Systemic Concentrations Can Limit the Oral Absorption of Poorly Soluble Drugs: An Investigation of Non-Sink Permeation Using Physiologically Based Pharmacokinetic Modeling

In the early drug discovery environment, poorly soluble compounds with suboptimal potency are often used in efficacy studies to demonstrate in vivo preclinical proof-of-concept for new drug discovery targets and in preclinical toxicity studies to assess chemical scaffold safety. These compounds present a challenge to formulation scientists who are tasked with improving their oral bioavailability because high systemic concentrations are required. Despite the use of enabling formulations, increases in systemic exposure following oral delivery are often not achieved. We hypothesize that in some cases non-sink intestinal permeation can occur for poorly soluble compounds where their high systemic concentrations can act to inhibit their own oral absorption. Rats were given a 30 mg/kg oral dose of 1,3-dicyclohexyl urea (DCU) alone or concurrently with deuterated DCU (D8-DCU) intravenous infusions at rates of 13, 17, and 22 mg/kg/h. D8-DCU infusions dose dependently inhibited DCU oral absorption up to a maximum of 92%. Physiologically based pharmacokinetic modeling was utilized to understand the complex interaction between high DCU systemic concentrations and its effect on its own oral absorption. We show that high systemic concentrations of DCU act to suppress its own absorption by creating a condition where intestinal permeation occurs under non-sink conditions. More importantly, we identify relevant DCU concentrations that create the concentration gradient driving the intestinal permeation process. A new parameter, the maximum permeation extraction ratio, is proposed and provides a simple means to assess the extent of non-sink permeation

Excitatory synaptic function is selectively enhanced in CA1 of hippocampal brain slices following <i>in vitro</i> SAHA treatment.

<p>(A) The median amplitude of mEPSCs was significantly increased in SAHA-treated slices (p<0.05, n = 14 vehicle, 14 SAHA), while there was no significant change to mEPSC frequency as measured by the median interval between events (p>0.05). Example mEPSC traces from vehicle and SAHA treated slices are shown inset (scale bar represents 10 pA and 250 ms). (B) SAHA treatment did not significantly alter the amplitude or frequency of mIPSCs (p>0.05, n = 14, 11). Example mIPSC traces from vehicle and SAHA treated slices are shown inset (scale bar represents 10 pA and 500 ms). All data points are plotted as mean ±SEM.</p

SAHA treated slices exhibit enhanced induction of LTP and impaired LTD.

<p>(A) An induction protocol that was subthreshold in vehicle treated slices readily evoked LTP in SAHA treated slices (p<0.05, n = 5,5). Example traces before and after LTP induction are shown in red for SAHA and black for vehicle treated slices (scale bars represent 20 pA and 20 ms). (B) An induction protocol that readily induced LTD in vehicle treated slices could not produce LTD in SAHA treated slices (p<0.05, n = 9 vehicle, 8 SAHA). Example traces before and after LTD induction are shown in red for SAHA and black for vehicle treated slices (scale bars represent 25 pA and 20 ms). Data are plotted as mean ±SEM.</p

Acute or chronic SAHA treatment does not produce significant drug class activity signatures as assessed by the SmartCube®.

<p>A. Groups of mice were treated acutely with a single injection of 50 mg/kg or 150 mg/kg SAHA or vehicle. In addition, a group was treated with valproate (225 mg/kg). Both does of SAHA were behaviorally inactive without a clear therapeutic signal. In contrast valproate was behaviorally active (p<0.001, discrimination index = 100%) with a strong anxiolytic signature and a mild psychostimulant signature. B. Groups of mice were treated daily for 14 days with SAHA or Valproate. While the lower dose of SAHA appeared behaviorally active (p<0.001, discrimination index = 88%), the activity was not consistent with any known therapeutic signal and the higher dose was not behaviorally active. In contrast valproate showed a strong behavioral activity (p<0.001, discrimination index = 98%) with a predominantly anxiolytic signature. C. The legend shows the 15 classes of behavioral activity that were assessed.</p

Intrinsic membrane properties are unaltered by SAHA treatment.

