JOURNAL OF
`
`MEDICINAL
`CH EM ISTRY
`
`@ Copyright 1991 by the American Chemical Society
`
`Volume 34, Number 3
`
`March 1991
`
`Perspective
`
`Reevaluating Equilibrium and Kinetic Binding Parameters for Lipophilic Drugs
`Based on a Structural Model for Drug Interaction with Biological Membranes
`
`R. Preston Mason,tJll David G. Rhodes,tJ and Leo G. Herbette*>t**-*i*
`Departments of Radiology, Medicine, and Biochemistry, the Travelers Center on Aging, and the Biomolecular Structure
`Analysis Center, Uniuersity of Connecticut Health Center, Farmington, Connecticut 06032. Received July 27, 1990
`concentration" of the drug, as measured experimentally.
`Introduction
`This difference in the Kd values is related to the membrane
`Structure-function activity relationships of drugs that
`partition coefficient of the drug.
`bind to certain membrane-associated receptors must take
`In addition to affinity constants, drug interaction with
`into account the local membrane bilayer environment
`the membrane should be considered for other pharmaco-
`where the binding event occurs. The partitioning of drugs
`logical parameters such as pICs and association rate con-
`in an isotropic two-phase bulk solvent system such as
`stants. These parameters are important considerations for
`octanol/buffer apparently is not a good model for drug
`designing new therapeutic agents that have a dominant
`interaction with the lipid bilayer of membranes. Knowl-
`interaction with a cell membrane and a specific component
`edge of these membrane-based partition coefficients then
`of a cell membrane.
`necessitates reanalysis of other physical, chemical, and
`functional parameters.
`Molecular Models for Drug Binding to Membrane
`In this Perspective, we have reexamined the model used
`Receptors
`in the equilibrium dissociation constant (Kd) determination
`Generally, the mechanism for drug binding to a plasma
`for certain lipid-soluble drugs based on recent experimental
`membrane receptor has been considered to be analogous
`data describing the interaction of these drugs with the
`to that of endogenous ligands such as hormones, growth
`membrane bilayer. Because several lines of experimental
`factors, neurotransmitters, etc. These agonists are gen-
`evidence suggest that some lipophilic drugs bind to hy-
`erally water soluble and thought to bind to an extracellular
`drophobic, intramembrane receptor sites via the membrane
`portion of the receptor. For example, the charged ace-
`bilayer, the concentration of such drugs in the membrane
`tylcholine neurotransmitter and ita competitive antagonist
`bilayer compartment in equilibrium with the receptor
`bind to an extracellular portion of the cy subunit near the
`needs to be considered for Kd dculations. In other words,
`opening of the ion channel.'
`instead of expressing the "free" and "bound" concentra-
`In contrast to ligand binding directly from the aqueous,
`tions of the drug in terms of a total aqueous volume (moles
`extracellular environment, there is experimental support
`of drug per liter of solution), these quantities should be
`for highly lipophilic drugs to bind via the membrane bi-
`expressed as a function of the membrane lipid volume
`layere2 For example, local anesthetics that are noncom-
`(moles of drug per liter of membrane lipid). The results
`petitive blockers (NCB) bind to the acetylcholine receptor
`of this analysis indicate that Kd values calculated on the
`at a site distinct from that of the agonist.s Photoaffinity
`labeling experiments suggest that the binding site for NCB
`basis of an aqueous concentration of the drug are signif-
`icantly different from those using the "membrane
`(1) Changeux, J.; Devillers-Thiery, A,; Chemouilli, P. Acetyl-
`* Send correspondence to Dr. Leo Herbette, Biomolecular
`choline Receptor, An Allosteric Protein. Science 1984, 225,
`Structure Analysis Center, University of Connecticut Health
`1345.
`Center, Farmington, CT 06032.
`(2) Hille, K. B. Local Anesthetics, Hydrophilic and Hydrophobic
`* Department of Medicine.
`Department of Radiology.
`Pathways for the Drug Receptor Reaction. J. Gen. Physiol.
`1977,69, 497-515.
`(3) Peper, K.; Bradley, R. J.; Dreyer, F. The Acetylcholine Re-
`Department of Biochemistry.
`ceptor at the Neuromuscular Junction. Physiol. Rev. 1982,62,
`I Travelers Center on Aging.
`* Biomolecular Structure Analysis Center.
`1271-1340.
