Impact of the molar activity and PSMA expression level on [18F]AlF-PSMA-11 uptake in prostate cancer.

This two-part preclinical study aims to evaluate prostate specific membrane antigen (PSMA) as a valuable target for expression-based imaging applications and to determine changes in target binding in function of varying apparent molar activities (MAapp) of [18F]AlF-PSMA-11. For the evaluation of PSMA expression levels, male NOD/SCID mice bearing prostate cancer (PCa) xenografts of C4-2 (PSMA+++), 22Rv1 (PSMA+) and PC-3 (PSMA-) were administered [18F]AlF-PSMA-11 with a medium MAapp (20.24 ± 3.22 MBq/nmol). SUVmean and SUVmax values were respectively 3.22 and 3.17 times higher for the high versus low PSMA expressing tumors (p < 0.0001). To evaluate the effect of varying MAapp, C4-2 and 22Rv1 xenograft bearing mice underwent additional [18F]AlF-PSMA-11 imaging with a high (211.2 ± 38.9 MBq/nmol) and/or low MAapp (1.92 ± 0.27 MBq/nmol). SUV values showed a significantly increasing trend with higher MAapp. Significant changes were found for SUVmean and SUVmax between the high versus low MAapp and medium versus low MAapp (both p < 0.05), but not between the high versus medium MAapp (p = 0.055 and 0.25, respectively). The effect of varying MAapp was more pronounced in low expressing tumors and PSMA expressing tissues (e.g. salivary glands and kidneys). Overall, administration of a high MAapp increases the detection of low expression tumors while also increasing uptake in PSMA expressing tissues, possibly leading to false positive findings. In radioligand therapy, a medium MAapp could reduce radiation exposure to dose-limiting organs with only limited effect on radionuclide accumulation in the tumor.

Quantitative and Qualitative Analyses of Biodistribution and PET Image Quality of a Novel Radiohybrid PSMA, 18F-rhPSMA-7, in Patients with Prostate Cancer.

Radiohybrid PSMA (rhPSMA) ligands, a new class of theranostic prostate-specific membrane antigen (PSMA)-targeting agents, feature fast 18F synthesis and utility for labeling with radiometals. Here, we assessed the biodistribution and image quality of 18F-rhPSMA-7 to determine the best imaging time point for patients with prostate cancer. Methods: In total, 202 prostate cancer patients who underwent a clinically indicated 18F-rhPSMA-7 PET/CT were retrospectively analyzed, and 12 groups based on the administered activity and uptake time of PET scanning were created: 3 administered activities (low, 222-296 MBq; moderate, 297-370 MBq; and high, 371-444 MBq) and 4 uptake time points (short, 50-70 min; intermediate, 71-90 min; long, 91-110 min; and extra long, ≥111 min). For quantitative analyses, SUVmean and organ- or tumor-to-background ratio were determined for background, healthy organs, and 3 representative tumor lesions. Qualitative analyses assessed overall image quality, nonspecific blood-pool activity, and background uptake in bone or marrow using 3- or 4-point scales. Results: In quantitative analyses, SUVmean showed a significant decrease in the blood pool and lungs and an increase in the kidneys, bladder, and bones as the uptake time increased. SUVmean showed a trend to increase in the blood pool and bones as the administered activity increased. However, no significant differences were found in 377 tumor lesions with respect to the administered activity or uptake time. In qualitative analyses, the overall image quality was stable along with the uptake time, but the proportion rated to have good image quality decreased as the administered activity increased. All other qualitative image parameters showed no significant differences for the administered activities, but they showed significant trends with increasing uptake time: less nonspecific blood activity, more frequent background uptake in the bone marrow, and increased negative impact on clinical decision making. Conclusion: The biodistribution of 18F-rhPSMA-7 was similar to that of established PSMA ligands, and tumor uptake of 18F-rhPSMA-7 was stable across the administered activities and uptake times. An early imaging time point (50-70 min) is recommended for 18F-rhPSMA-7 PET/CT to achieve the highest overall image quality.

Radiation Dosimetry and Biodistribution of 18F-PSMA-11 for PET Imaging of Prostate Cancer.

