WO1999010522A1 - Drug discovery using multiple membrane mimetic affinities - Google Patents
Drug discovery using multiple membrane mimetic affinities Download PDFInfo
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- WO1999010522A1 WO1999010522A1 PCT/US1998/017398 US9817398W WO9910522A1 WO 1999010522 A1 WO1999010522 A1 WO 1999010522A1 US 9817398 W US9817398 W US 9817398W WO 9910522 A1 WO9910522 A1 WO 9910522A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/5432—Liposomes or microcapsules
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/89—Inverse chromatography
Definitions
- This invention relates to prediction of biological properties. More particularly, the present invention is directed to the measurement of multiple membrane affinities of test compounds, methods and compositions useful for acquiring data characteristic of such affinities, and a method and system for using such data alone or in combination with other molecular descriptors for the prediction of biological activity.
- the present invention is directed to a method of screening test compounds for probable biological properties based principally on correlation of numerical values characteristic of their interaction with two or more membrane mimetic surfaces with corresponding values for control compounds of known biological activities/function.
- the method is grounded on the premise that compounds with similar sets of membrane binding properties will have similar pharmacological properties and/or biological activities.
- the membrane binding properties of test compounds can be calculated, or they can be determined empirically with use of, for example, liposomes, immobilized artificial membranes, such as those described in U.S. Patent 4,931,498, the disclosure of which is incorporated herein by reference, Langmuir Blodget films, computer chips or similar devices with immobilized lipids, capillary zone electrophoresis columns coated with membrane lipids, and the like.
- vector calculus is utilized to pattern match membrane interaction values of test compounds with control compounds to predict biological properties.
- the pattern matching protocol can be applied to data sets containing only values characteristic of multiple membrane interactions, or such data sets can include other biologically significant molecular descriptors such as molecular surface area, molecular weight, dipole moment, octanol-water partition coefficients, molecular volume, membrane diffusion coefficients, metabolism rates, cell efflux rates, etc.
- membrane binding data are obtained for test compounds and control compounds for use in accordance with this invention using immobilized artificial membrane chromatographic substrates in high pressure liquid chromatographic systems using aqueous mobile phases.
- Data relevant to the thermodynamics and kinetics of compound/membrane interaction is reflected in retention time and peak width, respectively. All data are preferably normalized relative to a standard compound or a set of compounds, for example, a set of compounds having a common biological activity or function.
- This invention also provides novel carboxyl-functional, head group- protected phospholipids useful for preparing immobilized artificial membrane structures useful for acquiring membrane interaction data. They are prepared by novel high yielding transphosphitidylation of phosphatidylcholine derivatives using phospholipase D in the presence of protected alcohols.
- Fig. 1 is a 3-dimensional vector plot of membrane affinity data for compounds known to act at serotonin receptors.
- Fig. 2 is a 3 -dimensional vector plot of membrane affinity data for compounds known to act at dopamine receptors.
- membrane binding model was established to predict biological activity of compounds independent of both receptor binding assays and in vivo experiments.
- the membrane binding model was developed based on measurement of membrane binding constants of biologically active compounds on membrane mimetic surfaces.
- the membrane binding data demonstrate that compounds within therapeutic classes exhibit similar binding profiles on amphiphilic/membrane mimetic surfaces and concomitantly similar tendencies to distribute into tissue lipid pools where activity is elicited.
- Lipid heterogeneity in cell membranes functions to regulate the distribution and activity of many drugs. A drug compound with affinity for a particular lipid will accumulate in lipid pools enriched with that lipid.
- Examples include vinblastin and chlorpromazine which accumulate in the inner leaflet of plasma membranes because phosphatidyl serine (PS) and phosphatidyl inositol (PI) are enriched on the cytoplasmic side of cells.
- Drug-lipid interactions have been used to explain biological activity, the volume of distribution of drugs, drug binding to membrane receptors, and drug conformation in membranes. Boundary lipids near membrane receptors can play a key role in sequestering compounds and presenting membrane associated compounds to the receptor, or they can participate directly in regulating receptor function. Although such may not be true for all membrane receptors, it has been documented for several receptors. The results from the experimental work upon which this invention is based demonstrates that compounds with similar biological activity exhibit similar membrane binding properties.
- the drug discovery process traditionally focused on the structure of compounds as the dominant factor governing biological activity.
- structurally diverse compounds within a given therapeutic class exhibit similar membrane binding properties indicates a cooperative role between the receptor and its adjacent membrane.
- the in vitro membrane binding model presented in accordance with this invention provides new insight regarding the criteria needed for a compound to elicit a given biological activity. Since both the receptor and its surrounding membranes contribute to a compound eliciting a particular biological activity, receptor binding assays used in conjunction with the present in vitro membrane binding model provides a synergistic approach to screening potential therapeutic compounds.
- a method of screening test compounds for probable biological properties comprises the steps of identifying two or more membrane mimetic surfaces each having a unique amphiphilic composition.
- a set of control compounds is selected for comparison purposes.
- Each control compound has a known biological property, for example, a biological activity or interaction with a known receptor type.
- the control compounds can have a common property, or they can be selected to represent widely variant biological properties.
- For each control compound there is defined an ordered set of numerical values characterizing a biologically relevant interaction (e.g., affinity) of that compound with each of the selected membrane mimetic surfaces.
- the ordered set of numerical values for each control compound or each set of control compounds can be represented by the expression (C C 2 C n ) wherein n is the number of membrane mimetic surfaces identified and used in the screening method.
- C C 2 C n A similar ordered set of numerical values (T T 2 T n ) for each test compound characteristic of its biologically relevant interaction with each of the respective membrane mimetic surfaces is determined.
- the set of numerical values for the test compound is then compared with the sets of respective values for the control compounds, and the biological properties of those control compound having ordered sets of numerical values best matching the respective numerical values in the ordered set of values for the test compound is identified.
- the values C h C 2 C n and T,, T 2 T n can be determined by computer calculations or empirically by any one or more of a wide variety of art-recognized techniques for evaluating membrane interactions.
- the numerical values characteristic of membrane affinity are determined chromatographically using an aqueous mobile phase and a stationary phase comprising a membrane mimetic surface, for example, in a high performance liquid chromatographic system such as that described in U.S. Patent 4,931,498, expressly incorporated herein by reference.
- the term "membrane mimetic surface" as used in describing and defining the present invention refers to any surface bearing immobilized amphiphilic molecules (i.e., those having both lipophilic and hydrophilic portions capable of exhibiting some selective affinity for or otherwise interacting with a solute (e.g., a test or control compound) in a fluid phase in contact with the surface.
- a solute e.g., a test or control compound
- Preferred membrane mimetic surfaces are those described in the above-incorporated U.S. Patent No. 4,931,498.
