Lercanidipine: short plasma half-life, long duration of action and high cholesterol tolerance. Updated molecular model to rationalize its pharmacokinetic properties.

Calcium-channel antagonist drugs of the 1,4-dihydropyridine type have been shown to bind to the L-type calcium channel. These drugs are not only amphiphilic, but new molecular designs have become increasingly lipophilic and can readily transport across cell membranes, accessing both hydrophilic and hydrophobic environments, despite becoming more soluble in the membrane bilayer. This biophysical understanding appears not only to define the molecular pathways for drug binding to the calcium-channel receptor, but also to explain differences in the overall clinical pharmacokinetics observed for different drugs in this class. The pharmacokinetic profile of calcium antagonists, although influenced to some degree by interactions with their target calcium-channel receptor, appears to be largely dictated by their interactions with cell membranes at the molecular level. There appears to be a correlation between the duration of action of such membrane-active drugs and the membrane partition coefficient in conjunction with the washout rate. This class of drugs has evolved from a drug such as amlodipine, with a long duration of action related to prolonged plasma half-life, to lercanidipine, which has the shortest plasma half-life relative to its intrinsically long duration of action. Recently, it was discovered that membrane cholesterol reduces the amount of calcium-channel antagonist that can partition into the membrane. Atherosclerotic disease results in increased levels of membrane cholesterol in smooth muscle cells. Latest generation calcium antagonist, which have a long duration of action, can better overcome this negative effect. Lercanidipine has now been shown to have one of the highest measured tolerances to cholesterol, which may indicate its ability to treat a broad range of hypertensive patients with varying degrees of progressive atherosclerotic disease. On what criteria should the effectiveness of calcium antagonists be evaluated? A good calcium antagonist needs to exhibit a placebo-like side-effect profile, thus ensuring good patient compliance. However, an intrinsically long-lasting, once-a-day dose is also pharmacokinetically desirable. To be a truly optimal calcium antagonist, it should function and be efficacious over a broad range of hypertensive patients. It should be able to control blood pressure in light of other complications such as progressive atherosclerotic disease. Recent studies indicate that during the progression of atherosclerosis, cholesterol levels within cell membranes of the arterial wall increase, a process that can reduce the effective concentration of calcium antagonists in these membranes. What is needed is a calcium antagonist that is slow acting to reduce vasodilatory induced side-effects and intrinsically long lasting to ensure once-a-day dosage, and that possesses a high cholesterol tolerance factor to overcome the molecular and compositional changes taking place in the arterial wall, so that it can treat effectively a broad range of hypertensive patients.

X-ray diffraction analysis of cytochrome P450 2B4 reconstituted into liposomes.

Two general models of the membrane topology of microsomal cytochrome P450 have been proposed: (1) deep immersion in the membrane, and (2) a P450cam-like heme domain anchored to the membrane with one or two membrane-spanning helices. Lamellar X-ray diffraction of oriented membrane multilayers was employed to distinguish these alternatives. Cytochrome P450 2B4 was reconstituted into unilamellar phospholipid proteoliposomes (molar protein to lipid ratio 1:90). Sedimentation of the proteoliposomes produced an ordered stack of bilayers with a one-dimensional repeat distance (d) perpendicular to the plane of the bilayer. The stacked multilayers were exposed to an X-ray beam (lambda = 1.54 A) at near grazing incidence, and lamellar diffraction patterns were recorded. With proteoliposome multilayers, up to six diffraction orders could be observed. Their spacing corresponded to a d of 63.6 A, calculated according to Bragg's Law, comprising the lipid bilayer, the projection of the incorporated protein beyond the bilayer, and the intermembrane water layer. With liposome multilayers containing no P450, the observed d was 59.6 A. These data suggest that the increase of distance between successive bilayers in the stack due to the presence of P450 2B4 was only about 4 A. This distance is much less than would be expected with the "N-terminal membrane-anchor" model of the membrane topology, in which the P450 molecules largely extend beyond the surface of the membrane (> or = 35 A). Furthermore, the mass distribution deduced from Fourier synthesis confirms that the protein is deeply immersed in the membrane.

Single crystal X-ray structures of the two 4-heptadecyl derivatives of (1R,5S)-3,6,8-trioxabicyclo[3.2.1]octane.

