Functional Characterization of Undenatured Type II Collagen Supplements: Are They Interchangeable?

Undenatured (native) type II collagen is a dietary supplement ingredient reported to support joint health in healthy individuals by providing relief from symptoms of stiffness and discomfort and improving mobility. This benefit is thought to occur through oral tolerance, a mechanism whereby the immune system distinguishes between innocuous material in the gut and potentially harmful foreign invaders. The presence of antigenic epitopes in undenatured type II collagen, but not in denatured (hydrolyzed) collagen, is thought to be the basis for the therapeutic benefits. The purpose of this study was to investigate the physicochemical and analytical characteristics of type II collagen supplements currently available on the market and to explore whether they might be sufficiently similar in their physical properties to yield similar benefits in promoting joint health. Collagen type II supplement powders (raw material) and capsules (products in the market) were examined for color, particle size, quality profiles, fatty acid profiles, electron microscopy, and were analyzed for amino acid content as well as antigenic potential via an ELISA assay. Powders labeled as undenatured type II collagen were found to have markedly different properties, including the size of collagen fibers as per electron microscopy and antigenic configuration as per the ELISA assay. As significant differences were found between products, it allows consumers and practitioners to not assume that products labeled as undenatured (native) type II collagen are interchangeable.

Multiple mechanisms for exogenous heparin modulation of vascular endothelial growth factor activity.

Heparin and heparin-like molecules are known to modulate the cellular responses to vascular endothelial growth factor-A (VEGF-A). In this study, we investigated the likely mechanisms for heparin's influence on the biological activity of VEGF-A. Previous studies have shown that exogenous heparin's effects on the biological activity of VEGF-A are many and varied, in part due to the endogenous cell-surface heparan sulfates. To circumvent this problem, we used mutant endothelial cells lacking cell-surface heparan sulfates. We showed that VEGF-induced cellular responses are dependent in part on the presence of the heparan sulfates, and that exogenous heparin significantly augments VEGF's cellular effects especially when endogenous heparan sulfates are absent. Exogenous heparin was also found to play a cross-bridging role between VEGF-A(165) and putative heparin-binding sites within its cognate receptor, VEGFR2 when they were examined in isolation. The cross-bridging appears to be more dependent on molecular weight than on a specific heparin structure. This was confirmed by surface plasmon resonance binding studies using sugar chips immobilized with defined oligosaccharide structures, which showed that VEGF-A(165) binds to a relatively broad range of sulfated glycosaminoglycan structures. Finally, studies of the far-UV circular dichroism spectra of VEGF-A(165) showed that heparin can also modulate the conformation and secondary structure of the protein.

GLYX-13: a monoclonal antibody-derived peptide that acts as an N-methyl-D-aspartate receptor modulator.

We previously created a monoclonal antibody (MAb), B6B21, that acts as a partial agonist at the glycine site of the N-methyl-d-aspartate (NMDA) receptor [Moskal, J.R., Schaffner, A.E., 1986. Monoclonal antibodies to the dentate gyrus: immunocytochemical characterization and flow cytometric analysis of hippocampal neurons bearing a unique cell-surface antigen. J. Neurosci. 6, 2045-2053.]. The hypervariable region of the light chain of B6B21 was cloned and sequenced. Peptides were then synthesized based on this sequence information and screened using rat hippocampal membrane preparations to measure [(3)H]MK-801 binding in the presence of 7-chlorokynurenic acid, a glycine site-specific competitive inhibitor of NMDA receptor [Moskal, J.R., Yamamoto, H., Colley, P.A., 2001. The use of antibody engineering to create novel drugs that target N-methyl-d-aspartate receptors. Curr. Drug Targets 2, 331-345.]. Peptides that were able to increase [(3)H]MK-801 binding in a dose-dependent manner under these conditions were named Glyxins. Here we report that GLYX-13, a tetrapeptide (TPPT-amide), was found to readily cross the blood-brain barrier and modulate the NMDA receptor in a glycine-like fashion when examined pharmacologically and electrophysiologically. When GLYX-13 was administered to rats at 0.5-1.0mg/kg i.v., a significant enhancement in learning was observed using a hippocampus-dependent trace eye blink conditioning paradigm. These data indicate that the Glyxins are a new class of NMDA receptor modulators that may have therapeutic potential. Based on the broad agonist range in vitro and the potent cognitive-enhancing properties in a valid in vivo model of learning, GLYX-13 is a new drug candidate with potential for the treatment of cognitive disorders.

