Nanofitin as a New Molecular-Imaging Agent for the Diagnosis of Epidermal Growth Factor Receptor Over-Expressing Tumors.

Epidermal growth-factor receptor (EGFR) is involved in cell growth and proliferation and is over-expressed in malignant tissues. Although anti-EGFR-based immunotherapy became a standard of care for patients with EGFR-positive tumors, this strategy of addressing cancer tumors by targeting EGFR with monoclonal antibodies is less-developed for patient diagnostic and monitoring. Indeed, antibodies exhibit a slow blood clearance, which is detrimental for positron emission tomography (PET) imaging. New molecular probes are proposed to overcome such limitations for patient monitoring, making use of low-molecular-weight protein scaffolds as alternatives to antibodies, such as Nanofitins with better pharmacokinetic profiles. Anti-EGFR Nanofitin B10 was reformatted by genetic engineering to exhibit a unique cysteine moiety at its C-terminus, which allows the development of a fast and site-specific radiolabeling procedure with 18F-4-fluorobenzamido-N-ethylamino-maleimide (18F-FBEM). The in vivo tumor targeting and imaging profile of the anti-EGFR Cys-B10 Nanofitin was investigated in a double-tumor xenograft model by static small-animal PET at 2 h after tail-vein injection of the radiolabeled Nanofitin 18F-FBEM-Cys-B10. The image showed that the EGFR-positive tumor (A431) is clearly delineated in comparison to the EGFR-negative tumor (H520) with a significant tumor-to-background contrast. 18F-FBEM-Cys-B10 demonstrated a significantly higher retention in A431 tumors than in H520 tumors at 2.5 h post-injection with a A431-to-H520 uptake ratio of 2.53 ± 0.18 and a tumor-to-blood ratio of 4.55 ± 0.63. This study provides the first report of Nanofitin scaffold used as a targeted PET radiotracer for in vivo imaging of EGFR-positive tumor, with the anti-EGFR B10 Nanofitin used as proof-of-concept. The fast generation of specific Nanofitins via a fully in vitro selection process, together with the excellent imaging features of the Nanofitin scaffold, could facilitate the development of valuable PET-based companion diagnostics.

Evaluation of pharmacokinetic interactions between long-acting HIV-1 fusion inhibitor albuvirtide and lopinavir/ritonavir, in HIV-infected subjects, combined with clinical study and simulation results.

1. A clinical study to assess the interactions between albuvirtide (320 mg) and lopinavir/ritonavir (400/100 mg) was conducted in 10 HIV-1-infected subjects. Because albuvirtide requires a long period to achieve steady state, and extended monotherapy may lead to early resistance, it is unethical to take albuvirtide alone to achieve steady state. Therefore, a population pharmacokinetic model was developed to predict steady-state concentration-time curve of solely administered albuvirtide. 2. When albuvirtide and lopinavir/ritonavir were co-administered, the plasma concentration of albuvirtide when the infusion ended (Cend) increased by about 34%, but the geometric mean ratios and 90% confidence intervals (90% CIs) of AUC(0-t) [1.09 (0.96-1.24)] and Ctrough [1.00 (0.83-1.20)] were within the range of 0.8-1.25. For lopinavir, the ratios (90% CIs) of AUC(0-t), Cmax and Ctrough were 0.63 (0.49-0.82), 0.67 (0.53-0.86) and 0.65 (0.46-0.91); for ritonavir, those ratios (90% CIs) were 0.62 (0.42-0.91), 0.61 (0.38-0.99) and 0.72 (0.40-1.26), respectively. 3. Co-administration of albuvirtide with lopinavir/ritonavir has little effect on albuvirtide exposure, but it decreases the plasma exposures of lopinavir/ritonavir. However, the drug-drug interactions may not reduce the effectiveness of this co-therapy, the trough concentration of lopinavir may be sufficient and this combination could achieve similar clinical efficacy with marketed drugs. So, a phase 3 clinical trial without dose adjustment is underway to validate their effectiveness and safety.

Synthesis and preliminary characterization of radioiodinated benzofuran-3-yl-(indol-3-yl)maleimide derivatives as potential SPECT imaging probes for the detection of glycogen synthase kinase-3ß (GSK-3ß) in the brain.