The single crystal structures of the two diastereomeric 4-heptadecyl derivatives of (1R,5S)-3,6,8-trioxabicyclo[3.2.1]octane have been determined by X-ray diffraction to be (1R,4R,5S)-heptadecyl-3,6,8-trioxabicyclo[3.2.1]octane (I) and (1R,4S,5S)-4-heptadecyl[3,6,8-trioxabicyclo[3.2.1]octane (II), respectively, which have an exo or axial 4-heptadecyl group, and an endo or equatorial 4-heptadecyl group, respectively. The structures of I and II had been suggested by their phase-sensitive 2D NOESY 1H-NMR spectra, but are now established unambiguously. These optically pure non-ionic lipid-like amphipathic molecules (I and II) represent the first 3,6,8-trioxabicyclo[3.2.1]octanes for which single crystal structures have been solved. Crystals of both isomer I and isomer II were orthorhombic with space group P2(1)2(1)2(1), and had unit cell dimensions of a = 9.586, b = 43.14, c = 5.289 A, and a = 7.34, b = 51.8, c = 5.636 A, respectively. The structures of I and II were both solved by using direct methods to R = 0.045 and R = 0.086, respectively. Both I and II pack in stacked bilayers with interdigitating and tilting hydrocarbon chains. The molecular and hydrocarbon cross sections are I: S = 50.70 A2, sigma = 19.00 A2; and II: S = 41.37 A2, sigma = 18.26 A2.

Interaction of the NMDA receptor noncompetitive antagonist MK-801 with model and native membranes.

MK-801, a noncompetitive antagonist of the NMDA (N-methyl-D-aspartate) receptor, has protective effects against excitotoxicity and ethanol withdrawal seizures. We have determined membrane/buffer partition coefficients (Kp[mem]) of MK-801 and its rates of association with and dissociation from membranes. Kp[mem] (+/- SD) = 1137 (+/- 320) in DOPC membranes and 485 (+/- 99) in synaptoneurosomal (SNM) lipid membranes from rat cerebral cortex (unilamellar vesicles). In multilamellar vesicles, Kp[mem] was higher: 3374 (+/- 253) in DOPC and 6879 (+/- 947) in SNM. In cholesterol/DOPC membranes, Kp[mem] decreased as the cholesterol content increased. MK-801 associated with and dissociated from membranes rapidly. Addition of ethanol to SNM did not affect Kp[mem]. MK-801 decreased the cooperative unit size of DMPC membranes. The decrease was smaller than that caused by 1,4-dihydropyridine drugs, indicating a weaker interaction with the hydrocarbon core. Small angle x-ray diffraction, with multilayer autocorrelation difference function modeling, indicated that MK-801 in a cholesterol/DOPC membrane (mole ratio = 0.6) causes a perturbation at approximately 16.0 A from the bilayer center. In bilayers of cholesterol/DOPC = 0.15 (mole ratio) or pure DOPC, the perturbation caused by MK-801 was more complex. The physical chemical interactions of MK-801 with membranes in vitro are consistent with a fast onset and short duration of action in vivo.

Favorable amphiphilicity of nimodipine facilitates its interactions with brain membranes.

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 10(-10) M) near its Kd, 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.

The molecular basis for lacidipine's unique pharmacokinetics: optimal hydrophobicity results in membrane interactions that may facilitate the treatment of atherosclerosis.

Membrane-active drugs can be characterized by direct measurements of their membrane partition coefficients, washout rates from membranes, and washin rates into membranes. There appears to be a correlation between the duration of action of such membrane-active drugs and the membrane partition coefficient in conjunction with the washout rate. Lacidipine has a high membrane partition coefficient compared to other 1,4-dihydropyridine calcium-channel antagonists and a slow washout rate from membranes. Clinically, it also exhibits an extended duration of action. This control at the membrane molecular level may provide an optimal pharmacokinetic profile for lacidipine in the treatment of hypertension. In addition, these same properties may be important for lacidipine as an antiproliferative agent in the treatment of atherosclerosis.

X-ray diffraction analysis of brain lipid membrane structure in Alzheimer's disease and beta-amyloid peptide interactions.

