Peg Davis

Afferent Drive Elicits Ongoing Pain in a Model of Advanced Osteoarthritis

Summary Monosodium iodoacetate (MIA)‐induced ongoing pain depends on input from the joint; blockade of TRPV1 and TRPA1 channels fails to block MIA‐induced ongoing pain. Abstract Osteoarthritis (OA) is a chronic condition characterized by pain during joint movement. Additionally, patients with advanced disease experience pain at rest (ie, ongoing pain) that is generally resistant to nonsteroidal antiinflammatory drugs. Injection of monosodium iodoacetate (MIA) into the intraarticular space of the rodent knee is a well‐established model of OA that elicits weight‐bearing asymmetry and referred tactile and thermal hypersensitivity. Whether ongoing pain is present in this model is unknown. Additionally, the possible relationship of ongoing pain to MIA dose is not known. MIA produced weight asymmetry, joint osteolysis, and cartilage erosion across a range of doses (1, 3, and 4.8 mg). However, only rats treated with the highest dose of MIA showed conditioned place preference to a context paired with intraarticular lidocaine, indicating relief from ongoing pain. Diclofenac blocked the MIA‐induced weight asymmetry but failed to block MIA‐induced ongoing pain. Systemic AMG9810, a transient receptor potential V1 channel (TRPV1) antagonist, effectively blocked thermal hypersensitivity, but failed to block high‐dose MIA‐induced weight asymmetry or ongoing pain. Additionally, systemic or intraarticular HC030031, a TRPA1 antagonist, failed to block high‐dose MIA‐induced weight asymmetry or ongoing pain. Our studies suggest that a high dose of intraarticular MIA induces ongoing pain originating from the site of injury that is dependent on afferent fiber activity but apparently independent of TRPV1 or TRPA1 activation. Identification of mechanisms driving ongoing pain may enable development of improved treatments for patients with severe OA pain and diminish the need for joint replacement surgery.

Solubility of aromatic pollutants in mixed solvents

The solubility behavior of several aromatic and polynuclear aromatic pollutants was determined in a variety of water/cosolvent mixtures. The data was used to test a log-linear solubility model and develop a relationship for determining the slope of the log-linear solubility curve.

Topographically designed analogs of [cyclic] [D-Pen2,D-Pen5]enkephalin

The conformationally restricted, cyclic disulfide-containing delta opioid receptor selective enkephalin analogue [D-Pen2,D-Pen5]enkephalin (1, DPDPE) was systematically modified topographically by addition of a methyl group at either the pro-S or pro-R position of the beta carbon of an L-Phe4 or D-Phe4 residue to give [(2S,3S)-beta-MePhe4]DPDPE (2), [(2R,3R)-beta-MePhe4]DPDPE (3), [(2S,3R)-beta-MePhe4]DPDPE (4), and [(2R,3S)-beta-MePhe4]DPDPE (5). The four corresponding isomers were prepared in which the beta-methylphenylalanine residue was p-nitro substituted, that is with a beta-methyl-p-nitrophenylalanine (beta-Me-p-NO2Phe) residue, to give [(2S,3S)-beta-Me-p-NO2Phe4]DPDPE (6), [(2R,3R)-beta-Me-p-NO2Phe4]DPDPE (7), [(2S,3R)-beta-Me-p-NO2Phe4] DPDPE (8), and [(2R,3S)-beta-Me-p-NO2Phe4]DPDPE (9), respectively. The potency and selectivity (delta vs mu opioid receptor) were evaluated by radioreceptor binding assays in the rat brain using [3H]CTOP (mu ligand) and [3H]DPDPE (delta ligand) and by bioassay with mouse vas deferens (MVD, delta receptor assay) and guinea pig ileum (GPI, mu receptor assay). The eight analogues of DPDPE showed highly variable binding and bioassay activities particularly at the delta opioid receptor (4 orders of magnitude), but also at the mu opioid receptor, which led to large differences (3 orders of magnitude) in receptor selectivity. For example, [(2S,3S)-beta-MePhe4]DPDPE (2) is 1800-fold selective in binding to the delta vs mu receptor, making it one of the most selective delta opioid receptor ligands in the enkephalin series as assessed by the rat brain binding assay, whereas the corresponding (2R,3R)-beta-Me-p-NO2Phe-containing analogue 9 is only 4.5-fold selective (nonselective) in this same assay. On the other hand, in the bioassay systems, [(2S,3S)-beta-Me-p-NO2Phe4]DPDPE (5) is more potent than DPDPE and 8800-fold selective for the MVD (delta receptor) vs the GPI (mu receptor), making it the most highly selective ligand in this series for the delta opioid receptor on the basis of these bioassays. In these assay systems, the (2R,3S)-beta-MePhe4-containing analogue 5 had very weak potency and virtually no receptor selectivity (4.4-fold). These results demonstrate that topographical modification alone in a conformationally restricted peptide ligand can significantly modulate both potency and receptor selectivity of peptide ligands that have multiple sites of biological activity and suggest that this approach may have general application to peptide ligand design.