We report on the synthesis and preliminary characterization of two radioiodinated benzofuran-3-yl-(indol-3-yl)maleimides, 3-(benzofuran-3-yl)-4-(5-[(125) I]iodo-1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione ([(125) I]5), and 3-(5-[(125) I]iodo-1-methyl-1H-indol-3-yl)-4-(6-methoxybenzofuran-3-yl)-1H-pyrrole-2,5-dione ([(125) I]6), as the first potential SPECT imaging probes targeting glycogen synthase kinase-3ß (GSK-3ß). In this study, we used (125) I as a surrogate of (123) I because of its ease of use. The radioiodinated ligands were prepared from the corresponding tributyltin precursors through an iododestannylation reaction using hydrogen peroxide as an oxidant with a radiochemical yield of 10-30%. In vitro binding experiments suggested that both compounds show high affinity for GSK-3ß at a level similar to a known GSK-3ß inhibitor. Biodistribution studies with normal mice revealed that the radioiodinated compounds display sufficient uptake into (1.8%ID/g at 10 min postinjection) and clearance from the brain (1.0%ID/g at 60 min postinjection). These preliminary results suggest that the further optimization of radioiodinated benzofuran-3-yl-(indol-3-yl)maleimide derivatives may facilitate the development of clinically useful SPECT imaging probes for the in vivo detection of GSK-3ß.

A first-in-human phase I dose-escalation, pharmacokinetic, and pharmacodynamic evaluation of intravenous LY2090314, a glycogen synthase kinase 3 inhibitor, administered in combination with pemetrexed and carboplatin.

PURPOSE: LY2090314 (LY) is a glycogen synthase kinase 3 inhibitor with preclinical efficacy in xenograft models when combined with platinum regimens. A first-in-human phase 1 dose-escalation study evaluated the combination of LY with pemetrexed/carboplatin. PATIENTS AND METHODS: Forty-one patients with advanced solid tumors received single-dose LY monotherapy lead-in and 37 patients received LY (10-120 mg) plus pemetrexed/carboplatin (500 mg/m(2) and 5-6 AUC, respectively) across 8 dose levels every 21 days. Primary objective was maximum tolerated dose (MTD) determination; secondary endpoints included safety, antitumor activity, pharmacokinetics, and beta-catenin pharmacodynamics. RESULTS: MTD of LY with pemetrexed/carboplatin was 40 mg. Eleven dose-limiting toxicities (DLTs) occurred in ten patients. DLTs during LY monotherapy occurred at ≥ 40 mg: grade 2 visual disturbance (n = 1) and grade 3/4 peri-infusional thoracic pain during or shortly post infusion (n = 4; chest, upper abdominal, and back pain). Ranitidine was added after de-escalation to 80 mg LY to minimize peri-infusional thoracic pain. Following LY with pemetrexed/carboplatin therapy, DLTs included grade 3/4 thrombocytopenia (n = 4) and grade 4 neutropenia (n = 1). Best overall response by RECIST included 5 confirmed partial responses (non-small cell lung cancer [n = 3], mesothelioma, and breast cancer) and 19 patients having stable disease. Systemic LY exposure was approximately linear over dose range studied. Transient upregulation of beta-catenin measured in peripheral blood mononuclear cells (PBMCs) occurred at 40 mg LY. CONCLUSIONS: The initial safety profile of LY2090314 was established. MTD LY dose with pemetrexed/carboplatin is 40 mg IV every 3 weeks plus ranitidine. Efficacy of LY plus pemetrexed/carboplatin requires confirmation in randomized trials.

Rapid (18)F-labeling and loading of PEGylated gold nanoparticles for in vivo applications.

