Optimized Methods for the Production and Bioconjugation of Site-Specific, Alkyne-Modified Glucagon-like Peptide-1 (GLP-1) Analogs to Azide-Modified Delivery Platforms Using Copper-Catalyzed Alkyne-Azide Cycloaddition.

This study aimed to develop and optimize chemistries to produce alkyne-modified glucagon-like peptide-1(7-36)-amide (GLP-1(7-36)-NH2) libraries, which could be rapidly and efficiently conjugated to other components and screened to identify compounds with the best drug delivery properties, as potential treatments for type 2 diabetes or obesity. For this purpose, the Lys26 (K26) side-chain, and the amino (N)- and carboxy (C)-termini of a dipeptidyl peptidase 4 (DPPIV)-resistant GLP-1 sequence (GLP-1(7-36;A8G)-NH2), were modified with an alkyne (4-pentynoic acid or propiolic acid). These analogs were characterized with respect to human GLP-1 receptor (hGLP-1R) agonist activity, effects on cell viability and human serum stability, revealing that these modifications maintained low (N-terminal; EC50 1.5 × 10-9 M) to subnanomolar (C-terminal and K26, ∼4 × 10-10 M) agonist activity toward hGLP-1, had no effect on cell viability, and for the N-terminal and K26 modifications, increased human serum proteolytic stability (t1/2 > 24 h). Copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction conditions were investigated using the C-terminal modified GLP-1 analog and an azide-modified model lipid peptide, with respect to the effects of altering the azide/alkyne ratio, cosolvents, temperature, reducing agents, Cu(I)-stabilizing ligand, copper source, and the concentrations of reagents/reactants, in order to identify general conditions that provide fast reactions and high yields. A 1:2 azide-alkyne (lipid:GLP-1 peptide) and 4:1 sodium ascorbate/copper sulfate molar ratio in 65% v/v DMSO-water at room temperature, in the absence of Cu(I)-stabilizing ligands (THPTA or l-histidine) and buffers (phosphate, pH 7), provided the best yields. This work reports a library of characterized GLP-1 analogs and chemistries for their attachment to other species, providing useful tools to improve GLP-1 delivery and pharmacology (e.g., through conjugation to other species that lower blood glucose, increase the duration of action, or enable delivery via a nonparenteral route).

Inhibitory effects of dynorphin 3-14 on the lipopolysaccharide-induced toll-like receptor 4 signalling pathway.

Dynorphin 1-17 (DYN 1-17) is biotransformed rapidly to a range of fragments in rodent inflamed tissue with dynorphin 3-14 (DYN 3-14) being the most stable and prevalent. DYN 1-17 has been shown previously to be involved in the regulation of inflammatory response following tissue injury, in which the biotransformation fragments of DYN 1-17 may possess similar features. This study investigated the effects of DYN 3-14 on lipopolysaccharide (LPS)-induced nuclear factor-kappaB/p65 (NF-κB/p65) nuclear translocation and the release of pro-inflammatory cytokines interleukin-1beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) in differentiated THP-1 cells. Treatment with DYN 3-14 (10nM) resulted in 35% inhibition of the LPS-induced nuclear translocation of NF-κB/p65. Furthermore, DYN 3-14 modulated both IL-1ß and TNF-α release; inhibiting IL-1ß and paradoxically augmenting TNF-α release in a concentration-independent manner. A number of opioids have been implicated in the modulation of the toll-like receptor 4 (TLR4), highlighting the complexity of their immunomodulatory effects. To determine whether DYN 3-14 modulates TLR4, HEK-Blue™-hTLR4 cells were stimulated with LPS in the presence of DYN 3-14. DYN 3-14 (10µM) inhibited TLR4 activation in a concentration-dependent fashion by suppressing the LPS signals around 300-fold lower than LPS-RS, a potent TLR4 antagonist. These findings indicate that DYN 3-14 is a potential TLR4 antagonist that alters cellular signaling in response to LPS and cytokine release, implicating a role for biotransformed endogenous opioid peptides in immunomodulation.

Dynorphin 1-17 and Its N-Terminal Biotransformation Fragments Modulate Lipopolysaccharide-Stimulated Nuclear Factor-kappa B Nuclear Translocation, Interleukin-1beta and Tumor Necrosis Factor-alpha in Differentiated THP-1 Cells.

