Selected IAM Abstracts/Summaries

1. Warner-Lambert Company: Design of Peptidomimetic and Non-Peptide Src SH2 Ligands and Prodrugs

Authors: Charles J. Stankovic, Narayanan Surendran,2 Michael K. Connolly, Russell C. DuChene, Elizabeth A. Lunney, Mark S. Plummer, Kimberly S. Para, Aurash Shahripour, James H. Fergus,1 James S. Marks,1 Roman Herrera,3 Susan E. Hubbell,3 Christine Humblet, Alan R. Saltiel,3 Barbra H. Stewart,2 and Tomi K. Sawyer, Departments of Chemistry, Biochemistry,1 Pharmacokinetics and Drug Metabolism,2 and Cell Biology,3 Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI 48105 USA. Presented at the 26th National Medicinal Chemistry Symposium, Richmond, VA, June 1998.

Src homology-2 (SH2) domains consist of ~100 amino acids, which selectively bind phosphotyrosine containing sequences in proteins and peptides, and thus provide a fundamentally important mechanism for protein-protein recognition. Due to their ubiquitous occurrence in many intracellular proteins involved in signal transduction, SH2 domains serve a pivotal role in the orchestration of phosphotyrosine dependent cell signaling pathways. The ability to inhibit specific SH2 domain-phosphoprotein interactions, and thus specific cell signaling pathways, provides new possibilities for therapeutic intervention. Our efforts have focused on the design of selective peptide and peptidomimetic inhibitors of the pp60src SH2 domain, as one example of this approach. Using an iterative structure-based design approach, employing modeling and X-ray crystallography, we have designed a series of dipeptides, peptidomimetics, and novel non-peptides with micromolar to sub-micromolar affinities for the Src SH2 domain. Although potent in in vitro binding assays, these ligands still possess a phosphonate moiety which is expected to limit the cellular penetration. A prodrug strategy was used to develop cell permeable analogs of some of our best ligands. The cell permeability of these compounds was assessed using the Caco-2 model system and by using HPLC analysis of direct uptake studies in Balc3T3 cells. Correlation of these results with those for IAM.+ PC HPLC studies and with cLogP's will also be discussed. The effects of these ligands on various cell lines and their possible roles in cancer and osteoporosis will also be presented.

2. Synthesis and Evaluation of Compounds that Facilitate the Gastrointestinal Absorption of Heparin

Authors: Andrea Leone-Bay, Duncan R. Paton, John Freeman, Christine Lercara, Doris O’Toole, David Gschneidner, Eric Wang, Elizabeth Harris, Connie Rosado, Theresa Rivera, Aldona DeVincent, Monica Tai, Frank Mercogliano, Rajesh Agarwal, Harry Leipold, and Robert A. Baughman.

Publication: Journalof Medicinal Chemistry 1998, 41:7, 1163-1171.

Abstract/Summary: A family of novel compounds (delivery agents) that promote the gastrointestinal absorption of USP heparin in rats and primates has been discovered. The delivery agents in combination with heparin were administered either orally or intracolonically in an aqueous propylene glycol solution and caused dramatic increases in both plasma heparin concentrations (anti-Factor Xa) and clotting times (APTT). Using one of the most effective delivery agents in the series, and estimated relative bioavailability of 8% can be achieved following oral administration to cynomolgus monkeys. To establish a correlation between the in vivo data and an in vitro parameter, immobilized artificial membrane (IAM) chromatography was performed. Log relative k’ values were correlated to the efficiency of oral heparin delivery.

3. 4-[4-(2-Hydroxybenzoyl)aminophenyl]butyric Acid as a Novel Oral Delivery Agent for Recombinant Human Growth Hormone

Authors: Andrea Leone-Bay*, Koc-Kan Ho, Rajesh Agarwal, Robert A. Baughman, Kiran Chaudhary, Frenel DeMorin, Lise Genoble, Campbell McInnes, Christine Lercara, Sam Milstein, Doris O'Toole, Donald Sarubbi, Bruce Variano, Duncan R. Paton.

Publication: Journal of Medicinal Chemistry, 39, 2571-2578 (1996)

Abstract/Summary: A series of N-acylated, non-alpha, aromatic amino acids was prepared and shown to promote the absorption of recombinant human growth hormone (rhGH) from the gastrointestinal tract. Seventy compounds in this family were tested in vivo in rats. Of the compounds tested, 4-[4-(2-hydroxybenzoyl) aminophenyl]butyric acid was identified as a preclinical candidate and was used to demonstrate the oral delivery of rhGH in primates. A significant positive correlation was found between the relative log k'of the delivery agents, as determined by HPLC on an Immobilized Artificial Membrane (IAM) column, and serum rhGH concentrations following oral or colonic dosing in rats. Structure-activity relationships have also been developed based on electronic effects and hydrogen bonding characteristics of the aromatic amide substituents.