Water-soluble 3 nm maleimide-terminated PEGylated gold nanoparticles (maleimide-AuNP) were synthesized in both partially hydrolyzed and nonhydrolyzed forms. Both of these maleimide-AuNPs, when reacted with the silicon-fluorine prosthetic group [(18)F]SiFA-SH, resulted in radiolabeled AuNPs. These NPs were readily purified with high radiochemical yields (RCY) of 60-80% via size exclusion chromatography. Preliminary small animal positron emission tomography (PET) measurements in healthy rats gives information about the pathway of excretion and the stability of the radioactive label in vivo. The partially hydrolyzed [(18)F]SiFA-maleimide-AuNPs shows uptake in the brain region of interest (ROI) (> 0.13%ID/g) which was confirmed by ex vivo examination of the thoroughly perfused rat brain. The multiple maleimide end groups on the AuNP surface also allows for the simultaneous incorporation of [(18)F]SiFA-SH and a bioactive peptide (cysteine-modified octreotate, cys-TATE, which can bind to somatostatin receptor subtypes 2 and 5) in a proof-of-concept study. The well-defined Michael addition reaction between various thiol containing molecules and the multifunctionalized maleimide-AuNPs thus offers an opportunity to develop a new bioconjugation platform for new diagnostics as well as therapeutics.

Pharmacokinetics, metabolism, and excretion of the glycogen synthase kinase-3 inhibitor LY2090314 in rats, dogs, and humans: a case study in rapid clearance by extensive metabolism with low circulating metabolite exposure.

LY2090314 (3-[9-fluoro-2-(piperidin-1-ylcarbonyl)-1,2,3,4-tetrahydro[1,4]diazepino[6,7,1-hi]indol-7-yl]-4-imidazo[1,2-a]pyridin-3-yl-1H-pyrrole-2,5-dione) is an intravenous glycogen synthase kinase-3 inhibitor in oncology trials. Drug disposition was characterized after intravenous infusion of [(14)C]LY2090314 to rats and dogs, and was related to available clinical data. LY2090314 exhibited high clearance (approximating hepatic blood flow) and a moderate volume of distribution (∼1-2 l/kg) resulting in rapid elimination (half-life ∼0.4, 0.7, and 1.8-3.4 hours in rats, dogs, and humans, respectively). Scaled clearance from liver microsomes accurately predicted perfusion-limited clearance across species. LY2090314 was cleared by extensive metabolism, and the numerous metabolites were rapidly excreted into feces via bile (69-97% of dose; 62-93% within 0-24 hours); urinary recovery of drug-related material was low (≤3% of dose). Despite extensive metabolism, in rats and humans the parent compound was the sole identifiable drug-related moiety in plasma. Even in Mdr1a-, Bcrp-, and Mrp2-knockout rats, LY2090314 metabolites did not appear in circulation, and their urinary excretion was not enhanced, because the hypothesized impaired biliary excretion of metabolites in the absence of these canalicular transporters was not observed. Canine metabolite disposition was generally similar, with the notable exception of dog-unique LY2090314 glucuronide. This conjugate was formed in the dog liver and was preferentially excreted into the blood, where it accounted for the majority of circulating radioactivity at later times, and was predominantly recovered in urine (16% of dose). In conclusion, LY2090314 was rapidly cleared by extensive metabolism with negligible circulating metabolite exposures due to biliary excretion of metabolites into feces with no apparent intestinal reabsorption.

Evaluation of a maleimido derivative of NOTA for site-specific labeling of affibody molecules.

Radionuclide molecular imaging has the potential to improve cancer treatment by selection of patients for targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. The NOTA chelator forms stable complexes with a number of radionuclides suitable for SPECT or PET imaging. A maleimidoethylmonoamide NOTA (MMA-NOTA) has been prepared for site-specific labeling of Affibody molecules having a unique C-terminal cysteine. Coupling of the MMA-NOTA to the anti-HER2 Affibody molecule Z(HER2:2395) resulted in a conjugate with an affinity (dissociation constant) to HER2 of 72 pM. Labeling of [MMA-NOTA-Cys(61)]-Z(HER2:2395) with (111)In gave a yield of >95% after 20 min at 60 °C. In vitro cell tests demonstrated specific binding of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) to HER2-expressing cell lines. In mice bearing prostate cancer DU-145 xenografts, the tumor uptake of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) was 8.2 ± 0.9% IA/g and the tumor-to-blood ratio was 31 ± 1 (4 h postinjection). DU-145 xenografts were clearly visualized by a gamma camera. Direct in vivo comparison of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) and [(111)In-MMA-DOTA-Cys(61)]-Z(HER2:2395) demonstrated that both conjugates provided equal radioactivity uptake in tumors, but the tumor-to-organ ratios were better for [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) due to more efficient clearance from normal tissues. In conclusion, coupling of MMA-NOTA to a cysteine-containing Affibody molecule resulted in a site-specifically labeled conjugate, which retains high affinity, can be efficiently labeled, and allows for high-contrast imaging.