`0022-262319111834-0869$02.60/0 0 1991 American Chemical Society
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`870 Journal of Medicinal Chemistry, 1991, Vol. 34, No. 3
`to the acetylcholine receptor is deep in the pore of the
`
`channel, in a transmembrane r e g i ~ n . ~ ~ ~ In addition, the
`activity of some of these anesthetics parallel their hydro-
`phobicity. Electron spin resonance (ESR) studies exam-
`ined the binding of reversibly charged forms of an NCB
`anesthetic, 2-[N-methyl-N-(2,2,6,6-tetramethyl-l-oxy-
`piperidin-4-yl)amino] ethyl 4- (hexyloxy) benzoate (CGSL)
`to the receptor6 The charged form of the anesthetic binds
`only when the channel is open. However, when the
`channel is closed, only the uncharged form of the anesth-
`etic (as controlled by pH) can bind to the high affinity
`receptor, presumably through the lipid phase. ESR ex-
`periments indicate the uncharged compound is associated
`with the membrane hydrocarbon core and thereby binds
`to the receptor protein following diffusion through the
`membrane.6
`Anesthetic drug access to the acetylcholine receptor via
`the membrane bilayer is also supported by patch clamp
`studies. Despite a high-resistance membrane patch seal
`enclosing acetylcholine receptors, microperfusion of the
`anesthetic isoflurance into the medium outside of the patch
`resulted in altering channel activity within the patch.’
`The presence of the high-resistance seal suggested that the
`compound gained access to the receptor through the lipid
`phase.
`Evidence for an intrabilayer receptor site that must be
`accessed by diffusion through the lipid phase has also been
`implicated for the @-adrenergic receptor. The human genes
`for both the a2 and p2 adrenergic receptors have been
`cloned and expressed in Xenopus oocytes. The receptors
`are homologous and contain seven hydrophobic domains
`that have been modeled as seven transmembrane spanning
`segments.8 Deletion mutations have indicated that the
`seventh membrane spanning domain is necessary for ligand
`binding.8 These mutations give experimental support to
`a transmembrane, intrabilayer receptor site. Although
`certain @-adrenergic antagonists are formally charged, as
`in the case of propranolol, small angle neutron diffraction
`experiments have observed the drug’s time-averaged lo-
`cation to be in the hydrocarbon core, near the glycerol
`backbone, of biological membranes9 while the partition
`coefficient of propranolol into biological membrane was
`relatively high, Kp > lO3.lo
`
`(4) Heidmann, T.; Changeux, J.-P. Time-resolved Photolabeling
`by the Noncompetitive Blocker Chlorpromazine of the Ace-
`tylcholine Receptor in its Transiently Open and Closed Ion
`Channel Conformation. PNAS (USA) 1984,81, 1897-1901.
`(5) Giraudat, J.; Dennis, M.; Heidmann, T.; Haumont, P. Y.;
`Lederer, R.; Changeux, J. P. Structure of the High-Affinity
`Site for Noncompetitive Blockers of the Acetycholine Recep-
`tor. [3H] Chlorpromazine Labels Homologous Residues in the
`p and 6 Chains. Biochemistry 1987, 26, 2410-2418.
`(6) Blanton, M.; McCardy, E.; Gallaher, R.; Wang, H. H. Non-
`competitive inhibitors reach their binding sit in the acetyl-
`choline receptor by two different paths. Mol. Pharmacol. 1988,
`33,634-642.
`(7) Brett, R. S.; Dilger, J. P.; Yland, K. F. Isoflurane causes
`“Flickering” of the Acetylcholine Receptor Channel: Obser-
`vations using the Patch Clamp. Anesthesiology 1988, 69,
`161-170.
`(8) Kobilka, B. K.; Kobilka, T. S.; Daniel, K.; Regan, J. W.; Caron,
`M. G.; Lefkowitz, R. J. Chimeric a2, p2-Adrenergic Receptors:
`Delineation of Domains Involved in Effector Coupling and
`Ligand Binding Specificity. Science 1988, 240, 1310-1316.
`(9) Herbette, L. G.; Katz, A. M.; Sturtevant, J. M. Comparisons
`of the Interactions of Proparanol and Timolol with Model and
`Biological Membrane Systems. Mol. Pharmacol. 1983, 24,
`259-269.
`(10) Herbette, L. G.; Chester, D. W.; Rhodes, D. G. Structural
`Analysis of Drug Molecules in Biological Membranes. Bio-
`phys. J. 1986,49,91-94.