4. Immobilized Nicotinic Receptor Stationary Phase for Online Liquid Chromatographic Determination of Drug-Receptor Affinities

Authors: Y. Zhang, Y. Xiao, K. Kellar, and I. Wainer

Publication: Analytical Biochemistry 263, 1998 Article Number AB982828

Abstract/Summary: A series of immobilized receptor-based liquid chromatographic stationary phases has been developed for use in the on-line screening of drug candidates. The receptors were immobilized in the phospholipid monolayer of an immobilized artificial membrane (IAM) stationary phase and the resulting receptor-IAM particles packed in a column and used in a HPLC system. The initial stationary phase was based upon a subtype of the nicotinic acetylcholine receptor (nAChR), rat a 3/b 4 nAChRs prepared from a stably transfected KXa 3/b 4 R2 cell line. The resulting nAChR-IAM particles were used in the LC study of ligand-nAChR binding using frontal chromatographic techniques with [3H]-epibatidine as a marker ligand and with a LC on-line radioactivity detector to monitor the elution profiles. The dissociation constants of ligands were determined by chromatographing a series of concentrations of [3H]-epibatidine and by running 60 pM of [3H]-epibatidine in the presence of four other ligands in a series of concentration. Binding affinities were determined by frontal chromatography for (±)-epibatidine (Kd: 0.27 ± 0.05 nM) > A85380 (Kd: 17.2 ± 0.5 nM) > (-)-nicotine (Kd : 88 ± 33 nM) > carbachol (Kd: 1,280 ± 30 nM) > atropine (Kd: 14,570 ± 2600 nM). The rank order of these values showed a good correlation (r2 = 0.9991) with the rank order of the Kd values measured by standard binding assays. The immobilized receptor LC stationary phase was stable and reproducible. Additional stationary phases prepared from other nAChR subtypes demonstrated that the technique not only differentiates between ligands, but can also demonstrate differences between receptor subtypes. The phases can be placed on-line with mass spectrometers to provide isolation of high affinity ligands from a mixture of compounds and then structural information on the selected compounds

5. Immobilized Enzyme Reactors Based Upon Cofactor Dependent Enzymes for the ON-LINE Synthesis and Purification of Chiral Compounds

Authors: Irving W. Wainer, Dean V. Johnson, Daphne Wahnon and Vivian Sotolongo, Georgetown University, Washington, DC 20007, USA. HPLC 1998, St. Louis, MO

Abstract/Summary: An immobilized enzyme reactor (IMER) has been created through the non-covalent immobilization of the co-factor dependent enzyme horse liver alcohol dehydrogenase {HLADH} on an Immobilized Artificial Membrane (IAM) HPLC stationary phase. The HLADH catalyzed reduction of prochiral and chiral ketones is enantiospecific and enantioselective producing chiral alcohols with high enantiomeric and diastereomeric excesses. The HLADH-IMER was coupled directly to an OJR-CSP creating an on-line stereospecific synthesis and separation system. HLADH catalyzed reductions require the presence of the cofactor NADH and an economic utilization of the HLADH-IMER required an auxiliary system for regeneration of this cofactor. In order to accomplish this, an enzymatic method employing the oxidation of glucose-6-phosphate by glucose-6-phosphate dehydrogenase (G-6-PDH) from Leuconostoc mesenteroide was used for the regeneration of NADH. The enzyme and regenerating substrate are readily available and inexpensive. The regeneration system was connected in-line before the switching valve and the mobile phase was directed through the regeneration system during the course of the enzymatic reactions which were carried out for up to 48 h. The resuIts from studies with the HLADH-IMER the HLADH mediated reduction of (R,S)-2-phenyl- tetrahydropyran-4-one {R,S-1} into trans-(2S,4S)-2-phenyl-tetrahydropyran-4-ol {S,S-2} and is-(2R,4S)-2-phenyl-tetrahydropyran-4-ol {R,S-3} will be presented. After an 18 h reaction cycle on the HLADH-IMER, 50% of (S)-1 had been reduced to (S,S)-2 (ee 100%) compared to a 56% (ee 100%) conversion by free HLADH under the same conditions. In the same reaction, (R)-1 was converted to (R,S)-3 in an 18% yield (ee 100%). On the HLADH- IMER, the relative enantioselectivity of the conversion was (S)-1:(R)-1 » 3:1 after an 18 h incubation while the same ratio derived from free-HLADH was 14:1. The data demonstrates that co-factor dependent IMER-CSP systems can be used for the enantioselective synthesis and direct purification of chiral compounds reducing the time and manipulations involved in this process as well as conserving the enzyme.

6. Immobilized Artificial Membrane Chromatography: Surface Chemistry and Applications

Authors: C. Pidgeon*, C. Marcus*, and F. Alvarez**, Applications of Enzyme Biotechnology, Plenum Press, New York, 1991 *Purdue University, **Schering Plough Corporations Abstract/Summary: Immobilized Artificial Membranes are solid surfaces containing phospholipids immobilized on silica particles at surface densities similar to the ligand density of reversed phase chromatographic surfaces.

Chromatographic and non-chromatographic applications of Immobilized Artificial Membrane surfaces are reviewed and compared to the chromatographic and non-chromatographic applications of reversed phase columns. The methodology for synthesizing Immobilized Artificial Membranes and stability of Immobilized Artificial Membranes are also described. Several examples are presented regarding the ability of Immobilized Artificial Membrane surfaces to model biological processes. Examples include predicting the transport of solutes across human skin, predicting the transport of amino acids across the blood brain barrier, and predicting the binding of solutes to liposome membranes. In addition, the purification of several membrane proteins, including cytochrom P450 from rat adrenals and rat livers, NADH oxidase, and rabbit intestinal phospholipid binding protein are discussed.

Column: IAM Fast-Screen Mini Column

6. HPLC Separation Tools for Drug Membrane Permeability Prediction and Membrane Protein Purification

Phosphatidylcholine (PC) is the major phospholipid found in cell membranes. IAM Chromatography phases prepared from PC analogs closely mimic the surface of a biological cell membrane. Consequently, IAM phases display a high affinity for membrane proteins and are useful in membranes protein purification and in the study of drug-membrane interactions. The IAM surface is formed by covalently bonding the membrane-forming phospholipid to silica.