PET of insulinoma using ¹8F-FBEM-EM3106B, a new GLP-1 analogue.

Derived from endocrine pancreatic beta cells, insulinomas express glucagon-like peptide-1 (GLP-1) receptor with high density and incidence. In this study, we labeled a novel GLP-1 analogue, EM3106B, with (18)F and performed PET imaging to visualize insulinoma tumors in an animal model. A GLP-1 analogue that contains multiple lactam bridges, EM3106B, was labeled with (18)F through a maleimide-based prosthetic group, N-2-(4-(18)F-fluorobenzamido)ethylmaleimide ((18)F-FBEM). The newly developed radiotracer was characterized by cell based receptor-binding assay, cell uptake and efflux assay. The stability in serum was evaluated by radio-HPLC analysis. In vivo PET imaging was performed in nude mice bearing subcutaneous INS-1 insulinoma tumors and MDA-MB-435 tumors of melanoma origin. Ex vivo biodistribution study was performed to confirm the PET imaging data. EM3106B showed high binding affinity (IC(50) = 1.38 nM) and high cell uptake (5.25 ± 0.61% after 120 min incubation). (18)F-FBEM conjugation of EM3106B resulted in high labeling yield (24.9 ± 2.4%) and high specific activity (>75 GBq/µmol at the end of bombardment). EM3106B specifically bound and was internalized by GLP-1R positive INS-1 cells. After intravenous injection of 3.7 MBq (100 µCi) of (18)F-FBEM-EM3106B, the INS-1 tumors were clearly visible with high contrast in relation to the contralateral background on PET images, and tumor uptake of (18)F-FBEM-EM3106B was determined to be 28.5 ± 4.7 and 25.4 ± 4.1% ID/g at 60 and 120 min, respectively. (18)F-FBEM-EM3106B showed low uptake in MB-MDA-435 tumors with low level of GLP-1R expression. Direct tissue sampling biodistribution experiment confirmed high tracer uptake in INS-1 tumors and receptor specificity in both INS-1 tumor and pancreas. In conclusion, (18)F-FBEM-EM3106B exhibited GLP-1R-receptor-specific targeting properties in insulinomas. The favorable characteristics of (18)F-FBEM-EM3106B, such as high specific activity and high tumor uptake, and high tumor to nontarget uptake, demonstrate that it is a promising tracer for clinical insulinoma imaging.

Discovery of potent and bioavailable GSK-3beta inhibitors.

Here we report on the discovery of a series of maleimides which have high potency and good selectivity for GSK-3beta. The incorporation of polar groups afforded compounds with good bioavailability. The most potent compound 34 has an IC(50) of 0.6nM for GSK-3beta, over 100-fold selectivity against a panel of other kinases, and shows efficacy in rat osteoporosis models. The X-ray structure of GSK-3beta protein with 34 bound revealed the binding mode of the template and provided insights for future optimization opportunities.

Protein kinase C protects from DNA damage-induced necrotic cell death by inhibiting poly(ADP-ribose) polymerase-1.

The goal of the current study, conducted in freshly isolated thymocytes was (1) to investigate the possibility that the activation of poly(ADP-ribose) polymerase-1 (PARP-1) in an intact cell can be regulated by protein kinase C (PKC) mediated phosphorylation and (2) to examine the consequence of this regulatory mechanism in the context of cell death induced by the genotoxic agent. In cells stimulated by the PKC activating phorbol esters, DNA breakage was unaffected, PARP-1 was phosphorylated, 1-methyl-3-nitro-1-nitrosoguanidine-induced PARP activation and cell necrosis were suppressed, with all these effects attenuated by the PKC inhibitors GF109203X or Gö6976. Inhibition of cellular PARP activity by PKC-mediated phosphorylation may provide a plausible mechanism for the previously observed cytoprotective effects of PKC activators.

Pharmacokinetics and tolerability of a novel long-acting glucagon-like peptide-1 analog, CJC-1131, in healthy and diabetic subjects.