Prostate-specific membrane antigen (PSMA) is highly overexpressed in prostate cancer. Many PSMA analog radiotracers for PET/CT prostate cancer staging have been developed, such as 68Ga-PSMA-11. This radiotracer has achieved good results in multiple clinical trials, but because of the superior imaging characteristics of 18F-fluoride, 18F-PSMA-11 was developed. The aim of this study was to evaluate the administration safety and radiation dosimetry of 18F-PSMA-11. Methods: Six patients (aged 62-68 y; mean, 66 ± 2 y) with suspected prostate cancer recurrence after previous treatment were administered 2 MBq of 18F-PSMA-11 per kilogram of body weight and then underwent low-dose PET/CT imaging at 0, 20, 50, 90, and 300 min after injection. To evaluate the safety of administration, vital parameters were monitored. To assess toxicity, full blood count and biochemical parameters were determined. According to the latest International Commission on Radiological Protection recommendations, radiation dosimetry analysis was performed using IDAC-Dose 2.1. For blood activity measurement, small samples of venous blood were collected at various time points after injection. The unbound 18F-fluoride fraction was determined in plasma at 20, 50, and 90 min after administration to evaluate the defluorination rate of 18F-PSMA-11. Results: After injection, 18F-PSMA-11 cleared rapidly from the blood. At 5 h after injection, 29.0% ± 5.9% of the activity was excreted in urine. The free 18F fraction in plasma increased from 9.7% ± 1.0% 20 min after injection to 22.2% ± 1.5% 90 min after injection. The highest tracer uptake was observed in kidneys, bladder, spleen, and liver. No study drug-related adverse events were observed. The calculated mean effective dose was 12.8 ± 0.6 µSv/MBq. Conclusion:18F-PSMA-11 can be safely administered and results in a mean effective dose of 12.8 ± 0.6 µSv/MBq. Therefore, the total radiation dose is lower than for other PSMA PET agents and in the same range as 18F-DCFPyL.

Analysis of the Lipolytic Activity of Whole-Saliva and Site-Specific Secretions from the Oral Cavity of Healthy Adults.

It is currently unclear how the process of fat digestion occurs in the mouth of humans. This pilot study therefore aimed to quantify the levels of lipolytic activity at different sites of the mouth and in whole saliva. Samples of whole saliva and from 4 discrete sites in the oral cavity were collected from 42 healthy adult participants. All samples were analyzed for lipolytic activity using two different substrates (olive oil and the synthetic 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR)). Bland⁻Altman analyses suggested that the two assays gave divergent results, with 91% and 23% of site-specific and 40% and 26% of whole-saliva samples testing positive for lipolytic activity, respectively. Non-parametric multiple comparisons tests highlighted that median (IQR) of lipolytic activity (tested using the olive oil assay) of the samples from the parotid 20.7 (11.7⁻31.0) and sublingual 18.4 (10.6⁻47.2) sites were significantly higher than that of whole saliva 0.0 (0.0⁻35.7). In conclusion, lipolysis appears to occur in the oral cavity of a proportion of individuals. These findings give a preliminary indication that lipolytic agent activity in the oral cavity may be substrate-specific but do not discount that the enzyme is from sources other than oral secretions (e.g., microbes, gastric reflux).

Lipophilic nalmefene prodrugs to achieve a one-month sustained release.

Nalmefene is an opioid antagonist which as a once-a-day tablet formulation has recently been approved for reducing ethanol intake in alcoholic subjects. In order to address the compliance issue in this patient population, a number of potential nalmefene prodrugs were synthesized with the aim of providing a formulation that could provide plasma drug concentrations in the region of 0.5-1.0ng/mL for a one-month period when dosed intramuscular to dogs or minipigs. In an initial series of studies, three different lipophilic nalmefene derivatives were evaluated: the palmitate (C16), the octadecyl glutarate diester (C18-C5) and the decyl carbamate (CB10). They were administered intramuscularly to dogs in a sesame oil solution at a dose of 1mg-eq. nalmefene/kg. The decyl carbamate was released relatively quickly from the oil depot and its carbamate bond was too stable to be used as a prodrug. The other two derivatives delivered a fairly constant level of 0.2-0.3ng nalmefene/mL plasma for one month and since there was no significant difference between these two, the less complex palmitate monoester was chosen to demonstrate that dog plasma nalmefene concentrations were dose-dependent at 1, 5 and 20mg-eq. nalmefene/kg. In a second set of experiments, the effect of the chain length of the fatty acid monoester promoieties was examined. The increasingly lipophilic octanoate (C8), decanoate (C10) and dodecanoate (C12) derivatives were evaluated in dogs and in minipigs, at a dose of 5mg-eq. nalmefene/kg and plasma nalmefene concentrations were measured over a four-week period. The pharmacokinetic profiles were very similar in both species with Cmax decreasing and Tmax increasing with increasing fatty acid chain length and the target plasma concentrations (0.5-1.0ng/mL over a month-long period) were achieved with the dodecanoate (C12) prodrug. These data therefore demonstrate that sustained plasma nalmefene concentrations can be achieved in both dog and minipig using nalmefene prodrugs and that the pharmacokinetic profile of nalmefene can be tuned by varying the length of the alkyl group.

