Chronic treatment with the (iso-)glutaminyl cyclase inhibitor PQ529 is a novel and effective approach for glomerulonephritis in chronic kidney disease.

Glomeruli and renal tubule injury in chronic kidney disease (CKD) is reported to involve induction of macrophage activation through the CCL2/CCR2 axis. The effects of inhibitors of the CCL2/CCR2 axis, such as anti-CCL2 antibody and CCR2 antagonist, on kidney function in animal models or humans with kidney dysfunction have been demonstrated. The N-terminal glutamine on immature CCL2 is replaced with pyroglutamate (pE) by glutaminyl cyclase (QC) and isoQC. pE-CCL2 is stable and resistant to peptidases. We hypothesized that inhibiting QC/isoQC activity would lead to the degradation of CCL2, thereby ameliorating CKD and reducing kidney inflammation. To test this hypothesis, we investigated the renoprotective properties of the QC/isoQC inhibitor PQ529 in anti-glomerular basement membrane (GBM) antibody-induced glomerulonephritis Wistar Kyoto (WKY) rats. Three-week repeated administration of PQ529 (30 and 100 mg/kg, twice daily) significantly reduced the serum and urine CCL2 and urinary protein excretion in a dose-dependent manner. Correlations between the urinary protein level and serum or urinary CCL2 levels were confirmed in tested animals. Repeated administration of PQ529 significantly reduced the expression of CD68, a macrophage marker, in the kidney cortex and mononuclear infiltration into the tubulointerstitium. In addition, decreased levels of urinary KIM-1, ß2 microglobulin, and clusterin were detected, suggesting the inhibition of inflammation in both the proximal and distal tubules. These results suggest that PQ529 suppresses the progression of inflammation-induced renal dysfunction by inhibiting the CCL2/CCR2 axis. Inhibition of QC/isoQC may thus be a viable alternative therapeutic approach for treating glomerulonephritis and CKD patients.

Glutaminyl Cyclase Inhibitor PQ912 Improves Cognition in Mouse Models of Alzheimer's Disease-Studies on Relation to Effective Target Occupancy.

Numerous studies suggest that the majority of amyloid-ß (Aß) peptides deposited in Alzheimer's disease (AD) are truncated and post-translationally modified at the N terminus. Among these modified species, pyroglutamyl-Aß (pE-Aß, including N3pE-Aß40/42 and N11pE-Aß40/42) has been identified as particularly neurotoxic. The N-terminal modification renders the peptide hydrophobic, accelerates formation of oligomers, and reduces degradation by peptidases, leading ultimately to the accumulation of the peptide and progression of AD. It has been shown that the formation of pyroglutamyl residues is catalyzed by glutaminyl cyclase (QC). Here, we present data about the pharmacological in vitro and in vivo efficacy of the QC inhibitor (S)-1-(1H-benzo[d]imidazol-5-yl)-5-(4-propoxyphenyl)imidazolidin-2-one (PQ912), the first-in-class compound that is in clinical development. PQ912 inhibits human, rat, and mouse QC activity, with Ki values ranging between 20 and 65 nM. Chronic oral treatment of hAPPSLxhQC double-transgenic mice with approximately 200 mg/kg/day via chow shows a significant reduction of pE-Aß levels and concomitant improvement of spatial learning in a Morris water maze test paradigm. This dose results in a brain and cerebrospinal fluid concentration of PQ912 which relates to a QC target occupancy of about 60%. Thus, we conclude that >50% inhibition of QC activity in the brain leads to robust treatment effects. Secondary pharmacology experiments in mice indicate a fairly large potency difference for Aß cyclization compared with cyclization of physiologic substrates, suggesting a robust therapeutic window in humans. This information constitutes an important translational guidance for predicting the therapeutic dose range in clinical studies with PQ912.

Use of biopartitioning micellar chromatography and RP-HPLC for the determination of blood-brain barrier penetration of α-adrenergic/imidazoline receptor ligands, and QSPR analysis.

