Bulk Organic Solvent-Water Systems as a Possible Model To Predict Alkyl PAminobenzoate Partitioning in Liposomes L. MA,

c. RAMACHANDRAN, AND N. D. WElNER'

Received August 22, 1991, from the College of Pharmacy, University of Michigan, Ann Arbor, MI 48709. March 16, 1992. Abstract 0 This study compares the bilayer-water distribution coefficients of a homologous series of n-alkyl paminobenzoates in liposomes with their respective distribution coefficients in octanol-water, oleyl

alcohohater, and hexanwater systems. The data indicate that the bilayer-water distribution coefficient is quite sensitive to changes in solute structure and to the structural organization of the bilayer and that octanol, oleyl alcohol, and hexane are able to reflect the partitioning changes that occur in the liposomal bilayer with respect to increasing the alkyl chain length of malkyl paminobenzoates. For this particular homologous series, the hexanwater system tended to underestimate the bilayer-water distribution coefficients, whereas octanol-water and oleyl alcohohater systems overestimated solute partitioning into the bilayer. The similarity of the lipid environment, with respect to solute partitioning, in the organic solvent system and that in liposomes can be ascertained by using the Collander relationship.

Liposomes consist of one or more concentric spheres of lipid bilayers separated by water or aqueous buffer compartments. Because of this structural configuration, liposomes are being used increasingly as drug delivery systems and as model biological membranes. Studies with liposomes may increase our understanding of the interaction and pharmacological action of drug molecules on biological membranes. They are ideal drug delivery systems, because they are relatively nontoxic, biodegradable, and capable of entrapping solute within their aqueous compartments and lipid bilayers. The exact location of a drug in the liposome depends on various factors, including the relative solubility of drug in water and in the lipid phase and the relative amounts of aqueous compartments and lipid bilayers within the liposome. The bilayer-water distribution coefficient (DC) of a drug in liposomes is a valuable and important physicochemical parameter in the design of a liposomal drug delivery system. Meyerl and Overton2 were the first to show that a parallel relationship exists between the narcotic activity of drugs and their oil-water partition coefficient and initiated the use of partition coefficients as a means of defining the relative hydrophobicity of biologically active organic compounds. The determination of the bilayer-water DC of drugs in liposomes is a tedious, expensive, and time-consuming process. A simpler model to predict solute partitioning in liposomes is, therefore, desirable. The major objective of this study was to determine if bulk organic solvenbwater systems could serve as a model to predict solute partitioning into liposomes. This research attempts to correlate the bilayerwater DCs of a homologousseries of n-alkyl p-aminobenzoates (methyl to butyl) in liposomes with their DCs in octanolwater, oleyl alcohol-water, and hexan+water systems. This particular homologous series was chosen because its physicochemical properties have been well characterized by other investigators,%' they are biologically active, and they possess a strong UV chromophore. They also cover a wide range of hydrophobicities and, therefore, are well suited as a model for 1104 1 Journal of Pharmaceutical Sciences Vol. 87, No. 7 7 , November 7992

Accepted for publication

structure-activity studies. The three organic solventa were chosen because of their different chemical structures and degrees of hydrophobicity. Dipalmitoylphosphatidylchoiine (DPPC) was chosen as the model phospholipid, primarily because it is a neutral phospholipid and the effects of charge on solute partitioning could be excluded. Aa a saturated phospholipid, it is relatively stable because it is not as prone to free radical oxidation as are the naturally occurring unsaturated phospholipids.