Selective CNS Uptake of the GCP-II Inhibitor 2-PMPA following Intranasal Administration.

Glutamate carboxypeptidase II (GCP-II) is a brain metallopeptidase that hydrolyzes the abundant neuropeptide N-acetyl-aspartyl-glutamate (NAAG) to NAA and glutamate. Small molecule GCP-II inhibitors increase brain NAAG, which activates mGluR3, decreases glutamate, and provide therapeutic utility in a variety of preclinical models of neurodegenerative diseases wherein excess glutamate is presumed pathogenic. Unfortunately no GCP-II inhibitor has advanced clinically, largely due to their highly polar nature resulting in insufficient oral bioavailability and limited brain penetration. Herein we report a non-invasive route for delivery of GCP-II inhibitors to the brain via intranasal (i.n.) administration. Three structurally distinct classes of GCP-II inhibitors were evaluated including DCMC (urea-based), 2-MPPA (thiol-based) and 2-PMPA (phosphonate-based). While all showed some brain penetration following i.n. administration, 2-PMPA exhibited the highest levels and was chosen for further evaluation. Compared to intraperitoneal (i.p.) administration, equivalent doses of i.n. administered 2-PMPA resulted in similar plasma exposures (AUC0-t, i.n./AUC0-t, i.p. = 1.0) but dramatically enhanced brain exposures in the olfactory bulb (AUC0-t, i.n./AUC0-t, i.p. = 67), cortex (AUC0-t, i.n./AUC0-t, i.p. = 46) and cerebellum (AUC0-t, i.n./AUC0-t, i.p. = 6.3). Following i.n. administration, the brain tissue to plasma ratio based on AUC0-t in the olfactory bulb, cortex, and cerebellum were 1.49, 0.71 and 0.10, respectively, compared to an i.p. brain tissue to plasma ratio of less than 0.02 in all areas. Furthermore, i.n. administration of 2-PMPA resulted in complete inhibition of brain GCP-II enzymatic activity ex-vivo confirming target engagement. Lastly, because the rodent nasal system is not similar to humans, we evaluated i.n. 2-PMPA also in a non-human primate. We report that i.n. 2-PMPA provides selective brain delivery with micromolar concentrations. These studies support intranasal delivery of 2-PMPA to deliver therapeutic concentrations in the brain and may facilitate its clinical development.

Preclinical evaluation of BAY 1075553, a novel (18)F-labelled inhibitor of prostate-specific membrane antigen for PET imaging of prostate cancer.

PURPOSE: Prostate-specific membrane antigen (PSMA) is a transmembrane protein overexpressed in prostate cancer and is therefore being explored as a biomarker for diagnosing and staging of the disease. Here we report preclinical data on BAY 1075553 (a 9:1 mixture of (2S,4S)- and (2R,4S)-2-[(18)F]fluoro-4-phosphonomethyl-pentanedioic acid), a novel (18)F-labelled small molecule inhibitor of PSMA enzymatic activity, which can be efficiently synthesized from a direct radiolabelling precursor. METHODS: The (18)F-radiolabelled stereoisomers of 2-[(18)F]fluoro-4-(phosphonomethyl)-pentanedioic acid were synthesized from their respective isomerically pure precursors dimethyl 2-{[bis(benzyloxy)phosphoryl]methyl}-4-(tosyloxy)pentanedioate. In vivo positron emission tomography (PET) imaging and biodistribution studies were conducted in mice bearing LNCaP, 22Rv1 and PC-3 tumours. Pharmacokinetic parameters and dosimetry estimates were calculated based on biodistribution studies in rodents. For non-clinical safety assessment (safety pharmacology, toxicology) to support a single-dose human microdose study, off-target effects in vitro, effects on vital organ functions (cardiovascular in dogs, nervous system in rats), mutagenicity screens and an extended single-dose study in rats were conducted with the non-radioactive racemic analogue of BAY 1075553. RESULTS: BAY 1075553 showed high tumour accumulation specific to PSMA-positive tumour-bearing mice and was superior to other stereoisomers tested. Fast clearance of BAY 1075553 resulted overall in low background signals in other organs except for high uptake into kidney and bladder which was mainly caused by renal elimination of BAY 1075553. A modest uptake into bone was observed which decreased over time indicating organ-specific uptake as opposed to defluorination of BAY 1075553 in vivo. Biodistribution studies found highest organ doses for kidneys and the urinary bladder wall resulting in a projected effective dose (ED) in humans of 0.0219 mSv/MBq. Non-clinical safety studies did not show off-target activity, effects on vital organs function or dose-dependent adverse effects. CONCLUSION: BAY 1075553 was identified as a promising PET tracer for PSMA-positive prostate tumours in preclinical studies. BAY 1075553 can be produced using a robust, direct radiosynthesis procedure. Pharmacokinetic, toxicology and safety pharmacology studies support the application of BAY 1075553 in a first-in-man microdose study with single i.v. administration.

