Behavioral pharmacology of the mixed-action delta-selective opioid receptor agonist BBI-11008: studies on acute, inflammatory and neuropathic pain, respiration, and drug self-administration.

RATIONALE AND OBJECTIVES: The present study characterized the behavioral pharmacology of a novel, mixed-action delta-selective (78:1) opioid receptor agonist, BBI-11008. This glycopeptide drug candidate was tested in assays assessing antinociception (acute, inflammatory, and neuropathic pain-like conditions) and side-effect endpoints (respiratory depression and drug self-administration). RESULTS: BBI-11008 had a 78-fold greater affinity for the delta opioid receptor than the mu receptor, and there was no binding to the kappa opioid receptor. BBI-11008 (3.2-100; 10-32 mg kg-1, i.v.) and morphine (1-10; 1-3.2 mg kg-1, i.v.) produced antinociceptive and anti-allodynic effects in assays of acute thermal nociception and complete Freund's adjuvant (CFA)-induced inflammatory pain, with BBI-11008 being less potent than morphine in both assays. BBI-11008 (1-18 mg kg-1, i.v.) had similar efficacy to gabapentin (10-56 mg kg-1, i.v.) in a spinal nerve ligation (SNL) model of neuropathic pain. In the respiration assay, with increasing %CO2 exposure, BBI-11008 produced an initial increase (32 mg kg-1, s.c.) and then decrease (56 mg kg-1, s.c.) in minute volume (MV) whereas morphine (3.2-32 mg kg-1, s.c.) produced dose-dependent decreases in MV. In the drug self-administration procedure, BBI-11008 did not maintain self-administration at any dose tested. CONCLUSIONS: These results suggest that the glycopeptide drug candidate possesses broad-spectrum antinociceptive and anti-allodynic activity across a range of pain-like conditions. Relative to morphine or fentanyl, the profile for BBI-11008 in the respiration and drug self-administration assays suggests that BBI-11008 may have less pronounced deleterious side effects. Continued assessment of this compound is warranted.

Long-term morphine delivery via slow release morphine pellets or osmotic pumps: Plasma concentration, analgesia, and naloxone-precipitated withdrawal.

AIMS: Slow-release morphine sulfate pellets and osmotic pumps are common routes of chronic morphine delivery in mouse models, but direct comparisons of these drug delivery systems are lacking. In this study, we assessed the efficacy of slow-release pellets versus osmotic pumps in delivering morphine to adult mice. MAIN METHODS: Male C57BL/6NCr mice (8weeksold) were implanted subcutaneously with slow-release pellets (25mg morphine sulfate) or osmotic pumps (64mg/mL, 1.0µL/h). Plasma morphine concentrations were quantified via LC-MS/MS, analgesic efficacy was determined by tail flick assay, and dependence was assessed with naloxone-precipitated withdrawal behaviors (jumping) and physiological effects (excretion, weight loss). KEY FINDINGS: Morphine pellets delivered significantly higher plasma drug concentrations compared to osmotic pumps, which were limited by the solubility of the morphine sulfate and pump volume/flow rate. Within 96h post-implantation, plasma morphine concentrations were indistinguishable in pellet vs. pump-treated samples. While osmotic pump did not have an antinociceptive effect in the tail flick assay, pumps and pellets induced comparable dependence symptoms (naloxone-precipitated jumping behavior) from 24-72h post-implantation. SIGNIFICANCE: In this study, we compared slow-release morphine pellets to osmotic minipumps for morphine delivery in mice. We found that osmotic pumps and subcutaneous morphine sulfate pellets yielded significantly different pharmacokinetics over a 7-day period, and as a result significantly different antinociceptive efficacy. Nonetheless, both delivery methods induced dependence as measured by naloxone-precipitated withdrawal.

Heat-shock protein 90 (Hsp90) promotes opioid-induced anti-nociception by an ERK mitogen-activated protein kinase (MAPK) mechanism in mouse brain.

Recent advances in developing opioid treatments for pain with reduced side effects have focused on the signaling cascades of the µ-opioid receptor (MOR). However, few such signaling targets have been identified for exploitation. To address this need, we explored the role of heat-shock protein 90 (Hsp90) in opioid-induced MOR signaling and pain, which has only been studied in four previous articles. First, in four cell models of MOR signaling, we found that Hsp90 inhibition for 24 h with the inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) had different effects on protein expression and opioid signaling in each line, suggesting that cell models may not be reliable for predicting pharmacology with this protein. We thus developed an in vivo model using CD-1 mice with an intracerebroventricular injection of 17-AAG for 24 h. We found that Hsp90 inhibition strongly blocked morphine-induced anti-nociception in models of post-surgical and HIV neuropathic pain but only slightly blocked anti-nociception in a naive tail-flick model, while enhancing morphine-induced precipitated withdrawal. Seeking a mechanism for these changes, we found that Hsp90 inhibition blocks ERK MAPK activation in the periaqueductal gray and caudal brain stem. We tested these signaling changes by inhibiting ERK in the above-mentioned pain models and found that ERK inhibition could account for all of the changes in anti-nociception induced by Hsp90 inhibition. Taken together, these findings suggest that Hsp90 promotes opioid-induced anti-nociception by an ERK mechanism in mouse brain and that Hsp90 could be a future target for improving the therapeutic index of opioid drugs.