Experimental Section MaterialgDPPC was purchased from Avanti Polar Lipids, Birmingham, AL, and used without further purification. Cholesterol (CHOL), obtained from Sigma Chemical Company, St. Louis, MO, was recrystallized from ethanol solution three times prior to use. Methyl paminobenzoate was obtained from Aldrich Chemical Company, Milwaukee, "I,and ethyl and butyl p-aminobenzoate were obtained from Sigma. Propyl paminobenzoate was obtained from ICN Biomedicals, Inc., K & K Labs, Costa Mesa, CA. (-)Tyrosine HCL, chlorophenol, anisole, acetanilide, quinoline, chlorobenzene, n-decylamine, l-octanol, and N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonicacid (HEPES) were all purchased from Sigma. Pyridine was obtained from MCB, Cincinnati, OH, and phenol (reagent grade) was purchased from EM Chemicals, Gibbetown, NJ. Aniline was purchased from Fisher Chemicals, Fairlawn, NJ, and acridine was obtained from Aldrich. L-a-[2-Palmitoyl-9,103H(N)lphosphatidylcholine, obtained from New England Nuclear (specific activity, 58 Ci/mmol), was diluted with a 1:l (v/v) ratio of ethano1:toluene to a specific activity of 2.9 Ci/mmol. [14ClInulin (total activity, 250 pCi), diluted with water to a specific activity of -3 pCi/mL, and Ecolite+ scintillation solvent were both obtained from ICN Radiochemicals. Absolute ethanol (200 pro00 was purchased from Aaper Alcohol and Chemical Company, Louisville, KY. Methanol (HPLC grade) was obtained from Mallinckrodt Chemicals, Paris, NY. All chemicals were analytical grade and used without further purification. Preparation of Multilamellar Vesicles (MLVskMLVs were prepared by a slight modification of the method described by Bangham et a1.8 DPPC, with or without CHOL, and the alk lpaminobenmate of interest were first dissolved in chloroform. [ HIDPPC, used as the lipid marker, also was added to the chloroform solution (the DPPC:[3HlDPPC molar ratio in all experiments was 1:l x lo-'). All liposome preparations with CHOL were prepared with a DPPC:CHOL molar ratio of 2:l. This lipid mixture was deposited as a uniform thin film on the sides of a round-bottom flask by removal of the organic solvent, chloroform, at 50 "C under reduced pressure in a Buchi rotary evaporator (Buchi Technical Laboratories, Flawil, Switzerland). Residual chloroform was removed by placing the flask under reduced pressure for at least 12 h, and the dried film was then hydrated with 0.05 M HEPES buffer (pH 7.4) containing trace amounts of ["C-carboxyl]inulin (0.015 &i/mL of buffer), which served as the aqueous marker. In all systems, the dried lipid film was initially hydrated with buffer for 15 min at 50 "C, and the resulting phospholipid concentration was 68 pmol/mL. Further incubation of the multilamellar dispersion, by gentle mechanical agitation, was continued for 72 h a t 50 "C. After incubation at 60 "C, the lipomme dispersions were equilibrated at 25 "C for a t least 24 h. Preparation of Liposomes by the Extrusion T e c h n i q u e D P P C MLVs (without CHOL) were prepared, as described earlier, by a

r

0022-3!549/92'7 7 0 0 - 7 704$02.50/0 0 7992, American PharmaceuticalA s m a t i o n