Salt and cocrystals of sildenafil with dicarboxylic acids: solubility and pharmacokinetic advantage of the glutarate salt.

Sildenafil is a drug used to treat erectile dysfunction and pulmonary arterial hypertension. Because of poor aqueous solubility of the drug, the citrate salt, with improved solubility and pharmacokinetics, has been marketed. However, the citrate salt requires an hour to reach its peak plasma concentration. Thus, to improve solubility and bioavailability characteristics, cocrystals and salts of the drug have been prepared by treating aliphatic dicarboxylic acids with sildenafil; the N-methylated piperazine of the drug molecule interacts with the carboxyl group of the acid to form a heterosynthon. Salts are formed with oxalic and fumaric acid; salt monoanions are formed with succinic and glutaric acid. Sildenafil forms cocrystals with longer chain dicarboxylic acids such as adipic, pimelic, suberic, and sebacic acids. Auxiliary stabilization via C-H···O interactions is also present in these cocrystals and salts. Solubility experiments of sildenafil cocrystal/salts were carried out in 0.1N HCl aqueous medium and compared with the solubility of the citrate salt. The glutarate salt and pimelic acid cocrystal dissolve faster than the citrate salt in a two hour dissolution experiment. The glutarate salt exhibits improved solubility (3.2-fold) compared to the citrate salt in water. Solubilities of the binary salts follow an inverse correlation with their melting points, while the solubilities of the cocrystals follow solubilities of the coformer. Pharmacokinetic studies on rats showed that the glutarate salt exhibits doubled plasma AUC values in a single dose within an hour compared to the citrate salt. The high solubility of glutaric acid, in part originating from the strained conformation of the molecule and its high permeability, may be the reason for higher plasma levels of the drug.

Pharmacokinetics and pharmacodynamics of the glutamate carboxypeptidase II inhibitor 2-MPPA show prolonged alleviation of neuropathic pain through an indirect mechanism.

Glutamate carboxypeptidase II (GCP II) is a therapeutic target in neurologic disorders associated with excessive activation of glutamatergic systems. The potent, orally bioavailable GCP II inhibitor 2-(3-mercaptopropyl) pentanedioic acid (2-MPPA) is effective in preclinical models of diseases where excess glutamate release is implicated, including neuropathic pain, and was the first GCP II inhibitor to be administered to man. The relationships between dosing regimen, pharmacokinetics, and analgesia in a neuropathic pain model were examined in rats to aid development of clinical dosing. The efficacy of oral 2-MPPA in the chronic constrictive injury model was not simply related to plasma concentrations. Even though maximal concentrations were observed within 1 hour of dosing, the analgesic effect took at least 8 days of daily dosing to become significant. The delay was not due to tissue drug accumulation since inhibitory concentrations of the drug were achieved in the nerve within 1 hour of dosing. There was also no accumulation of drug in plasma or tissue after multiple daily dosing. Effects were dependent on reaching a threshold concentration since dividing the daily dose led to a loss of effect. The analgesic effect outlasted plasma exposure and was maintained for days even after daily dosing was halted. The delayed onset, dependence on threshold plasma concentration, and sustained effects after exposure support the hypothesis that an indirect, long-lived mechanism of action exists. Although these longer lasting secondary mechanisms are not yet identified, daily clinical dosing of a GCP II inhibitor seems justified.

Synthesis and evaluation of diastereoisomers of 1,4,7-triazacyclononane-1,4,7-tris-(glutaric acid) (NOTGA) for multimeric radiopharmaceuticals of gallium.

