Preclinical Evaluation of 11C-Sarcosine as a Substrate of Proton-Coupled Amino Acid Transporters and First Human Application in Prostate Cancer.

Sarcosine is a known substrate of proton-coupled amino acid transporters (PATs), which are overexpressed in selected tissues and solid tumors. Sarcosine, an N-methyl derivative of the amino acid glycine and a metabolic product of choline, plays an important role for prostate cancer aggressiveness and progression. Methods:11C-radiolabeled sarcosine was tested as a new PET imaging probe in comparison with 11C-choline in 2 prostate cancer tumor xenograft models (DU-145 and PC-3). We characterized 11C-sarcosine transport in PC-3 and LNCaP tumor cells and performed 11C-sarcosine PET with CT in the first human subject with localized Gleason 4 + 3 prostate cancer. Target metabolite analyses of sarcosine and its natural precursors, glycine and choline, were performed from independent human prostate tissues. Results: In vitro assays indicated blockage of 11C-sarcosine uptake into PC-3 and LNCaP tumor cells by excess unlabeled (cold) sarcosine. 5-hydroxy-l-tryptophan, but not 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, competitively inhibited 11C-sarcosine tumor cell uptake, confirming PAT-mediated transport. In vivo tumor-to-background ratios (TBRs) obtained from 11C-sarcosine PET were significantly elevated compared with 11C-choline in DU-145 (TBR: 1.92 ± 0.11 for 11C-sarcosine vs. 1.41 ± 0.13 for 11C-choline [n = 10; P < 0.002]) and PC-3 tumors (TBR: 1.89 ± 0.2 for 11C-sarcosine vs. 1.34 ± 0.16 for 11C-choline [n = 7; P < 0.002]). 11C-sarcosine produced high-contrast images in 1 case of localized clinically significant prostate cancer. Target metabolite analyses revealed significant stepwise increases of sarcosine, glycine, and choline tissue levels from benign prostate tissue to localized prostate cancer and subsequently metastatic disease. 11C-sarcosine showed a favorable radiation dosimetry with an effective dose estimate of 0.0045 mSv/MBq, resulting in 2.68 mSv for a human subject (600-MBq dose). Conclusion:11C-sarcosine is a novel radiotracer for PATs and shows initial utility for prostate cancer imaging, with potential benefit over commonly used 11C-choline.

Indocyanine Green-Labeled Polysarcosine for in Vivo Photoacoustic Tumor Imaging.

Photoacoustic (PA) imaging has been considered an attractive imaging modality for sensitive and in-depth imaging of biomolecules with a high resolution in vivo. PA imaging probes utilizing fluorescence dyes, including indocyanine green (ICG), have been proposed to enhance PA signal intensity. On the other hand, nanomicelles modified with polysarcosine (PSar), a biocompatible hydrophilic polymer, on their surface have previously achieved rapid tumor uptake, suggesting active transport of PSar into tumor tissues. Thus, we hypothesized that PSar-based materials might be utilized as diagnostic probes for targeting tumors and therefore evaluated the potential of PSar labeled with an ICG derivative, ICG-PSar, as a PA imaging probe for targeting cancer. In this study, ICG-PSars with differing molecular weights (10, 20, and 30 kDa) were synthesized. In vitro cellular uptake studies using ICG-PSar demonstrated rapid uptake in colon26 tumor cells partially via macropinocytosis-mediated endocytosis. In vivo fluorescence imaging and biodistribution study indicated that ICG-PSar30k exhibited high accumulation in the tumor (8.4% dose/g), with high tumor-to-blood ratios reaching 4.6 at 24 h post injection of the probe. Finally, in vivo PA imaging studies showed that PA signal increased in tumors (251%) but not in blood vessels, achieving high contrast tumor imaging at 24 h after ICG-PSar30k probe injection. These results suggest that ICG-PSar has potential as a tumor-targeting PA imaging probe.

Gold nanoparticles coated with polysarcosine brushes to enhance their colloidal stability and circulation time in vivo.