- membrane binding properties of a test compound of unknown biological activity are compared to the membrane binding properties of compounds having known in vivo biological activity to assess the probability that the test compound will exhibit one or more biological activities in vivo.
- the analysis is made by pattern matching membrane binding constants measured on immobilized artificial membrane columns in a high performance liquid chromatography system. Two or more immobilized artificial membrane chromatographic columns are used. Each test compound is injected and chromatographed on each column and the peak width and the peak time of the test compound is recorded and normalized to an internal or external standard.
- experimentally measured selectivity values ⁇ k .
- Pattern matching using vector calculus, multivariate analysis or principal component analysis of the ⁇ k' and ⁇ sd values of the test compound and the control compounds allows comparison of the membrane binding properties of the test compounds and each of the control compounds or, if the control compounds all have a common biological activity/property, average or mean membrane binding values of the set of control compounds for each membrane surface.
- Important criteria for optimum implementation of the use of immobilized artificial membrane substrates for providing reliable values characteristic of membrane interaction include use of a stable membrane mimetic surface, preferably the same or a similar mobile phase composition and sample concentration, and use of a at least one common external standard.
- the external standard allows compensation for changes in measurements using the membrane surface from day-to-day and also lot-to- lot variation inherent in membrane mimetic preparations.
- the external standard should be stable, it should be soluble in the aqueous mobile phase (typically organic phase modified), and it should have a reasonable retention time using said mobile phase and a high UV extinction coefficient.
- the external standard is typically injected in the HPLC/IAM system frequently to reflect temporal fluctuation in retention time.
- chromatography on immobilized artificial membranes provides data characteristic of both equilibrium binding to the respective membrane mimetic surface (k 1 values) and the kinetics of mass transfer between the immobilized artificial membrane surface and the mobile phase ⁇ i.e. ⁇ values). Similar to all chromatography columns, columns utilizing immobilized artificial membranes exhibit column-to-column and lot-to-lot variation. This unavoidable variation in the experimentally determined k' and ⁇ values for each test compound and each control compound is eliminated, for pattern matching purposes, by normalizing k' and ⁇ values to an internal or external standard compound chromatographed on each immobilized artificial membrane column.
- k' and ⁇ values for the test compounds are each divided by the respective k' and ⁇ values of the standard to calculate ⁇ k , and ⁇ sd , the selectivity values and normalized standard deviations, respectively.
- a denotes the selectivity of the immobilized artificial membrane chromatographic surface for molecular recognition of the analyte
- ⁇ is a thermodynamic property and corresponds to a ratio of membrane partition coefficients.
- the values T,, T 2 , T n ( ⁇ k . or ⁇ for each test compound and the corresponding values C C 2 , C n for each control compound can be conveniently stored in any database management system for subsequent retrieval and analysis.
- the comparison of the set of numerical values for each test compound with the sets of respective values or the control compounds can be carried out using any of a wide variety of mathematical analytical techniques.
- the comparison is accomplished by multidimensional vector analysis of the data wherein the ordered set of numerical values for each test compound is compared to those corresponding values for each control compound, or, where the control compounds are all selected to have a common biological activity or biological function, an average or mean values for the selected set of control compounds.
- each ordered set of numerical values for the test compound and the values for each respective control compound can be displayed graphically as a vector in a 2- or 3 -dimensional coordinate system, respectively, to facilitate comparison of the numerical values of the test compound with those of the control compounds.
- the test compound data are displayed as a uniquely discernible vector quantity, i.e., in a color different than the control compounds.
- the control compounds comprise a set of compounds having a predefined biological property
- the numerical values for each member of said set of control compounds can be displayed as vector quantities uniquely discernible as a member of said set of control compounds and visibly distinguishable from the test compound.
- the data for the control compounds in a set of control compounds having a predefined biological property can be mathematically manipulated to define a mean or average membrane interaction value for each membrane species.
- the mean or average vector quantities calculated for the set of control compounds can be used in the above-described pattern matching analysis or they can be displayed graphically as uniquely discernible vector quantities with those of one or more test compounds.
- the membrane binding properties of six hallucinogens were measured on immobilized artificial membranes (IAMs) prepared from phosphatidylcholine (IAM.PC), phosphatidylserine (IAM.PS), phosphatidylethanolamine (IAM.PE), and sphingomyelin (IAM.SM).
- IAMs immobilized artificial membranes prepared from phosphatidylcholine (IAM.PC), phosphatidylserine (IAM.PS), phosphatidylethanolamine (IAM.PE), and sphingomyelin (IAM.SM).
- Mean vectors were calculated for several therapeutic classes of compounds. The calculated mean vector (i.e., the average vector representing a group of compounds with similar biological activity) is shown in Table 1 for hallucinogenic compounds.
- the average vector representing the membrane binding properties of hallucinogenic compounds is ⁇ 17.08 5.441 113.276 19.574 ⁇ which corresponds to the membrane binding properties on
- membrane binding constants are measured by injecting the drugs into high performance liquid chromatographs (HPLC) using immobilized artificial membrane (IAM) columns using the following IAM stationary phases:
- the peak position of the drug in the chromatogram is a measure of the affinity of the compound for the immobilized membrane surface.
- the retention time (peak time) was used to calculate the capacity factor, k', of the compound for the IAM surface.
- Capacity factors are proportional to equilibrium membrane binding constants K according to
- MAF ⁇ denotes a mean vector whereby the vector components are average membrane binding constants for a group of compounds (a training set) with similar biological activity or functionality, e.g., oral absorption.
- training set is a set of compounds of common biological properties used to define/calculate an MAF ⁇ value for that biological property.
- Normally distributed membrane/membrane mimetic binding affinities in N-dimensional space occur when bivariate log plots of ⁇ k' (i.e. PC vs PE, PC vs PS, etc.) have an ellipsoidal shape.
- the ellipse shape indicates that the affinity data for a given set of compounds having a known clinical efficacy or function are normally distributed in 2 dimensional space. In the case when a compound falls outside of the 0.95 quartile (3 standard deviations from the mean), it is considered to be an outlier.
- a composition comprising a mixture of compounds which have a common biological activity or function or clinical efficacy.
- the compounds in the training set composition are in a predetermined molar ratio.
- the control compounds in the training set composition are in a substantially equimolar ratio, optionally in combination with one or more external standards.
- the compounds in this training set selected so that the ellipse plot of the membrane mimetic binding data for the compounds in the training set composition are such that all compounds fall within the 0.95 quartile.
- the training set typically includes at least two, preferably at least three, most preferably 5 or more control compounds.