hydration of the lipid film for 15 min at 50 "C. The multilamellar lipomme dispersion (DPPC; 68 pmoVmL) was then injected into the central chamber of an extrusion device (Extruder; Lipex Biomembranes, Vancouver, British Columbia, Canada) located above two stacked polycarbonate filters (0.4 pm pore size; Nucleopore Corp., Pleasanton, CA), extruded through the filters with nitrogen gas at pressures of 2-5 psi (13,789.5-34,473.8 Pa), and collected via the outlet tube. To ensure a homogeneous size distribution, the collected dispersion was then reinjected into the Extruder, and the entire process was repeated in succession for a total of 10 cycles. Extrusion was carried out at 50 "C by immersing the entire assembly in a water bath. After extrusion the resulting liposome dispersion was incubated for 72 h at 50 "C and then equilibrated at 25 "C for 24 h. To compare partitioning effects at roughly equivalent thermodynamic activities, each ester was added at 16-208 saturation in the aqueous phase (Table I). Liposomes containing encapsulated ["Clinulin and the alkyl paminobenzoate of interest were separated from unentrapped ["Clinulin and the alkyl p-aminobenzoate by ultracentrifugation. Aliquots of the liposome dispersion (175 pL) were centrifuged at 160 000 x g a t 25 "C for 1h with a Beckman Airfuge (Spinco Division, Palo Alto, CA). The weights of the pellet and supernatant were determined immediately aRer centrifugation. Determination of Internal (Captured) Volume["C]Inulin was used to determine the internal aqueous volume of the liposomes. The assumption was that inulin will distribute only into water and will not interact with the phospholipid. It was assumed also that the concentration of free inulin was the same as its concentration in the internal water compartment of the liposome. Entrapped inulin was separated from free inulin as described earlier. The amount of lipid present in the dispersion was determined by using tritiated DPPC and nontritiated DPPC at a molar ratio of 1:l x lo-'. A h r ultracentrifugation, the supernatant and pellet were analyzed for ["Clinulin and [3H]DPPC radioactivity with a dual-labeled 3H- and %-counting program and a Beckman LS5000TD scintillation counter (Palo Alto, CA). Internal aqueous volumes, determined a t 23 "C, were calculated and expressed as microliters of trapped volume per micromole of phospholipid. Determination of Drug Concentration-The amount of alkyl p-aminobenzoate entrapped in the liposomes was determined by separatingfreedrugfrom entrapped drug by ultracentrifugation. The pellet and supernatant were analyzed for the presence of ester with a Perkin-Elmer 7 W-visible spectrophotometer. The supernatant and pellet were aeparated from each other, each was dissolved in a known amount of ethanol, and the UV absorbance8 of the supernatant and dissolved pellet were then measured at the wavelength of maximum absorbance of the alkyl p-aminobenzoate. Blank liposomes dissolved in ethanol were used as reference standards to correct for turbidity effects. Standard absorbance curves of the esters in ethanolic solutions were constructed. The concentration of solute in both phases was determined from the standard curves in the linear region of the Beer-Lambert plot. Calculation of Bilayer-Aqueous Compartment Distribution Coemeients-The of a drug between the aqueous compartments and lipid bilayers can be expressed as follows:

are the concentrations of the drug In eq 1, Cbilayer and Cagueous (expressed as weight ratio) in the bilayer and aqueous phase, respectively; w b and W, are the weights of each phase; and A,, and A, are the weights of h e drug in the bilayer and aqueous phase, respectively. A standard state of an infinitely dilute drug solution is assumed so that activity coefficients are equal to unity. This is

experimentally convenient, because concentration can then be used in place of activity. A,, cannot be directly determined experimentally without altering the original liposome system. However, ultracentrifugation of a liposome dispersion will yield a liposome pellet (containing the bilayer and aqueous phase) and a supernatant. A,b can be determined by subtracting the amount of drug associated with the trapped water in the liposome pellet (A,) from the amount of drug present in the pellet (Aap).A, can be determined by assuming that, at equilibrium, the trapped water inside the liposomes has the same physical properties as bulk water and that the concentration of drug in the entrapped water is the same as that in bulk water. Equation 1 can therefore be rewritten as follows:

