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SAHA Enhances Synaptic Function and Plasticity In Vitro but Has Limited Brain Availability In Vivo and Does Not Impact Cognition
Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) used for the treatment of cutaneous T cell lymphoma (CTCL) and under consideration for other indications. In vivo studies suggest reducing HDAC function can enhance synaptic function and memory, raising the possibility that SAHA treatment could have neurological benefits. We first examined the impacts of SAHA on synaptic function in vitro using rat organotypic hippocampal brain slices. Following several days of SAHA treatment, basal excitatory but not inhibitory synaptic function was enhanced. Presynaptic release probability and intrinsic neuronal excitability were unaffected suggesting SAHA treatment selectively enhanced postsynaptic excitatory function. In addition, long-term potentiation (LTP) of excitatory synapses was augmented, while long-term depression (LTD) was impaired in SAHA treated slices. Despite the in vitro synaptic enhancements, in vivo SAHA treatment did not rescue memory deficits in the Tg2576 mouse model of Alzheimer’s disease (AD). Along with the lack of behavioral impact, pharmacokinetic analysis indicated poor brain availability of SAHA. Broader assessment of in vivo SAHA treatment using high-content phenotypic characterization of C57Bl6 mice failed to demonstrate significant behavioral effects of up to 150 mg/kg SAHA following either acute or chronic injections. Potentially explaining the low brain exposure and lack of behavioral impacts, SAHA was found to be a substrate of the blood brain barrier (BBB) efflux transporters Pgp and Bcrp1. Thus while our in vitro data show that HDAC inhibition can enhance excitatory synaptic strength and potentiation, our in vivo data suggests limited brain availability may contribute to the lack of behavioral impact of SAHA following peripheral delivery. These results do not predict CNS effects of SAHA during clinical use and also emphasize the importance of analyzing brain drug levels when interpreting preclinical behavioral pharmacology
A Whole Cell Assay to Measure Caspase-6 Activity by Detecting Cleavage of Lamin A/C
Caspase-6 is a cysteinyl protease implicated in neurodegenerative conditions including Alzheimer's and Huntington's disease making it an attractive target for therapeutic intervention. A greater understanding of the role of caspase-6 in disease has been hampered by a lack of suitable cellular assays capable of specifically detecting caspase-6 activity in an intact cell environment. This is mainly due to the use of commercially available peptide substrates and inhibitors which lack the required specificity to facilitate development of this type of assay. We report here a 384-well whole-cell chemiluminescent ELISA assay that monitors the proteolytic degradation of endogenously expressed lamin A/C during the early stages of caspase-dependent apoptosis. The specificity of lamin A/C proteolysis by caspase-6 was demonstrated against recombinant caspase family members and further confirmed in genetic deletion studies. In the assay, plasma membrane integrity remained intact as assessed by release of lactate dehydrogenase from the intracellular environment and the exclusion of cell impermeable peptide inhibitors, despite the induction of an apoptotic state. The method described here is a robust tool to support drug discovery efforts targeting caspase-6 and is the first reported to specifically monitor endogenous caspase-6 activity in a cellular context
Role of P-Glycoprotein and Breast Cancer Resistance Protein-1 in the Brain Penetration and Brain Pharmacodynamic Activity of the Novel Phosphatidylinositol 3-Kinase Inhibitor GDC-0941
Differential Effects of Rifampin and Ketoconazole on the Blood and Liver Concentration of Atorvastatin in Wild-Type and Cyp3a and Oatp1a/b Knockout Mice
Evaluating the <i>In Vitro</i> Inhibition of UGT1A1, OATP1B1, OATP1B3, MRP2, and BSEP in Predicting Drug-Induced Hyperbilirubinemia
Hyperbilirubinemia
may arise due to inadequate clearance of bilirubin
from the body. Bilirubin elimination is a multifaceted process consisting
of uptake of bilirubin into the hepatocytes facilitated by OATP1B1
and OATP1B3. Once in the hepatocytes, it is extensively glucuronidated
by UGT1A1. Eventually, the glucuronide metabolite is excreted into
the bile via MRP2. UGT1A1 inhibition has been previously shown to
be linked with hyperbilirubinemia. However, because drug transporters
also contribute to bilirubin elimination, the purpose of this work
was to investigate the <i>in vitro</i> inhibition of OATP1B1,
OATP1B3, MRP2, and BSEP of select test drugs known to elicit hyperbilirubinemia.
