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Favorable amphiphilicity of nimodipine facilitates Its interactions with brain membranes
Pergamon
002%3908(93)EOOlS-6
Neuropharmacology Vol. 33, No. 2, pp. 241-249, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0028-3908/94 $6.00 + 0.00
Favorable Amphiphilicity of Nimodipine Facilitates Its Interactions with Brain Membranes L. G. HERBETTE,lm317
P. E. MASON,’
K. R. SWEENEY,’
M. W. TRUMBORE’
and R. P. MASON3,4,6.7 Departments of ‘Biochemistry, ‘Medicine, ‘Radiology, 4Psychiatry and ‘Pharmacy, 6Travelers Center on Aging, ‘Biomolecular Structure Analysis Center, University of Connecticut Health Center, Farmington, CT 06030, U.S.A. (Accepted 25 October 1993)
Summary-Nimodipine is a 1,4_dihydropyridine (DHP) calcium channel blocker which is used in the treatment of neurological deficits associated with subarachnoid hemorrhage. Small angle x-ray diffraction, differential scanning calorimetry, and equilibrium and kinetic binding techniques were used to study the interaction of nimodipine with bovine brain phosphatidylcholine (BBPC) membranes of varying cholesterol content. At concentrations (5 x lo-” M) near its &, the membrane partition coefficient of nimodipine was inversely related to the cholesterol to phospholipid (C: P) mole ratio in both model and native (rat synaptoneurosome) membranes. The nonspecific dissociation rate of nimodipine from BBPC was significantly slower at low C : P mole ratio (0.1: 1) than at high C: P mole ratio (0.6: 1). Calorimetric analysis showed that nimodipine decreased both the main phase transition temperature and cooperative unit size of melt for dimyristoyl phosphatidylcholine, dependent on membrane cholesterol content. Small angle x-ray diffraction analysis showed that nimodipine occupies a position in BBPC approx + 15 A from the center of the hydrocarbon core, near the hydrocarbon core/water interface. These data indicate that nimodipine is an amphiphilic molecule which rapidly washes out of and transports across membrane bilayers, facilitating its interactions with membranes and possibly its transport across the blood-brain barrier. KeywordsMembrane,
bilayer, cholesterol, Alzheimer’s, nimodipine, x-ray diffraction, kinetics, partitioning.
Nimodipine is a 1,Cdihydropyridine (DHP) which affects cerebrovascular and neuronal cell activity by modulating L-type voltage-sensitive Ca+* channels in the plasma membranes of these cells. Nimodipine initiates vasodilation by altering transmembrane influx of calcium across the plasma membrane of arterial smooth muscle cells. As a calcium channel antagonist, nimodipine binding in human neuronal membranes is specific and saturable with an affinity constant of 0.27 nM-’ while the maximal number of binding sites has been shown to be 5.8 pmol/g wet wt in human frontal cortex (Peroutka and Allen, 1983. In rat brain, the distribution of nimodipine binding sites is higher in the cerebral cortex than in the cerebellum (Skattebol and Triggle, 1987). Nimodipine is the only DHP approved in the United States for treatment of neurological deficits associated with subarachnoid hemorrhage (Janis and Triggle, 1991). There is also experimental evidence for nimodipine being effective in the treatment of ischemia (Steen et al., 1983, 1984, 1985), epilepsy (Hoffmeister et al., 1982; Meyer et al., 1986a, b, 1987) and most *To whom correspondence should be addressed: Dr. Leo G. Herbette, Biomolecular Structure Analysis Center, University of Connecticut Health Center, Farmington, CT 06030, U.S.A.
recently, degenerative dementia of the Alzheimer’s disease type (Tollefson, 1990). The CNS activity of nimodipine may be related, in part, to favorable physical and chemical properties which facilitate its transfer across the blood-brain barrier and its bioavailability in the brain. Compared to other DHPs, nimodipine exhibits cerebrovasodilatory and neuronal activity at concentrations less than that required to induce peripheral hypotension (for review, see Scriabine et al., 1989). For example, the apparent volume of distribution of nimodipine in rat brain was found to be several-fold greater than that of nifedipine, a closely related compound (van den Kerckhoff and Drewes, 1985). Once in the brain, nimodipine partitioning into the neuronal plasma membrane may precede receptor binding as has been previously proposed for vascular smooth muscle (Rhodes et al., 1985; Herbette et al., 1989). Thus, the membrane concentration of nimodipine, as opposed to its aqueous concentration, is likely to be in equilibrium with the plasma membrane Ca+* channel and this may have a critical effect on the potency/activity of the drug (Mason et al., 1991). A precise understanding of the true lipophilicity (or amphiphilicity) of nimodipine requires direct measurement of its interaction with membrane lipid bilayers 241
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HERBETTE et al.
of varying lipid composition (e.g. cholesterol content). Previous studies have shown that membrane partitioning for this class of drugs varies substantially and is highly dependent on the cholesterol content of the membrane (Mason er af., 1992a), an important factor when considering that neural membrane cholesterol composition changes with disease, including Alzheimer’s disease (Mason et al., 1992b). Moreover, membrane cholesterol heterogeneity is observed at the cellular and subcellular levels (for review, see Yeagle, 1987). The goal of this study, then, was to investigate the interaction of nimodipine with membranes of varying lipid composition, especially with respect to cholesterol, to reproduce the heterogeneity found in brain tissue. Specifically, the equilibrium and kinetic membrane interactions of nimodipine were measured as a function of cholesterol content using reconstituted and intact brain membranes. The thermodynamic properties associated with the membrane interactions of nimodipine were evaluated with calorimetry. The results of this work indicate that at concentrations near its Z&, the equilibrium and kinetic binding properties of nimodipine are highly dependent on membrane cholesterol content. Moreover, nimodipine insertion into the membrane perturbs the organization of the lipid bilayer, as evidenced by significant changes in membrane thermotropic properties. This latter effect was also highly dependent on membrane cholesterol content. These data may have important implications for understanding the ability of nimodipine to passively cross the blood-brain barrier in order to achieve a high volume of distribution in the brain for activity. METHODS
Preparation
of model phospholipid
membrane
vesicles
For kinetic experiments, multilamellar vesicles (MLV) and large unilamellar vesicles (LUV) formed from bovine brain phosphatidylcholine (BBPC) in the presence and absence of cholesterol were utilized. MLV and LUV are useful for filtration experiments as they efficiently bind to glass fiber filters utilized for nonspecific membrane kinetic experiments. BBPC and cholesterol, both solubilized in chloroform, were obtained from Avanti Polar Lipids Inc. (Alabaster, AL) and stored in a dessicator at -20°C. Thin layer chromatography was used to ascertain purity and the presence of degradative products in the lipid and cholesterol suspensions. Specific quantities of cholesterol and phospholipid in mole ratios (C: P) of 0.1: 1, 0.3 : 1, and 0.6: 1 were mixed and dried down in a glass test tube, while vortexing, to a thin film under a stream of N, gas, and placed under vacuum overnight to remove residual solvent. Tris buffer (150 mM NaCl, 10 mM Trizma HCl, pH 7.0) was then added to yield a lipid concentration of 2 mg/ml and the solution was vortexed at room temperature (above the thermal phase transition temperature) to form MLV. LUV (average 80 nm dia) were formed by extruding MLV through a 100 nm polycarbonate filter mounted in a mini-extruder (“Liposofast”, Avestin,
Ottawa, Canada) as previously described (MacDonald et al., 1991). The structural integrity of these vesicles was examined by transmission electron microscopy after staining the lipids with 0~0,. To confirm the final C: P mole ratio in the membrane vesicles, a portion of the vesicles was assayed for cholesterol and phosphate as described below. Preparation
of native membrane
vesicles
Synaptoneurosomes were isolated from the cerebral cortex of an adult Sprague-Dawley rat (Moring et al., 1990) and assayed for protein, phospholipid, and cholesterol content. Specifically, membrane protein was determined using bovine serum albumin as a standard (Lowry et al., 1951). The phospholipid phosphorus content of the samples was determined by previously described methods (Chen et al., 1956; Chester et al., 1987). Cholesterol was extracted from native membranes using a chloroform : methanol : water system (Folch et al., 1957). A calorimetric assay was used to measure total cholesterol in native membranes by a modification of the cholesterol oxidase method (Chester et al., 1986; Heider and Boyett, 1978). Drug/membrane
partition
coejicients