Small angle x-ray diffraction analysis of Alzheimer's disease (AD) lipid membranes reconstituted from cortical gray matter showed significant, reproducible structure changes relative to age-matched control samples. Specifically, there was an average 4 A reduction in the lipid bilayer width and marked changes in membrane electron density profiles of AD cortical samples. There were no significant structure differences in the membrane bilayers isolated from an unaffected region (cerebellum) of the AD brain. Lipid and protein analysis of six AD and six age-matched controls showed that the phospholipid:protein mass ratio was unchanged, but that the unesterified cholesterol:phospholipid (C:PL) mole ratio decreased by 30% in the AD temporal gyrus relative to age-matched controls. The C:PL mole ratio was not significantly different for samples prepared from cerebellum of AD versus control patients. X-ray diffraction analysis of a cholesterol-enriched AD sample demonstrated a virtual restoration of the normal membrane bilayer width and electron density profile, suggesting that the cholesterol deficit played a major role in the AD lipid membrane structure perturbation. Addition of beta-amyloid peptide to bovine brain phospholipid membranes significantly changed the electron density associated with the hydrocarbon core. Alterations in the composition and structure of the membrane bilayer may play an important role in the pathophysiology of AD by altering the activity and catabolism of membrane-bound proteins, including the beta-amyloid precursor protein.

Molecular interaction between lacidipine and biological membranes.

AIM: To examine the molecular basis for the unique pharmacokinetics of lacidipine by defining interactions between lacidipine and biological membranes, which may explain the long clinical half-life of this calcium channel antagonist. METHODS: Radiotracer analysis was used to determine the membrane partition coefficient and washout kinetics of lacidipine with membranes of different composition. Small-angle X-ray diffraction with angstrom resolution was used to determine the location of lacidipine in membranes. RESULTS: Lacidipine had a high membrane partition coefficient, which decreased as cholesterol in the membrane increased, and a slow rate of membrane washout. The drug was found deep within the membrane's hydrocarbon core, which was consistent with the other membrane drug parameters. CONCLUSIONS: Lacidipine's location and interaction within membranes may provide a longer duration of therapeutic action and can explain the unique pharmacokinetics of this drug.

Equilibrium and kinetic studies of the interactions of salmeterol with membrane bilayers.

The interaction of salmeterol with model membranes has been studied with regard to equilibrium and kinetic behavior, including determination of the membrane-based partition coefficient, the rate of dissociation of salmeterol from membranes, and the rate of association. These data were obtained in various membrane preparations and under various conditions (e.g., temperature, cholesterol content). The compound is very lipophilic, compared with other beta 2 agonists such as salbutamol, and has a rapid association rate and a moderate dissociation rate. The equilibrium data support the assertion that the salmeterol action measured in perfused tissue involves an exo-site for nonspecific binding that may be identified with or related to the lipid bilayer. The kinetic data in unilamellar and multilamellar liposomes of synthetic lipids further suggest that the approach to the exo-site and the active site may involve components in the native system other than the lipid bilayer in which the beta 2 receptor is located. These additional components may explain the slow onset and the extraordinarily long duration of action.

Molecular basis for the inhibition of 1,4-dihydropyridine calcium channel drugs binding to their receptors by a nonspecific site interaction mechanism.

The "membrane bilayer" pathway (Rhodes, D. G., J. G. Sarmiento, and L. G. Herbette. 1985. Mol. Pharmacol. 27:612-623.) for 1,4-dihydropyridine calcium channel drug (DHP) binding to receptor sites in cardiac sarcolemmal membranes has been extended to include the interaction of amphiphiles within the lipid bilayer. These studies focused on the ability of the Class III antiarrhythmic agents bretylium and clofilium to nonspecifically inhibit DHP-receptor binding in canine cardiac sarcolemma. Clofilium was found to inhibit nimodipine binding with an inhibition constant of approximately 5 microM, whereas bretylium had no effect on nimodipine binding. Small angle x-ray diffraction was then used to examine the differential ability of these two Class III agents to inhibit DHP-receptor binding. The time-averaged locations of bretylium, clofilium, and nimodipine in bovine cardiac phosphatidylcholine (BCPC) bilayers (supplemented with 13 mol% cholesterol) were determined to a resolution of 9 A. The location of bretylium as dominated by its phenyl ring in BCPC bilayers was found to be at the hydrocarbon core/water interface, similar to that of the dihydropyridine ring of nimodipine. The location of clofilium as dominated by its phenyl ring was found to be below the hydrocarbon/core water interface within the hydrocarbon chain region of the bilayer, similar to that of the phenyl ring of nimodipine. The location of the dihydropyridine ring portion of nimodipine has previously been shown by neutron diffraction to be located at the hydrocarbon core/water interface of native sarcoplasmic reticulum, consistent with the small angle x-ray data from model membranes in this paper. Therefore, we speculate that the nonspecific inhibition arises from the interaction of clofilium's phenyl ring with the site on the calcium channel receptor where the phenyl ring portion of nimodipine must interact. The DHP-receptor binding pathway would then involve both nonspecific (membrane) and specific (protein) binding components, both of which are necessary for receptor binding.