(2) In eq 2, W,,w b , and W,represent the weights of the supernatant, lipid bilayer, and trapped water, respectively (W,and W, are directly determined with radiolabels (['HIDPPC as the lipid phase marker and ["Clinulin as the aqueous phase marker), and A, and A,, represent the amount of solute in the supernatant and 1'ipoeome pellet, respectively. The above calculations for the DC of the n-alkyl paminobenzoates in liposomes is similar to that proposed by Katz and Diamond.0 Free Energy of Partitioning-The change in standard free energy of partitioning of drug from the aqueous phase to the lipid bilayer (AGO) can be calculated fmm the DC by wing the following equation: AG" = -2.3 RT log DC, where AGO is expressed in calories per mole, R is the universal gas constant, and T is absolute temperature. AG" therefore represents the change in free energy upon transferring 1 mol of solute from the aqueous phase to the lipid bilayer. Distribution Studies in t h e n-Octanol-HEPES Buffer System-A high-performance liquid chromatography technique was used to determine the octanol-HEPES buffer DCs of the n-alkyl paminobenzoates buffer. The method and its rationale are described in detail by Minick et a1.10 and Mirrless et al.11 The capacity factor (k') represents the partitioning characteristics of a solute between a mobile phase and a stationary phase and is defined as (tr-to)/to,where t, is the retention time of the compound and tois the retention time of a nonretained solute. The logarithm of k' correlates well with the logarithm of the partition coefficient of a solute between octanol and water (logPdw).l@-lz The t, was calculated by measuring the distance from the peak of (-)-tyrosine, the non-retained solute, to the peak of the sample. The void volume was determined by measuring the distance from the peak of the solute idection mark to the peak of the nonretained marker. A Brownlee 3.9 mm x 30 mm C,, stainless steel column was equilibrated with various mixtures (25-658, v/v) of methanol-0.05 M HEPES buffer (pH 7.4) containing 0.25% (v/v) 1-octanol and 0.15% (v/v) n-decylamine. n-Octanol was added to the mobile phase in order to change the partitioning characteristice of the column from an alkane-like phase to an octanol-like phase. Octanol binds strongly to octadecysilane and thus minimizes the number of free silanol sites present on the column and, therefore, minimizes hydrogen bonding influences on partitioning.loJs n-Decylamine also was added to the mobile phase to help mask silanophilic interactions.10 The apparatus for high-performance liquid chromatography consisted of a solvent delivery module (Beckman 114M), an absorbance detector (Waters model 440)operated at a wavelength of 254 nm, and a chart recorder (Linear). The flow rate (1.2-2 mumin) and chart speed were adjusted to obtain sharp, well-defined peaks. A series of standards (pyridine, aniline, acetanilide, phenol, quinoline, anisole, p-chlorophenol, chlorobenzene, andacridine) of known log PdW values and the alkyl p-aminobenzoates (methyl to butyl) were first dissolved in either 0.05 M HEPES buffer (pH7.4) or 2% methanol for the hydrophobic standards and then eluted through a CIScolumn

Table CDrug Concentratlon In Liporome Dlrperalon Prlor to !3eperatlon Procedure'

Liposome Type and Composttion

Methyl pAbab

Ethyl pAbaC

Propyl pAbad

Butyl pAbae

DPPC MLVs DPPC:CHOL MLVs Extruded DPPC

0.93 (9) 1.3 (13) 0.93 (9)

0.73 (14) 0.61 (10) 0.67 (11)

0.38(14) 0.41 (15)

0.14 (15) 0.19 (20) 0.18 (19)

0.45 (16)

Values in parentheses are percent saturation in aqueous phase; drug concentration is In millimolar. paminobenzoate. Propyl pamlnobenzoate. Butyl paminobenzoate.

Methyl paminobenzoate.

Ethyl

Journal of Pharmaceutical Sciences J 1105 Vol. 81, No. 1 1 , November 1992

with the four methanol-buffer mixtures (with methanol and buffer at various ratios). Samples were run in triplicate for each methanolbuffer mixture. Then, k' was determined for each sample for the four methanol-buffer mixtures. The lipophilicity (kJ of each sample was estimated by extrapolation of a plot of log k' versus percent methanol to 0% methanol. A standard curve, with log P,, and log k; for the standards, was then established. Then log P,, of the n-alkyl p-aminobenzoates was determined by interpolation of the regression line between log P,, and log k; of the standards.