The membrane partition coefficient is an equilibrium value expressing the relative amount, by mass, of drug in the membrane vs aqueous buffer. This distribution of drug between the membrane lipid and aqueous buffer is an expression of the drug’s true lipophilicity or affinity for the membrane bilayer. Nimodipine partition coefficients in model and native membranes (Kp,,em,) were experimentally measured using vacuum filtration as previously described in detail were filtered (Herbette et al., 1989). All solutions through Whatman GFjC glass fiber filters on a Brandel M-48 cell harvester (Brandel, Gaithersburg, MD). Using vesicles (20 pg/ml) radiolabelled with [ ‘HIcholesterol, the amount of membrane lipid retained on the filters during the filtration process was accurately and reproducibly measured. It was shown that 82 f 2% of MLV and 51 + 2% of LUV (mean f SD) were retained regardless of the C:P mole ratio in four trials consisting of 8 samples (n = 32) for each mole ratio. The filter retention of rat synaptoneurosomes labelled with [ 3H]cholesterol was 75% (1 trial consisting of 6 samples). In experiments measuring total nonspecific binding, the incubation reaction mixture consisted of a fixed phospholipid concentration of 20 pg/ml, with varying C: P mole ratios in the model membranes (0: 1, 0.3 : 1, 0.6 : 1). Radiolabelled [‘Hlnimodipine (New England Nuclear Research Products, Boston, MA) and unlabelled nimodipine (Miles Pharmaceuticals, West Haven, CT) dissolved in ethanol was added to produce a final concentration of drug in the reaction mixture of 5 x lo--” M. The KPtmemlvalues for nimodipine have been previously shown to be independent of drug concentrations between lo-” M and lop6 M (Herbette et al.,
Amphiphilicity
1989). A drug concentration of 5 x lo-” M was chosen because this value is similar to the K,, for nimodipine. Since the equilibrium partitioning of nimodipine into MLV and LUV is not measurably timedependent, incubation time for all reactions was set at 30 min. The filters for these experiments were not washed. Reaction mixtures containing drug, but no membrane, were used to correct the total nonspecific binding for binding of drug directly to filters. All filters were counted for radioactivity (Tracer Analytic Delta 300/689 1 Liquid Scintillation System). The amount of drug in the filtrate was determined by subtracting the number of moles of drug bound to the membrane from the total number of moles of drug added to the reaction mixtures. The amount of lipid bound to the filters was corrected for the recovery of membrane on the filters. Membrane partition coefficients were thus calculated for each trial using the following equation: K P[mem]=
(g of drug bound to membrane/g (g
of lipid)
of free drug/g of buffer)
Drug release C’ Washout”) from membranes
Time-dependence of nimodipine release from model membranes (MLV 0.6: 1 and 0.1: 1; LUV 0.6: 1) was measured by incubating [ 3H]nimodipine (5 x lo- ‘OM) with phospholipid (20 pg/ml) in Tris buffer (pH 7.0) for 30 min. All solutions were filtered through Whatman GF/C glass fiber filters on a Brandel cell harvester, but not washed. Control reaction mixtures contained drug, but no membranes. Three filters with membranes and 3 control filters were immediately counted for radioactivity, representing maximal binding of drug to membranes. The remaining filters were impaled, in groups of 3, on 25-gauge needles mounted on a flat Plexiglass “donut”. The filters on each needle were spaced with pieces of tubing (length 1 cm, o.d. 1 mm) to prevent filters from touching. The Plexiglass structure was then placed near the base of a 3-l round plastic container situated on a magnetic stirrer at medium speed, and the container was filled with Tris buffer. At specific time intervals, filters with and without membranes were removed from the buffer and counted for radioactivity. Control filters retained at least 90% of the lipid over the duration of these washout experiments for both multilamellar and unilamellar preparations, independent of cholesterol content. Experimental data were corrected to account for this loss of lipid. Statistical evaluation and computer modeling
Statistical analysis was used to evaluate nonspecific equilibrium binding parameters for nimodipine. For model membrane partition coefficient experiments, four trials consisting of 12 samples (n = 48) were carried out for each incremental change in the C : P mole ratio for both MLV and LUV. For each set of experiments, K PCmemI values were obtained and standard error of the mean was calculated. The Student’s t-test was used to determine significance of differences between KPLmemI val-
243
of nimodipine
ues found for MLV vs LUV at each C: P mole ratio. From the synaptoneurosome preparation 12 aliquots were used for determining partition coefficients; a mean K P~meml was determined. For nonspecific membrane dissociation rates, data were collected at a minimum of 13 (maximum 16) time points, ranging from 0 to 150 min depending on the C : P mole ratio. Each time point consisted of 3 experimental and 3 control determinations. Raw data from a minimum of 4 washout curves generated for each condition were analyzed using a least-squares fitting algorithm (PCNONLIN, SC1 Software, Lexington, KY). An empirical model was fitted to the observed data in an effort to describe the washout curves and obtain estimates of half-life of nimodipine release. DifSerential scanning calorimetry
Differential scanning calorimetry (DSC) was carried out on drug-containing and control MLV prepared by the following procedure. For control MLV, 100 ~1 aliquots of a 0.03 M solution of dimyristoylphosphatidylcholine (DMPC) in chloroform was dried to a thin film by evaporation under N, with the residual solvent being removed by overnight evacuation. The dried lipid was then rehydrated for 10 min at 50°C in 100 ~1 of sample buffer (0.05 mM Hepes, 150 mM NaCl, pH 7.0) and vortexed for 1 min to form MLV. Two different cholesterol : phospholipid mole ratios were used in this study: 1: 20 and 1: 10. In these cases, either 5.0 or 10.0 ~1 aliquots of 0.03 M cholesterol solution in chloroform were dried down with the lipid. For nimodipine-containing samples (1: 44 drug : DMPC), 2.25 ~1 of a 0.03 M stock solution of nimodipine in chloroform was dried down with the lipid and/or lipid/cholesterol at the two ratios stated above and vesicles were produced as described. Aliquots (15 ~1) of MLV containing 0.300 pg of DMPC were placed into DSC sample pans and hermetically sealed. DSC was carried out using a TA Instruments DSC 2910 Differential Scanning Calorimeter (TA Instruments, New Castle, DE) with the data being analyzed using a TA Instruments Thermal Analyst 2000 system. The scan rate was 2”C/min starting at 6°C and ending at 34.5”C. The reference was 15 ~1 of sample buffer. The change in cooperative unit size was determined by the following formula: ACUS = (AH&H*Fw),,,,
- (AH,,/AH*Fw),,,,,,
where ACUS is the change in the cooperative unit size, AHvh is the van? Hoff enthalpy, AH is the calorimetric enthalpy and Fw is the formula weight of the lipid undergoing transition. Preparation of multibilayer samples for x-ray dlrraction
The BBPC MLV were prepared in the presence of known amounts of nimodipine as follows. The desired amounts of lipid and cholesterol (C:P 0.6: 1) dissolved in CHCl, were placed in a glass test tube, dried to a thin film under a stream of N,, and placed under vacuum overnight to remove residual solvent. Buffer (0.5 mM
244
L.G. HERBETTE et al.
Hepes, 2.0 mM NaCl, pH 7.27) was added to yield a final phospholipid concentration of 5 mg/ml and the solution was vortexed to form a cloudy white suspension of MLV (Bangham et al., 1965). Nimodipine (dissolved in ethanol) was added to each sample aliquot to produce final drug: lipid mole ratios of 1: 15 and 1: 25. Ethanol concentration in these samples was ~5% prior to centrifugation. Multibilayer samples for small angle x-ray diffraction were prepared as described previously (Chester et al., 1987). Sample preparation and dehydration took approx 3 hr. The membrane samples were stored at 4°C. Salts used for controlling relative humidity (r.h.) were Na/Na tartrate (91%), ZnSO, (93%), KNO, (96%) and K,SO, (98%). Small qngle x-ray
dlflraction
CuK, x-rays produced by a GX-18 rotating anode generator (Enraf Nonius, Bohemia, NY) were either line focused for the small angle studies or point focused for the equatorial studies using Franks’ mirror assemblies. A nickel filter was used to select CuK, radiation (n = 1.54W). Diffraction
data collection
Data were recorded at 5°C on a Braun positionsensitive 1-D detector (Innovative Technologies, Inc., South Hamilton, MA) interfaced to a MicroVax II (Digital Equipment Corp., Maynard, MA). Data reduction for this method (background and geometrical corrections) has been described previously (Mason et al., 1989). Since the entire lamellar reflection for each observed intensity was collected by the detector, the lamellar intensity functions from the BBPC samples collected with the electronic detector were simply Lorentz corrected by a factor of s = 2 sin e/n. Data were recorded on Kodak DEF-5 (Eastman Kodak Co., Rochester, NY) film and qualitatively examined to determine the high angle acyl-chain packing of the samples and to verify the low angle detector data. Phasing
the data
A swelling analysis was used to phase the lamellar reflections for each experiment (Moody, 1963). Sets of intensity data obtained at different hydration states ranging from 91 to 98% relative humidity, each with unique unit cell repeat distances, were used to assign an unambiguous phase combination to the experimentally obtained structure factors (h = l-6). The final phase choice was the only physically reasonable combination, in terms of empirical examination of resultant electron density profiles. RESULTS Equilibrium kinetics
partitioning
AND DISCUSSION and
association /dissociation
Figure 1 summarizes the effect of membrane cholesterol content on the equilibrium membrane partition coefficients (KPtmeml) of nimodipine in BBPC and rat
n BBPC MLV
ka BBPC LUV q RAT SNM
0
0.55
0.3 Cholesterol
: Phospholipld
mol
0.6 ratio
Fig. I. Effect of membrane cholesterol content on nimodipine partitioning into mode1 and native membranes. The equilibrium Kpt,,,I of nimodipine was measured in membranes of varying cholesterol content, including bovine brain phosphatidylcholine (BBPC) multilamellar vesicles (MLV) and large unilamellar vesicles (LUV), as well as rat synaptoneurosomes. Lipid concentrations were maintained at 20pg/ml (pH 7.0, 2lC), and drug concentrations were maintained at 5 x 10.“M.