- the training set composition can be combined with test compounds (and optionally training set compositions for other biological properties) to prepare mixture for LC/MS analysis of membrane/membrane mimetic binding properties.
- the training set compositions in accordance with this invention provide ideal internal controls for direct comparison of test compound affinities with those exhibited by the compound members of the training set in LC/MS analytical protocols.
- the mean membrane binding affinities for each training set are calculated by averaging the PC, PE, PS, and SM as illustrated in Table 3 for selected hallucinogen compounds. Multivariate Analysis of Variance (MANOVA) confirmed that greater than 90% of the mean membrane binding affinities of all therapeutic classes and mechanism of actions were different at the 0.05 confidence level. In other words, the mean vector for each training set developed for use in accordance with this invention was unique compared to all other training sets.
- D 2 and Q values can also be used for classifying/comparing compounds by vector analysis of binding data (and other quantitative physicochemical properties).
- Membrane binding data for an unknown compound is an observation vector y
- the mean membrane binding data for the i* training set of compounds is denoted as the vector y,.
- the classification of an unknown compound involves calculating the mean squared distance, D 2 , between y and y,.
- S is the covariance matrix of the i* group of compounds.
- the unknown compound is assigned to the therapeutic class or receptor type with the smallest D 2 value.
- An alternate method for classifying compounds is by use of Q values.
- D 2 and Q were used to classify the activity of each compound comprising all of the training sets. A compound was considered to classify correctly when its known therapeutic class or mechanism of action was within the top two hits based on D 2 and Q.
- Table 4 shows the D 2 and Q values for the classification of MDA (a hallucinogen).
- the relative order of elution is a measure of the relative affinity of each compound for each membrane mimetic surface.
- theta, D 2 , and Q values for a compound of unknown activity do not necessarily reflect elution order.
- a low theta and D 2 or large Q may occur even though a compound does not have the same relative order of elution exemplified by the mean vector of the most comparable training set. Consequently, in vivo activity can be predicted using not only theta, D 2 , and Q values, but also the LC elution order of the unknown compound on the respective solid phase substrates vs that of the mean vector for a known training set.
- Peaks widths evaluate the on-off kinetics of drug membrane interactions.
- the unknown compound BDFA was evaluated for hallucinogenic activity. Its theta value relative to the hallucinogen MAF ⁇ was 3.6 and its order of elution matched that of the hallucinogen MAF ⁇ .
- the peak widths of BDFA on SM and PS are much larger than the corresponding peak width of MAF ⁇ .
- In vivo studies have shown that BDFA is inactive. Both the theta value and order of elution indicate that BDFA would be active in vivo. Only the difference in the peak widths provided evidence that BDFA would be inactive in vivo.
- a system for screening test compounds for probable biological properties comprises two or more membrane mimetic surfaces, each having a unique amphiphilic composition.
- the system includes means for quantifying the interaction of test compounds and control compounds with each of the surfaces and assigning a numerical value characteristic of said quantified interaction of the test compound and each respective membrane surface.
- the screening system also includes a database comprising numerical values characteristic of the quantified interaction of selected control compounds or training sets of control compounds with the membrane surfaces. At least a portion of the selected control compounds have a predefined biological property.
- the system includes an analyzer (e.g.
- the system includes a graphics algorithm for displaying the numerical values for the test compound and the numerical values for at least a portion of the control compounds as visibly distinguishable vector quantities.
- the analyzer includes an algorithm using vector calculus manipulation to identify the control compound or compounds having numerical value best matching those of the test compound.
- the system can be programmed to report all control compounds wherein ⁇ (see above) is less than 15°, more preferably less than 10°, or where D 2 is minimized and/or Q is maximized.
- Figs. 1 and 2 show vector plots in 3 -dimensional "membrane space" for compounds which act at serotonin receptors: buspirone, cinanserin, mescaline, methysergide, plamphetamine, psilocin, quipazine, meterogline and others.
- Fig. 1 shows vector plots in 3 -dimensional "membrane space" for compounds which act at serotonin receptors: buspirone, cinanserin, mescaline, methysergide, plamphetamine, psilocin, quipazine, meterogline and others.
- FIG. 2 shows vector plots in 3-dimensional "membrane space" for compounds that act at dopamine receptors: apomorphine, clozapine, domperidone, haloperidol, pridinol, SCH23390, SKF38393, spiperone, sulpiride, chlorpromazine, dihyrexidine, dopamine, pe henazine, prochlorperazine.
- That phenomenon forms the basis of the present invention and allows multiple membrane binding constants to serve as a predictor of biological activity.
- Figs. 1 and 2 are illustrated as the products of pattern matching by vector analysis, other art-recognized mathematical analytical techniques such as multivariant analysis and principal component analysis can be applied to membrane binding data to compare test compounds with control compounds with known biological function.
- Vector analysis is particularly useful for pattern matching in accordance with this invention in that it can be carried out, albeit not graphically represented, in more than three dimensions.
- Such techniques can also be applied to data sets that include, in addition to data characteristic of multiple membrane affinities, other biologically significant molecular descriptors.
- membrane binding data acquisition is illustrated using immobilized artificial membrane chromatography supports in a high performance liquid chromatography system
- other techniques can be used, for example, computer chips or similar devices with immobilized lipids, capillary zone electrophoresis columns coated with membrane lipids, Langmuir Blodget films, liposomes, and adsorbed monolayers of lipids on any surface, for example an AFM tip and evaluating the change in oscillation of the tip in the presence of test and control compounds.
- numerical values characteristic of both MAF and non-MAF parameters can be obtained by computer calculations.
- data sets for use in accordance with this invention can include calculated non-MAF parameters, and calculated MAF parameters including simulated MAF properties.
- Calculated non-MAF parameters are numerical quantities that can be derived from a known chemical structure. Examples include surface area, polar surface area, number of H- bonds, topological indices, solubility, etc. These parameters alone do not predict the general membrane binding properties, i.e., MAFs, of compounds.
- the term "calculated MAF parameters” refers to a combination of calculated non-MAF parameters, with or without individual membrane binding constants, that can be used to predict Membrane Affinity Fingerprints (MAF). Since HPLC retention times can be used to calculate membrane binding constants, methods used to calculate retention times of solutes are actually calculating MAF parameters. An example of a calculated retention time for 12 compounds can be found in (Amie, D.
- simulated MAF refers to a calculated MAF parameter obtained from Molecular Dynamics Simulations of compounds with IAMs, bilayer membranes, other membrane mimetic surfaces, or a force field characteristic of these membranes.
- W is the lipid residue of a phospholipid of the general formula W-OPO 2 OB, wherein W can be acylglyceryl, diacylglyceryl, or N- acyl 3-O-(protected) sphingosin-1-yl, wherein "acyl” is C 8 -C 24 alkanoyl or C 8 -C 24 alkenoyl.