Results and Discussion The DCs of a homologous series of n-alkyl p-aminobenmates in three organic solvent-water systems are presented in Table 11. The D C s in octanol-water were determined as described earlier. DCs in oleyl alcohol-water were obtained from the literature, and DCs in hexane-water were estimated by using the mole fraction solubility data of the alkyl esters (methyl to butyl) at 25 OC.5.7914 The ratio of the solubility of each ester in pure water to that in hexane was assumed to be equivalent to the DC.6016 n-Octanol was chosen as a solvent system because of its hydrogen-bonding ability and its solvating and associating properties. Water-saturated n-octanol-water systems presumably possess structural characteristics as a result of the formation of water-n-octanol clusters.16 Many of the quantitative structure-activity relationships reported in the medical literature are based on the use of partitioning in octanolwater systems as models for solute-drug partitioning into biological membranes. Oleyl alcohol was selected because it has been suggested by Collander17 to be a suitable model for biological membranes. Its hydrocarbon chain is similar in length to that of DPPC (CISversus c16).Hexane was chosen primarily because it is an apolar, nonhydroxylic solvent. In all three bulk organic solvent-water systems, the DC of the drug (alkyl p-aminobenzoate) increased linearly on a logarithmic scale with increasing alkyl chain length [determination coefficient (?) >0.96]. The slope (a)of the plot of log DC versus alkyl chain length represents the incremental methylene contribution to the DC (Table 111). For all three bulk organic solvent-water systems, a was -0.6. For most nonpolar solvents, the change in solubility with chain length is usually between 0.3and 0.6.1SThe highest DCs for the alkyl p-aminobenzoate are seen with the octanol-water system and the lowest with the hexane-water system. The DCs in the oleyl alcohol-water system were slightly lower than those in the octanol-water systems. The AGO of the esters in all three solvent systems increases linearly with alkyl chain length of the ester and is lower in the octanol-water system than in the hexane-water system (Figure 1). The incremental ACP due to a methylene unit is presented in Table IV; based on these values, partitioning in the hexane-water system appears to be more sensitive to changes in the structure of the n-alkyl Table ICValues of log DC of a Homologous Serles of rrAlkyl pAmlnobenzoates In Dlfferent Organlc Solvent-Water and Llpoeomal Systems Log DC of Indicated n-Alkyl pAminobenzoate Ester

System

Solvents Octanol Oleyl alcohola Hexaneb Liposomes DPPC MLVs DPPC:CHOL MLVs Extruded DPPC a

From ref 28.

Methyl

Ethyl

Propyl

Butyl

1.4 1.1 -0.57

2.1 1.6 0.026

2.4 2.3 0.63

3.2 2.8 1.3

0.40 1.1 1.1

1.1 1.9 1.7

1.7 2.3 2.1

0.03 0.81 0.77

From ref 7.

1106 I Journal of Pharmaceutical Sciences Vol. 81, No. 11, November 1992

Table Ill-Llnear Regresslon Parametem of Conelatlon between Log DC and Alkyl Chaln Length In Varlous Systems'

a

System Solvents Octanol Oleyl alcohol Hexane Liposomes DPPC MLVs DPPC:CHOL MLVs Extruded DPPC

rz

b

0.57 0.58 0.62

0.85 0.50 1.2

0.97 1.o 1.o

0.57

-0.62 0.21 0.27

0.99 0.97 0.99

0.53 0.46

aLinear regression equation: log DC = ax + b, where a is the contribution of a methylene unit to partitioning, x is the number of carbons on the alkyl side chain, and b is the y intercept. 2OOOl

h -4000

1

-5000

0

2

1

3

4

Number of Carbons on the Alkyl Side Chain

Flgure 1-A@ at 298 K in bulk organic solvent-water systems as a function of alkyl chain length. Key: (0)octanol-water; (A)oleyl alcoholwater;).( hexanwater. Table IV-Llnear Regresslon Parameters of Comlatlon between A@ and Alkyl Chaln Length In Varlous Systems' System

b

rz

-1154 -679 1638

0.97 1.o 1.o

841

0.99 0.97 0.99

a ~

Solvents Octanol Oleyl alcohol Hexane Liposomes DPPC MLVs DPPC:CHOL MLVs Extruded DPPC

-773 -787 -843

- 775 -715 -623

-285 -367

a Linear regression equation: A@ = ax + b, where a is the incremental change In free energy due to a methylene unit, xis the number of carbons on the alkyl side chain, and b is the y-intercept.