synaptoneurosomes. Results for model membranes (MLV and LUV) are shown as the mean f SEM and demonstrate an inverse relationship between membrane C: P mole ratio and KP,meml.For the rat synaptoneurosome preparation (in which the C: P mole ratio was determined to be 0.55: 1), the membrane partition coefficient was found to be consistent with the KPImemlvalue expected for model systems between C: P mole ratios 0.3: 1 and 0.6: 1 by interpolation. Thus, the interaction of nimodipine with membranes is highly sensitive to the amount of cholesterol in the membrane and the drug would be expected to preferentially partition into membranes that contain lower amounts of cholesterol. For each different C :P mole ratio, partition coefficients for MLV and LUV were not found to be statistically different. These data demonstrate that the drug comes to equilibrium with lipid vesicles independent of the surface area to volume ratio under experimental conditions in which the total amount of lipid is kept constant. These findings indicate that nimodipine gains access to all available lipid and is not restricted to the outermost layers of the MLV system. It was not possible to measure association (wash-in) kinetics for nimodipine in either MLV or LUV since equilibrium appeared to be achieved by the earliest time point feasible, viz. 30 sec. Moreover, the rates of nimodipine dissociation (washout) from MLV and LUV at the same C:P mole ratio (0.6: 1) were found to be similar, as evidenced by first-order washout half-lives (k SD) of 3.83 (+ 0.267) and 3.46 (& 0.332) min, respectively. Nimodipine thus appears to transport easily across lipid bilayers arranged in either a single or multilamellar system. The computer-generated curves in Fig. 2 represent the dissociation of nimodipine from MLV at C : P 0.1: 1 vs 0.6: 1. The nimodipine washout data from both lipid
Amphiphilicity
245
of nimodipine
0.6
Time (mid
0.4
0.2 --*_-___ 0
~------lD_____ L
J,
0
10
40
30
20
60
50
1.4
10
0.6
Time
15
20
(min)
0.4
0.2
0
d
3.
6b
g6
.
. 120
150
e
l.O-:
I
,i
(cl
o.8- 4
‘t: : IL
0.6
- i :‘: i ‘:,‘*.,
_E
i
0.4
*
\ \
‘l\ ‘. *... \
---
15
20
(min)
c:P
0.6:1
--A______ -----A___
0
10
------- c:p 0.1:1
‘L\
‘\__ o-,
5
Time
\ 0.2-
-0
, 30
------______ -------_______ --------_______ -----___ . I_ I 1 . I -__ 60 90 120 15c Time
(mid
Fig. 2. Effect of membrane cholesterol content on the release of nimodipine from model membranes. The release of nimodipine from bovine brain phosphatidylcholine (BBPC) multilamellar vesicles was measured as a function of time in the presence of 0.6: 1 (a and c) and 0.1: 1 (b and c) cholesterol: phospholipid mole ratios. Membranes (20 pg/ml phospholipid, pH 7.0, 21”C), were incubated with nimodipine (5 x lo-” M) for 30 minutes prior to To.Data were plotted as mean f SD in (a) and (b), and analyzed (c) using a least-squares fitting algorithm (PCNONLIN).
246
L. G. HERBETTE et al.
systems were fitted with a sum of first-order and zeroorder terms. The first-order half-life (*SD) of nimodipine dissociation was 3.83 (+ 0.267) minutes for C : P 0.6:1 vesicles and 13.7 (f2.74)min for C:P O.l:l vesicles. Integration of the equation describing release of nimodipine was performed in order to calculate area under the washout curve (AUC) from time 0 to 100 min. One hundred minutes was chosen as the upper limit of the time frame since this time represents greater than 99% of the total washout of drug in the first-order portion of the curve for both MLV and LUV. Total AUC was strikingly different: AUC for C : P 0.1 : 1 (lower cholesterol) was more than 2-fold higher than the AUC for C: P 0.6: 1 (26.9 vs 11.5 fraction*minutes, respectively). The percent of total AUC attributable to the first-order release portion of the curve was similar for each condition, 46.1% and 42.0% for C : P 0.1 : 1 and 0.6: 1, respectively. The total AUC due to first-order release from C : P 0.1: 1 vesicles was 2.5fold higher than from C : P 0.6 : 1 vesicles (12.4 vs 4.83 fraction*minutes, respectively). Similarly, the total AUC due to zero-order release from C : P 0.1 : 1 vesicles more than 2-fold greater than from C: P 0.6: 1 vesicles. It appears that cholesterol content of the lipid bilayer has a profound effect on nimodipine washout, affecting both the first-order and zero-order portions of the washout curve. These experiments demonstrate that nimodipine can rapidly wash into a single membrane bilayer compartment (LUV) as well as across a series of membrane bilayers (MLV). These results are consistent with the very rapid wash-in of nimodipine in both model and native membranes (Herbette et al., 1989). Nimodipine partition coefficients in both MLV and LUV are inversely related to cholesterol content; furthermore, nonspecific dissociation rates are much slower at low C : P (0.1 : 1) than at high C:P mole ratios (0.6: 1). Thus, the kinetics of nonspecific drug interactions with MLV and LUV are highly dependent on membrane cholesterol content, and indicate that nimodipine may not gain equal access to all available sites within the lipid bilayer in the presence of elevated levels of cholesterol. The ability of nimodipine to partition into the membrane may be a critical determinant of its bioavailability; that is, changes in membrane cholesterol content could substantially modulate the concentration of drug in membrane sites. In brain tissue, perturbations of membrane cholesterol composition during aging (increased cholesterol content) and in Alzheimer’s disease (decreased cholesterol content) may affect the bioavailability of calcium channel blockers such as nimodipine (Mason et al., 1992a, b). D$erential
scanning
calorimetry
In the absence of cholesterol, nimodipine, as do other dihydropyridines, strongly perturbed the thermal properties of DMPC membranes. These results have been summarized in Table 1. At the drug : lipid ratio used in this study (1:44), nimodipine in the absence of cholesterol decreased the main phase transition temperature by
Table
1. The effects of cholesterol on the nimodipine/lipid brane interaction
Sample
C:L
L L/N L/C L/C/N L/C L/C/N
mole ratio 0 0 I:20 I : 20 1: 10 1: 10
Tm 22.19 22.27 22.43 21.62 22.18 21.01
ATm -0.52 -0.81 -1.17
CUS 332 205 325 191 542 220
memACUS -127 -133 -322
Tm, temperature of main phase transition; CUS, cooperative unit size; L, DMPC (dimyristoylphosphatidylcholine); N, nimodipine; C, cholesterol. The mole ratio of nimodipine to DMPC was I :44.
0.52”C, going from 22.79”C in the absence of drug to 22.27”C in the presence of nimodipine. In addition, nimodipine caused a decrease in the number of molecules simultaneously undergoing the phase transition by 127 molecules, the cooperative unit size decreasing from 332 molecules in the absence of nimodipine to 205 molecules when nimodipine was present. As the amount of cholesterol in the membrane increased, nimodipine exhibited increased membrane perturbing activity. At a cholesterol: DMPC mole ratio of 1 : 20, nimodipine decreased the Tm by 0.81”C and the cooperative unit size by 133 molecules as compared to the DMPC : cholesterol control. At a ratio of 1: 10 the decrease in Tm was 1.17”C and the decrease in cooperative unit size was 322 molecules. From these data it is clear that cholesterol modulates the ability of nimodipine to interact with a membrane, thereby enhancing its ability to perturb the thermal properties of a membrane. As was shown in Table 1, the presence of cholesterol in DMPC membranes strongly affects the magnitude of the membrane perturbation caused by nimodipine. Cholesterol serves to enhance the effects of nimodipine leading to a much larger change in membrane thermal properties than would be expected. It appears that as the cholesterol content of the DMPC membranes increases, nimodipine causes a greater disruption of membrane structure. Based on these results one might expect that it would be easier to remove nimodipine from membranes with increased levels of cholesterol, which is what was observed in nonspecific dissociation experiments. As was stated previously, cholesterol alters the structure of acyl chains in the vicinity of the molecule. As nimodipine partitions into the membrane it perturbs the packing characteristics of the lipid acyl chains in its vicinity. Because cholesterol occupies a similar position in the membrane, the effect of nimodipine is magnified, causing an even greater perturbation. Therefore, by occupying a position similar to that of nimodipine, cholesterol could reduce the free volume available for nimodipine to partition into, thereby reducing its KPlmeml,while at the same time enhancing the amount of perturbation to the membrane structure caused by nimodipine, leading to an increased rate of “washout”. Structural
model
The primary structure of the DHP-sensitive (L-type) calcium channel has been determined by molecular biology techniques. The site of photoreactive DHP
Amphiphilicity of nimodipine covalent binding to the CI,subunit has been mapped with site-directed antipeptide antibodies; these studies indicate that amino acid sequences associated with two transmembrane helices near the extracellular membrane surface of the CL,subunit contribute to the DHP binding site (for review, see Catterall and Striessnig, 1992). The amino acids which constitute this binding site region are both hydrophobic and hydrophilic, at the hydrocarbon core/water interface. Figure 3 shows the superposition of two electron density profile structures of the BBPC membrane bilayer with different amounts of nimodipine. From this comparison, the time-averaged center of mass location of Brain membrane with nimodipine 0.6: I cholesterol
I
-20
1 110
0
: lipid
10
20
30
Fig. 3. Membrane bilayer electron density profiles on a relative scale. Electron density profiles for BBPC membranes (0.6: 1 cholesterol : phospholipid mole ratio) in the presence of 1: 25 (solid line) and 1: 15 (dashed line) nimodipine : phospholipid mole ratios. The profiles were arbitrarily scaled to one another to match the electron density maxima and minima. The profiles were correlated to a highly schematic representation of a cholesterol-containing membrane, as shown in the lower panel. The two maxima of electron density at x = + 20 A correspond to the electron-dense phosphate headgroups, whereas the minimum of electron density in the center at x = 0 8, corresponds to the center of the membrane hydrocarbon core. The small electron density difference centered at x = + 15 8, indicates the probable location of nimodipine, similar to previous studies with the agonist, Bay K 8644 (Mason et al., 1989). Data shown for T = 37°C at 96% relative humidity.