- novel carboxyl-functional, head group protected phospholipids of the formula HOOC-W-OPO 2 OZ are prepared in high yield by phospholipase D (PLD) transphosphatidylation of compounds of the formula HOOC-W-OPO 2 OCH 2 CH 2 N + (CH 3 ) 3 in the presence of an excess of a protected alcohol of the formula ZOH wherein Z is protected glyceryl, 2-(protected amino)ethyl, 2-protected carboxyl-2-amino ethyl or the residue of an acid protecting group, for example, 4-nitrobenzyl or 4-nitrophenylethyl.
- PLD phospholipase D
- W is a lipid reside as defined above with the proviso that it is selected so that the starting compounds serve as a substrate for phospholipase D activity.
- a buffered aqueous organic solvent for example chloroform or ethyl acetate
- carboxyl-functional compounds are useful for preparing novel head group-protected immobilized artificial membranes on surfaces of solid substrates having carboxyl reactive functional groups such as hydroxy, amino, and thiol groups. Removal of the protecting groups on the immobilized phospholipids provide the corresponding deprotected artificial membrane structures.
- protected or "protecting group” as used in describing the present invention refers to those chemical moieties that 1) can be temporarily bonded to a reactive functional group (e.g., phospho, carboxyl, hydroxy or amino) to prevent subsequent unwanted reaction of the reactive functional group during other chemical modification of the compound bearing said functional group and that 2) can be removed from the functional group or groups at a subsequent synthesis step without unwanted reaction of other portions of the protected molecule.
- a reactive functional group e.g., phospho, carboxyl, hydroxy or amino
- Protecting groups for amino, carboxyl and hydroxy functionalities are well known in the art, as are their respective synthesis and conditions for removal (deprotection).
- Preferred protected alcohols useful for the preparation of the carboxyl functional, head group-protected phospholipids in accordance with this invention are ispropylidene glycerol, allyl serine, 2-(t-butoxycarbonylamino)ethyl, 4-nitrobenzyl alcohol, and 2-(4-nitrophenyl)ethanol.
- Another artificial membrane stationary phase useful for assessing membrane interaction and predicting biological properties in accordance with this invention is a novel immobilized ceramide-based membrane.
- the ceramide stationary phase is prepared by covalently bonding N-(13- carboxyltridecanoyl)-D-erythro-sphingosine through the ⁇ -carboxyl group (via the ceramide-imidazolide) to silica propylamine and subsequent C-3 and C-10 endcapping of residual propylamine groups.
- the resulting stationary phase can be used in HPLC systems to predict skin permeability constants.
- the immobilized artificial membranes used in accordance with this invention to predict biological activities can be prepared using one or more than one ⁇ -carboxyl substituted lipids.
- Multilipid structures useful as a stationary phase for HPLC chromatographic determination of membrane interaction and having a predefined ratio or stoichiometry of phospholipid components can be prepared to simulate those lipid/phospholipid ratios known to exist in a predetermined biological membrane.
- the binding affinities of test compounds and control compounds to both homologous and heterologous membrane substrates can be measured and compared to assess/predict biological function.
- Membrane binding data (numerical values relating to interaction with membranes or membrane mimetic surfaces) were obtained using HPLC chromatography on a multiplicity of membrane mimetic surfaces, preferably immobilized artificial membranes (IAMs).
- the membrane mimetic surfaces used to quantitate membrane lipid interactions were prepared from analogs of phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), and sphingomyelin (SM). These membrane lipids were chosen because of their established asymmetry in plasma membranes. Synthetic methods for preparing immobilized membranes are well established. Experimentally, drugs were injected into high performance liquid chromatography (HPLC) columns. The peak positions in the chromatograms measure the affinity of compounds for the immobilized membrane lipid surfaces.
- HPLC high performance liquid chromatography
- membrane binding must significantly contribute to the regulation of CNS transport because all CNS compounds tested have unique membrane binding compared to non-CNS compounds.
- the region occupied by non-CNS compounds differs from that of CNS compounds both in vector direction and vector magnitude.
- sets of membrane binding data can be used to determine if a given compound is likely to penetrate the blood brain barrier.
- membrane binding properties unique to each therapeutic class regulates the localization of compounds within the CNS. Specifically, when compound localization within the CNS is regulated primarily by membrane binding properties, then drug vectors within therapeutic classes are expected to occupy a unique region of membrane space. This clustering was observed for all therapeutic classes of compounds tested which suggests that the in vitro membrane binding model has significant utility in modeling the distribution of compounds within the CNS.
- the five PLW 00 "* ligands prepared were analogs of phosphatidylglycerol (PG 2 ⁇ :OOH p , la), phosphatidylserine (PS 2 ⁇ ⁇ COOH p , lb), phosphatidylethanolamine (PE 2 ⁇ - COOH p , lc), phosphatidic acid (PA 2 ⁇ 00 ⁇ , Id) and sphingomyelin (SM' ,HCOOH,P I 2).
- PLD transphosphatidylation reactions using PC- COOH were typically ⁇ 90-95% complete.
- Scheme 1 Structures of protected diacylated phospholipid analogs of PG, PA, PE and PS. After immobilization and following deprotection, these ester PLs have identical interfacial functional groups to endogenous membrane phospholipids (PG, PA, PE, or PS).
- Scheme 1 shows the protected diacylated P 2 ⁇ "COOH p ligands prepared during this work.
- the protecting groups, R P shown in Scheme 1 are the same protecting groups used to prepare single chain ether PLs that were immobilized except for the serine analog.
- Bee venom PLA 2 was reported to have high activity in low water- organic solvent systems (1.7% buffer/chloroform V/V). Preliminary studies were intended to determine if 1.7 % V/V buffer/CHCl 3 was sufficient for scale up reactions whereby several gram quantities of 5 could be produced from a single reaction.
- the first reaction was a 100 fold scale up from the published procedure and Tris buffer was used instead of HEPES.
- DMPC (1.0 g) incubated with bee venom PLA 2 (1000 units) in 1.7% V/V Tris buffer (50mM, pH 7.2)/CHCl 3 did not react over 4 hours, and when the aqueous phase was adjusted to 5% V/V buffer/CHCl 3 the reaction still did not proceed.
- PCs were also hydrolyzed in quantitative yields using PLA 2 indicating the procedure may be used as a general synthetic step for obtaining lysophosphatidylcholine intermediates.