p-aminobenzoates than in the octanol-water and oleyl alcohol-water systems. These differences are attributable to the fact that the hexan+water system lacks the semipolar nature to account for dipole interactions between the alkyl p-aminobenzoates and the organic phase, thus resulting in a disproportionate emphasis on methylene group contributions. The bilayer-water DCs of alkyl p-aminobenzoates (methyl to butyl) were determined in three liposome systems: DPPC MLVs, DPPC:CHOL (2:l molar ratio) MLVs, and extruded DPPC liposomes (Table 11). For the three liposome systems studied, the log DC of the alkyl p-aminobenzoates into

liposomes increased linearly as the chain length of the ester increased (?, >0.97). Partitioning into the bilayers of extruded liposomes and in DPPC:CHOL liposomes was slightly less dependent on chain length than partitioning in DPPC MLVs (Figure 2), as also reflected in the free energies of partitioning (AGO) from the aqueous phase to the lipid bilayer (Figure 3). In all three liposome systems, AGO decreased linearly with increasing alkyl chain length, but the incremental AGO due to a methylene residue was greater in DPPC MLVs (Table IV). Nonextruded DPPC:CHOL and extruded DPPC liposomes gave the highest bilayer-water DCs. This result can be attributed to changes in the structural organization of the bilayer caused by the presence of CHOL or the extrusion process.19-20Both CHOL incorporation into the bilayer and the extrusion process cause a decrease in the surface density of the bilayer, thus favoring solute accommodation into the bilayer. The highly ordered anisotropic state of the bilayer in DPPC MLVs is, in essence, a deterrent to solute partitioning, particularly for the more hydrophilic solutes such as methyl and ethyl p-aminobenzoate where partitioning into the bilayer would rely on a balance of hydrophilic and hydrophobic interactions. The bilayer-water DCs of the alkyl p-aminobenzoates were compared with their DCs in three organic solvent-water systems to determine if the latter systems could serve as models to predict drug partitioning in liposomes. Examination of the data in Table I1 reveals that, regardless of liposome type and composition, the use of octanol-water and oleyl alcohol-water systems would overestimate the partitioning of the alkyl p-aminobenzoates into the liposomal bilayer, whereas the use of the hexane-water system would underestimate partitioning into the bilayer. However, the organic solvent-water systems adequately reflect relative partitioning changes that occur in the liposomal bilayer with respect to increasing chain length (Figure 1). The hexane-water system appears to be the most sensitive to solute structural changes and the DPPC extruded liposomes and the DPPC: CHOL multilamellar liposomes the least sensitive to changes in solute structure (Table III). The observations that the organic solvent-water systems do not exactly predict the partitioning of alkyl p-aminoben-

A

-1.0 4 0

1

2

3

4

Number of Carbons on the AlkyI Side Chain

Flgure 2-Bilayer-water DCs at 25 “C as a function of alkyl chain length. Key: (A) DPPC MLV; (B) DPPC:CHOL MLV (2:l molar ratio); (0) extruded DPPC liposornes. Points represent the average of three independentdeterminations t standard error. Drug concentrations used are listed in Table I.

-1

0

1

2

3

4

Number of Carbons on the Alkyl Side Chain

Figure S A G O at 298 K as a function of alkyl chain length. Key: (0) DPPC MLV; (B) DPPC:CHOL MLV (2:l molar ratio); (A) extruded DPPC liposornes. Points represent the average of three independent determinations t standard error. Drug concentrations used are listed in Table

I.

