This study explores the role of mevalonate inhibitors in the activation of NF-k ␤ and the induction of inducible nitric oxide synthase (iNOS) and cytokines (TNF-␣ , IL-1 ␤ , and IL-6) in rat primary astrocytes, microglia, and macrophages. Lovastatin and sodium phenylacetate (NaPA) were found to inhibit LPS-and cytokine-mediated production of NO and expression of iNOS in rat primary astrocytes; this inhibition was not due to depletion of end products of mevalonate pathway (e.g., cholesterol and ubiquinone). Reversal of the inhibitory effect of lovastatin on LPS-induced iNOS expression by mevalonate and farnesyl pyrophosphate and reversal of the inhibitory effect of NaPA on LPS-induced iNOS expression by farnesyl pyrophosphate, however, suggests a role of farnesylation in the LPS-mediated induction of iNOS. The inhibition of LPS-mediated induction of iNOS by FPT inhibitor II, an inhibitor of Ras farnesyl protein transferase, suggests that farnesylation of p21 ras or other proteins regulates the induction of iNOS. Inhibition of LPSmediated activation of NF-k ␤ by lovastatin, NaPA, and FPT inhibitor II in astrocytes indicates that the observed inhibition of iNOS expression is mediated via inhibition of NF-k ␤ activation. In addition to iNOS, lovastatin and NaPA also inhibited LPS-induced expression of TNF-␣ , IL-1 ␤ , and IL-6 in rat primary astrocytes, microglia, and macrophages. This study delineates a novel role of the mevalonate pathway in controlling the expression of iNOS and different cytokines in rat astrocytes, microglia, and macrophages that may be important in developing therapeutics against cytokineand NO-mediated neurodegenerative diseases. ( J. Clin. Invest. 1997. 100:2671-2679.)

BackgroundVRC01 is a human broadly neutralizing monoclonal antibody (bnMAb) against the CD4-binding site of the HIV-1 envelope glycoprotein (Env) that is currently being evaluated in a Phase IIb adult HIV-1 prevention efficacy trial. VRC01LS is a modified version of VRC01, designed for extended serum half-life by increased binding affinity to the neonatal Fc receptor.Methods and findingsThis Phase I dose-escalation study of VRC01LS in HIV-negative healthy adults was conducted by the Vaccine Research Center (VRC) at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD). The age range of the study volunteers was 21–50 years; 51% of study volunteers were male and 49% were female. Primary objectives were safety and tolerability of VRC01LS intravenous (IV) infusions at 5, 20, and 40 mg/kg infused once, 20 mg/kg given three times at 12-week intervals, and subcutaneous (SC) delivery at 5 mg/kg delivered once, or three times at 12-week intervals. Secondary objectives were pharmacokinetics (PK), serum neutralization activity, and development of antidrug antibodies. Enrollment began on November 16, 2015, and concluded on August 23, 2017. This report describes the safety data for the first 37 volunteers who received administrations of VRC01LS. There were no serious adverse events (SAEs) or dose-limiting toxicities. Mild malaise and myalgia were the most common adverse events (AEs). There were six AEs assessed as possibly related to VRC01LS administration, and all were mild in severity and resolved during the study. PK data were modeled based on the first dose of VRC01LS in the first 25 volunteers to complete their schedule of evaluations. The mean (±SD) serum concentration 12 weeks after one IV administration of 20 mg/kg or 40 mg/kg were 180 ± 43 μg/mL (n = 7) and 326 ± 35 μg/mL (n = 5), respectively. The mean (±SD) serum concentration 12 weeks after one IV and SC administration of 5 mg/kg were 40 ± 3 μg/mL (n = 2) and 25 ± 5 μg/mL (n = 9), respectively. Over the 5–40 mg/kg IV dose range (n = 16), the clearance was 36 ± 8 mL/d with an elimination half-life of 71 ± 18 days. VRC01LS retained its expected neutralizing activity in serum, and anti-VRC01 antibody responses were not detected. Potential limitations of this study include the small sample size typical of Phase I trials and the need to further describe the PK properties of VRC01LS administered on multiple occasions.ConclusionsThe human bnMAb VRC01LS was safe and well tolerated when delivered intravenously or subcutaneously. The half-life was more than 4-fold greater when compared to wild-type VRC01 historical data. The reduced clearance and extended half-life may make it possible to achieve therapeutic levels with less frequent and lower-dose administrations. This would potentially lower the costs of manufacturing and improve the practicality of using passively administered monoclonal antibodies (mAbs) for the prevention of HIV-1 infection.Trial registrationClinicalTrials.gov NCT02599896

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Nitric oxide produced by inducible nitric-oxide synthase (iNOS) in different brain cells in response to various cytokines plays an important role in the pathophysiology of stroke and other neurodegenerative diseases. This study underlines the importance of cAMP in inhibiting the induction of NO production by lipopolysaccharide (LPS) and cytokines in rat primary astrocytes. Compounds (forskolin, 8-bromo-cAMP, and (S p )-cAMP) that increase cAMP and activate protein kinase A (PKA) were found to inhibit LPS-and cytokine-mediated production of NO as well as the expression of iNOS, whereas compounds (H-89 and (R p )-cAMP) that decrease cAMP and PKA activity stimulated the production of NO and the expression of iNOS in rat primary astrocytes. Forskolin, but not the inactive analogue 1,9-dideoxyforskolin, inhibited NO production and iNOS expression in a dose-dependent manner in astrocytes. The inhibition of LPS-and/or cytokine-induced NO production in rat C 6 glial cells by forskolin suggest that similar to astrocytes, iNOS expression in C 6 cells is also regulated by similar mechanisms. In contrast, in rat peritoneal macrophages the cAMP analogues stimulated the LPS-and cytokineinduced production of NO. In vitro, the PKA had no effect on iNOS activity in LPS-treated astrocytes or macrophages, suggesting that PKA modulates the intracellular signaling events associated with the induction of iNOS biogenesis rather than the post-translational modification of iNOS. The compounds which activate PKA activity, blocked the activation of NF-␤ in astrocytes but stimulated the activation of NF-␤ in macrophages. This differential regulation of NF-␤ activation in two different cell types (astrocytes and macrophages) by the same second messenger (cAMP) indicates that intracellular events or pathways in the activation of NF-␤ may be different. Moreover, this inhibition of iNOS expression in LPS-and cytokine-treated astrocytes by cAMP may be of therapeutic potential in NOmediated cytotoxicity in neurodegenerative diseases.

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