GABP transcription factor is required for myeloid differentiation, in part, through its control of Gfi-1 expression.

GABP is an ets transcription factor that regulates genes that are required for myeloid differentiation. The tetrameric GABP complex includes GABPα, which binds DNA via its ets domain, and GABPß, which contains the transcription activation domain. To examine the role of GABP in myeloid differentiation, we generated mice in which Gabpa can be conditionally deleted in hematopoietic tissues. Gabpa knockout mice rapidly lost myeloid cells, and residual myeloid cells were dysplastic and immunophenotypically abnormal. Bone marrow transplantation demonstrated that Gabpα null cells could not contribute to the myeloid compartment because of cell intrinsic defects. Disruption of Gabpa was associated with a marked reduction in myeloid progenitor cells, and Gabpα null myeloid cells express reduced levels of the transcriptional repressor, Gfi-1. Gabp bound and activated the Gfi1 promoter, and transduction of Gabpa knockout bone marrow with Gfi1 partially rescued defects in myeloid colony formation and myeloid differentiation. We conclude that Gabp is required for myeloid differentiation due, in part, to its regulation of the tran-scriptional repressor Gfi-1.

Characterization of early EDEM1 protein maturation events and their functional implications.

The endoplasmic reticulum (ER) quality control factor EDEM1 associates with a number of ER proteins and ER-associated degradation (ERAD) substrates; however, an understanding of its role in ERAD is unclear. The early maturation events for EDEM1 including signal sequence cleavage and glycosylation were analyzed, and their relationship to the function of EDEM1 was determined. EDEM1 has five N-linked glycosylation sites with the most C-terminal site recognized poorly cotranslationally, resulting in the accumulation of EDEM1 containing four or five glycans. The fifth site was modified post-translationally when bypassed cotranslationally. Signal sequence cleavage of EDEM1 was found to be a slow and inefficient process. Signal sequence cleavage produced a soluble form of EDEM1 that efficiently associated with the oxidoreductase ERdj5 and most effectively accelerated the turnover of a soluble ERAD substrate. In contrast, a type-II membrane form of EDEM1 was generated when the signal sequence was uncleaved, creating an N-terminal transmembrane segment. The membrane form of EDEM1 efficiently associated with the ER membrane protein SEL1L and accelerated the turnover of a membrane-associated ERAD substrate. Together, these results demonstrated that signal sequence cleavage functionally regulated the association of EDEM1-soluble and membrane-integrated isoforms with distinct ERAD machinery and substrates.

EDEM1 recognition and delivery of misfolded proteins to the SEL1L-containing ERAD complex.

Terminally misfolded or unassembled secretory proteins are retained in the endoplasmic reticulum (ER) and subsequently cleared by the ER-associated degradation (ERAD) pathway. The degradation of ERAD substrates involves mannose trimming of N-linked glycans; however, the mechanisms of substrate recognition and sorting to the ERAD pathway are poorly defined. EDEM1 (ER degradation-enhancing alpha-mannosidase-like 1 protein) has been proposed to play a role in ERAD substrate signaling or recognition. We show that EDEM1 specifically binds nonnative proteins in a glycan-independent manner. Inhibition of mannosidase activity with kifunensine or disruption of the EDEM1 mannosidase-like domain by mutation had no effect on EDEM1 substrate binding but diminished its association with the ER membrane adaptor protein SEL1L. These results support a model whereby EDEM1 binds nonnative proteins and uses its mannosidase-like domain to target aberrant proteins to the ER membrane dislocation and ubiquitination complex containing SEL1L.

Sweet bays of ERAD.

Proteins that improperly mature in the endoplasmic reticulum (ER) are dislocated to the cytoplasm for proteasome-mediated destruction. A recent study provides insight into the incompletely understood processes for selection and targeting of aberrant proteins for ER-associated protein degradation. The identification of the ER chaperones GRP94 and BiP as binding partners for the mannose-binding proteins OS-9 and XTP3-B, indicates that these protein complexes bind to aberrant proteins and direct them to the Hrd1 dislocation and ubiquitylation complex in the ER membrane.

EDEM1 reveals a quality control vesicular transport pathway out of the endoplasmic reticulum not involving the COPII exit sites.