`
`Perspective
`
`I
`
`Figure 1. This figure illustrates a hypothetical transmembrane
`ion channel with a hydrophobic, intrabilayer receptor site labeled
`“R”. Evidence for such a hydrophobic site is based on the DHP
`receptor sequence analysis (Tanabe et al., 1987) and photoaffinity
`labeling (Takahashi et al., 1987). Drugs that bind to this receptor
`site are indicated by oriented diamonds with an intrabilayer
`distribution profiie characterized by a Gaussian curve on the right.
`The center of the Gaussian curve, marked by an arrow and rep-
`resenting the location along the bilayer normal axis of highest
`drug concentration, is at a depth in the membrane coincident with
`the drug’s putative receptor site.
`Finally, a “membrane bilayer pathway’’ has been de-
`scribed for the binding of lipophilic 1,kdihydropyridine
`(DHP) Ca2+ channel blockers to voltage-dependent Ca2+
`channels in cardiac and smooth muscle sarcolemma. This
`would occur in a two-step process.ll First, the drug
`molecule must partition to a well-defined, energetically
`favorable location, orientation, and conformation in the
`membrane bilayer before laterally diffusing to an intra-
`bilayer receptor binding site (Figure 1).
`The primary structure of the DHP receptor subunit
`from rabbit skeletal muscle has been deduced from its
`DNA sequences. The polypeptide is structurally similar
`to the voltage-dependent sodium channel with four units
`of homology that comprise six putative transmembrane
`a-helices that may serve as the channel for Ca2+.l2~l3 In
`light of the high homology of the hydrophobic domains of
`Ca2+ channels with Na+ channels, it is interesting that
`DHPs have been shown to bind with high affinity and
`stereoselectivity to the cardiac sarcolemmal sodium
`~hanne1.l~ These data suggest that the specific receptor
`
`(11) Rhodes, D. G.; Sarmiento, J. G.; Herbette, L. G. Kinetics of
`Binding of Membrane-active Drugs to Receptor Sites. Diffu-
`sion Limited Rates for a Membrane Bilayer Approach of 1,4-
`Dihydropyridine
`Channel Antagonists to their Active
`Site. Mol. Pharmacol. 1985,27, 612-623.
`(12) Tanabe, T.; Takeshima, H.; Mikami, A.; Flockerzi, V.; Tak-
`ahashi, H.; Kangawa, K.; Kojima, M.; Matsuo, H.; Hirose, T.;
`Numa, S. Primary Structure of the Receptor for
`Channel
`Blockers from Skeletal Muscle. Nature 1987, 328, 313-318.
`(13) Ellis, S. B.; Williams, M. E.; Ways, N. R.; Brenner, R.; Sharp,
`A. H.; Leung, A. T.; Campbell, K. P.; McKenna, E.; Koch, W.
`J.; Hui, A.; Schwartz, A.; Harpold, M. M. Sequence and Ex-
`pression of mRNAs Encoding the a1 and a2 Subunits of a
`Channel. Science 1988,241,1661-1664.
`DHP-Sensitive
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`Perspective
`site for the DHPs common to both the Ca2+ and Na+
`channel is a hydrophobic, transmembrane domain.
`Moreover, the DHP receptor subunit can be heavily la-
`beled by a hydrophobic photoaffiniy probe, indicating that
`the protein consists of multiple transmembrane helices.ls
`The probability that DHPs interact with the bulk lipid
`phase in the cardiac sarcolemma is high in light of high
`partition coefficients measured for several DHPs (Kp. >
`lo3, refs 10, 16-19) and the very low receptor density
`(approximately one receptor site per square micron in the
`cardiac sarcolemmal membrane; ref 20). Diffusion-limited
`rates calculated for a membrane pathway are approxi-
`mately 3 orders of magnitude greater than those for an
`aqueous approach in which the drug reaches the receptor
`by diffusion through the bulk solvent." The two-dimen-
`sional component of this process, lateral diffusion through
`the bilayer, has a significant rate advantage if the ligand
`has the appropriate location and orientation for binding
`to the receptor site.21
`Experimental suppott for the first step of this pathway,
`namely DHP partitioning to a discrete, time-averaged lo-
`cation in the membrane bilayer, has been shown by using
`small-angle X-ray and neutron diffraction with several
`representative DHPS.'OJ~~~ The second step of the mem-
`brane bilayer pathway, namely DHP lateral diffusion
`through the membrane, was measured by using florescence
`redistribution after photobleaching (FRAP). With use of
`an active rhodamine labeled DHP analogue, the micro-
`scopic rate of drug lateral diffusion was measured in canine
`cardiac sarcolemmal lipid multilayers over a wide range
`At the highest relative hu-
`of relative h u m i d i t i e ~ . ~ ~ ~ ~ ~
`midity, the rate of lateral diffusion for the DHP was
`identical with that measured for phospholipid analogs (3.8
`X lo4 cm2/s). These rapid rates of diffusion suggest that
`
`(14) Yatani, A.; Kuntze, D. L.; Brown, A. M. Effecta of Dihydro-
`pyridine Ca+2 Channel Modulators on Cardiac Sodium Chan-
`nels. Am. J. Physiol. 1988, 254, H140-Hl47.