The cancer recognition (CARE) antibody test.

The cancer recognition (CARE) antibody (Ab) test is a serologic assay for a specific IgM that is elevated in cancer patients. All tests are measured using an indirect enzyme-linked immunosorbent assay (ELISA) of human serum. The target polypeptide in the CARE Ab test is the IgM binding epitope (LT-11) of the CARE antigen (Ag) consisting of a 16 mer structure that has been produced synthetically. The mean relative concentration (MRC) is determined relative to standard, normalized human plasma. Non-parametric analysis showed median MRC values of healthy volunteers (HVs) with no history of cancer (n =47), family history of cancer (n = 126) and a previous cancer history (n = 24) to be 26, 34 and 46, respectively. It was determined that there was no significance found among the medians of the three HV groups (P = 0.53). The specificity of the HV types was between 87 and 98%. Benign/non-cancer surgical patients (n = 27) had a median value of 20 with a specificity of 96%. The cancer patients (n = 61) had a median value of 246 with a sensitivity of 89%. There was a significant difference between the HV and cancer patients (P < 0.0001) as well as between the benign/surgical non-cancerous group and cancer patients (P < 0.0001). The IgM antibody is heat stable at room temperature for two days versus being frozen at -80 degrees C (r2 = 0.97). Either serum or plasma samples may be used in the CARE Ab test (r2 = 0.92). The CARE Ab was almost exclusively IgM with no serum conversion to IgG in sequential measurements of patients with cancer over a six-month period. Preliminary data from patients undergoing post-operative cancer treatment showed that decreasing Ab levels revealed patients negative for residual cancer or undergoing remission, while relapsing patients show an increase in Ab levels. A return to a positive Ab level shortly after treatment is a poor prognostic sign while in advanced cancers the Ab levels may be depressed significantly.

Reversal of heparin-induced increases in aPTT in the rat by PM102, a novel heparin antagonist.

Protamine is the only agent approved to reverse heparin-induced anticoagulation. Due to the significant adverse effects of protamine there is an important need for an alternative agent with an improved safety profile. The pharmacodynamics of PM102, a novel peptide-based heparin antagonist, was evaluated and compared to protamine in a rat model. Rats were dosed with intravenous heparin (50U/kg) and 4min later with protamine (0.25, 0.75mg/kg single intravenous bolus) or PM102 (0.1, 0.3, 1, 3, 30mg/kg single intravenous bolus). Blood samples were collected though 60min for assessment of activated partial thromboplastin time (aPTT) and plasma concentration of PM102. Both doses of protamine markedly lowered the elevated aPTT to baseline values within 1 to 5min after administration. PM102 (0.3-30mg/kg) also rapidly and completely reversed heparin-induced increases in aPTT within 1 to 5min. The effects of PM102 administered as an infusion over 10min also reversed aPTT with similar potency to that observed for bolus administration. The onset of reversal with infusion was delayed relative to the same total dose given as a bolus; however, the maximum effect was similar. PM102 rapidly (T(max) 1-2.6min) appeared in plasma after dosing. Concentrations of PM102 generally declined rapidly after reaching T(max) with a mean T(1/2) of 4 to 31min. PM102 is a novel synthetic peptide that effectively reverses the anticoagulant effect of heparin. It's utility as a bolus injection as well as infusion, its rapid efficacy and its rapid clearance make this an ideal candidate for clinical development.