241
nimodipine is approx 15 8, from the center of the hydrocarbon core near the hydrocarbon core/water interface. This location in BBPC membranes with relatively high cholesterol (0.6: 1 C : P mole ratio) is similar to that observed for nimodipine in sarcoplasmic reticulum membranes with lower cholesterol content [ ~0.2: 1 C: P mole ratio] (Herbette, 1985). Thus, in membrane preparations with varying cholesterol content (which affects absolute partition coefficients as well as dissociation rates), the site for structural interaction appears to be similar. Due to its unique chemical structure, cholesterol effects an increase in the time-averaged extension of fatty acyl chains in membrane bilayers by reducing the number of gauche conformers on average. This results in changes in the cross-sectional area per lipid molecule, and hence, the packing constraints of the fatty acyl chains. Previous neutron diffraction work, utilizing nimodipine deuterated at the 2,6-methyl substituents on the pyridine ring, demonstrated that this portion of the drug molecule was located close to the hydrocarbon core/water interface of the native sarcoplasmic reticulum membrane (Herbette, 1985), which is low (< 10 mole%) in cholesterol. Small angle x-ray diffraction results utilizing model lipid bilayer membranes without cholesterol and fully protonated nimodipine were also consistent with these neutron diffraction studies (unpublished data). Thus, nimodipine’s time-averaged center of mass location is within the hydrocarbon core near to the glycerol backbone structure, a location which may be in equilibrium with an intrabilayer receptor site as evidenced by previous biochemical studies (Catterall and Striessnig, 1992). One explanation for the difference in washout rates for nimodipine from low vs high cholesterol-containing membranes may be related to where the molecule is situated in the membrane and the dynamic stability of the molecule to be retained at that location. Under low cholesterol conditions, the packing constraints of the lipid fatty acyl chains may be comparatively relaxed with a greater number of gauche conformers on average, resulting in a greater free volume per lipid (White et al., 1981; Stockton and Smith, 1976). This could result in nimodipine being located slightly deeper in the hydrocarbon core, making it resistant to washout resulting in slower washout rates from the membrane. In contrast, under high cholesterol conditions, the packing constraints could result in more trans conformers, lowering the free volume per lipid, and resulting in nimodipine’s time-averaged location to be nearer the surface of the membrane. In addition, packing constraints of the fatty acyl chains may make it less favorable for nimodipine to be retained in the membrane, resulting in faster washout rates. This structural model then would be consistent with the kinetic model presented above. Thus, pharmacokinetic control of the molecule would be dependent, in part, on the composition and resulting structure of the membrane bilayer. Nimodipine has neither an obvious hydrophilic nor hydrophobic membrane “anchoring”moiety in its
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structure. Interestingly, nimodipine is a short-acting drug in contrast to amlodipine (Mason et al., 1992a) and lacidipine (Herbette et al., 1993) two longer-acting drugs. However, as with many other DHP antagonists, it has both polar and apolar regions and as such possesses a strong amphiphilic character. The structure of nimodipine is rather compact, which along with its chemical properties may enable nimodipine to rapidly wash out of a membrane bilayer compartment and to transport rapidly across membrane bilayers, in contrast to other DHPs. It would appear that nimodipine possesses favorable amphiphilic properties that result in facilitating its interactions with membranes and possibly its transport across the blood-brain barrier. CONCLUSIONS
(1) Nimodipine partitions rapidly into, transports rapidly across and washes rapidly out of reconstituted brain lipid (phosphatidylcholine) membranes; kinetic parameters characterizing the interactions of nimodipine with other membrane systems are consistent with the relatively high apparent rate constants obtained with these brain lipid membranes. These kinetic processes are dependent on the cholesterol content of the membrane. (2) The membrane equilibrium partition coefficient for nimodipine is highly sensitive to the cholesterol content of model and native brain membranes whereby partitioning decreases as the cholesterol content increases. This physical chemical assessment of the interactions of nimodipine with membranes is consistent with its physical location in brain membranes. (3) Based on previous findings in our laboratory (Mason et al., 1992b), the C: P mole ratios in neural membranes from certain affected cortical regions of Alzheimer’s disease brains are approx 30% less than corresponding areas of age-matched controls. Data from the present study suggest that nimodipine will interact more favorably (i.e. have greater bioavailability) in certain neural membranes from Alzheimer’s disease vs age-matched control cortical brain tissue. Extrapolation
of conclusions
Nimodipine possesses favorable amphiphilicity to facilitate interactions with cell membrane “barriers” and may have a preferential interaction with certain neural membranes containing lower cholesterol, as in the case of Alzheimer’s disease. Acknowledgements-We would like to thank the following people for their excellent technical assistance: Emma Hughes (x-ray diffraction), Negar Mahooti (partitioning and washout kinetics), and Lesley Niego (preparation of rat synaptoneurosome samples). Jill Moring, Ph.D. provided animal tissue for these studies. REFERENCES Bangham A. D., Standish M. M. and Watkins J. C. (1965) Diffusion of univalent ions across the lamellae of swollen phospholipids. J. MoIec. Biol. 13: 238-252.
Catterall W. A. and Striessnig J. (1992) Receptor sites for Ca2+ channel antagonists. Trends Pharmac. Sri. 13: 256262. Chen P. S. Jr., Toribara T. Y. and Warner H. (1956) Microdetermination of phosphorus. Analyt. Chem. 28: 1756-l 758. Chester D. W., Tortelotte M. E., Molchior D. L. and Roman0 A. H. (1986) The influence of saturated fatty acid modulation of bilayer physical state on cellular and membrane structure and function. Biochim. Biophys. Acta 860: 383-398. Chester D. W., Herbette L. G., Mason R. P., Joslyn A. F., Triggle D. J. and Koppel D. E. (1987) Diffusion of dihydropyridine calcium channel antagonists in cardiac sarcolemmal lipid multibilayers. Biophys. J. 52: 1021-1030. Folch J., Lees M. and Sloane Stanley G. A. (1957) A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226: 4977509. Heider J. G. and Boyett R. L. (1978) The picomcte determination of free and total cholesterol in cells in culture. J. Lipid Res. 19: 514-518. Herbette L. G. (1985) X-ray and neutron diffraction for probing the interactions of small molecules with biological membrane structures. Curr. Top. Bioenerg. 14: 21-52. Herbette L. G., Vant Erve Y. M. H. and Rhodes D. G. (1989) Interaction of 1,4_dihydropyridine Ca2+ channel antagonists with biological membranes, lipid bilayer partitioning could occur before drug binding to receptors. J. Molec. Cell. Curdiol. 21: 1877201. Herbette L. G., Gaviraghi G., Tulenko T. N. and Mason R. P. (1993) Molecular interactions between lacidipine and biological membranes. J. Hyperten. 11: S13-Sl9. Hoffmeister F., Benz U., Heise A., Krause H. P. and Neuser V. (1982) Behavioral effects of nimodipine in animals. Arzneim.-Forsch. 32: 3477360. Janis R. A. and Triggle D. J. (1991) Drugs acting on calcium channels. In: Calcium Channels: Their Properties, Functions, Regulation and Clinical Relevance, pp. 197-249. CRC Press, West Palm Beach, FL. Kerckhoff W. van den and Drewes L. R. (1985) Transfer of the Ca-antagonists nifedipine and nimodipine across the blood-brain barrier and their regional distribution in viva. J. Cereb. Blood Flow Metab. 5: Suppl. 1, 459460. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (195 1) Protein determination with the phenol reagent. J. Biol. Chem. 193: 265-275. MacDonald R. C., MacDonald R. I., Menco B. Ph. M., Takeshita K., Subbarao N. K. and Hu L. (1991) Smallvolume extrusion apparatus for preparation of large, unilamellar vesicles. Biochim. Biophys. Acta 1061: 2977303. Mason R. P., Gonye G. E., Chester D. W. and Herbette L. G. (1989) Partitioning and location of Bay K 8644, 1,4-dihydropyridine Ca2+ channel agonist, in model and biological lipid membranes. Biophys. J. 55: 769-778. Mason R. P., Rhodes D. G. and Herbette L. G. (1991) Reevaluating equilibrium and kinetic binding parameters for lipophilic drugs based on a structural model for drug interaction with biological membranes. J. Med. Chem. 34: 869-877. Mason R. P., Moisey D. M. and Shajenko L. (1992a) Cholesterol alters the binding of Ca’+-channel blockers to the membrane lipid bilayer. Molec. Pharmac. 41: 315-321. Mason R. P., Shoemaker W. J., Shajenko L., Chambers T. C. and Herbette L. G. (1992b) Structure changes in Alzheimer’s disease cortical membranes mediated by alterations in the cholesterol content. Neurobiol. Aging 13: 413419. Meyer F. B., Tally P. W., Anderson R. E., Sundt T. M. Jr. and