In the conventional synthesis of 1,4,7-tris-(glutaric acid)-1,4,7-triazacyclononane (NOTGA), four isomeric species are usually generated by the alkylation of 1,4,7-triazacyclononane with α-bromoglutaric acid diester. To estimate their biological efficacies as well as their stability and radiochemistry, the RRR/SSS and RRS/SSR NOTGA-(t)Bu prochelators were isolated and the corresponding cyclic RGDfK (RGD) conjugates with triethylene glycol linkages were prepared. The RRR/SSS and RRS/SSR diastereomers were obtained in 69% and 17% yields, respectively. In the complexation reaction with (67)GaCl(3), both diastereomers provided >98% radiochemical yields at pH 5 within 10 min when the reaction was conducted at room temperature. However, the RRR/SSS diastereomer exhibited more pH-sensitive radiochemical yields between pH 3.5 to 4.5. Despite their diasteromeric nature, both (67)Ga-labeled RGD-NOTGA remained stable during the apo-transferrin challenge, exhibiting similar affinity for integrin α(v)ß(3) and biodistribution with predominant renal excretion. Similar tumor uptake was also observed in mice bearing U87MG tumor xenograft, which resulted in impressively high contrast SPECT/CT images. These findings indicate that the RGD-NOTGA conjugates of both diastereomers presented here possess equivalent biological efficacies and their combined usage would be feasible. It is worth noting that specific properties of a given biomolecule, cell expression levels of the corresponding target molecule, and presence or absence of a pharmacokinetic modifier would affect the structural differences between diastereomers on the ligand-receptor interactions and pharmacokinetics. Thus, the preparation of corresponding conjugates and evaluation of their chemical and biological performances still remains important for applying NOTGA to other biomolecules of interest using the diastereomerically pure NOTGA-(t)Bu prochelator.

Phase I, pharmacokinetic and biological correlative study of OSI-7904L, a novel liposomal thymidylate synthase inhibitor, and cisplatin in patients with solid tumors.

PURPOSE: To evaluate the safety and describe the pharmacokinetic profile of OSI-7904L, a novel liposomal thymidylate synthase inhibitor, in combination with cisplatin (CDDP) in adults with advanced solid tumors. EXPERIMENTAL DESIGN: CDDP was administered as a 2-h intravenous infusion followed by OSI-7904L intravenously over 30 min, both given every 3 weeks. Doses of each drug were escalated in separate cohorts of patients. Five dose levels of CDDP/OSI-7904L were explored: 60/6, 60/9, 60/12, 60/7.5, and 75/7.5 mg/m2. Pharmacokinetic samples, baseline plasma homocysteine, and genotype polymorphisms were evaluated. RESULTS: Twenty-seven patients were treated with 101 total courses of CDDP/OSI-7904L. Dose-limiting toxicity was observed in 2 patients in the CDDP/OSI-7904L 60/12 mg/m2 cohort. One patient experienced rash, stomatitis, dehydration, renal failure, hyperbilirubinemia, and fatal neutropenic sepsis, whereas the other patient experienced grade 3 nausea, vomiting, and ileus. Therefore, the CDDP/OSI-7904L 60/9 mg/m2 cohort was expanded, with 2 of 6 patients reporting significant fatigue. Other toxicities were mild or moderate. Intermediate dose levels of 60/7.5 and 75/7.5 mg/m2 were evaluated, and the latter was identified as the recommended dose for phase II studies. No major pharmacokinetic interactions between CDDP and OSI-7904L were observed. Three patients had partial responses (gastric adenocarcinoma and heavily pretreated breast cancer). There was no significant relationship between baseline homocysteine and toxicity. CONCLUSIONS: The recommended doses for CDDP and OSI-7904L administered once every 3 weeks are 75 and 7.5 mg/m2, respectively. Pharmacokinetic interaction between the agents was not apparent. Preliminary clinical activity was observed in breast and gastric cancer.

Glutaric acid administration impairs energy metabolism in midbrain and skeletal muscle of young rats.

A genetic mice model of glutaric acidemia type I (GAI) has recently been developed, however affected animals do not develop the striatal damage characteristic of patients with this disorder. Therefore, the initial aim of the present work was to induce high glutaric acid (GA) concentrations in rat brain similar to those found in GAI patients through subcutaneous injection of GA. High brain GA concentrations (up to 0.60 micromol/g congruent with 0.60mM) were achieved by a single subcutaneous injection of saline-buffered GA (5 micromol/g body weight) to Wistar rats of 7-22 days of life. GA brain levels were about 10-fold lower than in plasma and 5-fold lower than in skeletal and cardiac muscles, indicating that the permeability of the blood brain barrier to GA is low. We also aimed to use this model to investigate neurochemical parameters in the animals. Thus, we evaluated the effect of this model on energy metabolism parameters in midbrain, in which the striatum is localized, as well as in peripheral tissues (skeletal and cardiac muscles) of 22-day-old rats. Control rats were treated with saline in the same volumes. We verified that CO2 production from glucose was not altered in midbrain of rats treated with GA, indicating a normal functioning of the tricarboxylic acid cycle. Creatine kinase activity was also not changed in midbrain, skeletal and cardiac muscles. In contrast, complex I-III activity of the respiratory chain was inhibited in midbrain (25%), while complexes I-III (25%) and II-III (15%) activities were reduced in skeletal muscle, with no alterations found in cardiac muscle. These data indicate that GA administration moderately impairs cellular energy metabolism in midbrain and skeletal muscle of young rats.