`Takahashi, M.; Seagar, M. J.; Jones, J. F.; Reber, B. F. X.;
`Catterall, W. A. Subunit Structure of Dihydropyridine-sensi-
`tive Ca+2 Channels from Skeletal Muscle. PNAS (USA) 1987,
`84,5478-5482.
`Herbette, L. G.; Vant Erve, Y. M. H.; Rhodes, D. G. Interac-
`tion of 1,4-Dihydropyridine Ca+2 Channel Antagoniata with
`Biological Membranes, Lipid Bilayer Partitioning Could Occur
`Before Drug Binding to Receptors. J. Mol. Cell. Cardiol. 1989,
`21, 187-201.
`Boer, R.; Grassegger, A.; Schudt, C.; Glossman, H. (+)-Nigul-
`dipine Binds With Very High Affinity to Ca+2 Channels and
`to a Subtype of a,-Adrenoceptors. Eur. J. Pharmacol. 1989,
`272, 131-145.
`Mason, R. P.; Gonye, G. E.; Chester, D. W.; Herbette, L. G.
`Partitioning and Location of Bay K 8644,1,4-Dihydropyridine
`Ca+2 Channel Agonist, in Model and Biological Lipid Mem-
`branes. Biophys. J. 1989,55, 769-778.
`Mason, R. P.; Campbell, S.; Wang, S.; Herbette, L. G. A Com-
`parison of Bilayer Location and Binding for the Charged 1,4-
`Dihydropyridine Ca+2 Channel Antagonist Amlodipine with
`Uncharged Drugs of this Class in Cardiac and Model Mem-
`branes. J. Mol. Pharmacol. 1989,36,634-640.
`Colvin, R. A.; Ashavaid, T. F.; Herbette, L. G. Structure-
`function Studies of Canine Cardiac Sarcolemmal Membranes.
`I. Estimation of Receptor Site Densities. Biochim. Biophys.
`Acta 1986,822,601-608.
`McCloskey, M.; Poo, M.-M. Rates of Membrane Associated
`Reactions, Reduction in Demensionality Revisited. J. Cell
`Biol. 1986, 102, 88-96.
`Mason, R. P.; Chester, D. W. Diffusional Dynamics of an Ac-
`tive Rhodamine-Labeled l,4-Dihydropyridine in Sarcolemmal
`Lipid Multibilayers. Biophys. J. 1989, 56, 1193-1201.
`Chester, D. W.; Herbette, L. G.; Mason, R. P.; Joslyn, A. F.;
`niggle, D. J.; Koppel, D. E. Diffusion of Dihydropyridine Ca+2
`Channel Antagonists in Cardiac Sarcolemmal Lipid Multibi-
`layers. Biophys. J. 1987,52, 1021-1030.
`
`Journal of Medicinal Chemistry, 1991, Vol. 34, No. 3 871
`
`octanol/ buffer
`40
`
`Table I. l,4-Dihydropyridine Partition Coefficienta into
`Biological Membranes and Octanol/BuffeP
`biological membranes*
`(sarcoplasmic reticulum)
`drug
`Bay P 8851
`125000
`26 OOO
`iodipine
`19 000
`amlodipine
`13 000
`nisoldipine
`Bay K 8644
`11 000
`6 300
`nimodipine
`3000
`nifedipine
`Some of the data in this table were reproduced from ref 10,16,
`18, and 19. *Similar values were obtained with cardiac sarcolem-
`mal lipid extracts, indicating a primary interaction of the drug
`with the membrane bilayer component of these biological mem-
`branes.