Test drugs investigated in this study were atazanavir and indinavir,
which are associated with hyperbilirubinemia and elevations in serum
transaminase; ritonavir and nelfinavir, which are not associated with
hyperbilirubinemia; and bromfenac, troglitazone, and trovafloxacin,
which are associated with severe idiosyncratic hepatotoxicity exhibiting
elevations in serum bilirubin and transaminase. Due to limited solubility
and poor ionization of bilirubin and its glucuronide, the formation
of estradiol 3-glucuronide was used as a surrogate to assess UGT1A1
activity, while the transport of pitavastatin, CDCF, and taurocholate
were used as surrogate probe substrates to monitor the function of
OATP1B1/OATP1B3, MRP2, and BSEP, respectively. It was assumed that
any inhibition of the surrogate probe substrates by test drugs is
indicative of the potential impact of test drugs to modulate the function
of proteins involved in bilirubin disposition. <i>In vitro</i> inhibition was determined by calculating IC<sub>50</sub>. Moreover, <i>C</i><sub>max</sub> and <i>C</i><sub>max,free</sub> were integrated with IC<sub>50</sub> values to calculate <i>R</i> and <i>R</i><sub>free</sub>, respectively, which
represents the ratio of probe drug glucuronidation/transport in the
absence and presence of test drugs. Analysis of the data showed that <i>R</i><sub>free</sub> demonstrated the best correlation to hyperbilirubinemia.
Specifically, <i>R</i><sub>free</sub> was above the 1.1
target threshold against UGT1A1, OATP1B1, and BSEP for atazanavir
and indinavir. In contrast, <i>R</i><sub>free</sub> was
below this threshold for ritonavir and nelfinavir as well as for bromfenac,
troglitazone, and trovafloxacin. For all test drugs examined, only
minor inhibition against OATP1B3 and MRP2 were observed. These data
suggest that the proposed surrogate probe substrates to evaluate the <i>in vitro</i> inhibition of UGT1A1, OATP1B1, and BSEP may be
suitable to assess bilirubin disposition. For protease inhibitors,
inclusion of OATP1B1 and BSEP inhibition may improve the predictability
of hyperbilirubinemia
Preclinical Assessment of the Absorption and Disposition of the Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor GDC-0980 and Prediction of Its Pharmacokinetics and Efficacy in Human
Role of P‑Glycoprotein on the Brain Penetration and Brain Pharmacodynamic Activity of the MEK Inhibitor Cobimetinib
Cobimetinib
is a MEK inhibitor currently in clinical trials as
an anticancer agent. The objectives of this study were to determine
in vitro and in vivo if cobimetinib is a substrate of P-glycoprotein
(P-gp) and/or breast cancer resistance protein (Bcrp1) and to assess
the implications of efflux on cobimetinib pharmacokinetics (PK), brain
penetration, and target modulation. Cell lines transfected with P-gp
or Bcrp1 established that cobimetinib was a substrate of P-gp but
not a substrate of Bcrp1. In vivo, after intravenous and oral administration
of cobimetinib to FVB (wild-type; WT), <i>Mdr1a/bÂ(−/−)</i>,<i> Bcrp1 (−/−)</i>, and <i>Mdr1a/bÂ(−/−)/BcrpÂ(−/−)</i> knockout (KO) mice, clearance was similar in WT (35.5 ± 16.7
mL/min/kg) and KO animals (22.0 ± 3.6 to 27.6 ± 5.2 mL/min/kg);
oral exposure was also similar between WT and KO animals. After an
oral 10 mg/kg dose of cobimetinib, the mean total brain to plasma
ratio (Kp) at 6 h postdose was 0.3 and 0.2 in WT and <i>Bcrp1Â(−/−)</i> mice, respectively. In <i>Mdr1a/bÂ(−/−)</i> and <i>Mdr1<i>a</i>/1b/Bcrp1Â(−/−)</i> KO mice and WT mice treated with elacridar (a P-gp and BCRP inhibitor),
Kp increased to 11, 6, and 7, respectively. Increased brain exposure
in <i>Mdr1a/bÂ(−/−)</i> and <i>Mdr1<i>a</i>/1b/Bcrp1Â(−/−)</i> KO and elacridar
treated mice was accompanied by up to ∼65% suppression of the
target (pErk) in brain tissue, compared to WT mice. By MALDI imaging,
the cobimetinib signal intensity was relatively high and was dispersed
throughout the brain of <i>Mdr1<i>a</i>/1b/Bcrp1Â(−/−)</i> KO mice compared to low/undetectable signal intensity in WT mice.