The central nervous system effects, pharmacokinetics and safety of the NAALADase-inhibitor GPI 5693.

AIM: The aim was to assess the central nervous system (CNS) effects, pharmacokinetics and safety of GPI 5693, an inhibitor of a novel CNS-drug target, NAALADase which is being evaluated for the treatment of neuropathic pain. METHODS: This was a double-blind, placebo-controlled, exploratory study in healthy subjects receiving oral GPI 5693 single ascending doses of 100, 300, 750, 1125 mg with a placebo treatment randomly interspersed. An open-label, parallel extension examined the effects of food and sex on the pharmacokinetics of 750, 1125 and 1500 mg doses. Blood samples were collected for pharmacokinetic and biochemical/haematological safety analysis, vital signs, ECG and adverse event checks were performed regularly up to 48 h postdose. Postdose CNS effects were assessed using eye movements, adaptive tracking, electroencephalography (EEG), body sway and Visual Analogue Scales (VAS). RESULTS: CNS effects were mainly observed after the 1125 mg dose, showing a significant decrease of adaptive tracking performance, VAS alertness and VAS mood, and an increase of EEG occipital alpha and theta power. Gastro-intestinal (GI) adverse effects were frequent at higher doses. No clinically significant changes in vital signs or ECG were noted during any of the treatments. The therapeutically relevant concentration range (950-11 100 ng ml(-1)) as determined from animal experiments was already reached after the 300 mg dose. C(max) after the 300 mg and 750 mg dose was 2868 and 9266 ng ml(-1) with a t(1/2) of 2.54 and 4.78 h, respectively. Concomitant food intake (with the 750 mg and 1125 mg doses) reduced C(max) by approximately 66% and AUC by approximately 40%. With concomitant food intake, the dose-normalized C(max) also decreased significantly by -5.6 (CI: -2.6 to -8.7) ng ml(-1) mg(-1). The pharmacokinetic variability was largest after the 300 mg and 750 mg dose, resulting in a SD of approximately 50% of the C(max). CONCLUSION: NAALADase inhibition with GPI 5693 was safe and tolerable in healthy subjects. Plasma concentrations that were effective in the reversal of hyperalgesia in the chronic constrictive injury animal model of neuropathic pain were obtained at doses of 300, 750 and 1125 mg in the fasted state. Comcomitant food intake reduced C(max) and AUC. CNS effects and GI AEs increased in incidence over placebo only at the 1125 mg dose.

Neuromuscular pharmacology of TAAC3, a new nondepolarizing muscle relaxant with rapid onset and ultrashort duration of action.

UNLABELLED: We selected bis [N-(3,4-diacetoxybenzyl) tropanium-3alpha-yl] glutarate dibromide (TAAC3) from many new tropinyl diester derivatives to evaluate its neuromuscular blocking (NMB) and autonomic side effects on anesthetized rats, rabbits, guinea pigs, cats, pigs, dogs, and monkeys. NMB potency, onset, recovery index, and duration of action were determined. Comparisons of these pharmacologic variables were made between TAAC3 and rocuronium. In the cat, the degrees of train-of-four and tetanic fade, posttetanic potentiation, and pharmacologic antagonism were evaluated. For determination of the NMB maintenance dose, TAAC3 was also given to rabbits and pigs in the initial dose/maintenance infusion mode. Cardiac vagal block was evaluated in the rat, pig, cat, and guinea pig on the basis of the inhibition of the bradycardia to stimulation of the vagus nerve. Sympathetic ganglion block was studied on the superior cervical ganglion-nictitating membrane preparation of the cat. TAAC3 produced nondepolarizing NMB. Its NMB 90% effective doses ranged from 90 to 425 microg/kg, depending on the species. TAAC3 had a faster onset (0.8-1.0 min), shorter recovery index (0.6-1.1 min), and shorter duration of action (1.8-3.5 min) than rocuronium. It produced a slight cumulative effect on infusion, but not on repeated single-dose administration. Cardiac vagal block was present at doses exceeding the NMB 90% effective dose. In the cat and pig at equipotent NMB doses, the degree of cardiac vagal block was similar to that of rocuronium. There was no demonstrable sympathetic ganglion block in the cat. In view of its favorable NMB characteristics, TAAC3 is now undergoing detailed preclinical studies. IMPLICATIONS: We developed a new nondepolarizing muscle relaxant, TAAC3, and investigated it in several animal models. TAAC3 has shown a very rapid onset and an ultrashort duration of neuromuscular blocking action. A minor degree of cardiac vagal block was observed. TAAC3 is promising for further studies.