Immature and nonnative proteins are retained in the endoplasmic reticulum (ER) by the quality control machinery. Folding-incompetent glycoproteins are eventually targeted for ER-associated protein degradation (ERAD). EDEM1 (ER degradation-enhancing alpha-mannosidase-like protein 1), a putative mannose-binding protein, targets misfolded glycoproteins for ERAD. We report that endogenous EDEM1 exists mainly as a soluble glycoprotein. By high-resolution immunolabeling and serial section analysis, we find that endogenous EDEM1 is sequestered in buds that form along cisternae of the rough ER at regions outside of the transitional ER. They give rise to approximately 150-nm vesicles scattered throughout the cytoplasm that are lacking a recognizable COPII coat. About 87% of the immunogold labeling was over the vesicles and approximately 11% over the ER lumen. Some of the EDEM1 vesicles also contain Derlin-2 and the misfolded Hong Kong variant of alpha-1-antitrypsin, a substrate for EDEM1 and ERAD. Our results demonstrate the existence of a vesicle budding transport pathway out of the rough ER that does not involve the canonical transitional ER exit sites and therefore represents a previously unrecognized passageway to remove potentially harmful misfolded luminal glycoproteins from the ER.

Yos9p: a sweet-toothed bouncer of the secretory pathway.

In this issue of Molecular Cell, Bhamidipati et al., (2005) and Kim et al., (2005), and Szathmary et al. (2005), and demonstrate that Yos9p selectively binds to aberrant glycoproteins in the endoplasmic reticulum (ER) and targets them for destruction through the ER-associated protein degradation pathway.

Analysis of creatine, creatinine, creatine-d3 and creatinine-d3 in urine, plasma, and red blood cells by HPLC and GC-MS to follow the fate of ingested creatine-d3.

Creatine, which is increasingly being used as an oral supplement, is naturally present in the body. Studies on the fate of a particular dose of creatine require that the creatine be labeled, and for studies in humans the use of a stable isotopic label is desirable. The concentrations of total creatine and total creatinine were determined using HPLC. Creatine and creatinine were then separated using cation exchange chromatography and each fraction was derivatized with trifluoroacetic anhydride and the ratio of the deuterated:undeuterated species determined using GC-MS. Ratios of creatine:creatine-d(3), and creatinine:creatinine-d(3), and the concentrations of each of these species, were able to be determined in urine, plasma and red blood cells. Thus, the uptake of labeled creatine into plasma and red blood cells and its excretion in urine could be followed for a subject who ingested creatine-d(3). Creatine-d(3) was found in the plasma and red blood cells 10 min after ingestion, while creatine-d(3) and creatinine-d(3) were found in the urine collected after the first hour.

Donor APCs are required for maximal GVHD but not for GVL.

Graft-versus-host disease (GVHD) is a major source of morbidity in allogenic stem cell transplantation. We previously showed that recipient antigen-presenting cells (APCs) are required for CD8-dependent GVHD in a mouse model across only minor histocompatibility antigens (minor H antigens). However, these studies did not address the function of donor-derived APCs after GVHD is initiated. Here we show that GVHD develops in recipients of donor major histocompatibility complex class I-deficient (MHC I(-)) bone marrow. Thus, after initial priming, CD8 cells caused GVHD without a further requirement for hematopoietic APCs, indicating that host APCs are necessary and sufficient for GHVD. Nonetheless, GVHD was less severe in recipients of MHC I(-) bone marrow. Therefore, once initiated, GVHD is intensified by donor-derived cells, most probably donor APCs cross-priming alloreactive CD8 cells. Nevertheless, donor APCs were not required for CD8-mediated graft-versus-leukemia (GVL) against a mouse model of chronic-phase chronic myelogenous leukemia. These studies identify donor APCs as a new target for treating GVHD, which may preserve GVL.

Graft-versus-leukemia in a retrovirally induced murine CML model: mechanisms of T-cell killing.

The graft-versus-leukemia (GVL) effect, mediated by donor T cells, has revolutionized the treatment of leukemia. However, effective GVL remains difficult to separate from graft-versus-host disease (GVHD), and many neoplasms are GVL resistant. Murine studies aimed at solving these problems have been limited by the use of leukemia cell lines with limited homology to human leukemias and by the absence of loss-of-function leukemia variants. To address these concerns, we developed a GVL model against murine chronic-phase chronic myelogenous leukemia (mCP-CML) induced with retrovirus expressing the bcr-abl fusion cDNA, the defining genetic abnormality of chronic-phase CML (CP-CML). By generating mCP-CML in gene-deficient mice, we have studied GVL T-cell effector mechanisms. mCP-CML expression of Fas or tumor necrosis factor (TNF) receptors is not required for CD8-mediated GVL. Strikingly, maximal CD4-mediated GVL requires cognate interactions between CD4 cells and mCP-CML cells as major histocompatibility complex-negative (MHC II(-/-)) mCP-CML is relatively GVL resistant. Nevertheless, a minority of CD4 recipients cleared MHC II(-/-) mCP-CML; thus, CD4 cells can also kill indirectly. CD4 GVL did not require target Fas expression. These results suggest that CPCML's GVL sensitivity may in part be explained by the minimal requirements for T-cell killing, and GVL-resistance may be related to MHC II expression.