`
`30
`40
`290
`730
`
`Table 11. Drug Partition Coefficients into Biological Membranes
`and OctanollBuffeP
`
`biological membranes
`octanol/ buffer
`(sarcoplasmic reticulum)
`drua
`350
`921 OOO
`amiodarone
`120
`12 500
`beta X-61
`250
`3 200
`beta X-67
`1200
`propranolol
`18
`3
`350
`beta X-57
`300
`cimetidine
`1
`0.7
`16
`timolol
`Some of the data in this table were reproduced from ref 10, 16,
`18, and 19.
`the overall binding rate by a membrane bilayer pathway
`is generally not rate-limited by the drug's diffusion through
`the membrane.l'
`Recently, Boer and co-workers" in the laboratory of H.
`Glossman have also observed high membrane partition
`coefficients for DHP analogues. However, their inter-
`pretation of the relationship of these findings to the 'true"
`& for DHP binding to Ca2+ channels did not consider the
`possibility of the membrane bilayer pathway as a model
`for DHP receptor binding. They view the high partitioning
`into the membrane as effecting a depletion of the active
`drug available in the surrounding medium for binding to
`an exposed receptor site by an aqueous pathway. Thus,
`they proposed that the true Kd was inversely related to the
`DHP's partition coefficient. By contrast, we propose, from
`a variety of studies including our own, that the relevant
`concentration of drug in equilibrium with the DHP re-
`ceptor site is within the membrane bilayer compartment,
`and that there is a direct relationship between the true &
`and the DHP partition coefficient.
`Drug Partition Coefficients into Biological
`Membranes Differ Dramatically from Those
`Measured in Octanol/Buffer Systems
`Data in Table I of drug partition coefficients highlight
`the fact that drug interactions with both model and bio-
`logical membranes are complex and cannot be mimicked
`by isotropic model systems, e.g. octanol/buffer. The
`charged DHP Ca2+ channel antagonist amlodipine is a case
`in point. The partition coefficient measured in octanol/
`buffer, KPlisol, for amlodipine was nearly 1 order of mag
`nitude lower than that of the uncharged DHP nimodipine.
`By contrast, its partition coefficient Kp in a biological
`membrane, KPImem, is over 3-fold higher than that of ni-
`modipine. The di#ferences in drug partitioning into oc-
`tanol/buffer versus membranes were also observed for a
`wide variety of drugs including antiarrhythmic agents, H2
`antagonists, and @-adrenergic blockers (Table 11).
`Once it has been recognized that the bilayer environ-
`ment is important to drug/lipid interactions and that drugs
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`872 Journal of Medicinal Chemistry, 1991, Vol. 34, No. 3
`assume a well defined location in membrane bilayers, it
`is not surprising to find that modulating the physical (e.g.,
`thermal phase transition; ref 18) or chemical (e.g., chole-
`sterol content; ref 24) characteristics of the membrane
`substantially affects the DHP KPlmeml. These changes in
`the composition of native plasma membranes and their
`effect on drug pharmacodynamics have clinical relevance
`when considering the membrane compositional changes,
`especially in the cholesterol content, associated with ag-
`ing,En chronic cigarette smoking,% experimental diabe-
`and hypercholesterolemiam$* In our current studies,
`t e ~ , ~
`we have shown that an increase in membrane cholesterol
`from a 0 1 cholesterokphospholipid mole ratio (C:Pl) to a
`0.6:l C:Pl mole ratio resulted in a 11-fold decrease in the
`KPImeml of the DHP Ca2+ channel antagonist nimodipine
`(data not shown). Thus, the drug interacts with a chem-
`ically and structurally anisotropic environment in a man-
`ner that cannot be predicted from Kp[isoj.
`Structural Implications of the Membrane Bilayer
`Model for Drug Binding: Drug-Design Concepts
`The mechanism of binding for DHP calcium channel
`antagonists and agonists to voltage-sensitive calcium
`channels in the cardiac sarcolemma is a complex reaction
`that may involve interaction with the membrane bilayer.
`The hypothesis that the DHP receptor site may be within
`the membrane bilayer compartment is indicated from
`genetic studies that suggest that the DHP receptor is a
`hydrophobic, transmembrane protein. Thus, DHP par-
`titioning to a discrete, energy favorable location, orienta-
`tion, and conformation may be prerequisite for subsequent
`intrabilayer receptor recognition and binding. By reducing
`the degrees of freedom of the drug by limiting it to a
`specific region of the membrane, the phospholipid bilayer
`can effectively increase the efficiency of binding for low
`concentrations of drug to an intrabilayer receptor site.