11C-MCG: synthesis, uptake selectivity, and primate PET of a probe for glutamate carboxypeptidase II (NAALADase).

Imaging of glutamate carboxypeptidase II (GCP II), also known as N-acetylated alpha-linked L-amino dipeptidase (NAALADase), may enable study of glutamatergic transmission, prostate cancer, and tumor neovasculature in vivo. Our goal was to develop a probe for GCP II for use with positron emission tomography (PET). Radiosynthesis of 11C-MeCys-C(O)-Glu or 11C-(S)-2-[3-((R)-1-carboxy-2-methylsulfanyl-ethyl)-ureido]-pentanedioic acid (11C-MCG), an asymmetric urea and potent (Ki = 1.9 nM) inhibitor of GCP II, was performed by C-11 methylation of the free thiol. Biodistribution of 11C-MCG was assayed in mice, and quantitative PET was performed in a baboon. 11C-MCG was obtained in 16% radiochemical yield at the end of synthesis with specific radioactivities over 167 GBq/mmol (4000 Ci/mmol) within 30 min after the end of bombardment. At 30 min postinjection, 11C-MCG showed 33.0 +/- 5.1%, 0.4 +/- 0.1%, and 1.1 +/- 0.2% ID/g in mouse kidney (target tissue), muscle, and blood, respectively. Little radioactivity gained access to the brain. Blockade with unlabeled MCG or 2-(phosphonomethyl)pentanedioic acid (PMPA), another potent inhibitor of GCP II, provided sevenfold and threefold reductions, respectively, in binding to target tissue. For PET, distribution volumes (DVs) were 1.38 then 0.87 pre- and postblocker (PMPA). Little metabolism of 11C-MCG occurred in the mouse or baboon. These results suggest that 11C-MCG may be useful for imaging GCP II in the periphery.

Cationic amino acids involved in dicarboxylate binding of the flounder renal organic anion transporter.

Three conserved cationic amino acids in predicted transmembrane domains 1, 8, and 11, respectively, of the flounder renal organic anion transporter, fROAT, were changed by site-directed mutagenesis and the resulting mutants functionally characterized in Xenopus laevis oocytes. Uptake of p-aminohippurate (PAH) in oocytes that expressed mutant H34I, K394A, or R478D was markedly reduced compared with oocytes that expressed wild-type fROAT, but was still several-fold higher than that in water-injected control oocytes. Immunocytochemically, no decrease in cell surface expression of the mutants could be detected. Only mutant R478D appeared to have a lower PAH affinity than the wild type. Similar to wild-type-dependent PAH transport, uptake induced by mutant H34I was sensitive to glutarate (GA) cis-inhibition. In contrast, mutants K394A and R478D could not be significantly affected by up to 10 mM GA, although the cRNA-dependent PAH uptake could still be almost completely suppressed by probenecid. Moreover, again in contrast to the wild type, neither PAH influx nor PAH efflux mediated by these two mutants could be trans-stimulated by GA, nor did they induce GA transport. These data suggest that amino acids K394 and R478 in fROAT are required for dicarboxylate binding and PAH/dicarboxylate exchange.

Role of the calcium/calmodulin-dependent protein kinase II in the regulation of the renal basolateral PAH and dicarboxylate transporters.

The aim of the present study was to investigate whether the activities of the renal basolateral organic anion transporter (PAH transporter) and the sodium-dependent dicarboxylate transporter are modulated by the calcium/calmodulin-dependent multifunctional protein kinase II (CaM kinase II). The studies were performed on isolated S2 segments of proximal tubules microdissected from rabbit kidneys without the use of enzymatic agents. 3H-PAH was used as marker substance of the anion transporter, and 14C-glutarate as a marker of the sodium/dicarboxylate cotransporter. Because the tubules were not perfused, and hence were collapsed, the tubular uptake of the marker substances reflects transport across the basolateral cell membrane. To obtain uptake rates most closely related to initial transport rates, 30 s tubular uptake measurements were performed. The results show that a selective inhibitor of CaM kinase II, KN93, inhibited tubular PAH uptake. The smallest effective dose was 10(-7) M. An inactive analogue of KN93, KN92, was without effect, even at the high concentration of 10(-5) M. In contrast to PAH transport, tubular 14C-glutarate uptake was not affected by KN93 (10(-5) M). PAH transport was also inhibited after elevation of intracellular Ca2+ by the Ca(2+)-ionophore A 23187 and by the polycationic antibiotic neomycin, but not by the intracellular Ca2+ modulators thapsigargin and ryanodine. The effect of the Ca(2+)-ionophore could be abolished by KN93, but not by Rp-cAMPs, an inhibitor of protein kinase A, indicating that this event was mediated by CaM kinase II, but not by PKA. The results provide the first evidence that, in addition to the protein kinases A and C (previous studies from this lab), CaM kinase II has a role in the regulation of the renal basolateral PAH transporter, whereas the renal basolateral dicarboxylate transporter does not depend on CaM kinase II activity.