Dynorphin 1-17, (DYN 1-17) opioid peptide produces antinociception following binding to the kappa-opioid peptide (KOP) receptor. Upon synthesis and release in inflamed tissues by immune cells, DYN 1-17 undergoes rapid biotransformation and yields a unique set of opioid and non-opioid fragments. Some of these major fragments possess a role in immunomodulation, suggesting that opioid-targeted therapeutics may be effective in diminishing the severity of inflammatory disorders. This study aimed to examine the immunomodulatory effects of DYN 1-17 and major N-terminal fragments found in the inflammatory environment on nuclear factor-kappaB/p65 (NF-κB/p65) nuclear translocation and the release of interleukin-1beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) from lipopolysaccharide (LPS)-stimulated, differentiated THP-1 cells. The results demonstrate that NF-κB/p65 nuclear translocation was significantly attenuated following treatment with DYN 1-17 and a specific range of fragments, with the greatest reduction observed with DYN 1-7 at a low concentration (10 nM). Antagonism with a selective KOP receptor antagonist, ML-190, significantly reversed the inhibitory effects of DYN 1-17, DYN 1-6, DYN 1-7 and DYN 1-9, but not other DYN 1-17 N-terminal fragments (DYN 1-10 and 1-11) on NF-κB/p65 nuclear translocation. DYN 1-17 and selected fragments demonstrated differential modulation on the release of IL-1ß and TNF-α with significant inhibition observed with DYN 1-7 at low concentrations (1 nM and 10 pM). These effects were blocked by ML-190, suggesting a KOP receptor-mediated pathway. The results demonstrate that DYN 1-17 and certain N-terminal fragments, produced in an inflamed environment, play an anti-inflammatory role by inhibiting NF-κB/p65 translocation and the subsequent cytokine release through KOP receptor-dependent and independent pathways.

The effects of pH on beta-endorphin and morphine inhibition of calcium transients in dorsal root ganglion neurons.

UNLABELLED: During inflammation, immune cells migrate into inflamed tissue and release opioid peptides that activate opioid receptors on peripheral sensory neurons to reduce pain. A characteristic of the inflamed environment in which these opioids act is acidic pH. Activation of opioid receptors leads to a decrease in the calcium component of neuronal action potentials. We investigated the hypothesis that inhibitory effects of opioids on intracellular calcium transients in dorsal root ganglion neuronal cultures are potentiated at acidic extracellular pH. Intracellular calcium responses to stimulation with capsaicin were measured in untreated neurons or after preincubation with beta-endorphin or morphine. beta-Endorphin significantly inhibited calcium responses to 300 nmol/L capsaicin at the lowest experimental extracellular pH (6.1, 6.5, and 7.2), whereas morphine inhibited capsaicin (300 nmol/L) responses significantly at pH 6.1 with a trend of inhibition at pH 6.5. The effect of pH on morphine inhibition of K+ -evoked calcium responses was also assessed. Morphine inhibition of calcium responses was significantly enhanced at pH 6.8 compared with pH 7.2 and pH 7.6. The inhibitory effects were reversed by naloxone, an opioid receptor antagonist. In conclusion, low extracellular pH potentiated beta-endorphin and morphine inhibition of calcium transients and might contribute to improved opioid efficacy during inflammation. PERSPECTIVE: The results of the current study suggest that acidic pH might contribute to increased opioid efficacy in inflamed tissue. This highlights the therapeutic potential of endogenous opioid analgesia, whereby opioid peptides are delivered locally in inflamed tissues, as well as the use of locally applied opioids in inflammatory conditions.

Reduction of beta-endorphin-containing immune cells in inflamed paw tissue corresponds with a reduction in immune-derived antinociception: reversible by donor activated lymphocytes.

UNLABELLED: The functional integrity of the immune system is essential for peripheral antinociception. Previous studies have demonstrated that immune cells elicit potent antinociception in inflamed tissues and that corticotropin-releasing factor-induced antinociception is significantly inhibited in animals that have undergone cyclosporin A (CsA)-induced immunosuppression. In this study, we examined the effect of a single bolus of CsA on inflammatory nociception. CsA-treated rats had substantially increased nociception compared with nonimmunosuppressed rats, consistent with a reduction in circulating and infiltrating lymphocytes. Furthermore, CsA-treated rats had inhibition of corticotropin-releasing factor-induced immune-derived antinociception, which was dose-dependently reversed by IV injection of concanavalin A-activated donor lymphocytes (1.0-7.0 x 10(6) cells/0.1 mL). In conclusion, our findings provided further evidence that opioid-containing immune cells are essential for peripheral analgesia. It is evident from these findings that control of inflammatory pain relies heavily on a functioning immune system. IMPLICATIONS: The immune system not only contributes to inflammation, but also provides localized analgesia. A depleted immune system results in a reduction of immune-derived analgesia and a potentiation of inflammatory pain. Donor activated lymphocytes reverse these effects, highlighting the importance of a functional immune system in inflammatory pain.