasma cell tumours in BALB/c mice with 2,6,10,14 parenteral administration, either i.v., i.m., or i.p., oral tetramethylpentadecane (pristane). 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Mutation Research 18, 15-24. W. (1989) Biochim. Biophys. Acta 980, 69-76. 73. Miller, R. M. & Bartha, R. (1989) Evidence from 51. White, S. H. (1977) Ann. N. Y. Acad. Sci. 303, liposome encapsulation for transport-limited micro 243-265. SO bial metabolism of solid alkanes. Applied and Envi 52. White, S. H., King, G. I. and Cain, J. E. (1981) ronmental Microbiology 55, 269-274. Nature 290, 161-163. 74. Zajic, J. E. & Mahomedy, A. Y. (1984). Biosurfact 53. Ward, A. J. I., Friberg, S. E., Larsen, D. W. and ants: Intermediates in the biosynthesis of amphipathic Rananavare, S. B. (1984) J. Phys. Chem. 88,826-827. molecules in microbes. In Petroleum microbiology. 54. McIntosh, T. J., Simon, S. A. and MacDonald, R. 55 Edited by R. M. Atlas. pp. 221-297, Macmillan Pub C. (1980) Biochim. Biophys. Acta 597, 445-463. lishing Company, New York. 55. Janz, S., Birkenfeld, T., Herzschuh, R. and Storch, 75. Pitha, J., Irie, T., Sklar, P. B. & Nye, J. S. (1988). H. (1989) Arch. Geschwulstforsch. 59, 415-422. Drug solubilizers to aid pharmacologists: amorphous 56. Gruen, D. W. R. and Haydon, D. A. (1980) Biophys. cyclodextrin derivatives. Life Sciences 43, 493-502. J. 30, 129-136. 60 76. Seibicke, S., Zimmermann, H. P. & Haeffner, E. W. 57. McCoy, G. D., Rosenkranz, H. S. and Klopman, G. (1988). Fusion of lipid vesicles with ascites tumor (1990) Carcinogenesis 11, 11 11-1117. cells and their lipid-depleted variants. Studies with 58. Baxter, C. S. and Miller, M. L. (1987) Carcinogene Tadioactive- and fluorescent-labeled vesicles. Bio sis 8, 1787-1790. chimica et Biophysica Acta 944, 487-496. 59. Slaga, T. J., Solanki, V. and Logani, M. (1983) in 65 77. Nichols, J. W. (1988) Kinetics of fluorescent-labeled Radioprotectors and Anticarcinogens (Nygaard, O. phosphatidylcholine transfer between nonspecific F. and Simic, M. G., eds.) pp. 471-485, Academic lipid transfer protein and phospholipid vesicles. Bio Press, Orlando. chemistry 27, 1889-1896. 5,321,014 29 30 78. Herrmann, A., Zachowski, A. & Devaux, P. F. 11. A complex of cyclodextrin and a compound se (1990). Protein-mediated phospholipid translocation lected from the group consisting of an alkane having at in the endoplasmic reticulum with a low lipid speci least twelve carbon atoms, an alkene and an alkyne ficity. Biochemistry 29, 2023-2027. which further comprises one or more biologically ac 79. Kimelberg, H. K. & Mayhew, E. G. (1978). Proper tive molecules other than said alkane having at least ties and biological effects of liposomes and their uses twelve carbon atoms, alkene or alkyne. in pharmacology and toxicology. Critical Reviews in 12. The complex of claim 11, wherein said biologi Toxicology 6, 25-79. cally active molecule is indomethacin. 80. Bar, R. (1989). Cyclodextrin-aided bioconversions 13. The complex of claim 12, wherein said cyclodex and fermentations. TIBTECH 7, 2-4. O trin is y-cyclodextrin. 81. Uekama, K. & Otagiri, M. (1986). Cyclodextrins in 14. The complex of claim 12, wherein said alkane is drug carrier systems. CRC Critical Reviews in Ther an n-alkane or an i-alkane. apeutic Drug Carrier Systems 3, 1-40. 15. The complex of claim 14, wherein said alkane is 82. Inaba, K., Wakuda, T. & Uekama, K. (1984). Pros n-dodecane, n-tetradecane, n-hexadecane, or prostane. taglandins and their cyclodextrin complexes. Journal 15 16. The complex of claim 11, wherein said complex of Inclusion Phenomena 2, 467-474. comprises y-cyclodextrin, pristane, and indomethacin. 83. Raabe, F., Janz, S. & Wolff, G. (1990). The effect of 17. A method for preparing a cyclodextrin inclusion non-ionic surfactants on the SOS-inducing potency of complex containing an alkane having at least twelve 4-nitroquinoline-1-oxide in Escherichia coli PQ37. carbon atoms, an alkene, or an alkyne, comprising the Journal of Basic Microbiology 30, 435-442. 