OBJECTIVE: The safety, tolerability, pharmacokinetics and preliminary pharmacodynamics of single rising doses of a novel GLP-1 analog, CJC-1131, was evaluated. METHODS: CJC-1131 was subcutaneously injected in 8 groups (1.5 - 20.5 microg/kg) of healthy subjects (each group of six subjects included 1 placebo per dose level). CJC-1131 was also injected subcutaneously in 6 groups (1.5 - 12 microg/kg) of Type 2 diabetic patients after a 9-day washout period from their own anti-diabetic medication. Each group of 8 patients included 2 placebo-treated patients. Seven blood glucose measurements were taken daily, and meal tolerance tests were performed on the day before dosing and on Day 3. RESULTS: CJC-1131 was quickly absorbed from the subcutaneous space, and a less than dose-proportional increase was found in Cmax. The half-life of CJC-1131 varied from 8.9 - 14.7 days in healthy subjects and from 9.1 - 13.8 days in patients. The maximum tolerated dose in healthy subjects was established at 12 microg/kg with nausea and vomiting being the dose-limiting events. These events occurred generally in the morning after dosing. Blood glucose levels in patients decreased on Day 1 in proportion with dose, with a maximum average decrease of 4.1 mmol/l in the highest dose group. Higher doses appeared to be related to a slight weight loss in patients. CONCLUSIONS: Conjugation to albumin led to a major prolongation of the half-life of GLP-1. The tolerability of this potential antidiabetic drug seems to be limited only by gastrointestinal complaints.

Mucoadhesive maleimide-functionalised liposomes for drug delivery to urinary bladder.

Intravesical drug administration is used to deliver chemotherapeutic agents via a catheter to treat bladder cancer. The major limitation of this treatment is poor retention of the drug in the bladder due to periodic urine voiding. In this work, maleimide-functionalised PEGylated liposomes (PEG-Mal) were explored as mucoadhesive vehicles for drug delivery to the urinary bladder. The retention of these liposomes on freshly excised porcine bladder mucosa in vitro was compared with conventional liposomes, PEGylated liposomes, two controls (dextran and chitosan), and evaluated through Wash Out50 (WO50) values. PEG-Mal liposomes exhibited greater retention on mucosal surfaces compared to other liposomes. The penetration abilities of conventional, PEG-Mal-functionalised and PEGylated liposomal dispersions with encapsulated fluorescein sodium into the bladder mucosa ex vivo were assessed using a fluorescence microscopy technique. PEGylated liposomes were found to be more mucosa-penetrating compared to other liposomes. All liposomes were loaded with fluorescein sodium salt as a model drug and the in vitro release kinetics was evaluated. Longer drug release was observed from PEG-Mal liposomes.

In situ low-immunogenic albumin-conjugating-corona guiding nanoparticles for tumor-targeting chemotherapy.

Nanoparticles (NPs) are unavoidably covered by a layer of immunogenic proteins upon injection into blood, such as immunoglobins and complements, which buries the active-targeting ligands and triggers the rapid clearance of NPs by the mononuclear phagocytic system. Low antifouling polyethylene glycol is used to inhibit the formation of the immunogenic corona but it leads to poor cellular uptake and the immunogen-related accelerated blood clearance (ABC) phenomenon in multiple administrations. Here, we develop surface maleimide-modified NPs that covalently conjugate in vivo plasma albumin in its corona upon exposure to blood. The in situ recruited low-immunogenic albumin-enriching corona is capable of protecting maleimide-decorated NPs from phagocytosis in the bloodstream, preventing the ABC phenomenon in the second administration, facilitating NP accumulation in the tumor site/cells by the passive EPR effect and albumin receptor-mediated active targeting, and finally improving the antitumor activity. Such findings suggest that the facile strategy, based on the in situ anchored albumin-enriching corona, is efficient at enabling maleimide-decorated NPs to acquire stealth and tumor-targeting ability.

Labeling of low-density lipoproteins using the 18F-labeled thiol-reactive reagent N-[6-(4-[18F]fluorobenzylidene)aminooxyhexyl]maleimide.