`The strong interaction of DHPs with membrane bilayers
`may also be helpful in understanding their side effects.
`DHPs may utilize a "membrane bilayer pathway" in their
`reactions with voltage-sensitive calcium channels in other
`tissues in a manner analogous to that described for the
`heart. For example, the cardiac drug Bay K 8644's various
`negative psychopharmacologic effects may result from
`
`Mason, R. P.; Moring, J.; Herbette, L. G. Cholesterol/Drug
`Molecular Interactions with Model and Native Membranes.
`Biophys. J. 1990,57, 523a.
`Hitzemann, R. J.; Johnson, D. A. Developmental Changes in
`Synaptic Membrane Lipid Composition and Fluidity. Neuro-
`chem. Res. 1983,8, 121-131.
`Shinitzky, M.; Heron, D. S.; Samuel, D. Restoration of Mem-
`brane Fluidity and Serotonin Receptors in the Aged Mouse
`Brain. In Aging of the Brain. Samuel et al., Ed.; Raven Press:
`New York, 1983; pp 329-336.
`Tulenko, T. N.; Lapotofsky, D.; Cox, R. H. Alterations in
`Membrane Phospholipid Bilayer Composition with Age in the
`Fisher 344 Rat. Physiologist 1988, 31, A138.
`Tulenko, T. N.; Rabinowitz, J. L.; Cox, R. H.; Santamore, W.
`P. Altered Na+/K+-ATPase, Cell Na+ and Lipid Profiles in
`Canine Arterial Wall with Chronic Cigarette Smoking. Eur.
`J. Biochem. 1988,20, 285-289.
`Roth, D. M.; Reibel, D. K.; Lefer, A. M. Vascular Respon-
`siveness and Eicosenoid Production in Diabetic Rate. Diabe-
`tologia 1983,24, 372-376.
`McMurchie, E. J.; Patten, G. S. Dietary Cholesterol Influencee
`Cardiac Beta-Adrenergic Receptor Adenylate C y c h Activity
`in the Marmoset Monkey by Changes in Membrane Chole-
`sterol Status. Biochim. Biophys. Acta 1988, 942, 324-332.
`McMurchie, E. J.; Patten, G. S.; Charnock, J. S.; McLennan,
`P. L. The Interaction of Dietary Fatty Acid and Cholesterol
`on Catecholamine-stimulated Adenylate Cyclase Activity in
`the Rat Heart. Biochim. Biophys. Acta 1987,898, 137-153.
`
`MEMBRANE MONOLAYER
`
`Perspec tiue
`
`\
`Phosphollpld
`Head Group
`
`,c*i
`,W
`H ,CH,
`: \CH,/q~~,
`Reglon of Phosphate Ester
`2, Negative Charge
`i
`cCI,
`
`a
`NlMODlPlNE $Nl13
`
`Figure 2. This figure s&arizes
`amlodipine's interaction with
`the membrane bilayer in light of ita determined center-of-mase
`location and crystal structure. The drug molecules are positioned
`next to a phcapholipid molecule to indicate the potential chemical
`interactions between the molecules in this two-dimensional rep.
`resentation. Amlodipine's location near the hydrocarbon core!
`water interface can facilitate both a hydrophobic interaction with
`the phospholipid acyl chains and an ionic interaction between
`the protonated amino function of the drug and the charged anionic
`oxygen of the phosphate headgroup. The dihydropyridine ring
`of amlodipine was superimposed on that of nimodipine (using
`structures obtained from crystallographic analysis) at the mem-
`brane location experimentally determined by neutron diffraction
`for nimodipine. The nimodipine structure and location is con-
`sistent with only hydrophobic interactions with the phospholipid
`acyl chains and not an electrostatic interaction with the phos-
`pholipid headgroup as in the case of amlodipine. (Reprinted with
`permission from Mol. Pharmacol. 1989, 36, 634-640.)
`
`binding to DHP sites in the central nervious system.32
`These data demonstrate that drug interactions with the
`native membrane bilayer are complex. Clearly, the chem-
`ical and crystal structure of the drug alone does not provide
`sufficient information with which to predict certain
`drug-membrane interactions. Moreover, traditional sci-
`entific methods to assess the "lipophilicity" of drugs by
`measuring partition coefficients into nonpolar alkane so-
`lutions such as octanol/buffer appeared to be inadequate
`for certain drugs on the basis of the results of this study.