The efflux of cobimetinib by P-gp may have implications for the treatment
of patients with brain tumors/metastases
SAHA enhances synaptic function and plasticity in vitro but has limited brain availability in vivo and does not impact cognition.
Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) used for the treatment of cutaneous T cell lymphoma (CTCL) and under consideration for other indications. In vivo studies suggest reducing HDAC function can enhance synaptic function and memory, raising the possibility that SAHA treatment could have neurological benefits. We first examined the impacts of SAHA on synaptic function in vitro using rat organotypic hippocampal brain slices. Following several days of SAHA treatment, basal excitatory but not inhibitory synaptic function was enhanced. Presynaptic release probability and intrinsic neuronal excitability were unaffected suggesting SAHA treatment selectively enhanced postsynaptic excitatory function. In addition, long-term potentiation (LTP) of excitatory synapses was augmented, while long-term depression (LTD) was impaired in SAHA treated slices. Despite the in vitro synaptic enhancements, in vivo SAHA treatment did not rescue memory deficits in the Tg2576 mouse model of Alzheimer's disease (AD). Along with the lack of behavioral impact, pharmacokinetic analysis indicated poor brain availability of SAHA. Broader assessment of in vivo SAHA treatment using high-content phenotypic characterization of C57Bl6 mice failed to demonstrate significant behavioral effects of up to 150 mg/kg SAHA following either acute or chronic injections. Potentially explaining the low brain exposure and lack of behavioral impacts, SAHA was found to be a substrate of the blood brain barrier (BBB) efflux transporters Pgp and Bcrp1. Thus while our in vitro data show that HDAC inhibition can enhance excitatory synaptic strength and potentiation, our in vivo data suggests limited brain availability may contribute to the lack of behavioral impact of SAHA following peripheral delivery. These results do not predict CNS effects of SAHA during clinical use and also emphasize the importance of analyzing brain drug levels when interpreting preclinical behavioral pharmacology
Comparison of potency of peptide-derived caspase inhibitors in cellular lamin cleavage assay, enzymatic activity and cell permeability.
<p>nd = not determined</p><p>a = z-VEID-TFPM enzymatic IC<sub>50</sub> is less than 0.0017 due to limit of enzymatic assay detection.</p><p>*Enzymatic IC<sub>50</sub> values were determined after a 15 minute enzyme/inhibitor preincubation and 40 minute enzyme reaction.</p
Effect of peptide-based caspase inhibitors on SKNAS cells as determined using the Caspase-Glo® 6 assay.
<p>SKNAS cells were treated with Ac-VEID-CHO (•) or Ac-DEVD-CHO (▪) prior to addition of 3 µM staurosporine for 6 hours and detection of VEID-ase activity as described in Experimental Procedures. Concentration inhibition curves were performed in duplicate and represent 1 of at least 3 experiments with similar results. Concentration-response curves for each inhibitor were normalized to zero and 100% based on no staurosporine or DMSO, respectively. The mean and standard error of the mean are reported.</p