Functional differences between rabbit and human Na(+)-dicarboxylate cotransporters, NaDC-1 and hNaDC-1.

The rabbit and human Na(+)-dicarboxylate cotransporters, NaDC-1 and hNaDC-1, were expressed in Xenopus oocytes, and the transport of succinate, citrate, and glutarate was compared. Both transporters had similar affinities for succinate and glutarate, with Michaelis-Menten constant (K(m)) values of approximately 0.5- 0.8 mM (succinate) and 6-7 mM (glutarate), verifying that they are low-affinity sodium-dependent dicarboxylate transporters. The two transporters differed in their handling of citrate. At pH 7.5, the K(m) value for citrate was 0.9 mM in the rabbit NaDC-1 and 7 mM in the human hNaDC-1. However, the human transporter was more sensitive to pH than the rabbit. At pH 5.5, the K(m) value for citrate decreased to 1.2 mM in hNaDC-1 and decreased to 0.3 mM in the rabbit transporter. Both transporters had Hill coefficients between 1.6 and 2.1, suggesting that multiple sodium ions are coupled to the transport of divalent anions. However, the human transporter, hNaDC-1, had a lower apparent affinity for sodium (KNa, 78 mM) than the rabbit transporter (KNa, 41 mM). In addition, the human hNaDC-1 was relatively insensitive to inhibition by lithium, furosemide, and flufenamate compared with the rabbit NaDC-1. The differences between the human and rabbit transporters may account for observed differences in renal handling of citrate between species.

Basolateral glutarate transport by isolated S2 segments of rabbit kidney proximal tubules.

The properties of tubular glutarate uptake and the coupling to p-aminohippurate (PAH) transport were studied on isolated nonperfused S2 segments of proximal tubules, microdissected from rabbit kidneys without the use of enzymatic agents. Because the tubules were totally collapsed, the tubular glutarate uptake may be assumed to represent the quantity transported across the basolateral membrane. The results show that the S2 segments effectively accumulated 14C glutarate (500 micron). The cell/bath 14C-glutarate concentration ratio reached maximum values of about 20 after a 20-min incubation period. The tubular 14C glutarate accumulation could be markedly depressed by lithium (5 mM) but not by probenecid (1 mM), which, however, inhibited tubular 3H-PAH (1 microM ) uptake. External PAH (0.1 mM) stimulated efflux of 14C-glutarate from S2 segments preloaded with 14C-glutarate (500 microM), and external glutarate stimulated tubular uptake of 3H-PAH (1 microM), providing evidence for glutarate-PAH countertransport in proximal S2 segments. The phorbol ester, phorbol 12-myristate 13-acetate (0.1 microM), did not affect steady-state cell/bath 14C-glutarate concentration ratio nor the initial 14C-glutarate transport rate. Protein kinase C may, therefore, not be a regulator of basolateral glutarate transport in renal S2 proximal tubules.

Ferrioxamine B derivatives as hepatobiliary contrast agents for magnetic resonance imaging.

Succinyl (SDF), phenylsuccinyl (PSDF), glutaryl (GDF), and phenylglutaryl (PGDF) derivatives of desferrioxamine B (DF) have been synthesized. In rats given the 59Fe(III) chelates of each these ligands at tracer levels, 82-94% of the 59Fe was eliminated within 1-2 days. 59Fe given as DF, SDF, and GDF chelates was excreted primarily in the urine, while nearly 50% of that given as PSDF and PGDF was excreted in the feces. Correspondingly, Fe-DF, Fe-SDF, and Fe-GDF (0.2 mmol/kg) produced early, marked renal, but no gastrointestinal magnetic resonance imaging (MRI) enhancement. Fe-PSDF and Fe-PGDF (0.2 mmol/kg) produced marked and rapid MRI enhancement of the upper small intestine. In animals with cannulated bile ducts, 59Fe from 59Fe-PGDF (carrier added, 0.1 mmol/kg) appeared rapidly in the collected bile, but not in the intestinal contents, proving that the contrast agent reaches the bowel via the bile. These changes in the excretion and MRI enhancement patterns brought about by the presence of a phenyl substituent apparently were not related to changes in lipophilicity or protein binding.