Immuno-PET Imaging to Assess Target Engagement: Experience from 89Zr-Anti-HER3 mAb (GSK2849330) in Patients with Solid Tumors.

PET imaging with radiolabeled drugs provides information on tumor uptake and dose-dependent target interaction to support selection of an optimal dose for future efficacy testing. In this immuno-PET study of the anti-human epidermal growth factor receptor (HER3) mAb GSK2849330, we investigated the biodistribution and tumor uptake of 89Zr-labeled GSK2849330 and evaluated target engagement as a function of antibody mass dose. Methods:89Zr-GSK2849330 distribution was monitored in 6 patients with HER3-positive tumors not amenable to standard treatment. Patients received 2 administrations of 89Zr-GSK2849330. Imaging after tracer only was performed at baseline; dose-dependent inhibition of 89Zr-GSK2849330 uptake in tumor tissues was evaluated 2 wk later using increasing doses of unlabeled GSK2849330 in combination with the tracer. Up to 3 PET scans (2 hours post infusion [p.i.] and days 2 and 5 p.i.) were performed after tracer administration. Biodistribution and tumor targeting were assessed visually and quantitatively using SUV. The 50% and 90% inhibitory mass doses (ID50 and ID90) of target-mediated antibody uptake were calculated using a Patlak transformation. Results: At baseline, imaging with tracer showed good tumor uptake in all evaluable patients. Predosing with unlabeled mAb reduced the tumor uptake rate in a dose-dependent manner. Saturation of 89Zr-mAb uptake by tumors was seen at the highest dose (30 mg/kg). Despite the limited number of patients, an exploratory ID50 of 2 mg/kg and ID90 of 18 mg/kg have been determined. Conclusion: In this immuno-PET study, dose-dependent inhibition of tumor uptake of 89Zr-GSK2849330 by unlabeled mAb confirmed target engagement of mAb to the HER3 receptor. This study further validates the use of immuno-PET to directly visualize tissue drug disposition in patients with a noninvasive approach and to measure target engagement at the site of action, offering the potential for dose selection.

Effects of vercirnon on the activity of CYP3A4, CYP2C19 and CYP2C8 enzymes and BCRP and OATP1B1 transporters using probe substrates.

PURPOSE: Vercirnon is a CCR9 chemokine receptor antagonist being developed for the treatment of Crohn's disease. As a variety of concomitant medications are often required for the treatment of Crohn's disease, it is important to characterise the drug interaction profile of vercirnon. To confirm the results of previous in vitro inhibition studies, this study assessed the in vivo effect of vercirnon on the activity of cytochrome P450 enzymes (CYP3A4, CYP2C19 and CYP2C8) and drug transport proteins (BCRP and OATP1B1) using probe substrates. METHODS: This was an open-label, single-sequence, repeat-dose study conducted in 24 healthy adult subjects. On days 1-4, subjects received probe substrates (midazolam, pioglitazone, omeprazole and rosuvastatin; in that order), followed by administration of vercirnon 500 mg twice daily (BID) on days 5-14. On days 11-14, in addition to vercirnon 500 mg BID, subjects also received probe substrates as on days 1-4. Blood samples were collected for pharmacokinetic analysis of probe substrates, vercirnon and two of its metabolites. RESULTS: Geometric least-squares mean ratios (90 % confidence interval) of area under the concentration-time curve from time zero to infinity for probe administered with vercirnon (test) compared with probe alone (reference) for midazolam, pioglitazone, omeprazole and rosuvastatin were 0.92 (0.85, 0.99), 1.01 (0.95, 1.07), 0.99 (0.76,1.31) and 0.98 (0.88, 1.09), respectively. CONCLUSIONS: Co-administration of probe substrates midazolam, pioglitazone, omeprazole, and rosuvastatin following repeat dosing of vercirnon 500 mg BID demonstrated vercirnon had no clinically significant effect on CYP3A4, CYP2C8, CYP2C19 enzyme activity or BCRP or OATP1B1 transporter activity.

Single-Dose Pharmacokinetics of the CCR9 Receptor Antagonist Vercirnon in Healthy US and Japanese Subjects.

Two randomized, single-dose, crossover studies were carried out to assess different formulations and doses of the CCR9 receptor antagonist vercirnon in healthy subjects. US study (n = 24): a five-period crossover study in healthy US subjects to assess the bioavailability of four new GlaxoSmithKline formulations compared with a "reference" formulation. Each subject received a single 500 mg dose of each of the five vercirnon formulations in a fed state. Primary pharmacokinetic (PK) endpoints were maximum plasma concentration, (Cmax ), and exposure as assessed by area under the curve (AUC). There was no significant difference in PK parameters and bioavailability between the formulations tested. Japanese study (n = 30): a four-period crossover study in healthy Japanese male subjects to assess PK and dose proportionality following single, ascending, oral doses of 250, 500, and 1,000 mg vercirnon under fasted and fed conditions. Vercirnon Cmax and AUC parameters in the fasted state increased in a less than dose proportional manner and were on average 20% higher in fed subjects compared with fasted subjects. Overall, these results support the premise that vercirnon has similar PK/safety profiles within US and Japanese populations. There was no evidence to preclude the use of the new vercirnon formulation in future studies.

Endothelium-dependent vasodilation in myogenically active mouse skeletal muscle arterioles: role of EDH and K(+) channels.