mates into liposomes is not unexpected. This is because two different types of “lipid phases are being compared. The bulk organic solvent phases are isotropic in their physical properties, whereas the phospholipid bilayer is anisotropic. The lipid bilayer can be considered as a n interfacial phase of matter in which physical properties vary with distance from the interface. A gradient of chain disorder exists in the hydrocarbon core of the bilayer; chain ordering increases with distance from the midbilayer toward the interface.21-29 Properties of interfacial phases of matter depend on surface density, whereas properties of bulk phases do not. The Collander equation17 has been used to correlate the DC of solutes in bilayer-water systems with the values for the same solutes in organic solvent-water systems9824-? log DC, = a + b log DCI1, where DCI is the DC of the solute in liposomes and DCII is the DC of the same solute in a bulk organic solvent system. The slope (b) of this double logarithmic plot quantifies the sensitivity of the system to perturbation by hydrophobic effectsl4Je and reflects the similarity of the solvent environment in the two systems.9.24 Leo et al.14 have suggested that the intercept (a) represents the “sensitivity of the biochemical system and the intrinsic activity of a set of congeners”. Leo et al.14 also indicate that the intercept correlates well with solvent lipophilicity and with water content a t saturation. On the other hand, Beezer et a1.28have proposed that the intercept has no fundamental physical meaning. The regression parameters of this double logarithmic plot for the various partition systems studied are presented in Table V. All three liposome systems exhibit good correlation with the three bulk organic solventwater systems. The best 1.2 values are seen with the oleyl alcohol-water systems. This may be due to the similarity of the oleyl alcohol system to the DPPC bilayer in regard to both hydrocarbon chain length as well as interfacial polarity. Similar correlations using the Collander relationship for organic solvents and liposomes have been found for other solutes.9.24,29 The slopes and intercepts of the Collander correlation between a given liposomal system and the three bulk organic solventwater systems provide useful information (Table V). The slope reflects differences in the nonaqueous-solvent properties of the two systems being compared. For example, for the Journal of Pharmaceutical Sciences I 1107 Vol. 81, No. 11, November 1992

Table V-Llnear Regresslon Parameters of Correlatlonr between l in DPPC Liporomem and in log DCr of ~ A l k ypAmlnobenzoate8 Various Bulk Organlc Solvent-Water Syrtems'

Solvent System Ii

ab

bC

P

DPPC MLV

Octanol Oleyl alcohol Hexane

-1.40 -1.10 0.49

0.97 0.99 0.92

0.95 0.99 0.99

DPPC:CHOL MLV

Octanol Oleyl alcohol Hexane

-0.47 -0.26 1.23

0.88 0.92 0.85

0.90 0.99 0.97

Extruded DPPC

Octanol Oleyl alcohol Hexane

-0.34 -0.13 1.20

0.77 0.79 0.74

0.94 1.0 0.99

Solvent System I

Linear regression parameters were calculated by using the Collander equation17 (seetext). Intercept. Slope. a

DPPC MLVs, the slope values are 0.97,0.99, and 0.92 when compared with octanol-water, oleyl alcohol-water, and hexane-water systems, respectively. This suggesta that partitioning of the n-alkyl p-aminobenzoates into the bilayers of DPPC MLVs is largely into a region that has the same degree of hydrophobicity as octanol and oleyl alcohol but slightly less hydrophobic than hexane. The slope values for DPPC:CHOL MLVs and extruded DPPC liposomes compared with the same solvent systems are consistently lower than those for DPPC MLVs. This suggests that the extruded DPPC liposomes and the nonextruded DPPC:CHOL MLVs have bilayers that are much less hydrophobic than octanol and oleyl alcohol and much more hydrophilic than hexane. These results can be explained largely by the differences in phospholipid organization of the different types of liposome systems. The phospholipids in the bilayers of DPPC MLVs are highly organized and tightly packed, thus creating a very dense bilayer, whereas the presence of CHOL or extrusion increases the spacing between the phospholipid headgroups and forms a bilayer having a lower surface density.lQ.20This increase in phospholipid headgroup spacing promotes an environment more amicable to the inclusion of semipolar solutes. The intercepts from Collander plots (Table V) associated with liposomal correlations with octanol-water and oleyl alcohol-water systems were all negative, whereas they were positive for those with hexane-water systems. These results imply that hexane-water systems are more lipophilic than the bilayer-water systems. The intercepts for the correlation with the hexanewater system increased from 0.49 for DPPC MLVs to 1.23 and 1.16 for DPPC:CHOL MLVs and extruded DPPC liposomes, respectively. Because the surface densities of the bilayers of the latter two liposome systems are lower and hence more hydrophilic than the surface density of DPPC MLVs at 25 "C, it is expected that the intercept would be larger for these two systems. The same reasoning also applies

1108 I Joumal of Pharmaceutical Sciences Vol. 81, No. 11, November 1992

to the correlations of liposomal systems with octanol-water and oleyl alcohol-water systems. The intercept values for DPPC: CHOL MLV liposomes and extruded DPPC lipoeomes were less negative than that for the MLVs; this result once again implies that the CHOL-containing and extruded liposoma1 bilayers have a phospholipid packing arrangement that is different from that in the DPPC multilamellar bilayer. In conclusion, organic solvent-water systems can reflect partitioning trends that occur in the liposomal bilayer with respect to increasing the alkyl chain length of alkyl p a m i nobenzoates. However, the isotropic nature of these systems limits their usefulness in predicting solute partitioning into the highly anisotropic lipid bilayer.