Evidence for changes in the Alzheimer's disease brain cortical membrane structure mediated by cholesterol.

Small angle X-ray diffraction analysis of Alzheimer's disease (AD) lipid membranes extracted from cortical gray matter showed significant, reproducible structure changes relative to age-matched control samples. Specifically, there was an average 4 A reduction in the lipid bilayer width and significant changes in the membrane electron density profiles of AD cortical samples. There were no significant structure differences in the membrane bilayers isolated from an unaffected region (cerebellum) of the AD brain. Lipid and protein analysis of 6 AD and 6 age-matched controls showed that the phospholipid:protein mass ratio was unchanged but that the unesterified cholesterol:phospholipid (C:PL) mole ratio decreased by 30% in the AD temporal gyrus relative to age-matched controls. By contrast, the C:PL mole ratio in the cerebellum did not change significantly. X-ray diffraction analysis of a cholesterol enriched AD sample demonstrated a virtual restoration of the normal membrane bilayer width and electron density profile, suggesting that the cholesterol deficit played a major role in the AD lipid membrane structure perturbation. Alterations in the composition and structure of the membrane bilayer may play an important role in the pathophysiology of AD by altering the activity and catabolism of membrane-bound proteins, including the beta-amyloid precursor protein.

New approaches to drug design and delivery based on drug-membrane interactions.

In this review, the complex physical and chemical interactions of drugs with model and biological membranes under normal and pathological conditions are examined at the molecular level. The results of our own published and unpublished structural studies are discussed and correlated with kinetic binding studies to assess the potential role of nonspecific drug interaction with the membrane bilayer in the overall receptor binding mechanism for membrane-bound receptors in heart and brain.

Large-scale structural changes in the sarcoplasmic reticulum ATPase appear essential for calcium transport.

Model refinement calculations utilizing the results from time-resolved x-ray diffraction studies indicate that specific, large-scale changes (i.e., structural changes over a large length scale or long range) occur throughout the cylindrically averaged profile structure of the sarcoplasmic reticulum ATPase upon its phosphorylation during calcium active transport. Several physical-chemical factors, all of which slow the kinetics of phosphoenzyme formation, induce specific, large-scale changes throughout the profile structure of the unphosphorylated enzyme that in general are opposite to those observed upon phosphorylation. These results suggest that such large-scale structural changes in the ATPase occurring upon its phosphorylation are required for its calcium transport function.

Rat cerebral cortical synaptoneurosomal membranes. Structure and interactions with imidazobenzodiazepine and 1,4-dihydropyridine calcium channel drugs.

Small angle x-ray scattering has been used to investigate the structure of synaptoneurosomal (SNM) membranes from rat cerebral cortex. Electron micrographs of the preparation showed SNM with classical synaptic appositions intact, other vesicles, occasional mitochondria, and some myelin. An immunoassay for myelin basic protein placed the myelin content of normal rat SNM at less than 2% by weight of the total membrane present. X-Ray diffraction patterns showed five diffraction orders with a unit cell repeat for the membrane of 71 to 78 A at higher hydration states. At lower hydration, 11 orders appeared; the unit cell repeat was 130 A, indicating that the unit cell contained two membranes. Electron density profiles for the 130-A unit cell were determined; they clearly showed the two opposed asymmetrical membranes of the SNM vesicles. SNM membrane/buffer partition coefficients (Kp) of imidazobenzodiazepine and 1,4-dihydropyridine (DHP) calcium channel drugs were measured; Kp's for DHP drugs were approximately five times higher in rabbit light sarcoplasmic reticulum than in SNM. Ro 15-1788 and the DHP BAY K 8644 bind primarily to the outer monolayer of vesicles of intact SNM membranes. Nonspecific equilibrium binding of Ro 15-1788 occurs mainly in the upper acyl chain of the bilayer in lipid extracts of SNM membrane.