Amphiphilicity Yaksh T. (1986a) Inhibition of electrically induced seizures by a dihydropyridine calcium channel blocker. Brain Res. 384: 18&183. Meyer F. B., Anderson R. E., Sundt T. and Sharbrough F. W. (198613) Selective central nervous system calcium channel blockers-a new class of anticonvulsant agents. Mayo Clin. Proc. 61: 239-247. Meyer F. B., Anderson R. E., Sundt T. M. Jr., Yaksh T. L. and Sharbrough F. W. (1987) Suppression of pentylenetetrazole seizures by oral administration of a dihydropyridine Ca’+-antagonist. Epilepsy 28: 409414. Moody M. F. (1963) X-ray diffraction pattern of nerve myelin: a method for determining the phases. Science 142: 1173-l 174. Moring J., Shoemaker W. J. Skita V., Mason R. P., Hayden H. C., Salomon R. M. and Herbette L. G. (1990) Rat cerebral cortical synaptoneurosomal membranes: structure and interactions with imidazobenzodiazepine and 1,4dihydropyridine drugs. Biophys. J. 58: 5 13-53 1. Peroutka S. J. and Allen G. S. (1983) Calcium channel antagonist binding sites labeled by [‘HI-nimodipine in human brain. J. Neurosurg. 59: 933-937. Rhodes D. G., Sarmiento J. G. and Herbette L. G. (1985) Kinetics of binding of membrane-active drugs to receptor sites. Diffusion limited rates for a membrane bilayer approach of 1,4_dihydropyridine Ca2+ channel antagonists to their active site. Mofec. Pharmac. 27: 612-623. Scriabine A., Schuurman T. and Traber J. (1989) Pharmacological basis for the use of nimodipine in central nervous system disorders. FASEB J. 3: 1799-1806. Skattebol A. and Triggle D. J. (1987) Regional distribution of
of nimodipine
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calcium channel ligand (1 ,Cdihydropyridine) binding sites and 45Ca2+ uptake process in rat brain. Biochem. Pharmac. (1987) 36: 41634166. Steen P. A., Newberg L. A,, Milde J. H. and Michenfelder J. D. (1983) Nimodipine improves cerebral blood flow and neurological recovery after complete cerebral ischemia in the dog. J. Cereb. Blood Flow Metab. 3: 3843. Steen P. A., Newberg L. A. Milde J. H. and Michenfelder J. D. (1984) Cerebral blood flow and neurologic outcome when nimodipine is given after complete cerebral ischemia in the dog. J. Cereb. Blood Flow Metab. 4: 82-87. Steen P. A., Gisvold S. E., Milde J. H., Newburg L. A., Scheithauer B. W., Lanier W. L. and Michenfelder J. D. (1985) Nimodipine improves outcome when given after complete cerebral ischemia in primates. Anesthesiology 62: 406-411. Stockton G. W. and Smith I. C. P. (1976) A deuterium nuclear magnetic resonance study of the condensing effect of cholesterol on egg phosphatidylcholine bilayer membranes. I. Perdeuterated fatty acid probes. Chem. Phys. Lipids 17: 251-263. Tollefson G. D. (1990) Short-term effects of the calcium channel blocker nimodipine (Bay-e-9736) in the management of primary degenerative dementia. Biol. Psych. 27: 1133-l 142. White S. H., King G. I. and Cain J. E. (1981) Location of hexane in lipid bilayers determined by neutron diffraction. Nature (Lond.) 290: 161-163. Yeagle P. (1987) Cholesterol and cell membranes. In: The Membranes of Cells, pp. 120-138. Academic Press, New York.
002%3908(93)EOOlS-6
Neuropharmacology Vol. 33, No. 2, pp. 241-249, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0028-3908/94 $6.00 + 0.00
Favorable Amphiphilicity of Nimodipine Facilitates Its Interactions with Brain Membranes L. G. HERBETTE,lm317
P. E. MASON,’
K. R. SWEENEY,’
M. W. TRUMBORE’
and R. P. MASON3,4,6.7 Departments of ‘Biochemistry, ‘Medicine, ‘Radiology, 4Psychiatry and ‘Pharmacy, 6Travelers Center on Aging, ‘Biomolecular Structure Analysis Center, University of Connecticut Health Center, Farmington, CT 06030, U.S.A. (Accepted 25 October 1993)
Summary-Nimodipine is a 1,4_dihydropyridine (DHP) calcium channel blocker which is used in the treatment of neurological deficits associated with subarachnoid hemorrhage. Small angle x-ray diffraction, differential scanning calorimetry, and equilibrium and kinetic binding techniques were used to study the interaction of nimodipine with bovine brain phosphatidylcholine (BBPC) membranes of varying cholesterol content. At concentrations (5 x lo-” M) near its &, the membrane partition coefficient of nimodipine was inversely related to the cholesterol to phospholipid (C: P) mole ratio in both model and native (rat synaptoneurosome) membranes. The nonspecific dissociation rate of nimodipine from BBPC was significantly slower at low C : P mole ratio (0.1: 1) than at high C: P mole ratio (0.6: 1). Calorimetric analysis showed that nimodipine decreased both the main phase transition temperature and cooperative unit size of melt for dimyristoyl phosphatidylcholine, dependent on membrane cholesterol content. Small angle x-ray diffraction analysis showed that nimodipine occupies a position in BBPC approx + 15 A from the center of the hydrocarbon core, near the hydrocarbon core/water interface. These data indicate that nimodipine is an amphiphilic molecule which rapidly washes out of and transports across membrane bilayers, facilitating its interactions with membranes and possibly its transport across the blood-brain barrier. KeywordsMembrane,
bilayer, cholesterol, Alzheimer’s, nimodipine, x-ray diffraction, kinetics, partitioning.
Nimodipine is a 1,Cdihydropyridine (DHP) which affects cerebrovascular and neuronal cell activity by modulating L-type voltage-sensitive Ca+* channels in the plasma membranes of these cells. Nimodipine initiates vasodilation by altering transmembrane influx of calcium across the plasma membrane of arterial smooth muscle cells. As a calcium channel antagonist, nimodipine binding in human neuronal membranes is specific and saturable with an affinity constant of 0.27 nM-’ while the maximal number of binding sites has been shown to be 5.8 pmol/g wet wt in human frontal cortex (Peroutka and Allen, 1983. In rat brain, the distribution of nimodipine binding sites is higher in the cerebral cortex than in the cerebellum (Skattebol and Triggle, 1987). Nimodipine is the only DHP approved in the United States for treatment of neurological deficits associated with subarachnoid hemorrhage (Janis and Triggle, 1991). There is also experimental evidence for nimodipine being effective in the treatment of ischemia (Steen et al., 1983, 1984, 1985), epilepsy (Hoffmeister et al., 1982; Meyer et al., 1986a, b, 1987) and most *To whom correspondence should be addressed: Dr. Leo G. Herbette, Biomolecular Structure Analysis Center, University of Connecticut Health Center, Farmington, CT 06030, U.S.A.
recently, degenerative dementia of the Alzheimer’s disease type (Tollefson, 1990). The CNS activity of nimodipine may be related, in part, to favorable physical and chemical properties which facilitate its transfer across the blood-brain barrier and its bioavailability in the brain. Compared to other DHPs, nimodipine exhibits cerebrovasodilatory and neuronal activity at concentrations less than that required to induce peripheral hypotension (for review, see Scriabine et al., 1989). For example, the apparent volume of distribution of nimodipine in rat brain was found to be several-fold greater than that of nifedipine, a closely related compound (van den Kerckhoff and Drewes, 1985). Once in the brain, nimodipine partitioning into the neuronal plasma membrane may precede receptor binding as has been previously proposed for vascular smooth muscle (Rhodes et al., 1985; Herbette et al., 1989). Thus, the membrane concentration of nimodipine, as opposed to its aqueous concentration, is likely to be in equilibrium with the plasma membrane Ca+* channel and this may have a critical effect on the potency/activity of the drug (Mason et al., 1991). A precise understanding of the true lipophilicity (or amphiphilicity) of nimodipine requires direct measurement of its interaction with membrane lipid bilayers 241
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HERBETTE et al.