The novel thiol-group-selective bifunctional 18F-labeling agent N-[6-(4-[18F]fluoro-benzylidene)aminooxyhexyl]maleimide ([18F]FBAM) has been developed. The bifunctional labeling precursor N-(6-aminoxyhexyl)maleimide containing a thiol-reactive maleimide group and a carbonyl-group-reactive aminooxy group was prepared in only three steps in a total chemical yield of 59%. Subsequent radiolabeling with 4-[18F]fluorobenzaldehyde gave the bifunctional 18F-labeling agent [18F]FBAM in a radiochemical yield of 29%. In a typical experiment, 3.88 GBq of [18F]fluoride could be converted into 723 MBq of [18F]FBAM within 69 min. Conjugation of [18F]FBAM with thiol groups was exemplified with the cysteine-containing tripeptide glutathione and with various apolipoproteins of human low-density lipoprotein (LDL) subfractions. The latter was evaluated with respect to the uptake of [18F]FBAM-LDL subfractions in human hepatoma cells (HepG2) in vitro. In vivo biodistribution studies in rats revealed high stability for [18F]FBAM-LDL subfractions. Moreover, the metabolic fate of [18F]FBAM-LDL subfractions in vivo was delineated by dynamic positron emission tomography studies using a dedicated small animal tomograph. Data were compared to former studies that used the NH2-reactive 18F-labeling agent N-succinimidyl-4-[18F]fluorobenzoate. The compound [18F]FBAM can be considered as an excellent prosthetic group for the selective and mild 18F labeling of thiol-group-containing biomolecules suitable for subsequent investigations in vitro and in vivo.

Biodistribution of 211At labeled HER-2 binding affibody molecules in mice.

The size of affibody molecules makes them suitable as targeting agents for targeted radiotherapy with the alpha-emitter 211At, since their biokinetic properties match the short physical half-live of 211At. In this study, the potential for this approach was investigated in vivo. Two different HER-2 binding affibody molecules were radiolabeled with 211At using both the linker PAB (N-succinimidyl-para-astatobenzoate) and a decaborate-based linker, and the biodistribution in tumor-bearing nude mice was investigated. The influence of L-lysine and Na-thiocyanate on the 211At uptake in normal tissues was also studied. Based on the biokinetic information obtained, the absorbed dose was calculated for different organs. Compared with a previous biodistribution with 125I, the 211At biodistribution using the PAB linker showed higher uptake in lungs, stomach, thyroid and salivary glands, indicating release of free 211At. When the decaborate-based linker was used, the uptake in those organs was decreased, but instead, high uptake in kidneys and liver was found. The uptake, when using the PAB linker, could be significantly reduced in some organs by the use of L-lysine and/or Na-thiocyanate. In conclusion, affibody molecules have suitable blood-kinetics for targeted radionuclide therapy with 211At. However, the labeling chemistry affects the distribution in normal organs to a high degree and needs to be improved to allow clinical use.

Oxidation and haem loss kinetics of poly(ethylene glycol)-conjugated haemoglobin (MP4): dissociation between in vitro and in vivo oxidation rates.

Haemoglobin-based oxygen carriers can undergo oxidation of ferrous haemoglobin into a non-functional ferric form with enhanced rates of haem loss. A recently developed human haemoglobin conjugated to maleimide-activated poly(ethylene glycol), termed MP4, has unique physicochemical properties (increased molecular radius, high oxygen affinity and low cooperativity) and lacks the typical hypertensive response observed with most cell-free haemoglobin solutions. The rate of in vitro MP4 autoxidation is higher compared with the rate for unmodified SFHb (stroma-free haemoglobin), both at room temperature (20-22 degrees C) and at 37 degrees C (P<0.001). This appears to be attributable to residual catalase activity in SFHb but not MP4. In contrast, MP4 and SFHb showed the same susceptibility to oxidation by reactive oxygen species generated by a xanthine-xanthine oxidase system. Once fully oxidized to methaemoglobin, the rate of in vitro haem loss was five times higher in MP4 compared with SFHb in the fast phase, which we assign to the beta subunits, whereas the slow phase (i.e. haem loss from alpha chains) showed similar rates for the two haemoglobins. Formation of MP4 methaemoglobin in vivo following transfusion in rats and humans was slower than predicted by its first-order in vitro autoxidation rate, and there was no appreciable accumulation of MP4 methaemoglobin in plasma before disappearing from the circulation. These results show that MP4 oxidation and haem loss characteristics observed in vitro provide information regarding the effect of poly(ethylene glycol) conjugation on the stability of the haemoglobin molecule, but do not correspond to the oxidation behaviour of MP4 in vivo.