Study of the topography of cannabinoids in model membranes using X-ray diffraction.

Small-angle X-ray diffraction was used to determine the topography of (-)-delta 8-tetrahydrocannabinol in partially hydrated dimyristoylphosphatidylcholine bilayers. Electron density profiles of lipid bilayers in the presence and absence of the cannabinoid were calculated using Fourier transform. Step-function equivalent profiles were then constructed to obtain the absolute electron density scale. We have compared the electron density profiles of the above preparations to determine the location of the drug molecule in the bilayer. By using (-)-5'-iodo-delta 8-tetrahydrocannabinol in parallel experiments, we were also able to locate the iodine atom in the bilayer and deduce the conformation of the cannabinoid side alkyl chain. All comparisons were made between different preparations having the same mesomorphic form and total period repeat distance. To achieve this, we have carried out X-ray diffraction experiments at various temperatures to cover the different mesomorphic phases and combined our data with the corresponding results from differential scanning calorimetry. Based on the results of this work and previous data on the orientation of the cannabinoid in model membranes, we concluded that the phenolic hydroxy group of the drug molecule exists near the carbonyl groups of DMPC and that the average position of the iodine atom is approx. 5.5 A from the center (terminal methyl region) of the DMPC bilayer. This requires the cannabinoid side-chain to assume an orientation parallel to the bilayer chains.

Structure of the calcium channel antagonist, nimodipine.

Isopropyl 2-methoxyethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydro-3,5- pyridinedicarboxylate, C21H26N2O7, Mr = 418.45, orthorhombic, P2(1)2(1)2(1), a = 12.5897 (6), b = 14.6410 (9), c = 11.636 (1) A, V = 2144.8 (2) A3, Z = 4, Dm = 1.29, Dx = 1.30 g cm-3, lambda(Cu K alpha) = 1.54178 A, mu = 7.77 cm-1, F(000) = 888, T = 298 K, R = 0.047 for 1629 observed reflections. The structure of the title compound is similar to that of related analogs, the nitrophenyl ring being roughly normal to the dihydropyridine ring, which is in a boat conformation (N1 is 10.75 degrees out of the C2-C3-C5-C6 plane; C4 is 19.55 degrees out of plane). The 3,5 substituents are in an extended conformation, away from the 2,6 methyl groups. The nitro group is distal to N1. Structure/activity relationships of 1,4-dihydropyridines are discussed in light of this structure.

Comparison of location and binding for the positively charged 1,4-dihydropyridine calcium channel antagonist amlodipine with uncharged drugs of this class in cardiac membranes.

The distinctive pharmacokinetic and pharmacodynamic activity of amlodipine, including long onset and duration of activity as a calcium channel antagonist, may be related to its interactions with membranes. We have used X-ray crystallography and small-angle X-ray scattering to examine and compare the crystal structure of amlodipine and its location in cardiac sarcolemmal lipid bilayers with that of uncharged dihydropyridines (DHPs) such as nimodipine. Crystallographic analysis demonstrated that the DHP ring of amlodipine is considerably more planar than that of nimodipine, that amlodipine has a greater torsion angle between the DHP and aryl rings, and that the protonated amino group extends away from the DHP ring structure. Despite the positive charge of amlodipine at physiological pH, membrane electron density profile structures showed amlodipine to have a time-averaged location near the hydrocarbon core/water interface similar to that observed for several uncharged DHPs. However, unlike uncharged DHPs, this location is consistent with an ionic interaction between the protonated amino function of amlodipine and the negatively charged phospholipid headgroup region, in addition to a hydrophobic interaction with the fatty acyl chain region near the glycerol backbone similar to other DHPs. This location may also provide an appropriate conformation and orientation for amlodipine binding to its receptor site at this depth in the membrane. Finally, we have measured the nonspecific partitioning of amlodipine into native sarcoplasmic reticulum membranes from rabbit skeletal muscle and compared these data with those for the uncharged DHPs. The partition coefficient into light sarcoplasmic reticulum for amlodipine was higher than that observed for most uncharged DHPs and rates of incorporation of amlodipine into membranes were very high, as with other DHPs, whereas the "washout time" of amlodipine from these membranes was longer by over 1 order of magnitude. These data suggest differences in membrane interactions for amlodipine, compared with uncharged DHPs, that may be correlated with its novel pharmacodynamic and pharmacokinetic profile.