0 steps of: 84. Issaq, H. J. (1988). The multimodal cyclodextrin introducing an alkane having at least twelve carbon bonded stationary phases for high performance liquid atoms, an alkene, or an alkyne into an aqueous chromataography. Journal of Lipid Chromataogra solution of a cyclodextrin; phy 11, 2131-2146. stirring said solution; and 85. Kano, K., Hashimoto, S., Imai, A. & Ogawa, T. 25 collecting the crystalline inclusion complexes which (1984). Three-component complexes of cyclodex precipitate from said solution. 18. A method for delivering an alkane having at least trins. Exciplex formation in cyclodextrin cavity. twelve carbon atoms, alkene, or alkyne to a cell, tissue, Journal of Inclusion Phenomena 2, 737-746. or organ, comprising contacting said cell, tissue, or 86. Potter, M., Wax, J. S. & Nordan, R. P. (1985) Inhi 30 organ with a complex of cyclodextrin and a compound bition of plasmacytoma development in BALB/c selected from the group consisting of an alkane having mice by indomethacin. Journal of Experimental Med at least twelve carbon atoms, an alkene or an alkyne. icine 161,996-012. 19. The method of claim 18, wherein said cell is a 87, Iranloye, T. A. (1985). Some factors affecting the eukaryotic cell. self emulsification of hydrocarbon oils. Progress in 35 20. The method of claim 19, wherein said eukaryotic Colloid Polymer Sciences 70, 119-126. cell is selected from the group consisting of a higher What is claimed is: plant cell, an animal cell, an alga, or a fungal cell. 1. A complex of a cyclodextrin and a compound 21. The method of claim 20, wherein said animal cell selected from the group consisting of an an alkene, and is a mammalian cell. an alkyne. 22. The method of claim 18, wherein said cell is a 2. The complex of claim 1, wherein said cyclodextrin prokaryotic cell. is selected from the group consisting of a-cyclodextrin, 23. The method of claim 22, wherein said prokaryotic B-cyclodextrin, y-cyclodextrin, and a substituted deriv cell is a bacterium or a virus. ative thereof. 24. The method of claim 18, wherein said contacting 3. The complex of claim wherein said alkene, or al 45 is carried out in vivo or in vitro. kyne is linear or branched. 25. The method of claim 24, wherein said contacting 4. The complex of claim 1, wherein said alkene, or is carried out in vitro by coating a tissue culture plate alkyne has at least twelve carbon atoms. with a complex of a cyclodextrin and an alkane, an 5. A complex of a cyclodextrin and an alkane wherein alkene, or an alkyne. said alkane has at least twelve carbon atoms. 50 26. A method for determining the effective selected 6. The complex of claim 5, wherein said alkane is an from the group consisting of a toxic effect, a mitogenic n-alkane or an i-alkane. effect, and a genotoxic effect of an alkane having at least 7. The complex of claim 6, wherein said alkane is twelve carbon atoms, alkene or alkyne or mammalian selected from the group consisting of n-C12 to n-C19, cells, comprising: and i-C12 to i-C19. 55 contacting said mammalian cells with a complex of 8. The complex of claim 7, wherein said alkane is cyclodextrin and a compound selected from the selected from the group consisting of n-dodecane n-tet group consisting of an alkane having at least twelve radecane, and n-hexadecane. carbon atoms, an alkene and an alkyne; and 9. The complex of claim 7, wherein said i-C19 alkane determining said effect of said complex on said cells. is pristane, 2,6,10,14-tetramethylpentadecane. 60 27. The method of claim 26, wherein said complex 10. The complex of claim 5, which is a complex of further contains a biologically active molecule other As-cyclodextrin and pristane, 2,6,10,14-tetramethylpen than said alkane, alkene, or alkyne. tadecane. s k k

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