A Structure–Activity Relationship Study and Combinatorial Synthetic Approach of C-Terminal Modified Bifunctional Peptides That Are δ/μ Opioid Receptor Agonists and Neurokinin 1 Receptor Antagonists

A series of bifunctional peptides with opioid agonist and substance P antagonist bioactivities were designed with the concept of overlapping pharmacophores. In this concept, the bifunctional peptides were expected to interact with each receptor separately in the spinal dorsal horn where both the opioid receptors and the NK1 receptors were found to be expressed, to show an enhanced analgesic effect, no opioid-induced tolerance, and to provide better compliance than coadministration of two drugs. Compounds were synthesized using a two-step combinatorial method for C-terminal modification. In the method, the protected C-terminal-free carboxyl peptide, Boc-Tyr( tBu)- d-Ala-Gly Phe-Pro-Leu-Trp(Boc)-OH, was synthesized as a shared intermediate using Fmoc solid phase chemistry on a 2-chlorotrityl resin. This intermediate was esterified or amidated in solution phase. The structure-activity relationships (SAR) showed that the C-terminus acted as not only a critical pharmacophore for the substance P antagonist activities, but as an address region for the opioid agonist pharmacophore that is structurally distant from the C-terminal. Among the peptides, H-Tyr- d -Ala-Gly-Phe-Pro-Leu-Trp-NH-Bzl ( 3) demonstrated high binding affinities at both delta and mu receptors ( K i = 10 and 0.65 nM, respectively) with efficient agonist functional activity in the mouse isolated vas deferens (MVD) and guinea pig isolated ileum (GPI) assays (IC 50 = 50 and 13 nM, respectively). Compound 3 also showed a good antagonist activity in the GPI assay with substance P stimulation ( K e = 26 nM) and good affinity for the hNK1 receptor ( K i = 14 nM). Consequently, compound 3 is expected to be a promising and novel type of analgesic with bifunctional activities.

Design of novel peptide ligands which have opioid agonist activity and CCK antagonist activity for the treatment of pain

Disease states such as neuropathic pain offer special challenges in drug design due to the system changes which accompany these diseases. In this manuscript we provide an example of a new approach to drug design in which we have modified a potent and selective peptide ligand for the CCK-2 receptor to a peptide which has potent agonist binding affinity and bioactivity at delta and mu opioid receptors, and simultaneous antagonist activity at CCK receptors. De novo design based on the concept of overlapping pharmacophores was a central hypothesis of this design, and led to compounds such as H-Tyr-DPhe-Gly-DTrp-NMeNle-Asp-Phe-NH(2) (i.e., RSA 601) which have the designed properties.

Development of potent μ and δ opioid agonists with high lipophilicity.

An SAR study on the Dmt-substituted enkephalin-like tetrapeptide with a N-phenyl-N-piperidin-4-ylpropionamide moiety at the C-terminal was performed and has resulted in highly potent ligands at μ and δ opioid receptors. In general, ligands with the substitution of D-Nle(2) and halogenation of the aromatic ring of Phe(4) showed highly increased opioid activities. Ligand 6 with good biological activities in vitro demonstrated potent in vivo antihyperalgesic and antiallodynic effects in the tail-flick assay.