References and Notes 1. Meyer, H. Arch. Exp. Pathol. Pharmakol. 1899,42,1OS118. 2. Overton, C.E. Studien iiber die Narkose; Fischer: Jena, Germany, 1901;p 195. 3. Adam, R.; Rideal, E. K.; Burnett, W. B.; Jenkins, R. L.;Dreiger, E. E.J . Am. Chem. SOC.1926,48,1758-1770. 4. Yalkowsky, S. H.; Amidon, G. L.;Zografi, G.; Flynn, G. L. J . Phurm. Scr. 1975,48-52. 5. Shah, A. C.;Nelson. K. G. J . Phurm. Sci.1980.69,210-212. 6. Yalkowsky, S. H.;Flynn, G . L.;Slunick, T. G; J . Phurm. Sci. 1972,61,852-857. 7. Neau, S. Ph.D. Thesis; University of Michigan, Ann Arbor, MI, 1988.

8. Bangham, A. D.; Standish, M. M.; Watkins, J. C. J . Mol. Bwl. 1965,13,238-252. 9. Katz. Y.: Diamond. J. M. J . Membr. Bwl. 1974.17.69-86. 10. Minick, D.J.; Frek, J. H.; Patrick, M. A.; Brent, D. A. J . Med. Chem. 1988,31,1923-1933. 11. Mirrless, M . S.;Moulton, S. J.; Murphy, C. T.; Taylor, P. J. J . Med. Chem. 1976,19,615-619. 12. Caron, J. C.;Shroot, B. J . Pharm. Sci. 1984,73, 1703-1706. 13. Unger, S.H. J . Phurm. Sci.1978,67,1364-1367. 14. Leo,A.; Hansch, C.; Elkins, D. Chem. Rev. 1971,71,525-616. 15. Yalkowaky, S.H.; Valvani, S. C.; Roseman, T. J. J . Phurm. Sci. 1983,72,8664370. 16. Smith, R. N.; Hansch, C.; Ames, M. J . Phurm. Sci. 1975, 64, 599-606. 17. Collander, R. Actu Chem. Scand. 1951,5,774-780. 18. Yalkowsky, S. H.;Flynn, G. L. J . Phurm. Sci. 1973,62,210-217. 19. Ma, L.;Ramachandran,C.; Weiner, N. D. Znt.J . Pharm. 1991,70, 209-218. 20. Ma, L.;Ramachandran,C.; Weiner, N. D. Znt. J . Pharm. 1991,77, 127-140. 21. Schindler, H.; Seelig, J. Biochemistry 1975,14,2283-2287. 22. Seelig, A.; Seelig, J. Biochemistry 1974,13,48394845. 23. Levine, Y. K.; Wilkins, M. H. F. Nature New Biol. 1971, 230, 69-72. 24. Katz, Y.; Diamond, J. M. J . Membr. Bwl. 1974,17,101-120. 25. Betageri, G.V.; Rodgers, J. A. Znt. J . Pharm. 1987,36,165-173. 26. Betageri, G.V.;Rodgers, J. A. Phurm. Res. 1989,6,399-403. 27. Jain, M. K.;Wu, N. Y.; Wray, L. W. Nature 1975,255,494-495. 28. Beezer, A. E.;Cooch, C. A.; Hunter, W. H.; Volpe, P. L.J . Phurm. Pharmmol. 1987,39,774-779. 29. Rodgers, J. A.; Davis, S. S. Biochim. Bwphye. Actu 1980, 598, 39-04.