of varying lipid composition (e.g. cholesterol content). Previous studies have shown that membrane partitioning for this class of drugs varies substantially and is highly dependent on the cholesterol content of the membrane (Mason er af., 1992a), an important factor when considering that neural membrane cholesterol composition changes with disease, including Alzheimer’s disease (Mason et al., 1992b). Moreover, membrane cholesterol heterogeneity is observed at the cellular and subcellular levels (for review, see Yeagle, 1987). The goal of this study, then, was to investigate the interaction of nimodipine with membranes of varying lipid composition, especially with respect to cholesterol, to reproduce the heterogeneity found in brain tissue. Specifically, the equilibrium and kinetic membrane interactions of nimodipine were measured as a function of cholesterol content using reconstituted and intact brain membranes. The thermodynamic properties associated with the membrane interactions of nimodipine were evaluated with calorimetry. The results of this work indicate that at concentrations near its Z&, the equilibrium and kinetic binding properties of nimodipine are highly dependent on membrane cholesterol content. Moreover, nimodipine insertion into the membrane perturbs the organization of the lipid bilayer, as evidenced by significant changes in membrane thermotropic properties. This latter effect was also highly dependent on membrane cholesterol content. These data may have important implications for understanding the ability of nimodipine to passively cross the blood-brain barrier in order to achieve a high volume of distribution in the brain for activity. METHODS
Preparation
of model phospholipid
membrane
vesicles
For kinetic experiments, multilamellar vesicles (MLV) and large unilamellar vesicles (LUV) formed from bovine brain phosphatidylcholine (BBPC) in the presence and absence of cholesterol were utilized. MLV and LUV are useful for filtration experiments as they efficiently bind to glass fiber filters utilized for nonspecific membrane kinetic experiments. BBPC and cholesterol, both solubilized in chloroform, were obtained from Avanti Polar Lipids Inc. (Alabaster, AL) and stored in a dessicator at -20°C. Thin layer chromatography was used to ascertain purity and the presence of degradative products in the lipid and cholesterol suspensions. Specific quantities of cholesterol and phospholipid in mole ratios (C: P) of 0.1: 1, 0.3 : 1, and 0.6: 1 were mixed and dried down in a glass test tube, while vortexing, to a thin film under a stream of N, gas, and placed under vacuum overnight to remove residual solvent. Tris buffer (150 mM NaCl, 10 mM Trizma HCl, pH 7.0) was then added to yield a lipid concentration of 2 mg/ml and the solution was vortexed at room temperature (above the thermal phase transition temperature) to form MLV. LUV (average 80 nm dia) were formed by extruding MLV through a 100 nm polycarbonate filter mounted in a mini-extruder (“Liposofast”, Avestin,
Ottawa, Canada) as previously described (MacDonald et al., 1991). The structural integrity of these vesicles was examined by transmission electron microscopy after staining the lipids with 0~0,. To confirm the final C: P mole ratio in the membrane vesicles, a portion of the vesicles was assayed for cholesterol and phosphate as described below. Preparation
of native membrane
vesicles
Synaptoneurosomes were isolated from the cerebral cortex of an adult Sprague-Dawley rat (Moring et al., 1990) and assayed for protein, phospholipid, and cholesterol content. Specifically, membrane protein was determined using bovine serum albumin as a standard (Lowry et al., 1951). The phospholipid phosphorus content of the samples was determined by previously described methods (Chen et al., 1956; Chester et al., 1987). Cholesterol was extracted from native membranes using a chloroform : methanol : water system (Folch et al., 1957). A calorimetric assay was used to measure total cholesterol in native membranes by a modification of the cholesterol oxidase method (Chester et al., 1986; Heider and Boyett, 1978). Drug/membrane
partition
coejicients
The membrane partition coefficient is an equilibrium value expressing the relative amount, by mass, of drug in the membrane vs aqueous buffer. This distribution of drug between the membrane lipid and aqueous buffer is an expression of the drug’s true lipophilicity or affinity for the membrane bilayer. Nimodipine partition coefficients in model and native membranes (Kp,,em,) were experimentally measured using vacuum filtration as previously described in detail were filtered (Herbette et al., 1989). All solutions through Whatman GFjC glass fiber filters on a Brandel M-48 cell harvester (Brandel, Gaithersburg, MD). Using vesicles (20 pg/ml) radiolabelled with [ ‘HIcholesterol, the amount of membrane lipid retained on the filters during the filtration process was accurately and reproducibly measured. It was shown that 82 f 2% of MLV and 51 + 2% of LUV (mean f SD) were retained regardless of the C:P mole ratio in four trials consisting of 8 samples (n = 32) for each mole ratio. The filter retention of rat synaptoneurosomes labelled with [ 3H]cholesterol was 75% (1 trial consisting of 6 samples). In experiments measuring total nonspecific binding, the incubation reaction mixture consisted of a fixed phospholipid concentration of 20 pg/ml, with varying C: P mole ratios in the model membranes (0: 1, 0.3 : 1, 0.6 : 1). Radiolabelled [‘Hlnimodipine (New England Nuclear Research Products, Boston, MA) and unlabelled nimodipine (Miles Pharmaceuticals, West Haven, CT) dissolved in ethanol was added to produce a final concentration of drug in the reaction mixture of 5 x lo--” M. The KPtmemlvalues for nimodipine have been previously shown to be independent of drug concentrations between lo-” M and lop6 M (Herbette et al.,
Amphiphilicity
1989). A drug concentration of 5 x lo-” M was chosen because this value is similar to the K,, for nimodipine. Since the equilibrium partitioning of nimodipine into MLV and LUV is not measurably timedependent, incubation time for all reactions was set at 30 min. The filters for these experiments were not washed. Reaction mixtures containing drug, but no membrane, were used to correct the total nonspecific binding for binding of drug directly to filters. All filters were counted for radioactivity (Tracer Analytic Delta 300/689 1 Liquid Scintillation System). The amount of drug in the filtrate was determined by subtracting the number of moles of drug bound to the membrane from the total number of moles of drug added to the reaction mixtures. The amount of lipid bound to the filters was corrected for the recovery of membrane on the filters. Membrane partition coefficients were thus calculated for each trial using the following equation: K P[mem]=
(g of drug bound to membrane/g (g
of lipid)
of free drug/g of buffer)
Drug release C’ Washout”) from membranes
Time-dependence of nimodipine release from model membranes (MLV 0.6: 1 and 0.1: 1; LUV 0.6: 1) was measured by incubating [ 3H]nimodipine (5 x lo- ‘OM) with phospholipid (20 pg/ml) in Tris buffer (pH 7.0) for 30 min. All solutions were filtered through Whatman GF/C glass fiber filters on a Brandel cell harvester, but not washed. Control reaction mixtures contained drug, but no membranes. Three filters with membranes and 3 control filters were immediately counted for radioactivity, representing maximal binding of drug to membranes. The remaining filters were impaled, in groups of 3, on 25-gauge needles mounted on a flat Plexiglass “donut”. The filters on each needle were spaced with pieces of tubing (length 1 cm, o.d. 1 mm) to prevent filters from touching. The Plexiglass structure was then placed near the base of a 3-l round plastic container situated on a magnetic stirrer at medium speed, and the container was filled with Tris buffer. At specific time intervals, filters with and without membranes were removed from the buffer and counted for radioactivity. Control filters retained at least 90% of the lipid over the duration of these washout experiments for both multilamellar and unilamellar preparations, independent of cholesterol content. Experimental data were corrected to account for this loss of lipid. Statistical evaluation and computer modeling
Statistical analysis was used to evaluate nonspecific equilibrium binding parameters for nimodipine. For model membrane partition coefficient experiments, four trials consisting of 12 samples (n = 48) were carried out for each incremental change in the C : P mole ratio for both MLV and LUV. For each set of experiments, K PCmemI values were obtained and standard error of the mean was calculated. The Student’s t-test was used to determine significance of differences between KPLmemI val-
243
of nimodipine
ues found for MLV vs LUV at each C: P mole ratio. From the synaptoneurosome preparation 12 aliquots were used for determining partition coefficients; a mean K P~meml was determined. For nonspecific membrane dissociation rates, data were collected at a minimum of 13 (maximum 16) time points, ranging from 0 to 150 min depending on the C : P mole ratio. Each time point consisted of 3 experimental and 3 control determinations. Raw data from a minimum of 4 washout curves generated for each condition were analyzed using a least-squares fitting algorithm (PCNONLIN, SC1 Software, Lexington, KY). An empirical model was fitted to the observed data in an effort to describe the washout curves and obtain estimates of half-life of nimodipine release. DifSerential scanning calorimetry
Differential scanning calorimetry (DSC) was carried out on drug-containing and control MLV prepared by the following procedure. For control MLV, 100 ~1 aliquots of a 0.03 M solution of dimyristoylphosphatidylcholine (DMPC) in chloroform was dried to a thin film by evaporation under N, with the residual solvent being removed by overnight evacuation. The dried lipid was then rehydrated for 10 min at 50°C in 100 ~1 of sample buffer (0.05 mM Hepes, 150 mM NaCl, pH 7.0) and vortexed for 1 min to form MLV. Two different cholesterol : phospholipid mole ratios were used in this study: 1: 20 and 1: 10. In these cases, either 5.0 or 10.0 ~1 aliquots of 0.03 M cholesterol solution in chloroform were dried down with the lipid. For nimodipine-containing samples (1: 44 drug : DMPC), 2.25 ~1 of a 0.03 M stock solution of nimodipine in chloroform was dried down with the lipid and/or lipid/cholesterol at the two ratios stated above and vesicles were produced as described. Aliquots (15 ~1) of MLV containing 0.300 pg of DMPC were placed into DSC sample pans and hermetically sealed. DSC was carried out using a TA Instruments DSC 2910 Differential Scanning Calorimeter (TA Instruments, New Castle, DE) with the data being analyzed using a TA Instruments Thermal Analyst 2000 system. The scan rate was 2”C/min starting at 6°C and ending at 34.5”C. The reference was 15 ~1 of sample buffer. The change in cooperative unit size was determined by the following formula: ACUS = (AH&H*Fw),,,,
- (AH,,/AH*Fw),,,,,,
where ACUS is the change in the cooperative unit size, AHvh is the van? Hoff enthalpy, AH is the calorimetric enthalpy and Fw is the formula weight of the lipid undergoing transition. Preparation of multibilayer samples for x-ray dlrraction
The BBPC MLV were prepared in the presence of known amounts of nimodipine as follows. The desired amounts of lipid and cholesterol (C:P 0.6: 1) dissolved in CHCl, were placed in a glass test tube, dried to a thin film under a stream of N,, and placed under vacuum overnight to remove residual solvent. Buffer (0.5 mM
244
L.G. HERBETTE et al.