- PC-COOH (4) was prepared from 5 as described. Earlier studies indicated that PLD from either vegetables or microbacterials has activity in organic/aqueous systems and it accepts unnatural substrates (including both primary and secondary alcohols) and generates phosphoester products. Therefore we extended PLD transphosphatidylation reactions to prepare the final ligands la-Id (Scheme 1). Ligands la and lb were prepared directly from PLD and did not require additional headgroup protection steps prior to ligand immobilization In contrast, ligands lc and Id require additional synthetic steps to protect the primary amines on each of these compounds so that these amines do not interfere with immobilization of the p 2 ⁇ 'COOH p ligands.
- PLD reactions The major side product in PLD reactions is the formation of PA because water is an efficient competitor to the alcohols used for the PLD reactions.
- one of the primary goals during PLD reactions is to minimize formation of PA side products regardless of PC analog subject to PLD transphosphatidylation.
- PLD reactions using compound 4 show either no or trace amounts of PA- COOH.
- ligand involved aminolysis of endogenous SM to form sphingosine-1-phosphocholine with a free amine.
- TBDPS tert-butyldiphenylsilane
- the TBDPS protecting groups can be removed with TBAF which does not degrade the silica surface after the ligand 2 is bonded to silica.
- the ester ligands were bonded to silica surfaces similar to procedures previously developed in our laboratory. See U.S. Patent No. 4,931,498.
- IAMs are prepared from 3 sequential bonding processes.
- the first bonding process links the piW 00 "* ligands to the surface using CDI as the activation agent and FTIR spectroscopy is used to verify that the headgroup protecting groups remain intact during the immobilization process. Residual amines on the silica surface are then endcapped with alkyl anhydrides.
- SPA silica propylamine starting material which becomes estCT IAM.PS p after immobilizing PS 2 ⁇ 00 TM, which then becomes es,CT IAM.PS p/C3 C3 after two sequential endcapping steps using C3 anhydride.
- the superscript p indicates that serine protecting groups have not been removed. After removal of all the protecting groups, ra,CT lAM.PS p/C3 C3 becomes the final product 10. This nomenclature, which is exemplified for the preparation of ratCT IAM.PS C3 C3 surfaces, is used for all IAMs.
- Bonding strategies of the P 2 '* COOH P ligands to the silica surfaces were the same as that of PC-COOH which did not require protecting groups.
- the pL2 ⁇ -cooH/ p ligands afe less soluble Jn CHCl3 than pc-COOH which caused longer CDI activation times.
- the CDI activated PL 2 ⁇ - COOH/p ligands are very soluble and bonding to silica propylamine was facile.
- IR band shifts with serine carboxyl group on """IAM.PS 0303 in the IR spectra under acidic and basic conditions were used to monitor the extent of deprotection.
- MtCT IAM.PS p C3/C3 with Bu 3 SnH and PdCl 2 (PPh 3 ) 3 IR spectra of the acidified IAM surface (serine-COOH) and basified IAM surface (serine-COO ' ) showed a decrease in the integrated ester region between 1780-1680 cm "1 by ⁇ 1.3 fold. This decrease in intensity was expected because the surface contains two interfacial esters from the immobilized phospholipid.
- Triethanolamine TAA
- Tris tris(hydroxymethyl)aminomethane
- EDTA ethylenediaminetetraacetic acid disodium salt
- TFA trifluoroacetic acid
- CDI 1,-1-carbonyldiimidazole
- CIO propionic anhydride
- Bu 3 SnH decanoic anhydride
- II bis(triphenylphosphine)palladium (II) chloride
- PdCl 2 (PPh 3 ) 3 alcohol-free anhydrous chloroform (stabilized with amylene), methylene chloride, 4-dimethylaminopyridine (DMAP), sodium acetate, di-tert-butyl dicarbonate (Boc anhydride), -nitrophenethyl alcohol (NPEA), triethylamine, tert- butyldiphenylsilyl chloride (TBDPSCI), imidazole, H-but
- Sphingomyelin (brain) and eggPC were ordered from Avanti Polar Lipids, Inc. (Alabaster, AL).
- Deuterated solvents for NMR spectroscopy including methanol and chloroform were from Cambridge Isotope Laboratories, Inc..(Andover, MA)
- Chloroform (alcohol free), acetone, isopropylalcohol, methanol, acetone, hexane, ethyl acetate, acetic acid, hydrochloride and sodium hydroxide were from Mallincrodt, Inc. (Paris, NY).
- Labmotor, stirring shaft and stirrer blades were purchased from ACE Glass, Inc. (Louisville, KY).
- Phospray reagent used for detecting phosphorus-containing organic compounds and ninhydrin reagent used for detecting amine-containing compounds were ordered from Supelco, Inc. (Bellefonte, PA).
- Silica propylamine (SPA) was provided by Rockland Technologies Inc. (Newport, DE).
- SPA (RN39-94) was used to prepare """IAM.PL surfaces and it is trifunctional and has a surface area of 180m 2 /g and a pore size of 80 Angstroms. The density of propylamine groups on RN39-94 is 3.09 ⁇ mol/m 2 .
- SPA (RN38-94) was used to prepare IAM.SM surfaces and it is monoflinctional and has the same surface area and pore size as RN3 -94.
- the density of propylamine groups on RN38-94 is 2.06 ⁇ g/m 2 .
- All infrared spectrum were recorded on a Nicolet magna-IR spectrometer interfaced with a IR Plan I microscope. Elemental analysis was performed on a Perkin-Elmer PE 240 in the Microanalytical Laboratory at the Purdue University Chemistry Department using approximately 10-15 mg of IAMs. l-myristoyl-s/ ⁇ -2-hydroxyphosphatidylchoIine (5, lysolecithin)
- DMPC DMPC (10.0 g) was weighed and dissolved in chloroform (300 ml). Then 32.0 ml of 50 mM TEA (pH 7.2) buffer containing 2.5 mM Ca ++ and 0.25 mM EDTA was added. Naja mocambique mocambique PLA 2 enzyme solution (1 ml, 1,000 units/ml in 50 mM TEA buffer) was pipetted into the DMPC suspension. The viscous white reaction was paddle-stirred at 100 rpm for either 7.5 hours (basic isozyme) or 11.0 hours (acidic isozyme). Isopropylalcohol (100 ml) was added to the reaction mixture before rotoevaporation to avoid bubbling during solvent removal.
- the residue was suspended in methanol (100 ml) and sonicated (1-2 min) to solubilize the residue.
- the methanol solution containing product was slowly pipetted into acetone ( ⁇ 500 ml) which caused a white precipitate (the product) to immediately form.
- N-(tert-butoxycarbonyl)-L-serine 25 g was suspended in anhydrous ethyl acetate (600 ml) using a r. b. flask (1000 ml) and then allyl bromide (60 ml) and triethylamine (24 ml) were added. After stirring for 24 hours, the reaction mixture was filtered, and the filtrate was concentrated via rotoevaporation to yield a yellow oil.
