Characterization of Toxicity with Spinal Opiates

描述（由申请人提供）：持续鞘内注射吗啡用于慢性疼痛患者。限制是吗啡会导致脑膜产生炎性细胞团块（肉芽肿）。我们在犬模型中总结了这些观察结果，其中腰椎脑脊液浓度导致肉芽肿与在人类中观察到的结果相当。在该基金的最后两个资助周期中，我们证明了肉芽肿是由吗啡和其他阿片类药物以浓度依赖的方式诱导的，而芬太尼或阿芬太尼则完全没有。我们假设肉芽肿是由脑膜肥大细胞（MMC）的脱肉芽引起的。因此：i)阿片类药物在体外使MMCs脱粒，皮下（SQ）给药后产生皮肤光斑，产生肉芽肿；ii)脑膜/皮下去肉芽/光斑和肉芽肿被MC稳定剂色莫利阻断，但不被阿片类拮抗剂阻断。这种不依赖阿片受体的MC脱粒的起源被假设为反映了非肽和肽阿片配体通过G蛋白偶联受体家族（如mas相关基因样受体（MrgX））作用于肥大细胞脱粒的阳离子性质。这些观察结果共同导致了假设的阐述，以表征和避免肉芽肿。假设1：阿片药物对肥大细胞的脱颗粒作用与阿片受体的激活无关，但可能依赖于阳离子电荷激活的受体（MrgX； HFPR）。假设2。阿片肉芽肿不依赖于阿片受体的激活，但会随非肽和肽阿片配体对肥大细胞脱颗粒的能力而变化。在假设1中，我们将检验阿片类药物（DAMGO、TAPP、DALGA和DMT- DALGA）和非阿片类肽（ziconotide）和非肽（如巴氯芬、clonidine、新斯的明）对狗的光斑、人原代肥大细胞培养物中肥大细胞脱颗粒和小鼠原代细胞培养物的浓度依赖性作用。利用人类肥大细胞培养，我们将使用shRNA来检查MgrX-r的作用，以减少该蛋白的表达，并确定该阳离子受体在肥大细胞脱颗粒中的作用。在假设2中，我们将i)对输注上述mu阿片肽的狗进行剂量反应曲线，以确定最大有效镇痛剂量：JMEAD和最大可耐受（如急性副作用有限）剂量（MTD）， ii)确定所选药物的鞘内PK， iii)确定输注这些肽的最大等效（镇痛）剂量是否会导致肉芽肿。

DESCRIPTION (provided by applicant): Continuous intrathecal (IT) morphine infusion is used in chronic pain patients. A limitation is that morphine results in an inflammatory cell mass (granuloma) arising from the meninges. We recapitulated these observations in a canine model wherein lumbar CSF concentrations leading to granulomas were comparable to those observed in humans. In the last two funding cycles of this grant, we showed that granulomas are induced in a concentration dependent fashion by morphine and other opiates and not at all by fentanyl or alfentanil. We hypothesized that the granuloma arises from the degranulation of meningeal mast cells (MMC). Thus: i) opiates that degranulate MMCs ex vivo and cutaneous flare after subcutaneous (SQ) delivery produce a granuloma; ii) the meningeal/subcutaneous degranulation/flare and the granuloma are blocked by the MC stabilizer cromolyn, but not by opiate antagonism. The origin of this opiate receptor-independent MC degranulation is hypothesized to reflect the cationic properties of the nonpeptide and peptide opioid ligands acting to degranulate mast cells through G protein coupled receptor families, such as the Mas-related gene like receptors (MrgX). These observations jointly lead to an elaboration of hypotheses to characterize and avoid the granuloma. Hypothesis 1: Degranulation of mast cells by opioid agents is independent of opiate receptor activation but potentially depend upon receptors activated by cationic charge (MrgX; HFPR). Hypothesis 2. The opiate granuloma is independent of opiate receptor activation but will covary with ability of the nonpeptide and peptide opioid ligands to degranulate mast cells. In hypothesis 1, we will examine concentration dependent effects of opioid (DAMGO, TAPP, DALGA, and DMT- DALGA) and nonopioid peptides (ziconotide) and non-peptides (e.g. baclofen, clonidine, neostigmine) on: flare in the dog, mast cell degranulation in human primary mast cell cultures and on murine primary cell cultures. Using the human mast cell cultures, we will examine the role of MgrX-r using shRNA to reduce that protein expression and define the role of that cationic receptor on mast cell degranulation. In hypothesis 2, we will i) undertake dose response curves in dogs with IT infusion of the above mentioned mu opioid peptides to define the just maximally effective analgesic dose: JMEAD and the maximum tolerable (e.g. acute side effect limited) dose (MTD), ii) define intrathecal PK of selected agent and iii) determine if infusion of the maximum equi-effective (analgesic) doses of these peptides lead to a granuloma.