Hepes, 2.0 mM NaCl, pH 7.27) was added to yield a final phospholipid concentration of 5 mg/ml and the solution was vortexed to form a cloudy white suspension of MLV (Bangham et al., 1965). Nimodipine (dissolved in ethanol) was added to each sample aliquot to produce final drug: lipid mole ratios of 1: 15 and 1: 25. Ethanol concentration in these samples was ~5% prior to centrifugation. Multibilayer samples for small angle x-ray diffraction were prepared as described previously (Chester et al., 1987). Sample preparation and dehydration took approx 3 hr. The membrane samples were stored at 4°C. Salts used for controlling relative humidity (r.h.) were Na/Na tartrate (91%), ZnSO, (93%), KNO, (96%) and K,SO, (98%). Small qngle x-ray
dlflraction
CuK, x-rays produced by a GX-18 rotating anode generator (Enraf Nonius, Bohemia, NY) were either line focused for the small angle studies or point focused for the equatorial studies using Franks’ mirror assemblies. A nickel filter was used to select CuK, radiation (n = 1.54W). Diffraction
data collection
Data were recorded at 5°C on a Braun positionsensitive 1-D detector (Innovative Technologies, Inc., South Hamilton, MA) interfaced to a MicroVax II (Digital Equipment Corp., Maynard, MA). Data reduction for this method (background and geometrical corrections) has been described previously (Mason et al., 1989). Since the entire lamellar reflection for each observed intensity was collected by the detector, the lamellar intensity functions from the BBPC samples collected with the electronic detector were simply Lorentz corrected by a factor of s = 2 sin e/n. Data were recorded on Kodak DEF-5 (Eastman Kodak Co., Rochester, NY) film and qualitatively examined to determine the high angle acyl-chain packing of the samples and to verify the low angle detector data. Phasing
the data
A swelling analysis was used to phase the lamellar reflections for each experiment (Moody, 1963). Sets of intensity data obtained at different hydration states ranging from 91 to 98% relative humidity, each with unique unit cell repeat distances, were used to assign an unambiguous phase combination to the experimentally obtained structure factors (h = l-6). The final phase choice was the only physically reasonable combination, in terms of empirical examination of resultant electron density profiles. RESULTS Equilibrium kinetics
partitioning
AND DISCUSSION and
association /dissociation
Figure 1 summarizes the effect of membrane cholesterol content on the equilibrium membrane partition coefficients (KPtmeml) of nimodipine in BBPC and rat
n BBPC MLV
ka BBPC LUV q RAT SNM
0
0.55
0.3 Cholesterol
: Phospholipld
mol
0.6 ratio
Fig. I. Effect of membrane cholesterol content on nimodipine partitioning into mode1 and native membranes. The equilibrium Kpt,,,I of nimodipine was measured in membranes of varying cholesterol content, including bovine brain phosphatidylcholine (BBPC) multilamellar vesicles (MLV) and large unilamellar vesicles (LUV), as well as rat synaptoneurosomes. Lipid concentrations were maintained at 20pg/ml (pH 7.0, 2lC), and drug concentrations were maintained at 5 x 10.“M.
synaptoneurosomes. Results for model membranes (MLV and LUV) are shown as the mean f SEM and demonstrate an inverse relationship between membrane C: P mole ratio and KP,meml.For the rat synaptoneurosome preparation (in which the C: P mole ratio was determined to be 0.55: 1), the membrane partition coefficient was found to be consistent with the KPImemlvalue expected for model systems between C: P mole ratios 0.3: 1 and 0.6: 1 by interpolation. Thus, the interaction of nimodipine with membranes is highly sensitive to the amount of cholesterol in the membrane and the drug would be expected to preferentially partition into membranes that contain lower amounts of cholesterol. For each different C :P mole ratio, partition coefficients for MLV and LUV were not found to be statistically different. These data demonstrate that the drug comes to equilibrium with lipid vesicles independent of the surface area to volume ratio under experimental conditions in which the total amount of lipid is kept constant. These findings indicate that nimodipine gains access to all available lipid and is not restricted to the outermost layers of the MLV system. It was not possible to measure association (wash-in) kinetics for nimodipine in either MLV or LUV since equilibrium appeared to be achieved by the earliest time point feasible, viz. 30 sec. Moreover, the rates of nimodipine dissociation (washout) from MLV and LUV at the same C:P mole ratio (0.6: 1) were found to be similar, as evidenced by first-order washout half-lives (k SD) of 3.83 (+ 0.267) and 3.46 (& 0.332) min, respectively. Nimodipine thus appears to transport easily across lipid bilayers arranged in either a single or multilamellar system. The computer-generated curves in Fig. 2 represent the dissociation of nimodipine from MLV at C : P 0.1: 1 vs 0.6: 1. The nimodipine washout data from both lipid
Amphiphilicity
245
of nimodipine
0.6
Time (mid
0.4
0.2 --*_-___ 0
~------lD_____ L
J,
0
10
40
30
20
60
50
1.4
10
0.6
Time
15
20
(min)
0.4
0.2
0
d
3.
6b
g6
.
. 120
150
e
l.O-:
I
,i
(cl
o.8- 4
‘t: : IL
0.6
- i :‘: i ‘:,‘*.,
_E
i
0.4
*
\ \
‘l\ ‘. *... \
---
15
20
(min)
c:P
0.6:1
--A______ -----A___
0
10
------- c:p 0.1:1
‘L\
‘\__ o-,
5
Time
\ 0.2-
-0
, 30
------______ -------_______ --------_______ -----___ . I_ I 1 . I -__ 60 90 120 15c Time
(mid
Fig. 2. Effect of membrane cholesterol content on the release of nimodipine from model membranes. The release of nimodipine from bovine brain phosphatidylcholine (BBPC) multilamellar vesicles was measured as a function of time in the presence of 0.6: 1 (a and c) and 0.1: 1 (b and c) cholesterol: phospholipid mole ratios. Membranes (20 pg/ml phospholipid, pH 7.0, 21”C), were incubated with nimodipine (5 x lo-” M) for 30 minutes prior to To.Data were plotted as mean f SD in (a) and (b), and analyzed (c) using a least-squares fitting algorithm (PCNONLIN).
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L. G. HERBETTE et al.
systems were fitted with a sum of first-order and zeroorder terms. The first-order half-life (*SD) of nimodipine dissociation was 3.83 (+ 0.267) minutes for C : P 0.6:1 vesicles and 13.7 (f2.74)min for C:P O.l:l vesicles. Integration of the equation describing release of nimodipine was performed in order to calculate area under the washout curve (AUC) from time 0 to 100 min. One hundred minutes was chosen as the upper limit of the time frame since this time represents greater than 99% of the total washout of drug in the first-order portion of the curve for both MLV and LUV. Total AUC was strikingly different: AUC for C : P 0.1 : 1 (lower cholesterol) was more than 2-fold higher than the AUC for C: P 0.6: 1 (26.9 vs 11.5 fraction*minutes, respectively). The percent of total AUC attributable to the first-order release portion of the curve was similar for each condition, 46.1% and 42.0% for C : P 0.1 : 1 and 0.6: 1, respectively. The total AUC due to first-order release from C : P 0.1: 1 vesicles was 2.5fold higher than from C : P 0.6 : 1 vesicles (12.4 vs 4.83 fraction*minutes, respectively). Similarly, the total AUC due to zero-order release from C : P 0.1 : 1 vesicles more than 2-fold greater than from C: P 0.6: 1 vesicles. It appears that cholesterol content of the lipid bilayer has a profound effect on nimodipine washout, affecting both the first-order and zero-order portions of the washout curve. These experiments demonstrate that nimodipine can rapidly wash into a single membrane bilayer compartment (LUV) as well as across a series of membrane bilayers (MLV). These results are consistent with the very rapid wash-in of nimodipine in both model and native membranes (Herbette et al., 1989). Nimodipine partition coefficients in both MLV and LUV are inversely related to cholesterol content; furthermore, nonspecific dissociation rates are much slower at low C : P (0.1 : 1) than at high C:P mole ratios (0.6: 1). Thus, the kinetics of nonspecific drug interactions with MLV and LUV are highly dependent on membrane cholesterol content, and indicate that nimodipine may not gain equal access to all available sites within the lipid bilayer in the presence of elevated levels of cholesterol. The ability of nimodipine to partition into the membrane may be a critical determinant of its bioavailability; that is, changes in membrane cholesterol content could substantially modulate the concentration of drug in membrane sites. In brain tissue, perturbations of membrane cholesterol composition during aging (increased cholesterol content) and in Alzheimer’s disease (decreased cholesterol content) may affect the bioavailability of calcium channel blockers such as nimodipine (Mason et al., 1992a, b). D$erential
scanning
calorimetry
In the absence of cholesterol, nimodipine, as do other dihydropyridines, strongly perturbed the thermal properties of DMPC membranes. These results have been summarized in Table 1. At the drug : lipid ratio used in this study (1:44), nimodipine in the absence of cholesterol decreased the main phase transition temperature by
Table
1. The effects of cholesterol on the nimodipine/lipid brane interaction
Sample
C:L
L L/N L/C L/C/N L/C L/C/N
mole ratio 0 0 I:20 I : 20 1: 10 1: 10
Tm 22.19 22.27 22.43 21.62 22.18 21.01
ATm -0.52 -0.81 -1.17
CUS 332 205 325 191 542 220
memACUS -127 -133 -322
Tm, temperature of main phase transition; CUS, cooperative unit size; L, DMPC (dimyristoylphosphatidylcholine); N, nimodipine; C, cholesterol. The mole ratio of nimodipine to DMPC was I :44.