- the crude product was purified by flash chromatography using a step hexane/ethyl acetate gradient from hexane (500 ml) followed by hexane/ethyl acetate (4:1, 1000 ml), hexane/ethyl acetate (2: 1, 1000 ml), and hexane/ethyl acetate (1: 1, 1000 ml). After drying in a vacuum oven overnight, 27.2 g of the intermediate N-fert-butoxycarbonyl- L-serine allyl ester was obtained (93% yield).
- N-fert-butoxycarbonyl-L-serine allyl ester (27.2 g) was placed in a 500 ml r. b. flask and cooled to 0 °C. To this yellow oil, 50% TFA/CH 2 C1 2 (100 ml) was added under positive N 2 pressure. The reaction mixture was stirred at 0 °C (30 min), warmed to room temperature, and stirred for 2 h. TFA and CH 2 C1 2 were removed by rotoevaporation to yield a yellow oil. The yellow oil was dissolved in ether (200 ml). After 3 h at 4 °C, the product completely precipitated.
- Sphingosine-1-phosphocholine was prepared by hydrolysis of SM with minor modification. Briefly, a solution of SM (10.5 g, 15 mmol), ⁇ -BuOH (150 ml), and HCI (45 ml, 6.0 N) was stirred at 100 °C for 1 hour followed by rotoevaporation and vacuum drying. The sphingosine-1-phosphocholine product was not purified, but, product formation was verified by FAB-MS m/z 465.2 [M+H + ]. Sphingosine-1- phosphocholine was dissolved in THF (500 ml) followed by the addition of TBDPSCI (23.4 ml, 90 mmol) and imidazole (12.2 g, 90 mmol) in that order.
- THF 500 ml
- TBDPSCI 23.4 ml, 90 mmol
- imidazole 12.2 g, 90 mmol
- reaction solvent was removed by rotoevaporation and product was purified using a step gradient mobile phases consisting of CH 2 C1 2 (500 ml), CH 2 C1 2 /THF (1:1, 500 ml), CH 2 Cl 2 /CH 3 OH (1: 1, 1000 ml), and CH 2 Cl 2 /CH 3 OH/H 2 O (65:35:4, 3000 ml).
- the chromatography fractions containing the product were pooled, and after rotoevaporation and vacuum oven drying 5.4 g of SM" "000 "* (2) was obtained (80.6 % yield).
- FTIR (thin film, cm “1 ) 3312.05, 3071.70, 3046.10, 2923.61, 2855.04, 1960.21, 1899.87, 1831.31, 1742.70, 1703.39, 1660.42, 1546.28, 1466.74, 1428.35, 1065.80, 967.63.
- PC-COOH (1.01 g, 1.43 mmol) was weighed and suspended in ethyl acetate (56 ml) and sonicated (5 min). The suspension was stirred (30 °C, 15 min) before pipetting 10.62 ml of NaOAc buffer (composed of 100 mM NaOAc and 50 mM CaCl 2 , pH 6.5) and 1.6 ml (12.10 mmol) of (R)-isopropylidene glycerol (IPG). After addition of TPG, the two-phase system immediately emulsified.
- NaOAc buffer Composed of 100 mM NaOAc and 50 mM CaCl 2 , pH 6.5
- IPG (R)-isopropylidene glycerol
- the product was both phospray positive and UV positive.
- the reaction mixture was acidified with KHSO 4 , (1.042 ml of IM), concentrated by rotoevaporation and the product purified by flash chromatography using silica gel.
- the solvent system was 700 ml of CHCl 3 /CH 3 OH (9: 1) followed by 800 ml of CHCl 3 /CH 3 OH/H 2 O (65:25:3) Chromatography fractions containing product were pooled, concentrated by rotoevaporated and vacuum dried; the yield after chromatography was 1.12 g of light yellow solid (75.1% yield).
- FTIR (thin film, cm "1 ) 2923.70, 2852.81, 1740.44, 1705.13, 1603.81, 1550.54, 1522.11, 1465.95,
- PE 2 ⁇ OOH P was preparecl simi ⁇ ar t0 PG 2O>-COOH/P e ⁇ cept that a mj ⁇ ed solvent system, ethyl acetate/chloroform (4.6: 1) was used. Chloroform was alcohol free. Based on scanning densitometry of TLC plates developed in CHCl 3 :CH 3 OH:NH 3 H 2 O (65:25:4), -88.0% of PC-COOH starting material was converted to the corresponding PE carboxyl analog. The reaction mixture was concentrated by rotoevaporation and the intermediate was not purified prior to protecting the primary PE amine with Boc.
- FTIR (thin film, cm “1 ) 3384.65, 2923.00, 2852.32, 1742.15, 1715.33, 1690.96, 1553.86, 1517.93, 1464.04, 1365.55, 1244.64, 1221.96, 1171.99, 1108.70, 1071.16.
- PC-COOH (7.0 g, 9.90 mmol) was dissolved in anhydrous chloroform (200 ml) using a 500 ml r.b. flask. Serine allyl ester (7.2g, 49.50 mmol) was added to the reaction mixture followed by 33.4 ml of buffer (composed of 100 mM NaOAc, 50mM CaCl 2 at pH 6.5). PLD (500 ⁇ l, 1.0 unit ⁇ l in NaOAc buffer) was added and the reaction was stirred at 30 °C under N 2 for 8 hours. The chemical intermediate, a carboxyl PS analog from the PLD reaction of PC-COOH, was concentrated via rotoevaporation and not purified prior to the protecting the serine amine with Boc.
- the silica was filtered, washed with CH 2 C1 2 (2 x 20 ml), CH 3 OH (3 x 20 ml), and acetone (20 ml), followed by vacuum drying (40 °C overnight).
- the deprotection step was repeated with 50% TF A/50% CH 2 C1 2 (V/V, 24 ml) for 4.0 hours.
- the silica was immersed in 50% 1 M Na 2 CO 3 /50% CH 3 OH (80 ml, 6 min) under constant stirring, followed by washing with water (600 ml), CH 3 OH (2 x 40 ml) and acetone (2 x 40 ml). The surface was finally vacuum-dried at 40 °C overnight.
- FTIR microscopy of surface (7) indicated that the IPG group was quantitatively removed because the characteristic IPG peak at 1370.0 cm "1 disappeared completely after the deprotection.
- the bonded ligand density[Ong, 1994 #14] (la) on es,CT IAM.PG p was 134.67 ⁇ mol g-silica based on elemental analysis of carbon content before (C, 2.13%) and after (C, 7.46%) ligand la bonding to silica surfaces.