Polysarcosine (PS), a non-ionic hydrophilic polypeptoid whose structure is similar to polypeptides, bearing repeating units of natural α-amino acid, has been used to stabilize gold nanoparticles (AuNPs) due to its excellent hydrophilicity and biocompatibility. Disulfide functionalized polysarcosines with different molecular weight were synthesized and used to cap AuNPs by traditional ligand exchange. The grafting of PS on AuNPs was evidenced by Fourier transform infrared (FTIR) spectroscopy and the alternation of surface zeta potential. The polysarcosine coated AuNPs (Au@PS) showed good stabilities in wide pH range and saline condition. They had strong resistance to ligand competition of dithiothreitol (DTT). They showed good stability in serum, with a molecular weight dependent interaction pattern with proteins. The Au@PS had very low cytotoxicity and cell uptake in vitro. Based on the results in vitro, polysarcosine with molecular weight of 5kD with the best ability to stabilize AuNPs was used for in vivo test. The Au@PS had a longer circulation time in blood after intravenous injection than that of Au@PEG, indicating a better stealth-like property of polysarcosine. The Au@PS did not cause obvious toxicity in vivo, suggesting potential applications in disease diagnosis and therapy.

Suppressive immune response of poly-(sarcosine) chains in peptide-nanosheets in contrast to polymeric micelles.

Nanoparticles are expected to be applicable for the theranostics as a carrier of the diagnostic and therapeutic agents. Lactosome is a polymeric micelle composed of amphiphilic polydepsipeptide, poly(sarcosine)64-block-poly(L-lactic acid)30, which was found to accumulate in solid tumors through the enhanced permeability and retention effect. However, lactosome was captured by liver on the second administration to a mouse. This phenomenon is called as the accelerated blood clearance phenomenon. On the other hand, peptide-nanosheet composed of amphiphilic polypeptide, poly(sarcosine)60-block-(L-Leu-Aib)6, where the poly(L-lactic acid) block in lactosome was replaced with the (L-Leu-Aib)6 block, abolished the accelerated blood clearance phenomenon. The ELISA and in vivo near-infrared fluorescence imaging revealed that peptide-nanosheets did not activate the immune system despite the same hydrophilic block being used. The high surface density of poly(sarcosine) chains on the peptide-nanosheet may be one of the causes of the suppressive immune response.

Evasion from accelerated blood clearance of nanocarrier named as "Lactosome" induced by excessive administration of Lactosome.

BACKGROUND: Nanoparticle of Lactosome, which is composed of poly(l-lactic acid)-base depsipeptide with diameter of 35nm, accumulates in solid tumors by the enhanced permeability and retention (EPR) effect. However, a pharmacokinetic alteration of Lactosome was observed when Lactosome was repeatedly administered. This phenomenon is named as the Lactosome accelerated blood clearance (ABC) phenomenon. In this study, the effect of Lactosome dose on the ABC phenomenon was examined and discussed in terms of immune tolerance. METHODS: To tumor transplanted mice, Lactosome (0-350mg/kg) was administrated. At 7days after the first administration, indocyanine green (ICG)-labeled Lactosome (ICG-Lactosome, 0-350mg/kg) was injected. Near-infrared fluorescence imaging was performed, and biodistribution of ICG-Lactosome was evaluated. Further, the produced amounts of anti-Lactosome IgM were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: ICG-Lactosome accumulated in the tumor region when the first Lactosome dose exceeded over 150mg/kg. The amounts of anti-Lactosome IgM were inversely correlated with the first Lactosome doses. Even after establishment of the Lactosome ABC phenomenon with the first Lactosome dose as low as 5.0mg/kg, the Lactosome ABC phenomenon can be evaded apparently by dosing ICG-Lactosome over 50mg/kg regardless of anti-Lactosome IgM production. CONCLUSIONS: There are two different mechanisms for evasion from the Lactosome ABC phenomenon before and after its establishment. In either mechanism, however, the Lactosome ABC phenomenon can be evaded by excessive administration of Lactosome. GENERAL SIGNIFICANCE: Lactosome is a potential nanocarrier for drug and/or imaging agent delivery, which can be used for frequent administrations without significant pharmacokinetic alterations.

First-time-in-human study of GSK923295, a novel antimitotic inhibitor of centromere-associated protein E (CENP-E), in patients with refractory cancer.

