A potent, synthetic cannabinoid was radiolabeled and used to characterize and precisely localize cannabinoid receptors in slide-mounted sections of rat brain and pituitary. Assay conditions for 3H-CP55,940 binding in Tris-HCl buffer with 5% BSA were optimized, association and dissociation rate constants determined, and the equilibrium dissociation constant (Kd) calculated (21 nM by liquid scintillation counting, 5.2 nM by quantitative autoradiography). The results of competition studies, using several synthetic cannabinoids, add to prior data showing enantioselectivity of binding and correlation of in vitro potencies with potencies in biological assays of cannabinoid actions. Inhibition of binding by guanine nucleotides was selective and profound: Nonhydrolyzable analogs of GTP and GDP inhibited binding by greater than 90%, and GMP and the nonhydrolyzable ATP analog showed no inhibition. Autoradiography showed great heterogeneity of binding in patterns of labeling that closely conform to cytoarchitectural and functional domains. Very dense 3H-CP55,940 binding is localized to the basal ganglia (lateral caudate-putamen, globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), cerebellar molecular layer, innermost layers of the olfactory bulb, and portions of the hippocampal formation (CA3 and dentate gyrus molecular layer). Moderately dense binding is found throughout the remaining forebrain. Sparse binding characterizes the brain stem and spinal cord. Densitometry confirmed the quantitative heterogeneity of cannabinoid receptors (10 nM 3H-CP55,940 binding ranged in density from 6.3 pmol/mg protein in the substantia nigra pars reticulata to 0.15 pmol/mg protein in the anterior lobe of the pituitary). The results suggest that the presently characterized cannabinoid receptor mediates physiological and behavioral effects of natural and synthetic cannabinoids, because it is strongly coupled to guanine nucleotide regulatory proteins and is discretely localized to cortical, basal ganglia, and cerebellar structures involved with cognition and movement.

ABSTRACT[3H]CP 55,940, a radiolabeled synthetic cannabinoid, which is 10-100. times more potent in vivo than 49-tetrahydrocannabinol, was used to characterize and localize a specific cannabinoid receptor in brain sections. The potencies of a series of natural and synthetic cannabinoids as competitors of [3HJCP 55,940 binding~correlated closely with their relative potencies in several biological assays, suggesting that the receptor characterized in our in vitro assay is the same receptor that mediates behavioral and pharmacological effects of cannabinoids, including human subjective experience. Autoradiography of cannabinoid receptors in brain sections from several mammalian species, including human, reveals a unique and conserved distribution; bing is most dense in outflow nuclei ofthe basal ganglia-the substantia nigra pars reticulata and globus pallidus-and in the hippocampus and cerebellum. Generally high densities in forebrain and cerebellum implicate roles, for cannabinoids in cognition and movement. Sparse densities in lower brainstem areas controlling cardiovascular and respiratory functions may explain why high doses of A9-tetrahydrocannabinol are not lethal.Marihuana (Cannabis sativa) is one of the oldest and most widely used drugs in the world (1, 2). The major psychoactive ingredient of the marihuana plant is A9-tetrahydrocannabinol (A9-THC) (3). A9-THC and other natural and synthetic cannabinoids produce characteristic motor, cognitive, and analgesic effects (4, 5). Early reports showing cannabinoid-like activity of 9f3-hydroxyhexahydrocannabinol (13-HHC) (6)(7)(8) inspired the synthesis of several distinct cannabinoids for studies of their potential use as analgesics (9) 55,940 was extracted, giving a radiochemical yield of 15% and a specific activity of 79 Ci/mmol (1 Ci = 37 GBq). Optimization and competition studies were carried out with slidemounted sections cut from unfixed frozen rat brains. Incubations were in plastic cytomailers (CMS), each containing eight 30-gm-thick "sausage" sections on four gelatin-coated slides in 5 ml of solution (13). The sausage sections were prepared by combining and mincing three whole rat brains to achieve relative homogeneity of receptor and protein conAbbreviations: A8-and A9-THC, A8_ and A9-tetrahydrocannabinol, respectively; BSA, bovine serum albumin; HHC, hydroxyhexahydrocannabinol; SNr, substantia nigra pars reticulata. tTo whom reprint requests should be addressed. 1932The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

T-20, a synthetic peptide corresponding to a region of the transmembrane subunit of the HIV-1 envelope protein, blocks cell fusion and viral entry at concentrations of less than 2 ng/ml in vitro. We administered intravenous T-20 (monotherapy) for 14 days to sixteen HIV-infected adults in four dose groups (3, 10, 30 and 100 mg twice daily). There were significant, dose-related declines in plasma HIV RNA in all subjects who received higher dose levels. All four subjects receiving 100 mg twice daily had a decline in plasma HIV RNA to less than 500 copies/ml, by bDNA assay. A sensitive RT-PCR assay (detection threshold 40 copies/ml) demonstrated that, although undetectable levels were not achieved in the 14-day dosing period, there was a 1.96 log10 median decline in plasma HIV RNA in these subjects. This study provides proof-of-concept that viral entry can be successfully blocked in vivo. Short-term administration of T-20 seems safe and provides potent inhibition of HIV replication comparable to anti-retroviral regimens approved at present.

Platinum-based DNA-damaging chemotherapy is standard-of-care for most patients with lung cancer but outcomes remain poor. This has been attributed, in part, to the highly effective repair network known as the DNA-damage response (DDR). ATR kinase is a critical regulator of this pathway, and its inhibition has been shown to sensitize some cancer, but not normal, cells in vitro to DNA damaging agents. However, there are limited in vivo proof-of-concept data for ATR inhibition. To address this we profiled VX-970, the first clinical ATR inhibitor, in a series of in vitro and in vivo lung cancer models and compared it with an inhibitor of the downstream kinase Chk1. VX-970 markedly sensitized a large proportion of a lung cancer cell line and primary tumor panel in vitro to multiple DNA damaging drugs with clear differences to Chk1 inhibition observed. In vivo VX-970 blocked ATR activity in tumors and dramatically enhanced the efficacy of cisplatin across a panel of patient derived primary lung xenografts. The combination led to complete tumor growth inhibition in three cisplatin-insensitive models and durable tumor regression in a cisplatin-sensitive model. These data provide a strong rationale for the clinical evaluation of VX-970 in lung cancer patients.

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