Ruboxistaurin: LY 333531.

Ruboxistaurin, an orally active protein kinase C beta (PKC beta) inhibitor, is a macrocyclic bisindolylmaleimide compound under development by Eli Lilly with potential as a therapy for diabetic macular oedema and other diabetic angiopathies, including diabetic retinopathy, diabetic peripheral neuropathy and diabetic nephropathy. Ruboxistaurin is awaiting approvals in the US and Europe for the treatment of diabetic retinopathy. Eli Lilly and Alcon entered into a long-term agreement to co-promote ruboxistaurin in the US and Puerto Rico for diabetic retinopathy. The agreement is subject to the US FDA's approval of the agent for this indication. Under the terms of the agreement, Alcon will assume primary responsibility for promotion to eye specialists including retinal specialists and general ophthalmologists, while Eli Lilly will be targeting endocrinologists and physicians. Subject to approval in the US, Eli Lilly will receive milestone and marketing payments from Alcon. Alcon in turn will receive compensation based on product sales. In December 2003, Eli Lilly signed a joint development and co-marketing agreement with Takeda Chemical Industries for ruboxistaurin in the Japanese market. Under the terms of the agreement, Eli Lilly Japan and Takeda will jointly develop ruboxistaurin in Japan, will file an NDA for diabetic peripheral neuropathy and diabetic retinopathy, and subsequently will market the drug in Japan. Ruboxistaurin was submitted for approval in Europe in the second quarter of 2006. The agent is also in phase II studies for the treatment of diabetic maculopathy (macular retinopathy) in Japan. Data from a phase III, 3-year study of ruboxistaurin in patients with moderate to severe diabetic retinopathy showed that ruboxistaurin markedly reduced the risk of sustained vision loss compared with placebo. This multicentre, randomised study, named PKC-DRS2 (Protein Kinase C-Diabetic Retinopathy Study 2), was conducted at 70 clinical sites and involved 685 patients with diabetic retinopathy. The agent is also in a phase II study in the US, Canada and Europe in patients with clinically significant macular oedema. The trial (B7A-MC-MBCU), which will evaluate oral administration of the drug using optical coherence tomography over a period of 18 months, is expected to enrol approximately 220 patients. This randomised, double-blind, placebo-controlled study was initiated in August 2005 and is expected to be completed in March 2008. Previously, results of the PKC-Diabetic Retinopathy Study (PKC-DRS) showed that ruboxistaurin at a dose of 32 mg/day has potential to reduce the risk of moderate vision loss especially in patients with diabetic macular oedema. This phase III, randomised, double-blind, multidose study in 252 patients with type 1 and type 2 diabetes receiving ruboxistaurin or placebo for 3-4 years evaluated the safety of the agent and its effect on progression of diabetic retinopathy, moderate vision loss and sustained moderate vision loss. The study was conducted at Joslin Diabetes Center and at centres in the US, Canada, Denmark, The Netherlands and the UK. In 2004, Eli Lilly presented new analysis of previously reported data for ruboxistaurin in diabetic macular oedema indicating that ruboxistaurin has the potential to decrease the progression of diabetic macular oedema involving the center of the macula. Positive results from the PKC Beta Inhibitor Diabetic Macular Edema (PKC-DMES) trial were reported in 2003. Eli Lilly expected to file for approval of ruboxistaurin for the treatment of diabetic peripheral neuropathy in the US and Europe in 2005. However, no development was reported for this indication. On 15 March 2007, Eli Lilly withdrew its marketing authorisation application for ruboxistaurin for diabetic retinopathy filed with EMEA in May 2006. Its current development status in the EU is unclear at this stage.

Regiospecific radiolabelling of Nanofitin on Ni magnetic beads with [18F]FBEM and in vivo PET studies.