As smooth muscle cell (SMC) membrane potential (E(m)) is critical for vascular responsiveness, and arteriolar SMCs are depolarized at physiological intraluminal pressures, we hypothesized that myogenic tone impacts on dilation mediated by endothelium-derived hyperpolarization (EDH). Studies were performed on cannulated mouse cremaster arterioles [diameter, 33+/-2 microm (n=23) at 60 mmHg; SMC Em -34.6+/-1.2 mV (n=7)]. Myogenic activity was assessed as tone developed in response to intraluminal pressure. Functional observations were related to mRNA, protein expression, and anatomy. Acetylcholine concentration-response curves showed a modest shift following indomethacin (10 microM) and L-NAME (100 microM), although maximal vasodilation was achieved. Residual dilation was removed by apamin (1 microM) in combination with TRAM-34 (1 microM) or charybotoxin (0.1 microM), indicating the requirement of small (S) and intermediate (I) calcium-activated potassium channels (K(Ca)). Charybdotoxin, but not TRAM-34, caused vasoconstriction, presumably through the inhibition of SMC BK(Ca). Expression of SK3 and IK1 was confirmed by immunohistochemistry and polymerase chain reaction, while myoendothelial junctions were common, suggesting a high degree of cell coupling. Also consistent with a role for endothelial K(Ca) channels, acetylcholine increased endothelium [Ca(2 +)](i). Apamin and TRAM-34 similarly blocked EDH-mediated dilation at intraluminal pressures of 30 and 90 mmHg, suggesting that in mouse arterioles, SK(Ca -) and IK(Ca -) mediated mechanisms predominate and operate independently of physiological levels of myogenic activation.

A role for heterocellular coupling and EETs in dilation of rat cremaster arteries.

OBJECTIVE: The authors probed endothelium-dependent dilation and endothelial cell Ca2+ handling in myogenically active resistance arteries. METHODS: First-order arteries were removed from rat cremaster muscles, cannulated, and pressurized (75 mmHg). Vessel diameter and endothelial cell Ca2+ were monitored using confocal microscopy, and arterial ultrastructure was determined using electron microscopy. RESULTS: Acetylcholine (ACh) stimulated elevations and oscillations in endothelial cell Ca2+, and concentration-dependently dilated arteries with myogenic tone. NO-independent dilation was blocked by 35 mM K+. Combined IK(Ca) (1 microM TRAM-34) and SK(Ca) (100 nM apamin) blockade partially inhibited NO-independent relaxations, with residual relaxations sensitive to BK(Ca) or cytochrome P-450 inhibition (100 nM iberiotoxin, and 20 microM 17-ODYA or 10 microM MS-PPOH). 11,12-EET stimulated iberiotoxin-sensitive dilation, but did not affect endothelial cell Ca2+. 15 mM K+ evoked dilation sensitive to inhibition of K(IR) (30 microM Ba2+) and Na+/K+-ATPase (10 microM ouabain), whereas these blockers did not affect ACh-mediated dilations. Homo- and heterocellular gap junctions were identified in radial sections through arteries. CONCLUSION: These data suggest that rises in endothelial cell Ca2+ stimulate SK(Ca) and IK(Ca) channels, leading to hyperpolarization and dilation, likely due to electrical coupling. In addition, a component was unmasked following SK(Ca) and IK(Ca) blockade, attributable to activation of BK(Ca) channels by cytochrome P-450 metabolites.

Endothelial cell Ca2+ increases are independent of membrane potential in pressurized rat mesenteric arteries.

In rat mesenteric arteries, the ability of ACh to evoke hyperpolarization of smooth muscle cells and consummate dilatation relies on an increase in endothelial cell cytosolic free [Ca2+] and activation of Ca2+-activated K+ channels (KCa). The time course of average and spatially organized rises in endothelial cell [Ca2+]i and concomitant effects on membrane potential were investigated in individual cells of pressurized arteries and isolated sheets of native cells stimulated with ACh. In both cases, ACh stimulated a sustained and oscillating rise in endothelial cell [Ca2+]i. Overall, the oscillations remained asynchronous between cells, yet occasionally localized intercellular coordination became evident. In pressurized arteries, repetitive waves of Ca2+ moved longitudinally across endothelial cells, and depended on Ca2+-store refilling. The rise in endothelial cell Ca2+ was associated with sustained hyperpolarization of endothelial cells in both preparations. This hyperpolarization was also evident when recording from smooth muscle cells in pressurized arteries, and from resting membrane potential, selective inhibition of small-conductance K Ca (SK Ca) with apamin (50 nM) was sufficient to inhibit this response. In the presence of phenylephrine-tone, both apamin and the selective inhibitor of intermediate conductance K Ca (IK Ca) TRAM-34 (1 microM) were required to inhibit the non-nitric oxide-mediated dilatation to ACh. When hyperpolarization of endothelial cells was fully prevented either with inhibitors of K Ca or in KCl (35 mM)-depolarized cells, both the time course and frequency of oscillations in endothelial cell [Ca2+]i to ACh were unaffected. Together, these data show that although a rise in endothelial cell [Ca2+]i stimulates hyperpolarization, depletion of intracellular stores with ACh stimulates Ca2+-influx which is not significantly influenced by the increase in cellular electrochemical gradient for Ca2+ caused by that hyperpolarization.