Differentiation between rat brain and mouse vas deferens δ opioid receptors

Certain enkephalin analogues, including those which contain the conformationally restricted amino acid E-(2R,3S)-cyclopropylphenylalanine [2R,3S)-delta E Phe), have been shown to have high affinity for brain delta opioid receptors but are much less active in mouse vas deferens bioassays. To investigate whether there are differences between delta opioid receptors in brain and mouse was deferens, the ability of a selective delta opioid compound, [D-Pen2,pCl-Phe4,D-Pen5]enkephalin (pCl-DPDPE), and [D-Ala2,(2R,3S)-delta E Phe4,Leu5]enkephalin methyl ester (CP-OMe), to inhibit [3H]pCl-DPDPE binding in both rat brain and mouse vas deferens were measured. pCl-DPDPE recognized brain and mouse vas deferens binding sites with equal affinity, however, CP-OMe showed 33 fold lower affinity in mouse vas deferens compared to brain. This suggests that mouse vas deferens delta opioid receptors may be distinct from brain delta opioid receptors.

Glycosylation Improves the Central Effects of DAMGO

A series of μ‐agonist DAMGO analogs were synthesized and pharmacologically characterized to test the ‘biousian’ hypothesis of membrane hopping. DAMGO was altered by incorporating moieties of increasing water solubility into the C‐terminus via carboxamide and simple glycoside additions. The hydrophilic C‐terminal moieties were varied from glycinol in DAMGO (1) to l‐serine amide (2), l‐serine amide β‐d‐xyloside (3), l‐serine amide β‐d‐glucoside (4), and finally to l‐serine amide β‐lactoside (5). Opioid binding and mouse tail‐flick studies were performed. Antinociceptive potency (intravenous) increased, passing through a maximum (A50 ≈ 0.2 μmol/kg) for 2 and 3 as membrane affinity versus water solubility became optimal, and dropped off (A50 ≈ 1.0 μmol/kg) for 4 and 5 as water solubility dominated molecular behavior. Intravenous A50 values were plotted versus hydrodynamic values (glucose units, g.u.) for the glycoside moieties, or the hydrophilic/hydrophobic Connolly surface areas (A50 versus e−Awater/Alipid), and provided either a V‐shaped or a U‐shaped curve, as predicted by the ‘biousian’ hypothesis. The μ‐selective receptor profile was maintained (Ki’s = 0.66–1.3 nm) upon modifications at the C‐terminus. The optimal ‘degree of glycosylation’ for the DAMGO peptide message appears to be between 1.25 and 1.75 g.u. (hydrodynamic g.u.), or 0.75 and 0.90 in terms of the surface‐derived amphipathicity values.

Dermorphin sequence with high δ-afinity by fixing the phe sidechain to trans at ξ1

Abstract The Phe sidechain in dermorphin was fixed into the trans conformation by linking the aromatic ring to the Gly nitrogen through a methylene bridge. The compound has high μ- and δ-opioid activities.

The Importance of Micelle-Bound States for the Bioactivities of Bifunctional Peptide Derivatives for δ/μ Opioid Receptor Agonists and Neurokinin 1 Receptor Antagonists

To provide new insight into the determining factors of membrane-bound peptide conformation that might play an important role in peptide-receptor docking and further biological behaviors, the dodecylphosphocholine (DPC) micelle-bound conformations of bifunctional peptide derivatives of delta-preferring opioid agonists and NK1 antagonists (1: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bzl(CF 3) 2; 2: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3,5-Bzl(CF 3) 2; 3: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bzl) were determined based on 2D NMR studies. Although the differences in the primary sequence were limited to the C-terminus, the obtained NMR conformations were unexpectedly different for each compound. Moreover, their biological activities showed different trends in direct relation to the compound-specific conformations in DPC micelles. The important result is that not only were the NK1 antagonist activities different (the pharmacophore located at the C-terminus)but the opioid agonist activities (this pharmacophore was at the structurally preserved N-terminus) also were shifted, suggesting that a general conformational change in the bioactive state was induced due to relatively small and limited structural modifications.