- the silica was filtered, washed with CHC1 3 (200 ml), CH 3 OH (100 ml), saline (1.0 M NaCl, 500 ml), H 2 O (500 ml) and acetone (100 ml) followed by vacuum drying (40 °C overnight).
- the bonded ligand density (lb) on e " CT IAM.PA p was 96.50 ⁇ mol/g-silica based on elemental analysis of carbon contents before (C, 2.13%) and after (C, 6.34%) ligand lb bonding to silica surfaces.
- the silica was filtered, washed with CH 2 C1 2 (2 x 20 ml), CH 3 OH (3 x 20 ml), and acetone (20 ml), followed by vacuum drying (40 °C, overnight).
- the first deprotection was - 80 % complete and a second deprotection step in 50% TF A/50% CH 2 C1 2 for 4.0 hours resulted in complete deprotection; the Boc IR band 1369.0 cm "1 completely disappeared.
- the bonded ligand density (lc) on es,CT IAM.PE p was 109.33 ⁇ mol/g-silica based on elemental analysis of carbon contents before (C, 2.13%) and after (C, 6.64%) ligand lc bonding to silica surfaces.
- estCT IAM.PS p/C3 C3 The bonding procedures for preparing estCT IAM.PS p/C3 C3 were the same as those used to prepare the "'"IAM.PG ⁇ 01003 except that double C3 endcapping was used. Deprotection of surface allyl ester group on estCT IAM.PS p/C3/C3 was based on the solution deprotection conditions. Briefly, cst ⁇ TAM.PS p/C3 C3 (22.44 g) was suspended in anhydrous CH 2 C1 2 (50 ml), nitrogen purged (5 min), sonicated (10 sec) and nitrogen purged (1 min) before adding the deprotection reagents.
- the surface was filtered, washed with CH 2 C1 2 (50 ml), CH 3 OH (5 x 50 ml), hexane (30 ml), ethyl acetate (30 ml) and acetone (40 ml), followed by vacuum drying (40 °C, overnight).
- the deprotection procedure of the allyl ester was repeated prior to Boc deprotection.
- Boc deprotection the conditions described above to prepare the "'TAM.PE 010 ' 03 surface were used except that the deprotection reaction was performed once. Based on FTIR microscopy, quantitative deprotection occurred.
- IAM.SM p cl0 C3 The bonding procedures for immobilizing SM ⁇ ooH p (2), CIO and C3 endcappings to prepare IAM.SM p cl0 C3 were the same as those used to prepare the estCT IAM.PG p cl0/C3 except the ligand coupling to silica propylamine was performed only once. Deprotection of TBDPS protecting group on the SM headgroup was done as described. Briefly, IAM.SM P C10/C3 (-2.1 g) was suspended in THF (20 ml) before TBAF (20 ml, 1.0 M solution in THF) was added to the suspension.
- the skin is the largest organ of the human body, a primary area contacted with the environment, and a route by which many chemical substances enter the body. Research has demonstrated that drug delivery through the skin is feasible for many simple potent drug molecules (less than 1000 Da) via transdermal drug delivery systems. Seven drugs marketed in United States including clonidine, estradiol, fentanyl, nicotine, nitroglycerin, scopolamine, and testosterone are delivered by transdermal systems. Through transdermal drug delivery systems, steady-state plasma concentrations of a drug can be achieved without the high peak levels associated with oral therapy. The avoidance of high peak levels may help minimize the side effects of certain drugs.
- the stratum corneum consists of dead cells surrounded by an extracellular matrix containing lipid lamellae.
- the major lipid components of this extracellular matrix is ceramides which comprise 50% of the total lipid and consist six structurally heterogeneous ceramides.
- ceramide 2 comprises 40% of the total ceramides.
- Ceramide consists of mainly 24- through 28- carbon fatty acids amide-linked to sphingosine and dihydrosphingosine bases.
- the ceramide ligand (N-f 13- carboxyltridecanoyl]-D-eryt/7ro sphingosine 2) used to prepare ceramide silica surface contains basic ceramide 2 functional moiety, and a free ⁇ -carboxyl group.
- the free ⁇ -carboxyl group functions as a tag to the silica propyl amine particles through 1,1'- carbonyldiimidazole (CDI) activation.
- CDI 1,1'- carbonyldiimidazole
- Anhydrides are used to endcap excess amine groups on the silica propyl amine particles. See Scheme 5.
- 1-pentadecyne 6 was synthesized from 1-bromotridecane and lithium acetylide ethylenediamine complex in dimethyl sulfoxide (DMSO) solution. Then oxazolidine aldehyde 5 was treated with lithium acetylide (from 1-pentadecyne 6 and n-butyl lithium) at -23 °C to produce the basic structure of sphingosine 7 in 84% yield after flash chromatography. High eryt ⁇ ro-selectivity (with diastereoselectivity (ds) ⁇ 89%) was observed in this reaction.
- DMSO dimethyl sulfoxide
- TBDPS- protected sphingosine 11 with dodecanedicarboxylic acid anhydride 12 (a cyclic anhydride, synthesized from diacid and dicyclohexylcarbodiimide (DCC)) in chloroform afforded N-[13-carboxyltridecanoyl]-D-eryt ⁇ ro-sphingosine 2 under catalysis of 4-dimethylaminopyridine (DMAP) in 87% yield after flash chromatography.
- DCC dicyclohexylcarbodiimide
- the ⁇ -carboxyl group of ceramide analog 2 was activated with CDI in chloroform. Following the activation, the ceramide-imidazolides were bonded to silica propylamine (SPA) particles with 24 hours shaking. After workup, ceramide- based silica stationary phase was obtained and subjected to CIO and C3 end-capping of the residual amines on the silica particle surface.
- the TBDPS protecting groups were removed with tetrabutylammonium fluoride(TBAF) in THF. The reactions were monitored by a Nicolet Magna 550 FT-IR spectrometer equipped with a Spectratech IR-Plan I microscope.
- Table 6 shows eight compounds eliciting low oral absorption that were used as a training set to calculate a low MAF ⁇ representative of low drug absorption.
- the small average D 2 value i.e., 3.5 ⁇ 1.24 relative to low MAF ⁇ (last column in Table 6) indicates that the group of compounds in the training set are statistically well defined.
- D 2 values based on low MAF ⁇ for the 13 compounds exhibiting complete oral absorption are very high (Table 7).
- the large average D 2 value i.e., 96.26 ⁇ 96.01 (last column in Table 7) indicates that compounds which are highly absorbed have statistically different membrane binding properties compared to compounds that exhibit low oral absorption.
- test compounds in Table 7 exhibited distinct D 2 values compared to the low MAF ⁇ vector, the compounds listed in Table 7 were used as a potential training set to generate a high MAF ⁇ vector.
