Allopregnanolone preclinical acute pharmacokinetic and pharmacodynamic studies to predict tolerability and efficacy for Alzheimer's disease.

To develop allopregnanolone as a therapeutic for Alzheimer's disease, we investigated multiple formulations and routes of administration in translationally relevant animal models of both sexes. Subcutaneous, topical (transdermal and intranasal), intramuscular, and intravenous allopregnanolone were bolus-administered. Pharmacokinetic analyses of intravenous allopregnanolone in rabbit and mouse indicated that peak plasma and brain levels (3-fold brain/plasma ratios) at 5min were sufficient to activate neuroregenerative responses at sub-sedative doses. Slow-release subcutaneous suspension of allopregnanolone displayed 5-fold brain/plasma ratio at Cmax at 30min. At therapeutic doses by either subcutaneous or intravenous routes, allopregnanolone mouse plasma levels ranged between 34-51ng/ml by 30min, comparable to published endogenous human level in the third trimester of pregnancy. Exposure to subcutaneous, topical, intramuscular, and intravenous allopregnanolone, at safe and tolerable doses, increased hippocampal markers of neurogenesis including BrdU and PCNA in young 3xTgAD and aged wildtype mice. Intravenous allopregnanolone transiently and robustly phosphorylated CREB within 5min and increased levels of neuronal differentiation transcription factor NeuroD within 4h. Neurogenic efficacy was achieved with allopregnanolone brain exposure of 300-500hr*ng/g. Formulations were tested to determine the no observable adverse effect level (NOAEL) and maximally tolerated doses (MTD) in male and female rats by sedation behavior time course. Sex differences were apparent, males exhibited ≥40% more sedation time compared to females. Allopregnanolone formulated in sulfobutyl-ether-beta-cyclodextrin at optimized complexation ratio maximized allopregnanolone delivery and neurogenic efficacy. To establish the NOAEL and MTD for Allo-induced sedation using a once-per-week intravenous regenerative treatment regimen: In female rats the NOAEL was 0.5mg/kg and MTD 2mg/kg. The predicted MTD in human female is 0.37mg/kg. In male rats the NOAEL and MTD were less than those determined for female. Outcomes of these PK/PD studies predict a safe and efficacious dose range for initial clinical trials of allopregnanolone for Alzheimer's disease. These findings have translational relevance to multiple neurodegenerative conditions

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism

Ozone exposure increased neutrophils in bronchoalveolar lavage (D closed bar).

<p>No other inflammatory cell type number was affected by either ozone or the dual p38/JNK MAPK inhibitors. *p<0.05 Significantly different from air exposed controls. Data are mean ± SEM. n = 3–6.</p

Blocking p38 and JNK MAPK completely prevented ozone-induced airway hyperreactivity mediated by the vagus nerves.

<p>In anesthetized and vagotomized guinea pigs, stimulation of the vagus nerves (10V, 0.2 ms pulse width, 1–25 Hz, 5 sec duration at 1 minute intervals) caused frequency dependent bronchoconstriction (A open circles; measured as an increase in inflation pressure in mmH₂O) that is significantly potentiated one day post-ozone exposure (A closed circles). Pretreatment with dual MAPK inhibitors V-05-013 (A closed squares), V-05-014 (B closed triangles), or V-05-015 (C closed inverted triangles) completely prevented ozone-induced airway hyperreactivity. All three dual MAPK inhibitors suppressed parasympathetic nerve activity (A open squares, B open triangles, C open inverted triangles). Ozone and air exposed control data are the same in A-C. *p<0.05, **p<0.01 Significantly different from air exposed controls. Data are mean ± SEM. n = 4–7.</p

Baseline cardiovascular and pulmonary parameters.

<p>Values are means ± SEM. Baseline pulmonary inflation pressure significantly increased after ozone exposure. Treatment with dual p38/JNK MAPK inhibitor V-05-013 significantly reduced the ozone-induced increase in baseline pulmonary inflation pressure. *p<0.05 Significantly different from air exposure. ^‡p<0.05 Significantly different from ozone exposure.</p

In control (air exposed) guinea pigs electrical stimulation of the vagus nerves (3–30V, 0.2 ms pulse width, 15 Hz, 5 sec duration at 1 minute intervals) resulted in vagally induced bronchoconstriction (measured as an increase in pulmonary inflation pressure; 16±1 mmH₂O).

<p>An M₂ receptor antagonist, gallamine, potentiated vagally induced bronchoconstriction up to 6-fold in air exposed animals (open circles) demonstrating that functional M₂ receptors were limiting acetylcholine release. The potentiation by gallamine was decreased in ozone-exposed animals, demonstrating M₂ receptors were dysfunctional after ozone exposure (closed circles). V-05-013 partially prevented M₂ receptor dysfunction (C closed squares), while V-05-014 (B closed triangles) and V-05-015 (C closed inverted triangles) completely protected M₂ receptor function. Vagally induced bronchoconstriction in the absence of gallamine was not different from control among all groups. Ozone and air exposed controls are the same in A–C. *p<0.05, **p<0.01 Significantly different from air exposed controls. Data are mean ± SEM. n = 4–7.</p

Ki values for dual p38 and JNK MAPK inhibitors.

<p>All compounds have a Ki greater than 1 µM for all other kinases tested.</p>*<p>This one value is an IC50, not a Ki.</p

Chemical structures of dual p38 and JNK MAPK inhibitors.

<p>Chemical structures of dual p38 and JNK MAPK inhibitors.</p