Characterization of Toxicity with Spinal Opiates

描述(申请人提供)：鞘内持续注射吗啡用于慢性疼痛患者。一个限制是，吗啡会导致脑膜产生炎性细胞团(肉芽肿)。我们在犬类模型中总结了这些观察结果，其中导致肉芽肿的腰椎脑脊液浓度与在人类中观察到的浓度相当。在这笔赠款的最后两个资助周期中，我们表明，吗啡和其他阿片类药物以浓度依赖的方式诱导肉芽肿，而芬太尼或阿芬太尼根本不诱导肉芽肿。我们推测肉芽肿源于脑膜肥大细胞(MMC)脱颗粒。因此：i)阿片类药物在体外使MMCs脱颗粒并在皮下注射(SQ)后产生肉芽肿；ii)脑膜/皮下脱颗粒/红斑和肉芽肿可被MC稳定剂克罗莫林阻断，但不能被阿片类药物拮抗。这种阿片受体非依赖性MC脱颗粒的起源被认为反映了非肽和多肽阿片配体通过G蛋白偶联受体家族作用于肥大细胞脱颗粒的阳离子性质，如Mas相关基因样受体(MRGX)。这些观察共同导致了描述和避免肉芽肿的假说的详细阐述。假设1：阿片类药物对肥大细胞的脱颗粒不依赖于阿片受体的激活，但可能依赖于阳离子电荷激活的受体(MRGX；HFPR)。假设2：阿片类肉芽肿不依赖于阿片受体的激活，但与非肽和多肽阿片配体脱颗粒肥大细胞的能力不同。在假设1中，我们将检验阿片类药物(DAMGO、TAPP、DULA和DMT-Dalga)、非阿片肽(齐康肽)和非肽(如巴氯芬、可乐定、新斯的明)对狗的闪光、人原代肥大细胞培养和小鼠原代肥大细胞培养的肥大细胞脱颗粒的浓度依赖性影响。利用人类肥大细胞培养，我们将使用shRNA检测MgrX-r在减少该蛋白表达方面的作用，并确定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)在IT输注上述MU阿片肽的狗身上进行剂量反应曲线，以定义JMEAD和最大可耐受(例如，急性副作用受限)剂量(MTD)，ii)定义选定药物的鞘内PK，iii)确定输注这些多肽的最大等效(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.