`The anisotropic bilayer structure, in contrast to a bulk
`phase solvent such as octanol with invariant properties
`throughout, has very different physical and chemical
`characteristics as a function of distance across the bilayer
`normal axis that will affect drug-lipid interaction. Drug
`partitioning and location in the bilayer appeared to exploit
`these differences to achieve an energetically favorable lo-
`cation, orientation, and conformation.
`Small-angle X-ray diffraction experiments also showed
`the "specificity" of nonspecific drug interactions for DHPs
`with the membrane bilayer. While in octanol, the DHP
`was randomly dispersed throughout the solution, in a
`membrane bilayer the DHP occupies a discrete, time-av-
`eraged location near the hydrocarbon core/water interface.
`This location can facilitate both hydrophobic and ionic
`interactions of amlodipine with neighboring phospholipid
`molecules (see Figure 2). These structural results were
`
`(32) Bolger, G . T.; Weiesman, B. A,; Skolnick, P. The Behavioral
`Effecte of the Calcium Agonist Bay K 8644 in the Mouse:
`Antagonism by the Calicium Antagonist Nifedipine. Nau-
`nyn.-Schmideberg's Arch. Pharmcol. 1986,328, 373-377.
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`Perspective
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`I
`7.5
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`1500
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`Journal of Medicinal Chemistry, 1991, Vol. 34, No. 3 873
`using crystal structure data to predict the drug structure
`in a membrane may not always be valid since the crystal
`and energy-minimized membrane bilayer structures of
`amlodipine may differ, as will be discussed in the next
`section. Further structure studies would be necessary to
`confirm amlodipine's orientation and conformation in the
`membrane for comparison to other uncharged DHPs.
`Nicardipine is also a positively charged DHP with a pK,
`(7.0) lower than that of amlodipine. Although at physio-
`logical pH approximately 30% of the nicardipine molecules
`are charged, this compound has a pharmacokinetic half-life
`similar to that of uncharged DHPs. The location of the
`protonated amino group of nicardipine is at the C3 position
`of the dihydropyridine ring, adjacent to the 4-phenyl
`substituent. If the DHP ring of nicardipine is at the same
`membrane location as that of nimodipine, the charged
`amino group may not be able to interact electrostatically
`with the charged headgroup of the membrane bilayer, even
`if fully extended. Further, the presence of a phenyl group
`adjacent to the charged tertiary amine of nicardipine would
`result in an energetically unfavorable interaction in the
`hydrophilic environment near the headgroup. Thus, de-
`spite its formal charge, nicardipine may not demonstrate
`the additional electrostatic interactions proposed for am-
`lodipine. This would result in a shorter residence time in
`the membrane and an observed duration of activity similar
`to that of uncharged DHPs.
`Drug Structure in a Crystal versus a Membrane
`Intuitively, the substantial differences in the drug's
`microenvironment in a crystal matrix versus the membrane
`bilayer would be expected to affect its molecular confor-
`mation substantially. To test this hypothesis, small-angle
`X-ray diffraction was used to identify the time-averaged
`location of the antiarrhythmic agent, amiodarone, in a
`synthetic lipid bilayer as shown in Figure 4." The location
`in the membrane was then used to assign an appropriate
`dielectric environment in which the determined crystal-
`lographic drug conformation could be energy minimized
`via the molecular mechanics program MMP2.= The drug
`was located -6 A from the center (terminal methyl region)
`of the lipid bilayer (Figure 4). Thus, a dielectric constant
`of K = 2, approximating that of the bilayer hydrocarbon
`core region was used to calculate a minimum-energy
`structure for membrane-bound amiodarone. The resulting
`calculated structure was significantly different when com-
`pared with the crystal structure of amiodarone. These
`calculations did not take into consideration specific steric
`interactions of the lipid acyl chains on the conformation
`of this lipophilic drug. Nevertheless, the results of this
`work suggest that the biologically active conformation of
`a drug that interacts with an intrabilayer receptor site, for
`example, may be quite distinct from its crystal structure
`conformation.