PURPOSE: GSK923295 is an inhibitor of CENP-E, a key cellular protein important in the alignment of chromosomes during mitosis. This was a Phase I, open-label, first-time-in-human, dose-escalation study, to determine the maximum-tolerated dose (MTD), safety, and pharmacokinetics of GSK923295. PATIENTS AND METHODS: Adult patients with previously treated solid tumors were enrolled in successive cohorts at GSK923295 doses ranging from 10 to 250 mg/m(2). GSK923295 was administered by a 1-h intravenous infusion, once weekly for three consecutive weeks, with treatment cycles repeated every 4 weeks. RESULTS: A total of 39 patients were enrolled. The MTD for GSK923295 was determined to be 190 mg/m(2). Observed dose-limiting toxicities (all grade 3) were as follows: fatigue (n = 2, 5%), increased AST (n = 1, 2.5%), hypokalemia (n = 1, 2.5%), and hypoxia (n = 1, 2.5%). Across all doses, fatigue was the most commonly reported drug-related adverse event (n = 13; 33%). Gastrointestinal toxicities of diarrhea (n = 12, 31%), nausea (n = 8, 21%), and vomiting (n = 7, 18%) were generally mild. Frequency of neutropenia was low (13%). There were two reports of neuropathy and no reports of mucositis or alopecia. GSK923295 exhibited dose-proportional pharmacokinetics from 10 to 250 mg/m(2) and did not accumulate upon weekly administration. The mean terminal elimination half-life of GSK923295 was 9-11 h. One patient with urothelial carcinoma experienced a durable partial response at the 250 mg/m(2) dose level. CONCLUSIONS: The novel CENP-E inhibitor, GSK923295, had dose-proportional pharmacokinetics and a low number of grade 3 or 4 adverse events. The observed incidence of myelosuppression and neuropathy was low. Further investigations may provide a more complete understanding of the potential for GSK923295 as an antiproliferative agent.

New 64Cu PET imaging agents for personalised medicine and drug development using the hexa-aza cage, SarAr.

The success of positron emission tomography (PET) in personalised medicine and drug development requires radioisotopes that provide high quality images and flexible chemistry for a broad application. 64Cu is arguably one of the most suitable PET isotopes for imaging with the evolving target agents, but there are not many appropriate chelating agents for 64Cu and this has limited its wider application. The bi-functional chelator, SarAr is known to bind 64Cu2+ quantitatively (i.e. one metal per ligand present) and rapidly (<2 min) at 10(-6) M over a range of pH (4-9). In this paper the conjugation of SarAr to the whole and fragmented antibody is described. Conjugation of the SarAr to the protein does not impair its coordination of the 64Cu. It complexes the 64Cu2+ rapidly, quantitatively and essentially irreversibly at pH 5. Animal studies show that the 64Cu-SarAr-immunoconjugates maintain their specificity for the target and are stable in vivo. Also, SarAr is a platform technology, is easy to use in a kit formulation and is readily adaptable for the wider application in 64Cu PET imaging.

Pharmacokinetics of oral betaine in healthy subjects and patients with homocystinuria.

AIMS: Large oral doses of betaine have proved effective in lowering plasma homocysteine in severe hyperhomocysteinaemia. The pharmacokinetic characteristics and metabolism of betaine in humans have not been assessed and drug monitoring for betaine therapy is not available. We studied the pharmacokinetics of betaine and its metabolite dimethylglycine (DMG) in healthy subjects and in three patients with homocystinuria. METHODS: Twelve male volunteers underwent an open-label study. After one single administration of 50 mg betaine kg-1 body weight and during continuous intake of twice daily 50 mg kg-1 body weight, serial blood samples and 24 h urines were collected to determine betaine and DMG plasma concentrations and urinary excretion, respectively. Patients were evaluated after one single dose of betaine. RESULTS: We found rapid absorption (t(1/2),abs 00.28 h, s.d. 0.17) and distribution (t(1/2), lambda1 00.59 h, s.d. 0.22) of betaine. A Cmax of 0.94 mmol l-1 (s.d. 0.19) was reached after tmax 00.90 h (s.d. 0.33). The elimination half life t(1/2), z was 14.38 h (s.d. 7.17). After repeated dosage, t(1/2), lambda1 (01.77 h, s.d. 0.75) and t(1/2), z (41.17 h, s.d. 13.50) increased significantly (95% CI 0.73, 01.64 h and 19.90, 33.70 h, respectively), whereas absorption remained unchanged. DMG concentrations increased significantly after betaine administration and accumulation occurred to the same extent as with betaine. Renal clearance was low and urinary excretion of betaine was equivalent to 4% of the ingested dose. Distribution and elimination kinetics in homocystinuric patients appeared to be accelerated. CONCLUSIONS: Betaine plasma concentrations change rapidly after ingestion. Elimination half-life increased during continuous dosing over 5 days. Betaine is mainly eliminated by metabolism. More pharmacokinetic and pharmacodynamic studies in hyperhomocysteinaemic patients are needed to refine the current treatment with betaine.

