A phase I study of the oral combination of CI-994, a putative histone deacetylase inhibitor, and capecitabine.

BACKGROUND: This study was conducted to determine the toxicity profile, maximum tolerated dose (MTD) and pharmacokinetics of the putative histone deacetylase inhibitor CI-994 in combination with capecitabine. PATIENTS AND METHODS: Fifty-four patients were treated according to three different dosing schemes in which the capecitabine dose was fixed and the CI-994 dose was escalated. Capecitabine was administered in twice daily divided doses, and CI-994 was given as a single daily dose. In schedule A, 26 patients were treated with capecitabine 1650 mg/m2/day and CI-994 for 2 weeks of a 3-week cycle. In schedule B, six patients received capecitabine 1650 mg/m2/day for two 3-week cycles and CI-994 for 5 of 6 weeks. In schedule C, 22 patients were treated with capecitabine 2000 mg/m2/day and CI-994 for 2 of 3 weeks. RESULTS: At the MTD, the principal dose-limiting toxicity was thrombocytopenia. The pharmacokinetics of CI-994 were unaltered by capecitabine, and there was no correlation between body surface area and major pharmacokinetic parameters. Platelet count nadir was best predicted by the observed maximal concentration (C(max)) of CI-994. CONCLUSIONS: The recommended phase II dose is 6 mg/m2 (or 10 mg) of CI-994 in combination with capecitabine 2000 mg/m2/day for 2 weeks of a 3-week cycle.

cAMP-dependent protein kinase inhibits mGluR2 coupling to G-proteins by direct receptor phosphorylation.

One of the primary physiological roles of group II and group III metabotropic glutamate receptors (mGluRs) is to presynaptically reduce synaptic transmission at glutamatergic synapses. Interestingly, previous studies suggest that presynaptic mGluRs are tightly regulated by protein kinases. cAMP analogs and the adenylyl cyclase activator forskolin inhibit the function of presynaptic group II mGluRs in area CA3 of the hippocampus. We now report that forskolin has a similar inhibitory effect on putative mGluR2-mediated responses at the medial perforant path synapse and that this effect of forskolin is blocked by a selective inhibitor of cAMP-dependent protein kinase (PKA). A series of biochemical and molecular studies was used to determine the precise mechanism by which PKA inhibits mGluR2 function. Our studies reveal that PKA directly phosphorylates mGluR2 at a single serine residue (Ser(843)) on the C-terminal tail region of the receptor. Site-directed mutagenesis combined with biochemical measures of mGluR2 function reveal that phosphorylation of this site inhibits coupling of mGluR2 from GTP-binding proteins

Protein kinase C and A3 adenosine receptor activation inhibit presynaptic metabotropic glutamate receptor (mGluR) function and uncouple mGluRs from GTP-binding proteins.

One of the most prominent roles of metabotropic glutamate receptors (mGluRs) in the CNS is to serve as presynaptic receptors that inhibit transmission at glutamatergic synapses. Previous reports suggest that the presynaptic effect of group II mGluRs at corticostriatal synapses can be inhibited by activators of protein kinase C (PKC). We now report that activation of PKC inhibits the ability of group II and group III mGluRs to regulate transmission at three major synapses in the hippocampal formation. Thus, this effect may be a widespread phenomenon that occurs at glutamatergic synapses throughout the CNS. We also report that this response is not limited to PKC-activating phorbol esters but that activation of A3 adenosine receptors induces a PKC-dependent inhibition of group III mGluR function at the Schaffer collateral-CA1 synapse. In addition to inhibiting mGluR modulation of excitatory synaptic transmission, we found that activation of PKC reduces inhibition of forskolin-stimulated cAMP accumulation by group II and group III mGluRs, suggesting that the effect of PKC on mGluR signaling is not specific to their effects on neurotransmitter release. This led us to test the hypothesis that PKC acts upstream from effector proteins regulated by mGluRs and acts at the level of the receptor or GTP-binding protein. Interestingly, we found that PKC inhibited mGluR-induced increases in [35S]-GTPgammaS binding in cortical synaptosomes. These data suggest that PKC-induced inhibition of mGluR signaling may be mediated by the inhibition of coupling of mGluRs to GTP-binding proteins.

Differential involvement of group II and group III mGluRs as autoreceptors at lateral and medial perforant path synapses.

1. Previous reports have shown that group III metabotropic glutamate receptors (mGluRs) serve as autoreceptors at the lateral perforant path, but to date there has been no rigorous determination of the roles of other mGluRs as autoreceptors at this synapse. Furthermore, it is not known which of the mGluR subtypes serve as autoreceptors at the medial perforant path synapse. With the use of whole cell patch-clamp and field excitatory postsynaptic potential (fEPSP) recording techniques, we examined the groups of mGluRs that act as autoreceptors at lateral and medial perforant path synapses in adult rat hippocampal slices. 2. Consistent with previous reports, the group III mGluR agonist (D,L)-2-amino-4-phosphonobutyric acid reduced fEPSPs and excitatory postsynaptic currents (EPSCs) in the dentate gyrus. However, the group-II-selective agonist (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) also reduced fEPSPs and EPSCs, suggesting that multiple mGluR subtypes may serve as autoreceptors at perforant path synapses. 3. Selective activation of either medial or lateral perforant pathways revealed that micromolar concentrations of (L)-2-amino-4-phosphonobutyric acid (L-AP4) reduce fEPSPs in lateral but not medial perforant path, suggesting group III involvement at the lateral perforant pathway. Conversely, DCG-IV and 2R, 4R-4-aminopyrrolidine-2,4-dicarboxylate, another group-II-selective mGluR agonist, potently reduced fEPSPs at the medial but not lateral perforant path, suggesting that a group II mGluR may act as an autoreceptor at the medial perforant path-dentate gyrus synapse. 4. Antagonist studies with group-selective antagonists such as (2S,3S,4S)-2-methyl-2-(carboxycyclpropyl)glycine (MCCG; group II) and alpha-methyl-L-AP4 (MAP4; group III) suggest differential involvement of each group at these synapses. The effect of L-AP4 at the lateral perforant path synapse was blocked by MAP-4, but not MCCG. In contrast, the effect of DCG-IV was blocked by application of MCCG, but not MAP4. 5. Previous studies suggest that the effect of L-AP4 at the lateral perforant path synapse is mediated by a presynaptic mechanism. In the present studies, we found that concentrations of DCG-IV that reduce transmission at the medial perforant path synapse reduce paired-pulse depression and do not reduce kainate-evoked currents recorded from dentate granule cells. This is consistent with the hypothesis that DCG-IV also acts by a presynaptic mechanism.