<p>(A) Representative traces from vehicle (black) and SAHA (red) treated slices during a series of hyperpolarizing and depolarizing current injection steps (scale bar represents 20 mV and 100 ms). There was no difference between vehicle and SAHA treated slices in the number of action potentials elicited by 500 ms current injection pulses at any of the current injection levels (p>0.05, n = 7,7). (B) Action potential threshold, input resistance, and membrane sag reflecting the hyperpolarization-induced inward current, were all unaltered following SAHA treatment (p>0.05). Data are plotted as mean ±SEM.</p

SAHA IC₅₀ values for each HDAC isozyme are shown as measured using <i>in vitro</i> enzymatic assays.

<p>Values are in µM and confidence intervals are in parentheses.</p

Pharmacokinetic analysis of SAHA following i.p. injection.

<p>A) Bioanalysis of the time course of total (top) and unbound (bottom) plasma, CSF, and brain levels of SAHA following a single 50 mg/kg ip injection (n = 3 mice/time point). The dotted red lines represent the SAHA concentration imposed on the <i>in vitro</i> slice cultures for the electrophysiological studies. B) Total (top) and unbound (bottom) SAHA levels are shown following a 150 mg/kg ip injection (n = 3/time point). All data is shown as mean ± SD.</p

Fear memory deficits in Tg2576 mice are not rescued by SAHA treatment.

<p>A) Compared to non-transgenic littermates (n = 15), Tg2576 (n = 14) mice showed significantly less freezing than wt mice when returned to the context in which conditioning occurred (context, p<0.001), when placed in an altered context (altered, p<0.01), or in response to the cue used for conditioning (cue, p<0.05). B) Tg2576 mice were treated daily for 33 days prior to, as well as during fear conditioning with either vehicle (n = 14), 25 mg/kg SAHA (n = 13), or 50 mg/kg SAHA (n = 13). There was no effect of treatment on the percentage of time Tg2576 mice spent freezing in response to the context, altered context, or cue (p>0.05). C) There was no effect of treatment on the percentage of time spent freezing during conditioning (p>0.05). D) There was no effect of treatment on the distance traveled in the open field test (total distance = 38.0±7.7 m for vehicle, 39.6±6.1 m for 25 mg/kg SAHA, and 34.2±5.2 m for 50 mg/kg SAHA, p>0.05). All data are plotted as mean ±SEM.</p

Leveraging the Pre-DFG Residue Thr-406 To Obtain High Kinase Selectivity in an Aminopyrazole-Type PAK1 Inhibitor Series

To increase kinase selectivity in an aminopyrazole-based PAK1 inhibitor series, analogues were designed to interact with the PAK1 deep-front pocket pre-DFG residue Thr-406, a residue that is hydrophobic in most kinases. This goal was achieved by installing lactam head groups to the aminopyrazole hinge binding moiety. The corresponding analogues represent the most kinase selective ATP-competitive Group I PAK inhibitors described to date. Hydrogen bonding with the Thr-406 side chain was demonstrated by X-ray crystallography, and inhibitory activities, particularly against kinases with hydrophobic pre-DFG residues, were mitigated. Leveraging hydrogen bonding side chain interactions with polar pre-DFG residues is unprecedented, and similar strategies should be applicable to other appropriate kinases

Discovery of 1‑{4-[3-Fluoro-4-((3<i>S</i>,6<i>R</i>)‑3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone (GNE-3500): a Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor C (RORc or RORγ) Inverse Agonist

Retinoic acid receptor-related orphan receptor C (RORc, RORγ, or NR1F3) is a nuclear receptor that plays a major role in the production of interleukin (IL)-17. Considerable efforts have been directed toward the discovery of selective RORc inverse agonists as potential treatments of inflammatory diseases such as psoriasis and rheumatoid arthritis. Using the previously reported tertiary sulfonamide <b>1</b> as a starting point, we engineered structural modifications that significantly improved human and rat metabolic stabilities while maintaining a potent and highly selective RORc inverse agonist profile. The most advanced δ-sultam compound, GNE-3500 (<b>27</b>, 1-{4-[3-fluoro-4-((3<i>S</i>,6<i>R</i>)-3-methyl-1,1-dioxo-6-phenyl-[1,2]­thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone), possessed favorable RORc cellular potency with 75-fold selectivity for RORc over other ROR family members and >200-fold selectivity over 25 additional nuclear receptors in a cell assay panel. The favorable potency, selectivity, in vitro ADME properties, in vivo PK, and dose-dependent inhibition of IL-17 in a PK/PD model support the evaluation of <b>27</b> in preclinical studies