Comparative studies on the pharmacokinetics of hydrophilic prolinedithiocarbamate, sarcosinedithiocarbamate and the less hydrophilic diethyldithiocarbamate.

The pharmacokinetics of the antitoxic and anticarcinogenic compounds diethyldithiocarbamate, prolinedithiocarbamate and sarcosinedithiocarbamate were compared in rats. The bioavailability, the distribution in the organism, the oxidation to thiuramdisulfides, the cleavage to CS2 and the excretion in urine and bile were investigated. The results showed different behaviour of the three compounds. The more toxic diethyldithiocarbamate had a short in vivo half-life, was oxidized to tetraethylthiuramdisulfide in blood, and was metabolized to high yields of CS2 in 24 h. In contrast, prolinedithiocarbamate was more stable in vivo, was found predominantly in the urinary tract and was excreted in urine. The differences could not be explained by the presence of the carboxy group in the latter dithiocarbamate, since sarcosinedithiocarbamate, which also contains a carboxy group, behaved like diethyldithiocarbamate.

Lack of evidence for a high-affinity sarcosinamide carrier or a catecholamine carrier in Calu-1 lung-cancer cells, HT-29 colon-cancer cells, and DHF fibroblasts.

We have previously demonstrated that uptake of the amino acid amide sarcosinamide by the glioma cell line SK-MG-1 occurs via the catecholamine carrier that accommodates epinephrine (Km = 0.284 mM; Vmax = 0.154 nmol/10(6) cells/min). Sarcosinamide chloroethylnitrosourea (SarCNU), a new anticancer agent that exerts increased in vitro antitumor activity against gliomas as compared with BCNU (bis-chloroethylnitrosourea), the standard agent of choice, competitively inhibits sarcosinamide uptake by SK-MG-1 cells [inhibition constant (Ki) = 3.26 mM]. Using radiolabeled N-[3H]-sarcosinamide, we determined the transport of sarcosinamide in HT-29 colon-cancer cells, in Calu-1 lung-cancer cells, and in normal foreskin DHF fibroblasts. Sarcosinamide transport was linear for up to 1 min at 22 degrees C. In HT-29 cells and DHF fibroblasts, the uptake of sarcosinamide followed Michaelis-Menten kinetics of carrier-mediated transport. In HT-29 cells the Michaelis constant (Km) was 2.76 +/- 0.1 mM and the maximal velocity (Vmax) was 2.03 +/- 0.1 nmol/10(6) cells/min, whereas in DHF fibroblasts the respective values were 6.58 +/- 3.90 mM and 12.08 +/- 8.20 nmol/10(6) cells/min. In these two cell lines, neither epinephrine nor leucine significantly reduced sarcosinamide transport. In Calu-1 cells there was no evidence of carrier-mediated transport of either sarcosinamide or epinephrine. These nonglial cell lines lack a high-affinity catecholamine carrier. The increased cytotoxicity of SarCNU in gliomas may correlate with the presence of a high-affinity catecholamine carrier.

Chromium-cage complex as contrast agent in MR imaging--biodistribution studies of the [57Co]cobalt analogue.

The synthesis and T1 and T2 relaxivities of the Cr(III)-(NH2)-Sar-cage complex is reported. An outer-sphere relaxation mechanism is postulated for the relaxivity of the complex. Tissue distribution studies in mice using a [57Co]cobalt analogue as a radioactive tracer showed that the complex is excreted rapidly in the urine. Some renal uptake of the complex is seen. Appreciable uptake of labelled cage complex was observed in 3-methylcholanthrene induced murine rhabdomyosarcoma.