The pharmacodynamic and mechanistic foundation for the antineoplastic effects of GFH009, a potent and highly selective CDK9 inhibitor for the treatment of hematologic malignancies.

To evade cell cycle controls, malignant cells rely upon rapid expression of select proteins to mitigate proapoptotic signals resulting from damage caused by both cancer treatments and unchecked over-proliferation. Cyclin-dependent kinase 9 (CDK9)-dependent signaling induces transcription of downstream oncogenes promoting tumor growth, especially in hyperproliferative 'oncogene-addicted' cancers, such as human hematological malignancies (HHMs). GFH009, a potent, highly selective CDK9 small molecule inhibitor, demonstrated antiproliferative activity in assorted HHM-derived cell lines, inducing apoptosis at IC50 values below 0.2 µM in 7/10 lines tested. GFH009 inhibited tumor growth at all doses compared to controls and induced apoptosis in a dose-dependent manner. Twice-weekly injections of GFH009 maleate at 10 mg/kg significantly prolonged the survival of MV-4-11 xenograft-bearing rodents, while their body weight remained stable. There was marked reduction of MCL-1 and c-MYC protein expression post-drug exposure both in vitro and in vivo. Through rapid 'on-off' CDK9 inhibition, GFH009 exerts a proapoptotic effect on HHM preclinical models triggered by dynamic deprivation of crucial cell survival signals. Our results mechanistically establish CDK9 as a targetable vulnerability in assorted HHMs and, along with the previously shown superior class kinome selectivity of GFH009 vs other CDK9 inhibitors, strongly support the rationale for currently ongoing clinical studies with this agent in acute myeloid leukemia and other HHMs.

CEP-26401 (irdabisant), a potent and selective histamine H3 receptor antagonist/inverse agonist with cognition-enhancing and wake-promoting activities.

CEP-26401 [irdabisant; 6-{4-[3-((R)-2-methyl-pyrrolidin-1-yl)-propoxy]-phenyl}-2H-pyridazin-3-one HCl] is a novel, potent histamine H3 receptor (H3R) antagonist/inverse agonist with drug-like properties. High affinity of CEP-26401 for H3R was demonstrated in radioligand binding displacement assays in rat brain membranes (K(i) = 2.7 ± 0.3 nM) and recombinant rat and human H3R-expressing systems (K(i) = 7.2 ± 0.4 and 2.0 ± 1.0 nM, respectively). CEP-26401 displayed potent antagonist and inverse agonist activities in [³5S]guanosine 5'-O-(γ-thio)triphosphate binding assays. After oral dosing of CEP-26401, occupancy of H3R was estimated by the inhibition of ex vivo binding in rat cortical slices (OCC50 = 0.1 ± 0.003 mg/kg), and antagonism of the H3R agonist R-α-methylhistamine- induced drinking response in the rat dipsogenia model was demonstrated in a similar dose range (ED50 = 0.06 mg/kg). CEP-26401 improved performance in the rat social recognition model of short-term memory at doses of 0.01 to 0.1 mg/kg p.o. and was wake-promoting at 3 to 30 mg/kg p.o. In DBA/2NCrl mice, CEP-26401 at 10 and 30 mg/kg i.p. increased prepulse inhibition (PPI), whereas the antipsychotic risperidone was effective at 0.3 and 1 mg/kg i.p. Coadministration of CEP-26401 and risperidone at subefficacious doses (3 and 0.1 mg/kg i.p., respectively) increased PPI. These results demonstrate potent behavioral effects of CEP-26401 in rodent models and suggest that this novel H3R antagonist may have therapeutic utility in the treatment of cognitive and attentional disorders. CEP-26401 may also have therapeutic utility in treating schizophrenia or as adjunctive therapy to approved antipsychotics.

Detection of low level histamine H3 receptor occupancy by autoradiography.

Histamine H(3) receptor antagonists have been proposed as a novel approach to the treatment of cognitive, attentional, and sleep disorders. It is apparent that H(3) receptor antagonists produce in vivo effects in preclinical animal models of central diseases across a wide dose range. In order to characterize the relationship between efficacy in the preclinical models and H(3) receptor occupancy, a brain slice receptor autoradiography method was used. Brain slice receptor autoradiography requires less in vitro tissue processing, preserves brain structure, and provides anatomical localization of compound in the brain. Consistent with H(3) receptor distribution, in vitro autoradiography experiments demonstrated specific binding of [(3)H]NAMH (N-alpha-methylhistamine) in rat cortex, and other brain regions, but not in cerebellum. Ex vivo H(3)R brain slice autoradiography was able to detect H(3) receptor occupancy by reference antagonists at doses lower than previously found using a homogenate assay format. The method is relatively quick with image acquisition on a beta-imager and is capable of detecting receptor occupancy in different brain regions simultaneously. Furthermore, the increased sensitivity should be useful in providing dosing guidelines for H(3) antagonists in both preclinical and clinical settings.

Correlation between ex vivo receptor occupancy and wake-promoting activity of selective H3 receptor antagonists.

The histamine H3 receptor (H3R) modulates the release of neurotransmitters that are involved in vigilance, cognition, and sleep-wake regulation. H3R antagonism has been proposed as a novel approach to the treatment of cognitive and attention deficit as well as sleep disorders. It is apparent that H3R antagonists produce pharmacological effects in preclinical animal models across a wide dose range. Several H3R antagonists were reported to be effective at producing cognitive enhancing effects at low doses, while producing robust wake enhancement at higher doses. To better understand the effect of H3R antagonists across a broad dose range, an ex vivo receptor binding assay has been used to estimate the degree of H3R occupancy in vivo. The H3R antagonists ciproxifan, thioperamide, GSK189254 (6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride), and ABT-239 ([4-(2-{2-[(2R)-2-methylpyrrolidinyl]ethyl}-benzofuran-5-yl)benzonitrile) produced wake-promoting activity in vivo and a dose-dependent inhibition of H3R binding ex vivo. For ciproxifan, thioperamide, and GSK189254, a relatively low level of cumulative wake activity was linearly correlated with up to 80% of the receptor occupancy. In contrast, an abrupt break from linearity and a robust increase of waking activity was observed at doses that produce greater than 80% occupancy. Our results suggest a relatively small increase of waking activity at low levels of receptor occupancy that may be consistent with reported enhancement of attention and cognitive function. Robust waking activity at higher levels of H3R occupancy may be mechanistically different from activities at low levels of H3R occupancy.

JNK-mediated BIM phosphorylation potentiates BAX-dependent apoptosis.

Trophic factor deprivation (TFD) activates c-Jun N-terminal kinases (JNKs), culminating in coordinate AP1-dependent transactivation of the BH3-only BCL-2 proteins BIM(EL) and HRK, which in turn are critical for BAX-dependent cytochrome c release, caspase activation, and apoptosis. Here, we report that TFD caused not only induction but also phosphorylation of BIM(EL). Mitochondrially localized JNKs but not upstream activators, like mixed-lineage kinases (MLKs) or mitogen-activated protein kinase kinases (MKKs), specifically phosphorylated BIM(EL) at Ser65, potentiating its proapoptotic activity. Inhibition of the JNK pathway attenuated BIM(EL) expression, prevented BIM(EL) phosphorylation, and abrogated TFD-induced apoptosis. Conversely, activation of this pathway promoted BIM(EL) expression and phosphorylation, causing BIM- and BAX-dependent cell death. Thus, JNKs regulate the proapoptotic activity of BIM(EL) during TFD, both transcriptionally and posttranslationally.