For this study, 31 compounds, including 16 imidazoline/α-adrenergic receptor (IRs/α-ARs) ligands and 15 central nervous system (CNS) drugs, were characterized in terms of the retention factors (k) obtained using biopartitioning micellar and classical reversed phase chromatography (log kBMC and log kwRP, respectively). Based on the retention factor (log kwRP) and slope of the linear curve (S) the isocratic parameter (φ0) was calculated. Obtained retention factors were correlated with experimental log BB values for the group of examined compounds. High correlations were obtained between logarithm of biopartitioning micellar chromatography (BMC) retention factor and effective permeability (r(log kBMC/log BB): 0.77), while for RP-HPLC system the correlations were lower (r(log kwRP/log BB): 0.58; r(S/log BB): -0.50; r(φ0/Pe): 0.61). Based on the log kBMC retention data and calculated molecular parameters of the examined compounds, quantitative structure-permeability relationship (QSPR) models were developed using partial least squares, stepwise multiple linear regression, support vector machine and artificial neural network methodologies. A high degree of structural diversity of the analysed IRs/α-ARs ligands and CNS drugs provides wide applicability domain of the QSPR models for estimation of blood-brain barrier penetration of the related compounds.

Results of the Phase I Trial of RG7112, a Small-Molecule MDM2 Antagonist in Leukemia.

PURPOSE: RG7112 is a small-molecule MDM2 antagonist. MDM2 is a negative regulator of the tumor suppressor p53 and frequently overexpressed in leukemias. Thus, a phase I study of RG7112 in patients with hematologic malignancies was conducted. EXPERIMENTAL DESIGN: Primary study objectives included determination of the dose and safety profile of RG7112. Secondary objectives included evaluation of pharmacokinetics; pharmacodynamics, such as TP53-mutation status and MDM2 expression; and preliminary clinical activity. Patients were divided into two cohorts: Stratum A [relapsed/refractory acute myeloid leukemia (AML; except acute promyelocytic leukemia), acute lymphoblastic leukemia, and chronic myelogenous leukemia] and Stratum B (relapsed/refractory chronic lymphocytic leukemia/small cell lymphocytic leukemia; CLL/sCLL). Some Stratum A patients were treated at the MTD to assess clinical activity. RESULTS: RG7112 was administered to 116 patients (96 patients in Stratum A and 20 patients in Stratum B). All patients experienced at least 1 adverse event, and 3 dose-limiting toxicities were reported. Pharmacokinetic analysis indicated that twice-daily dosing enhanced daily exposure. Antileukemia activity was observed in the 30 patients with AML assessed at the MTD, including 5 patients who met International Working Group (IWG) criteria for response. Exploratory analysis revealed TP53 mutations in 14% of Stratum A patients and in 40% of Stratum B patients. Two patients with TP53 mutations exhibited clinical activity. p53 target genes were induced only in TP53 wild-type leukemic cells. Baseline expression levels of MDM2 correlated positively with clinical response. CONCLUSIONS: RG7112 demonstrated clinical activity against relapsed/refractory AML and CLL/sCLL. MDM2 inhibition resulted in p53 stabilization and transcriptional activation of p53-target genes. We provide proof-of-concept that MDM2 inhibition restores p53 function and generates clinical responses in hematologic malignancies.

Biological profile and bioavailability of imidazoline compounds on morphine tolerance modulation.

Tolerance to opioid administration represents a serious medical alert in different chronic conditions. This study compares the effects of the imidazoline compounds 1, 2, and 3 on morphine tolerance in an animal model of inflammatory pain in the rat. 1, 2, and 3 have been selected in that, although bearing a common scaffold, preferentially bind to α2-adrenoceptors, imidazoline I2 receptors, or both systems, respectively. Such compounds have been tested in vivo by measuring the paw withdrawal threshold to mechanical pressure after complete Freund's adjuvant injection. To determine the ligand levels in rat plasma, an HPLC-mass spectrometry method has been developed. All the compounds significantly reduced the induction of morphine tolerance, showing different potency and duration of action. Indeed, the selective imidazoline I2 receptor interaction (2) restored the analgesic response by maintaining the same time-dependent profile observed after a single morphine administration. Differently, the selective α2C-adrenoceptor activation (1) or the combination between α2C-adrenoceptor activation and imidazoline I2 receptor engagement (3) promoted a change in the temporal profile of morphine analgesia by maintaining a mild but long lasting analgesic effect. Interestingly, the kinetics of compounds in rat plasma supported the pharmacodynamic data. Therefore, this study highlights that both peculiar biological profile and bioavailability of such ligands complement each other to modulate the reduction of morphine tolerance. Based on these observations, 1-3 can be considered useful leads in the design of new drugs able to turn off the undesired tolerance induced by opioids.

Clinical pharmacology characterization of RG7112, an MDM2 antagonist, in patients with advanced solid tumors.