- Table 8 shows D 2 values relative to ⁇ MAF* 1 for each compound in the high drug absorption training set. D 2 values based on ⁇ MAF 11 for the 8 compounds exhibiting low oral absorption are high (Table 9).
- **D 2 values are the calculated relative to the hieh MAF ⁇ vector.
- Mass spectrometers can be used as detectors for identification of compound eluting from a liquid chromatograph (LC) instrument.
- the only requirement for detection is that the compound can be ionized without composition.
- one preferred method for determining membrane binding constants is to use an MS as a detector for compounds eluting from LC columns.
- the general advantages of using MS detection are (1) compounds without chromophores can easily be detected, (2) multiple compounds can be uniquely identified from a single injection, and (3) small amounts of compound can be used for determination of binding constants chromatographically measured on immobilized artificial membranes (IAMs) or other solid phase chromatographic substrates. These three advantages allow simultaneous injection of very small amounts of hundreds of compounds with detection of essentially all compounds eluting from the column.
- IAMs immobilized artificial membranes
- Such procedure allows for direct comparison of binding affinities of a large number of test compounds with multiple compounds comprising one or more training sets, thereby eliminating inherent data variability deriving from column-to-column variability and variability in other experimental parameters.
- An example of data acquisition using LC with MS detection is given below.
- Test Mixture A mixture containing 0.100 ⁇ moles of 155 compounds was prepared.
- At least a portion of the compounds are compounds of known clinical significance; they serve as internal standards against which the binding affinities of the test compounds are measured.
- the mixture was dissolved in 6.0 mL 15% acetonitrile and 85% 30mM ammonium acetate, pH - 7.4. 20 ⁇ L of this test mixture was loaded onto a 3 x 0.46 cm IAM column. This corresponds to loading ⁇ 16 ⁇ g of the test mixture and loading - 320 pmoles of each compound onto the column.
- a 3 x 0.46 cm IAM column was used.
- the mobile phase was 15% acetonitrile and 85% 30mM ammonium acetate, pH - 7.4.
- the flow rate was 1.0 mL per minute and no flow splitter was used.
- the run time was 2 hours followed by a 10 minute 100% acetonitrile wash.
- Typical MS instrument have the capacity to use flow rates of 2 mL/min and at least one instrument has a mechanism for washing salts from the inlet of the ion source.
- mobile phases cannot have salts because evaporation of the mobile phase in the ion source results in excess buildup of salts that restrict flow of ions into the ion source.
- Finnigan recently developed an LC/MS instrument in which salts are washed away during the chromatography process.
- Affinity data are collected and stored electronically in a storage device as output from the MS detector and thereafter processed, typically using a computer data accessible communication with the data storage device, and programmed with a vector analysis algorithm, to provide a user readable output in a preprogrammed format for facilitating comparison of the binding affinities of test compounds with those of control compounds or with mean vector quantities for one or more training sets.
Abstract
Description
Claims
Priority Applications (9)
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CA002300863A CA2300863A1 (en) | 1997-08-22 | 1998-08-21 | Drug discovery using multiple membrane mimetic affinities |
JP2000507830A JP2001514375A (en) | 1997-08-22 | 1998-08-21 | Drug discovery support using multiple membrane pseudo-affinity |
US09/486,168 US6829540B1 (en) | 1997-08-22 | 1998-08-21 | Drug discovery using multiple membrane mimetic affinities |
EP98944484A EP1015623A1 (en) | 1997-08-22 | 1998-08-21 | Drug discovery using multiple membrane mimetic affinities |
AU92018/98A AU736198B2 (en) | 1997-08-22 | 1998-08-21 | Drug discovery using multiple membrane mimetic affinities |
IL13445898A IL134458A0 (en) | 1997-08-22 | 1998-08-21 | Drug discovery using multiple membrane mimetic affinities |
DE1015623T DE1015623T1 (en) | 1997-08-22 | 1998-08-21 | DETECTING MEDICINES USING MULTIPLE MEMBRANES WITH MIMETIC AFFINITIES |
NO20000838A NO20000838L (en) | 1997-08-22 | 2000-02-21 | Discovery of drugs using affinity for characteristic multiple membranes |
GR20010300057T GR20010300057T1 (en) | 1997-08-22 | 2001-10-31 | Drug discovery using multiple membrane mimetic affinities |
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US60/056,833 | 1997-08-22 |
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JP (1) | JP2001514375A (en) |
AU (1) | AU736198B2 (en) |
CA (1) | CA2300863A1 (en) |
DE (1) | DE1015623T1 (en) |
ES (1) | ES2161203T1 (en) |
GR (1) | GR20010300057T1 (en) |
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WO (1) | WO1999010522A1 (en) |
Cited By (15)
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WO2000070344A2 (en) * | 1999-05-13 | 2000-11-23 | Admetric Biochem Inc. | Method for increasing the efficiency of experimental fractionation in activity profiling of compound mixtures |
WO2001088528A2 (en) * | 2000-05-12 | 2001-11-22 | Admetric Biochem Inc. | High throughput chromatographic systems |
US6562627B1 (en) | 1998-12-23 | 2003-05-13 | Bdc Pharma Llc | High throughput method for measurement of physicochemical values |
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EP1444515B1 (en) * | 2001-11-12 | 2010-03-10 | Analiza, Inc. | Characterization of molecules |
EP2305251A2 (en) | 1999-08-16 | 2011-04-06 | Revaax Pharmaceuticals LLC | Pharmaceutical compositions comprising clavulanic acid or derivative thereof for the treatment of sexual disorder |
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- 1998-08-21 JP JP2000507830A patent/JP2001514375A/en not_active Ceased
- 1998-08-21 WO PCT/US1998/017398 patent/WO1999010522A1/en not_active Application Discontinuation
- 1998-08-21 EP EP98944484A patent/EP1015623A1/en not_active Withdrawn
- 1998-08-21 IL IL13445898A patent/IL134458A0/en unknown
- 1998-08-21 ES ES98944484T patent/ES2161203T1/en active Pending
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Also Published As
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JP2001514375A (en) | 2001-09-11 |
IL134458A0 (en) | 2001-04-30 |
ES2161203T1 (en) | 2001-12-01 |
NO20000838L (en) | 2000-03-30 |
EP1015623A1 (en) | 2000-07-05 |
AU736198B2 (en) | 2001-07-26 |
AU9201898A (en) | 1999-03-16 |
NO20000838D0 (en) | 2000-02-21 |
DE1015623T1 (en) | 2002-02-07 |
GR20010300057T1 (en) | 2001-10-31 |
CA2300863A1 (en) | 1999-03-04 |
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