`A Membrane Bilayer Pathway Affects
`Assumptions for Kd Calculations: Rationale for
`Recalculating "Free" and "Bound" Concentration
`Terms
`To calculate the equilibrium dissociation constant for
`a given drug and receptor, the amount of drug bound
`
`"
`
`
`
`I
`0
`
`I
`I
`30
`120
`TIME (MINUTES)
`Figure 3. Nompecific dissociation of 1 X lo4 M [3H]amlodipine
`(solid circles) and 1 X lo4 M [3H]nimodipine (open circles) from
`light sarcoplasmic reticulum membrane vesicles. This figure shows
`the percentage of drug nonspecifically associated with the mem-
`branes as a function of time. (Reprinted with permission from
`Mol. Pharmacol. 1989, 36, 634-640.)
`fundamental to our understanding of amlodipine's un-
`usually high partition coefficient value into membranes
`versus octanol (Table I). As expected, amlodipine's charge
`resulted in a relatively low partitioning into octanol (Kproct1
`= 30) when compared with the uncharged DHP, nimodi-
`pine (Kp = 260). Amlodipine's high membrane parti-
`tion coefhcient (Kpimem1 = 19ooO), which exceeds by 4-fold
`the value obtained for nimodipine (KpImem1 = 5000), can
`be explained by both its hydrophobic interactions with the
`membrane hydrocarbon core in addition to its very fa-
`vorable ionic bonding with the animic oxygen of the
`phospholipid headgroups (Figure 2). These membrane
`interactions were deduced from the X-ray diffraction
`structure studies.lg
`In addition, amlodipine's membrane interactions may
`be a clue to understanding its novel pharcodynamic and
`pharmacokinetic profile, including a slow onset and long
`duraction of activity in vitro and in vivo relative to un-
`charged drugs of this class.33 For example, amlodipine
`remained bound to LSR membranes 1 order of magnitude
`longer than the uncharged DHP, nimodipine (Figure 3).
`The location of amlodipine at the hydrocarbon core/water
`interface of the membrane is similar to that observed by
`X-ray and neutron diffraction for the uncharged DHPs
`Bay K 8644l" and nimodipine,1° suggesting a common,
`energetically favorable hydrophobic interaction with the
`fatty acyl chain region near the glycerol backbone. In
`addition, however, amlodipine may have an ionic inter-
`action between its protonated amino function and the
`charged anionic oxygen of the phosphate headgroup.
`Specifically, if one superimposes the DHP ring of amlo-
`dipine with that of nimodipine (using structures obtained
`from crystallographic analysis) at the membrane location
`experimentally determined by neutron diffraction for ni-
`modipine, the charged amino function of amlodipine can
`be placed in a region for effective ionic interaction with
`the anionic oxygen atom of the phosphate ester (Figure
`2). This additional charge-charge interaction for amlo-
`dipine may be the basis for its longer, nonspecific asso-
`ciation with the membrane and its unusual pharmacody-
`namics and pharmacokinetics described above. However,
`
`(33) Burges, R. A,; Gardiner, D. G.; Gwilt, M.; Higgins, A. J.;
`Blackburn, K. J.; Campbell, S. F.; Cross, P. E.; Stubbs, J. K.
`Calcium Channel Blocking Properties of Amlodipine in Vas-
`cular Smooth Muscle and Cardiac Muscle In Vitro: Evidence
`for Voltage Modulation of Vascular Dihydropyridine Recep-
`tors. J. Cardiooasc. Pharmacol. 1987, 9, 110-119.
`
`(34) Trumbore, M.; Chester, D. W.; Moring, J.; Rhodes, D.; Her-
`bette, L. G. Structure and Location of Amiodarone in a Mem-
`brane Bilayer as Determined by Molecular Mechanics and
`Quantitative X-ray Diffraction. Biophys. J. 1988,54,53&643.
`(35) Allinger, N. L.; Flanagan, H. L. Isotope Effects in Molecular
`Mechanics (MM2) Calculations on Deuterium Compounds. J.
`Comput. Chem. 1983,4,399-403.
`
`5 of 9
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`PENN EX. 2065
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`874 Journal of Medicinal Chemistry, 1991, Vol. 34, No. 3
`
`Perspectiue
`
`for specific binding calculations.
`Reevaluation of DHP Equilibrium Constants
`Based on Drug Concentration in the Membrane
`+ D + RD. The overall association constant (including
`Consider the bimolecular ligand-receptor reaction: R
`the entire, aqueous volume), K,', is
`K,' = CRD/CRCD
`(1)
`where CR, CD, and CRD are the molar concentrations of free
`receptor sites, free ligand, and receptor boun