PURPOSE: RG7112, the first selective small-molecule MDM2 antagonist in clinical testing, is a non-genotoxic oral p53 activator. To optimize its dose and schedule, a number of clinical pharmacology characteristics were explored in this multicenter trial in patients with advanced solid tumors. METHOD: In part 1, the impact of high-energy/high-fat meal and formulations (crystalline and amorphous) on relative bioavailability was examined in single-dose crossover designs. In part 2, schedule optimization (4 schedules of drug administration under fasting condition and 2 cohorts with liquid supplementation) was investigated in parallel, dose escalation designs. Clinical endpoints were pharmacokinetics (PK), pharmacodynamics (PD) including MIC-1 elevation and platelet reduction, and safety/tolerability. RESULTS: With a single-dose treatment, a high-fat/high-energy meal and a new formulation under fasting condition, respectively, enhanced overall bioavailability of RG7112 slightly over twofold. Following multiple-dose administrations, all four schedules yielded the comparable per-cycle (28-d) exposure (AUC), as designed; liquid supplements also enhanced bioavailability. High-dose treatments of consecutive daily dosing for 5 and 3 days resulted in higher on-treatment-day exposure to RG7112 than both weekly and low-dose/long-duration (20-day) daily schedules. Serum MIC-1 and blood platelet profiles showed similar patterns to those of PK when the clinical pharmacology conditions were varied, suggesting the relative importance of treatment-day exposure than overall per-cycle AUC. CONCLUSION: Food (both high-fat and low-fat meals) and new formulation enhanced bioavailability. High-dose consecutive daily treatment for 3-5 days is superior to weekly and low-dose/long-duration (20-day) daily schedules in yielding the sufficiently high drug exposure and PD effects potentially required for cancer treatment efficacy.

In vivo evaluation of a new ¹8F-labeled PET ligand, [¹8F]FEBU, for the imaging of I2-imidazoline receptors.

INTRODUCTION: The functions of I2-imidazoline receptors (I2Rs) are unknown, but evidence exists for their involvement in various neuropsychiatric disorders. Although a few positron emission tomography (PET) I2R ligands have been developed, of which [(11)C]FTIMD and [(11)C]BU99008 were evaluated as PET I2R imaging ligands in monkeys, no human PET imaging study using an I2R-selective PET ligand has been conducted yet. Thus, we synthesized an (18)F-labeled I2R-selective ligand (BU99018 or FEBU, Ki for I2Rs=2.6 nM), and evaluated its application using rodents in PET imaging in vivo toward the development of a clinically-useful I2R PET imaging ligand. METHODS: [(18)F]FEBU was synthesized by the reaction of its precursor and [(18)F]fluoroethyl bromide. A biodistribution and brain PET study were conducted in mice and rats respectively. RESULTS: [(18)F]FEBU was successfully synthesized yielding a radioactivity suitable for injection (10.1 ± 5.3% at the end of the irradiation (n=10) based on (18)F(-)). The specific activity at end of synthesis (EOS) was 40-147 TBq/mmol (n=10). The radiochemical purity was >99% at EOS and remained >99% for 90 min after EOS. In mice brain uptake was relatively high. In the blocking study with the co-injection of the high-affinity I2R ligand BU224 (1 mg/kg b.w.) brain uptake was significantly decreased 30 min post-injection. In the PET studies the radioactivity was highly accumulated in the I2R-rich hypothalamus. Pretreatment with BU224 (1 mg/kg b.w.) significantly decreased the radioactivity in the hypothalamus to 23% of that of the control from 60 to 90 min post-injection. CONCLUSION: [(18)F]FEBU was sufficiently stable as a PET ligand and had a relatively high specific binding affinity for I2Rs in rats and mice.

Preclinical optimization of MDM2 antagonist scheduling for cancer treatment by using a model-based approach.

