histogranin and Pain

Cell-based therapy for neuropathic pain could provide analgesics to local pain modulatory regions in a sustained, renewable fashion. In order to provide enhanced analgesic efficacy, transplantable cells may be engineered to produce complementary or increased levels of analgesic peptides. In addition, genetic labeling of modified cells is desirable for identification and tracking, but it should be retained intracellularly as desired analgesic peptides are secreted. Usually constructs encode proteins destined for either extra- or intracellular compartments, as these pathways do not cross. However, interactions between intracellular destinations provide a window of opportunity to overcome this limitation. In this report, we have explored this approach using a potential supplementary analgesic peptide, [Ser1]-histogranin (SHG), the stable synthetic derivative of a naturally occurring peptide with N-methyl D-aspartate (NMDA) antagonistic properties. A synthetic SHG gene was combined with (i) nerve growth factor-beta (NGF-beta) amino-terminal signal peptide to enable secretion, and (ii) a fluorescent cellular label (mRFP) with intervening cathepsin L cleavage site, and subcloned into a lentiviral vector. In addition, an endoplasmic retention signal, KDEL, was added to enable retrieval of mRFP. Using immunocytochemistry and confocal microscopic profile analysis, cells transduced by such lentiviruses were shown to synthesize a single SHG-mRFP polypeptide that was processed, targeted to expected subcellular destinations in several cell types. Dot blot and Western analysis revealed stable transduction and long-term secretion of SHG from PC12 cells in vitro. Transplantation of such cells provided modest analgesia in a rodent pain model consistent with low levels of SHG peptide in the cerebrospinal fluid (CSF). These results suggest that it is possible to deliver proteins with different final destinations from a single construct, such as pharmacologically active peptide for secretion and intracellular label for identifying transplantable cells.

Topics: Analgesics; Animals; Cell Line; Cell Transplantation; Humans; Oligopeptides; Pain; Peptides; Protein Sorting Signals; Proteins; Rats; Recombinant Fusion Proteins; Staining and Labeling

Opiates may be used to attenuate chronic pain, but long-term use is complicated by the possible increase in pain over time, escalating dose requirements, and untoward side effects. Adjuncts such as ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, may be added to opiates to provide more consistent analgesia. However, the unwanted motor side effects of NMDA receptor antagonists prevent their widespread clinical usage. In the current study, an analogue of the naturally-derived peptide histogranin, [Ser(1)]histogranin (SHG), an NMDA receptor modulator without adverse side effects like those in current clinical use, was evaluated for its potential to enhance the antinociceptive effect of intrathecal morphine in the rat formalin test. Intrathecal injection of a combination of SHG and morphine resulted in significantly reduced hind paw flinching compared with morphine alone in the first and second phases. The effective dose of SHG used in the combination had no efficacy when tested alone. These results were similar to the increased efficacy that was obtained with a combination of ketamine and morphine. Thus, enhancement of opiate efficacy is possible using a novel peptide NMDA receptor modulator with a potentially improved safety profile.

Topics: Analgesics, Opioid; Analysis of Variance; Animals; Behavior, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Interactions; Excitatory Amino Acid Antagonists; Formaldehyde; Injections, Spinal; Ketamine; Male; Morphine; Pain; Proteins; Rats; Rats, Sprague-Dawley; Time Factors

Histogranin (HN)-like nonpeptides were designed and synthesized using benzimidazole (compound 1) and o-phenylenediamine (compounds 2-7) as scaffolds for the attachment of phenolic hydroxyl and basic guanidino pharmacophoric elements present in HN. The benzimidazole derivative N-5-guanidinopentanamide-(2R)-yl-2-(p-hydroxybenzyl)-5-carboxybenzimidazole (1) and the o-phenylenediamine derivative N-5-guanidinopentanamide-(2S)-yl-2-N-(p-hydroxyphenylacetyl) phenylenediamine (2) were more potent analgesics than HN in both the mouse writhing (5.5 and 3.5 as potent as HN, respectively) and tail-flick (11.8 and 8.0 as potent as HN, respectively) pain assays. Improvements in the potencies and times of action of compound 2 in the mouse writhing test were obtained by attaching carboxyl (6)or p-Cl-benzoyl (7) groups at position 4 of the (2R) o-phenylenediamine derivative (5). In rats, compounds 2 (80 nmol i.t.), 6 (36 nmol i.t.), and 7 (18 nmol i.t.) were effective in blocking both persistent inflammatory pain in the formalin test and hyperalgesia in the complete Freund adjuvant assay. Compounds 2, 6, and 7, but not compound 1 at 10 nmol (i.c.v.) also mimicked the HN (60 nmol i.c.v.) blockade of N-methyl-D-aspartate (NMDA)-induced convulsions in mice. Finally, in primary cultures of rat alveolar macrophages, HN and compounds 1, 2, 6, and 7 (10(-8) M) significantly blocked lipopolysaccharide-induced cyclooxygenase-2 induction and prostaglandin E(2) secretion. These studies indicate that both derivatives of benzimidazole and o-phenylenediamine mimic the in vivo antinociceptive and in vitro anti-inflammatory effects of HN, but the HN protection of mice against NMDA-induced convulsions is mimicked only by the o-phenylenediamine derivatives.

Topics: Analgesics; Animals; Anti-Inflammatory Agents; Benzimidazoles; Cyclooxygenase 2; Dinoprostone; Disease Models, Animal; Drug Interactions; Isoenzymes; Lipopolysaccharides; Macrophages; Male; Mice; Pain; Phenylenediamines; Prostaglandin-Endoperoxide Synthases; Proteins; Rats; Rats, Sprague-Dawley

The aim of this study was to assess whether a natural peptide, histogranin, isolated from chromaffin cells and possessing NMDA receptor inhibitory activity, could reduce tonic pain. Rats received intrathecal injections of the stable analog [Ser1]histogranin (SHG), prior to induction of the formalin response. SHG markedly suppressed the second tonic phase of the formalin response compared with saline vehicle. A U-shaped dose-response curve was obtained. SHG had no effect on phase 1 acute pain responses. These findings indicate that SHG acts in a similar fashion as other, non-peptide, NMDA antagonists in suppressing tonic, but not acute pain. The presence of the natural peptide in chromaffin cells may contribute to the analgesic effects of adrenal medullary implants.

Topics: Animals; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Excitatory Amino Acid Antagonists; Injections, Spinal; Male; Pain; Pain Measurement; Proteins; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate