Heparin and heparan sulfates (H/HS) are a class of highly sulfated glycoamino-polysaccharide biopolymers found on the membranes of cells in the body. They have a large diversity in sugar modifications and in sulfate density (aninonic density) along the polysaccharide chains. This structural diversity provides H/HS with a sufficiently large number of unique sequences to account for its known ability to modulate precisely the functions of many diverse proteins and biological systems in normal and disease processes. E.g., cell growth, secretion, development, blood coagulation, and infections by virus and other human pathogens.. Due to the diversity of sequences within H/HS chains, however, libraries of the unique H/HS oligoS have not been readily obtainable for research. We based a strategy to obtain a macro combinatorial type of heparin-mimetic family (library) on the structure-function model for anticoagulant heparin (Rosenberg and coworkers), and selected a chemically sulfated natural xylan German phamaceutical comprised of S-oligoS that mimicked heparin in almost all its known biological actions, at similar uM concentrations, to obtain a pilot heparin-mimetic family of S-OligoS HD 01315-01-08-10. This enabled us to show that the in vitro inhibitions of HIV-1 cytotoxicity and syncytium-formation were each governed by a degree of structural specificity as well as separability from the anticoagulant S-oligoS of the pharmaceutical, two essential indicators of their usefulness for further drug development AL Stone, et al 1998 Structure-function relations of heparin-mimetic sulfated xylan oligosaccharides: inhibition of Human Immunodeiciency Virus-1 in vitro Glycoconjugate Journal 15:697-712. We devised enlarged procedures to enable the clinical preparation of a highly active HIV-1 virus fusion inhibitor free of anti-thrombin toxicity (PK II, SOLIS). SOLIS, POTENTIAL HIV-1 VIRAL ENTRY INHIBITOR: With HIV-infection in Americans at appx. one million and 50 times that worldwide, no cure or fully effective vaccine, the successful long term control of viral load and deadly effects in developed countries threatened by toxicity and multi-drug resistant strains, and most drugs approved by the FDA being protease or reverse transcriptase inhibitors with one inhibitor of virus entry, broad consensus counsels that inhibitors against viral components which have other functions in HIV-1 attack are critically needed. We are working on completing a clinical preparation of SOLIS for formulation in a small Phase I i.v.-administration safety trial. SOLIS would be an adjunct drug selected from the family library as an inhibitor of binding and entry of virus into target cells (e.g., it inhibits CD4 cell adherence to gp120-coated t.c. plates (binding assay) and is highly active against syncytium-formation of HIV-1 (gp41) with CD4 cell membrane (fusion assay). Heparin has long been known to affect conformation of proteins as a means of modulating their biological function. Thus, inhibition of the conformational changes in gp41, which are thought to be required for fusion progression, by SOLIS is a possible mechanism of action. ? SUMMARY OF PROPERTIES OF PK II:? IC 50 vs cell-killing (formazan assay) 0.20-0.3 ug/ml; -------- anticoagulation vs thrombin ----- less than 0.05-0.3 HU/mg? IC 50 vs vs virus-cell fusion 0.05-0.1 ug/ml;---------endotoxin content FDA LAL Assay ---less than 0.06 eu? (IC 50 may be 7-10 x greater in a commercial lab) ---;--------inhib. adherence CD4 cell to gp120-- concentration-dependent inhib. c? ? It has been known for many years that S-oligoS are very poorly absorbed by oral administration. Clinical studies of others using the sulfated xylan pharmaceutical were flawed by use of indirect means to assess blood levels of the drug during or after i.v. administraation, absent an available direct assay. Our strategy for the preparation of a biomarker is expected to enable a direct blood assay and accurate assessment of dosages of SOLIS relative to in vitro IC 50. I.v. administration of S-oligoS requires relatively large doses per person. An FDA licensed heparin-mimetic, similar to the European made starting source of SOLIS (PK II) is under our investigation as a possible ready source and was reported to be suitable in first stage preparative aspects last year. This product undergoes an additional purification step to obtain a whiter color. Two small preparations of components analogous to SOLIS are now completed and one exhibits inhibitory capacity in the formazan assay (cytopathology protection) similar to that of Pk II. Reproducibility of the chromatographic processes were not fully satisfactory, however. VIRUS LIGANDS: Viral proteins that enable HIV to attack, survive, and replicate in target human cells are available for in vitro study. We have proposed, when research staff would be available, to identify and isolate putative H/HS ligand(s) under conditions of HIV-1 tissue culture infection in the presence of a bifunctional SOLIS probe, and to achieve chemical bonding to endogenous viral/cell protein structures during virus attack. After cell membranes are purified, isolated membrane proteins would be analyzed for fluorescent probe. Such ligand(s) might be protective antigens and/or a means of obtaining endogenous H/HS receptors. STUDIES ON COMBINATION THERAPY: We are preparing Cp11 samples to elucidate 1.)whether the addition of increasing amounts of Cp11 (relatively inactive vs HIV-1) to the usual Pk II doses in the formazan cytotoxicity protection assay will effect the capacity of Pk II to protect cells, and 2.) whether constant molarity of total S-oligoS in usual dose range of Pk II, diminishing Pk II proportion, will yield the expected protection. Aliquots of Cp 8, highly active in the formazan assay, are in preparation for similar studies with Pk II if indicated. BIOMARKERS: Aliquots of Components 8 and 9 are under preparation to serve in devising the methods for obtaining a biomarker for direct measurement of i.v. administered SOLIS. Particular synthetic polypeptides will be selected for linkage to these S-OLIGOS based on our speculation of a mode of action of SOLIS, and immunologic biomarkers will be sought. STRUCTURE-FUNCTION (SF): Early on we showed that the mixture of S-oligoS remaining after chemical sulfation of the native xylan unexpectedly comprised a family of oligomers that range in mass from about 20 to less than 2 K on average. These contain decreasing numbers of a putative -D-glucuronyl-alpha 1,2 beta 1,4 D-(xylyl)3 (tetrasacchaide) motif and varying amount of axial sulfates (alterrnative chair conformation). (FTIR, HMQC heteronuclear two-dimensional NMR proton-13C correlation spectra, GlcA analysis published oral report abstracts: AL Stone and MO Longas 1999 Sulfated oligo-xylans .... : Characterization by FTIR spectroscopy FASEB J 13:A1385; AL Stone and MO Longas 1999 Structure- function relations ......: proton NMR studies Glycobiology 10:1119; MO Longas and AL Stone 2001 Structure-function relations ......: Study of alternate chair conformations by NMR spectroscopy Glycobiology 11:921-922. We are currently preparing samples of Cp II and Cp 8b for advanced FTIR spectral analysis and HMQC correlation spectra. SF underlying the above inhibitions of HIV-1 indicates that S-oligoS structure against gp120 binding to receptor(s) could contain a max of 4 motifs to retain maximum potency while that for inhibition of gp41 helical change could be 3. Capillary HPLC-mass spectroscopy will serve to analyze and model more specifically the various structures of Pk II. This information might help define structures for potential inhibitors that might have simpler chemistry.
| Wright, T E; Milam, K A; Rougee, L et al. (2011) Agreement of umbilical cord drug and cotinine levels with maternal self-report of drug use and smoking during pregnancy. J Perinatol 31:324-9 |