PURPOSE: Antitumor clinical activity has been demonstrated for the MDM2 antagonist RG7112, but patient tolerability for the necessary daily dosing was poor. Here, utilizing RG7388, a second-generation nutlin with superior selectivity and potency, we determine the feasibility of intermittent dosing to guide the selection of initial phase I scheduling regimens. EXPERIMENTAL DESIGN: A pharmacokinetic-pharmacodynamic (PKPD) model was developed on the basis of preclinical data to determine alternative dosing schedule requirements for optimal RG7388-induced antitumor activity. This PKPD model was used to investigate the pharmacokinetics of RG7388 linked to the time-course of the antitumor effect in an osteosarcoma xenograft model in mice. These data were used to prospectively predict intermittent and continuous dosing regimens, resulting in tumor stasis in the same model system. RESULTS: RG7388-induced apoptosis was delayed relative to drug exposure with continuous treatment not required. In initial efficacy testing, daily dosing at 30 mg/kg and twice a week dosing at 50 mg/kg of RG7388 were statistically equivalent in our tumor model. In addition, weekly dosing of 50 mg/kg was equivalent to 10 mg/kg given daily. The implementation of modeling and simulation on these data suggested several possible intermittent clinical dosing schedules. Further preclinical analyses confirmed these schedules as viable options. CONCLUSION: Besides chronic administration, antitumor activity can be achieved with intermittent schedules of RG7388, as predicted through modeling and simulation. These alternative regimens may potentially ameliorate tolerability issues seen with chronic administration of RG7112, while providing clinical benefit. Thus, both weekly (qw) and daily for five days (5 d on/23 off, qd) schedules were selected for RG7388 clinical testing.

From bradykinin B2 receptor antagonists to orally active and selective bradykinin B1 receptor antagonists.

The bradykinin (BK) B1 receptor is an attractive target for the treatment of chronic pain and inflammation. Starting from a dual B1 and B2 antagonist, novel antagonists were designed that display low-nanomolar affinity for human B1 receptor and selectivity over B2. Initially, potent imidazoline derivatives were studied, but these compounds suffered from low bioavailability. This issue could be overcome by the use of less basic amino derivatives leading to orally active compounds.

Identification of 2-oxo-N-(phenylmethyl)-4-imidazolidinecarboxamide antagonists of the P2X(7) receptor.

A backup molecule to compound 2 was sought by targeting the most likely metabolically vulnerable site in this molecule. Compound 18 was subsequently identified as a potent P2X(7) antagonist with very low in vivo clearance and high oral bioavailability in all species examined. Some evidence to support the role of P2X(7) in the etiology of pain is also presented.

Discovery of pyridone-containing imidazolines as potent and selective inhibitors of neuropeptide Y Y5 receptor.

A series of 2-pyridone-containing imidazoline derivatives was synthesized and evaluated as neuropeptide Y Y5 receptor antagonists. Optimization of the 2-pyridone structure on the 2-position of the imidazoline ring led to identification of 1-(difluoromethyl)-5-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoropyridin-3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-one (7m). Compound 7m displayed statistically significant inhibition of food intake in an agonist-induced food intake model in SD rats and no adverse cardiovascular effects in anesthetized dogs. In addition, markedly higher brain penetrability and a lower plasma Occ90 value were observed in P-gp-deficient mdr1a (-/-) mice compared to mdr1a (+/+) mice after oral administration of 7m.

Neuroprotective effects of alpha2-adrenergic receptor agonists.

Alpha2-adrenergic receptor agonists exert potent analgesic and sedative/hypnotic effects. In addition, they have been shown to be neuroprotective in various experimental models, but the molecular pathways leading to these actions are still poorly defined. This review summarizes the available literature and discusses potential mechanisms of alpha2-adrenergic receptor agonist mediated neuroprotection.

Tricyclic imidazoline derivatives as potent and selective adenosine A1 receptor antagonists.

Novel tricyclic imidazoline antagonists of the adenosine A1 receptor are described. For key compounds, the selectivity level over other adenosine receptor subtypes is examined along with their in vivo effects in a rat diuresis model. Compound 14, the (R)-isomer of 7,8-dihydro-8-ethyl-2-(4-bicyclo[2.2.2]octan-1-ol)-4-propyl-1H-imidazo[2,1-i]purin-5(4H)-one, is a particularly potent adenosine A1 receptor antagonist with good selectivity over the other three adenosine receptor subtypes: A1 (human) Ki=22 nM; A2A (human) Ki=4400 nM; A2B (human) Ki=580 nM; A3 (human) Ki>or=10,000 nM. Imidazoline 14 is a competitive adenosine A1 receptor antagonist with a pA2 value of 8.88 and is highly soluble in water (>100 mg/mL). In addition, it has an oral bioavailability of 84% and an oral half-life of 3.8 h in rats. When orally administered in a rat diuresis model, compound 14 promoted sodium excretion (ED50=0.01 mg/kg). This level of efficacy is comparable to that of BG9928, a selective adenosine A1 receptor antagonist that is currently in clinical trials as a treatment for congestive heart failure. Additional modifications to 14 also showed that the bridgehead hydroxyl group could be replaced with a propionic acid (compound 36) without a significant loss in binding affinity or in vivo activity.