The human immunodeficiency virus type-1 (HIV-1) encodes three enzymes essential for viral replication: a reverse transcriptase, a protease, and an integrase. The latter is responsible for the integration of the viral genome into the human genome and, therefore, represents an attractive target for chemotherapeutic intervention against AIDS. A drug based on this mechanism has not yet been approved. Benzyl-dihydroxypyrimidine-carboxamides were discovered in our laboratories as a novel and metabolically stable class of agents that exhibits potent inhibition of the HIV integrase strand transfer step. Further efforts led to very potent compounds based on the structurally related N-Me pyrimidone scaffold. One of the more interesting compounds in this series is the 2-N-Me-morpholino derivative 27a, which shows a CIC95 of 65 nM in the cell in the presence of serum. The compound has favorable pharmacokinetic properties in three preclinical species and shows no liabilities in several counterscreening assays.

Histone deacetylase (HDAC) inhibitors offer a promising strategy for cancer therapy, and the first generation HDAC inhibitors are currently in the clinic. Entirely novel ketone HDAC inhibitors have been developed from the cyclic tetrapeptide apicidin. These compounds show class I subtype selectivity and levels of cellular activity comparable to clinical candidates. A representative example has demonstrated tumor growth inhibition in a human colon HCT-116 carcinoma xenograft model comparable to known inhibitors.

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In vivo electroporation of plasmid DNA (DNA-EP) is an efficient and safe method for vaccines resulting in increased DNA uptake, enhanced protein expression and increased immune responses to the target antigen in a variety of species. To further enhance the efficacy of DNA-EP, we have evaluated the toll-like receptor7 (TLR7) agonist-2, 9, substituted 8-hydroxyadenosine derivative or SM360320-as an adjuvant to vaccines against HER2/neu and CEA in BALB-neuT and CEA transgenic mice (CEA.Tg), respectively. SM360320 induced in vivo secretion of interferon a (IFNa) and exerted a significant antitumor effect in CEA.Tg mice challenged with a syngenic tumor cell line expressing CEA and an additive effect with a CEA vaccine. Additionally, combination of SM360320 with plasmid encoding the extracellular and transmembrane domain of ratHER2/neu affected the spontaneous tumor progression in BALB-neuT mice treated in an advanced disease setting. The antitumor effect in mice treated with DNA-EP and SM360320 was associated with an anti-CEA and anti-p185 neu antibody isotype switch from IgG1 to IgG2a. These data demonstrate that SM360320 exerts significant antitumor effects and can act in association with DNA-EP for CEA-positive colon cancer and HER2-positive mammary carcinoma. These observations therefore emphasize the potential of SM360320 as immunological adjuvant for therapeutic DNA vaccines.

ABSTRACT:A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 3A4/5 in human liver microsomes is described. In contrast to the conventional testosterone 6␤-hydroxylation assay, the new method does not require high-performance liquid chromatography (HPLC) separation and mass spectrometry. The assay is based on the release of tritium as tritiated water that occurs upon CYP3A4/5-mediated 6␤-hydroxylation of testosterone labeled with tritium in the 6␤ position. The radiolabeled product is separated from the substrate using 96-well solid-phase extraction plates. The pharmacokinetic and toxicokinetic properties of pharmaceuticals depend in great part on their biotransformation by drug-metabolizing enzymes. The main drug-metabolizing system in mammals is cytochrome P450 (P450), a family of microsomal enzymes present predominantly in the liver. Multiple P450 enzymes catalyze the oxidation of chemicals of endogenous and exogenous origin, including drugs, steroids, prostanoids, eicosanoids, fatty acids, and environmental toxins (Ioannides, 1996). When a drug that is metabolized by a particular P450 enzyme is coadministered with an inhibitor of that same enzyme, changes in its pharmacokinetics can occur, which can give rise to adverse effects (Bertz and Granneman, 1997;Lin and Lu, 1998;Thummel and Wilkinson, 1998). It is therefore important to predict and prevent the occurrence of clearance changes due to metabolic inhibition. During the drug discovery process, it is routine practice in the pharmaceutical industry to assess the P450 inhibition potential of drug candidates to exclude potent inhibitors from further development (Lin and Lu, 1998;Crespi and Stresser, 2000;Riley, 2001). CYP3A4/5 is the most abundant P450 in human liver and is involved in the metabolism of about 50% of drugs used in human therapy (Guengerich, 1999). Inhibition of CYP3A4/5 activity can give rise to clinically significant and potentially life-threatening drug interactions (Thummel and Wilkinson, 1998). Several assay methods are currently used for determining the potential of drug candidates to inhibit CYP3A4/5 activity, and each of these methods presents distinct advantages and disadvantages. The most widely used method is the testosterone 6␤-hydroxylation assay, which is specific for enzymes of the CYP3A family (CYP3A4/5) (Waxman et al., 1988;Maenpaa et al., 1993;Wang et al., 1997;Yamazaki and Shimada, 1997). According to recent surveys conducted by reviewers in the Center for Drug Evaluation and Research of the United States Food and Drug Administration, the testosterone 6␤-hydroxylation assay represents the most commonly used probe reaction in support of new drug applications (Yuan et al., 1999(Yuan et al., , 2002. The practical challenge posed by this assay is that it requires HPLC separation of the reaction product from the substrate, followed by UV or mass spectrometric detection. This renders the assay relatively laborious, time-consumThis work was supported in part by a grant from t...