INTRODUCTION: Nanofitins are low molecular weight, single chain and cysteine-free protein scaffolds able to selectively bind a defined biological target. They derive from Sac7d bacterial protein family and are highly stable over a wide range of pH (0-13) and temperature (Tm ~80°C). Their extreme stability, low cost of production and high tolerability for chemical coupling make Nanofitins a very interesting alternative to antibodies and their fragments. Here, a hexahistidine tagged model Nanofitin (H4) directed against hen egg white lysozyme was radiolabelled and injected in mice to provide a baseline biodistribution and pharmacokinetic profiles to support future Nanofitin development programs. METHOD: A single cysteine residue has been genetically inserted in a model Nanofitin and its regioselective radiolabelling has been performed with 4-[18F]fluorobenzamido-N-ethylamino-maleimide ([18F]FBEM). The synthesis of [18F]FBEM has been completely implemented on a radiosynthesis unit (FastLab) including HPLC purification and formulation. Coupling with the [18F]FBEM has been achieved on a solid support (Ni magnetic beads) allowing rapid purification at room temperature without organic solvent. PET-MRI studies on C57BL/6 mice were conducted after injection of [18F]FBEM-Cys-H4 in order to access the biodistribution of this Nanofitin model. RESULTS: Radiochemical yield (decay corrected) of 54±7% (n=4) was obtained after optimization for coupling the [18F]FBEM to Nanofitin. Pharmacokinetics results of [18F]FBEM-Cys-H4 revealed a fast clearance through the liver and the kidneys. CONCLUSION: An efficient new method on Ni magnetic beads was developed to radiolabelled his-tagged biomolecules with [18F]FBEM. This procedure was applied on a Nanofitin model Cys-H4 and biodistribution kinetic studies were achieved to evaluate the potential use of Nanofitin for diagnostic imaging. Fast clearance indicates that Nanofitins represent very interesting tools for diagnostic imaging.

Scaffold Hopping and Optimization of Maleimide Based Porcupine Inhibitors.

Porcupine is an O-acyltransferase that regulates Wnt secretion. Inhibiting porcupine may block the Wnt pathway which is often dysregulated in various cancers. Consequently porcupine inhibitors are thought to be promising oncology therapeutics. A high throughput screen against porcupine revealed several potent hits that were confirmed to be Wnt pathway inhibitors in secondary assays. We developed a pharmacophore model and used the putative bioactive conformation of a xanthine inhibitor for scaffold hopping. The resulting maleimide scaffold was optimized to subnanomolar potency while retaining good physical druglike properties. A preclinical development candidate was selected for which extensive in vitro and in vivo profiling is reported.

Effects of chronic renal failure on the pharmacokinetics of ruboxistaurin and its active metabolite 338522.

BACKGROUND: Ruboxistaurin, a specific inhibitor of the beta(1) and beta(2) isoforms of protein kinase C, is currently in clinical development for the treatment of several diabetic microvascular complications. The major metabolite, N-desmethyl ruboxistaurin (metabolite 338522), is equipotent in its inhibitory activity. The elimination of ruboxistaurin and its metabolites is primarily through bile and the faecal route, with urinary excretion constituting only a minor route. OBJECTIVE: To assess the effects of chronic renal insufficiency on the pharmacokinetics of ruboxistaurin and metabolite 338522. METHODS: Six healthy subjects (creatinine clearance >80 mL/min/1.73 m(2)) and six end-stage renal disease (ESRD) subjects requiring long-term haemodialysis were studied. All subjects received a single oral dose of ruboxistaurin 32 mg followed by serial blood sampling up to 72 hours. ESRD subjects underwent haemodialysis approximately 58 hours after dosing, with blood samples obtained immediately before and after dialysis. RESULTS: No differences were observed in the pharmacokinetic parameters (area under the plasma concentration-time curve from time zero to infinity [AUC(infinity)], maximum plasma concentration [C(max)] and elimination half-life [t(1/2)]) of ruboxistaurin and metabolite 338522 between healthy and ESRD subjects Plasma concentrations of ruboxistaurin were below the lower limit of quantification by the time of haemodialysis. The predicted post-dialysis plasma concentrations of metabolite 338522 were not statistically different from the observed values (p=0.163). Ruboxistaurin was well tolerated in both groups of subjects. CONCLUSION: These results indicate that the kidney is not an important route of metabolism or excretion for ruboxistaurin and metabolite 338522. Based on the pharmacokinetic and tolerability findings, no formal dosage adjustment of ruboxistaurin should be required for patients with any degree of renal impairment who are undergoing haemodialysis.