0.52”C, going from 22.79”C in the absence of drug to 22.27”C in the presence of nimodipine. In addition, nimodipine caused a decrease in the number of molecules simultaneously undergoing the phase transition by 127 molecules, the cooperative unit size decreasing from 332 molecules in the absence of nimodipine to 205 molecules when nimodipine was present. As the amount of cholesterol in the membrane increased, nimodipine exhibited increased membrane perturbing activity. At a cholesterol: DMPC mole ratio of 1 : 20, nimodipine decreased the Tm by 0.81”C and the cooperative unit size by 133 molecules as compared to the DMPC : cholesterol control. At a ratio of 1: 10 the decrease in Tm was 1.17”C and the decrease in cooperative unit size was 322 molecules. From these data it is clear that cholesterol modulates the ability of nimodipine to interact with a membrane, thereby enhancing its ability to perturb the thermal properties of a membrane. As was shown in Table 1, the presence of cholesterol in DMPC membranes strongly affects the magnitude of the membrane perturbation caused by nimodipine. Cholesterol serves to enhance the effects of nimodipine leading to a much larger change in membrane thermal properties than would be expected. It appears that as the cholesterol content of the DMPC membranes increases, nimodipine causes a greater disruption of membrane structure. Based on these results one might expect that it would be easier to remove nimodipine from membranes with increased levels of cholesterol, which is what was observed in nonspecific dissociation experiments. As was stated previously, cholesterol alters the structure of acyl chains in the vicinity of the molecule. As nimodipine partitions into the membrane it perturbs the packing characteristics of the lipid acyl chains in its vicinity. Because cholesterol occupies a similar position in the membrane, the effect of nimodipine is magnified, causing an even greater perturbation. Therefore, by occupying a position similar to that of nimodipine, cholesterol could reduce the free volume available for nimodipine to partition into, thereby reducing its KPlmeml,while at the same time enhancing the amount of perturbation to the membrane structure caused by nimodipine, leading to an increased rate of “washout”. Structural
model
The primary structure of the DHP-sensitive (L-type) calcium channel has been determined by molecular biology techniques. The site of photoreactive DHP
Amphiphilicity of nimodipine covalent binding to the CI,subunit has been mapped with site-directed antipeptide antibodies; these studies indicate that amino acid sequences associated with two transmembrane helices near the extracellular membrane surface of the CL,subunit contribute to the DHP binding site (for review, see Catterall and Striessnig, 1992). The amino acids which constitute this binding site region are both hydrophobic and hydrophilic, at the hydrocarbon core/water interface. Figure 3 shows the superposition of two electron density profile structures of the BBPC membrane bilayer with different amounts of nimodipine. From this comparison, the time-averaged center of mass location of Brain membrane with nimodipine 0.6: I cholesterol
I
-20
1 110
0
: lipid
10
20
30
Fig. 3. Membrane bilayer electron density profiles on a relative scale. Electron density profiles for BBPC membranes (0.6: 1 cholesterol : phospholipid mole ratio) in the presence of 1: 25 (solid line) and 1: 15 (dashed line) nimodipine : phospholipid mole ratios. The profiles were arbitrarily scaled to one another to match the electron density maxima and minima. The profiles were correlated to a highly schematic representation of a cholesterol-containing membrane, as shown in the lower panel. The two maxima of electron density at x = + 20 A correspond to the electron-dense phosphate headgroups, whereas the minimum of electron density in the center at x = 0 8, corresponds to the center of the membrane hydrocarbon core. The small electron density difference centered at x = + 15 8, indicates the probable location of nimodipine, similar to previous studies with the agonist, Bay K 8644 (Mason et al., 1989). Data shown for T = 37°C at 96% relative humidity.
241
nimodipine is approx 15 8, from the center of the hydrocarbon core near the hydrocarbon core/water interface. This location in BBPC membranes with relatively high cholesterol (0.6: 1 C : P mole ratio) is similar to that observed for nimodipine in sarcoplasmic reticulum membranes with lower cholesterol content [ ~0.2: 1 C: P mole ratio] (Herbette, 1985). Thus, in membrane preparations with varying cholesterol content (which affects absolute partition coefficients as well as dissociation rates), the site for structural interaction appears to be similar. Due to its unique chemical structure, cholesterol effects an increase in the time-averaged extension of fatty acyl chains in membrane bilayers by reducing the number of gauche conformers on average. This results in changes in the cross-sectional area per lipid molecule, and hence, the packing constraints of the fatty acyl chains. Previous neutron diffraction work, utilizing nimodipine deuterated at the 2,6-methyl substituents on the pyridine ring, demonstrated that this portion of the drug molecule was located close to the hydrocarbon core/water interface of the native sarcoplasmic reticulum membrane (Herbette, 1985), which is low (< 10 mole%) in cholesterol. Small angle x-ray diffraction results utilizing model lipid bilayer membranes without cholesterol and fully protonated nimodipine were also consistent with these neutron diffraction studies (unpublished data). Thus, nimodipine’s time-averaged center of mass location is within the hydrocarbon core near to the glycerol backbone structure, a location which may be in equilibrium with an intrabilayer receptor site as evidenced by previous biochemical studies (Catterall and Striessnig, 1992). One explanation for the difference in washout rates for nimodipine from low vs high cholesterol-containing membranes may be related to where the molecule is situated in the membrane and the dynamic stability of the molecule to be retained at that location. Under low cholesterol conditions, the packing constraints of the lipid fatty acyl chains may be comparatively relaxed with a greater number of gauche conformers on average, resulting in a greater free volume per lipid (White et al., 1981; Stockton and Smith, 1976). This could result in nimodipine being located slightly deeper in the hydrocarbon core, making it resistant to washout resulting in slower washout rates from the membrane. In contrast, under high cholesterol conditions, the packing constraints could result in more trans conformers, lowering the free volume per lipid, and resulting in nimodipine’s time-averaged location to be nearer the surface of the membrane. In addition, packing constraints of the fatty acyl chains may make it less favorable for nimodipine to be retained in the membrane, resulting in faster washout rates. This structural model then would be consistent with the kinetic model presented above. Thus, pharmacokinetic control of the molecule would be dependent, in part, on the composition and resulting structure of the membrane bilayer. Nimodipine has neither an obvious hydrophilic nor hydrophobic membrane “anchoring”moiety in its
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structure. Interestingly, nimodipine is a short-acting drug in contrast to amlodipine (Mason et al., 1992a) and lacidipine (Herbette et al., 1993) two longer-acting drugs. However, as with many other DHP antagonists, it has both polar and apolar regions and as such possesses a strong amphiphilic character. The structure of nimodipine is rather compact, which along with its chemical properties may enable nimodipine to rapidly wash out of a membrane bilayer compartment and to transport rapidly across membrane bilayers, in contrast to other DHPs. It would appear that nimodipine possesses favorable amphiphilic properties that result in facilitating its interactions with membranes and possibly its transport across the blood-brain barrier. CONCLUSIONS
(1) Nimodipine partitions rapidly into, transports rapidly across and washes rapidly out of reconstituted brain lipid (phosphatidylcholine) membranes; kinetic parameters characterizing the interactions of nimodipine with other membrane systems are consistent with the relatively high apparent rate constants obtained with these brain lipid membranes. These kinetic processes are dependent on the cholesterol content of the membrane. (2) The membrane equilibrium partition coefficient for nimodipine is highly sensitive to the cholesterol content of model and native brain membranes whereby partitioning decreases as the cholesterol content increases. This physical chemical assessment of the interactions of nimodipine with membranes is consistent with its physical location in brain membranes. (3) Based on previous findings in our laboratory (Mason et al., 1992b), the C: P mole ratios in neural membranes from certain affected cortical regions of Alzheimer’s disease brains are approx 30% less than corresponding areas of age-matched controls. Data from the present study suggest that nimodipine will interact more favorably (i.e. have greater bioavailability) in certain neural membranes from Alzheimer’s disease vs age-matched control cortical brain tissue. Extrapolation
of conclusions
Nimodipine possesses favorable amphiphilicity to facilitate interactions with cell membrane “barriers” and may have a preferential interaction with certain neural membranes containing lower cholesterol, as in the case of Alzheimer’s disease. Acknowledgements-We would like to thank the following people for their excellent technical assistance: Emma Hughes (x-ray diffraction), Negar Mahooti (partitioning and washout kinetics), and Lesley Niego (preparation of rat synaptoneurosome samples). Jill Moring, Ph.D. provided animal tissue for these studies. REFERENCES Bangham A. D., Standish M. M. and Watkins J. C. (1965) Diffusion of univalent ions across the lamellae of swollen phospholipids. J. MoIec. Biol. 13: 238-252.
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