Design and Evaluation of 223Ra-Labeled and Anti-PSMA Targeted NaA Nanozeolites for Prostate Cancer Therapy—Part II. Toxicity,Pharmacokinetics and Biodistribution

Metastatic castration-resistant prostate cancer (mCRPC) is a progressive and incurable disease with poor prognosis for patients. Despite introduction of novel therapies, the mortality rate remains high. An attractive alternative for extension of the life of mCRPC patients is PSMA-based targeted radioimmunotherapy. In this paper, we extended our in vitro study of ²²³Ra-labeled and PSMA-targeted NaA nanozeolites [²²³RaA-silane-PEG-D2B] by undertaking comprehensive preclinical in vitro and in vivo research. The toxicity of the new compound was evaluated in LNCaP C4-2, DU-145, RWPE-1 and HPrEC prostate cells and in BALB/c mice. The tissue distribution of ¹³³Ba- and ²²³Ra-labeled conjugates was studied at different time points after injection in BALB/c and LNCaP C4-2 tumor-bearing BALB/c Nude mice. No obvious symptoms of antibody-free and antibody-functionalized nanocarriers cytotoxicity and immunotoxicity was found, while exposure to ²²³Ra-labeled conjugates resulted in bone marrow fibrosis, decreased the number of WBC and platelets and elevated serum concentrations of ALT and AST enzymes. Biodistribution studies revealed high accumulation of ²²³Ra-labeled conjugates in the liver, lungs, spleen and bone tissue. Nontargeted and PSMA-targeted radioconjugates exhibited a similar, marginal uptake in tumour lesions. In conclusion, despite the fact that NaA nanozeolites are safe carriers, the intravenous administration of NaA nanozeolite-based radioconjugates is dubious due to its high accumulation in the lungs, liver, spleen and bones.     

Pulmonary toxicity and translocation of nanodiamonds in mice

Because of the possible health threat of nanodiamonds (NDs) to organisms, the pulmonary toxicity and translocation of NDs in different sizes in mice were investigated after intratracheal instillation administration. Biochemical assays, ultrastructural and histopathological evaluations of the lungs of the control and the ND exposed mice were carried out at 1, 7, 14 or 28 days post-exposure. Exposure to 1.0 mg/kg NDs with an average diameter of 4 nm produced a temporary lung index increase at 1 day post-exposure. NDs did not have evident adverse effects in the lungs within the studied period according to histopathological and ultrastructural investigations. Furthermore, no lipid peroxidation of the lung was observed. On the whole, intratracheally instilled NDs are of low pulmonary toxicity. In addition, the amount of NDs in alveolar decreased with time elapsed and the macrophages burdened with NDs were clearly observed in the bronchia from 1 day to 28 days post-exposure. Thus we affirm the critical role of alveolar macrophages in the main excretion pathway of NDs from the lungs, i.e. they engulf the NDs, migrate upward to the trachea by escalator/mucociliary system and finally enter the pharynx.     

Biodistribution and accumulation of intravenously administered carbon nanotubes in mice probed by Raman spectroscopy and fluorescent labeling

The circulation, distribution and accumulation of intravenously administered single-walled carbon nanotubes (SWCNTs) in mice have been investigated by using the Raman spectroscopy and fluorescent labeling. The SWCNTs exhibited a rapid blood clearance with a half-life time of 3-4 h. However, rapid uptake and high levels of SWCNTs were observed in liver of mice. These SWCNTs accumulated in liver and were hard to excrete. The SWCNTs in liver led to pathological changes of liver, including injury of macrophages, cellular swelling, unspecific inflammation and blood coagulation. Similar SWCNT uptake behavior was found in spleen and kidney, whereas, no obvious pathological changes were observed in these organs.     

Size dependent cellular uptake, in vivo fate and light-heat conversion efficiency of gold nanoshells on silica nanorattles

Despite advances in photothermal therapy of gold nanoshells, reliable evaluations of their size dependence on the relative biological effects are needed. We report the size effects of PEGylated gold nanoshells on silica nanorattles (pGSNs) on their cellular uptake, in vivo fate and light-heat conversion efficiency in this study. The results indicate that smaller pGSNs have enhanced cellular uptake by the MCF-7 cells. For in vivo biodistribution study, pGSNs of different particle sizes (84-315 nm) distribute mainly in the liver and spleen in MCF-7 tumor-bearing BALB/c nude mice. Smaller pGSNs have a longer blood-circulation lifetime and higher light-heat conversion efficiency both in vitro and in vivo compared with larger ones. All three sizes of pGSNs can be excreted from the mice body at a slow rate and do not cause tissue toxicity after intravenous injection at a dosage of 20 mg kg(-1) for three times. The data support the feasibility of optimizing the therapeutic process for photothermal cell killing by plasmonic gold nanoshells.     

Ovarian Accumulation of Nanoemulsions: Impact of Mice Age and Particle Size

Nanotechnology in the field of drug delivery comes with great benefits due to the unique physicochemical properties of newly developed nanocarriers. However, they may come as well with severe toxicological side effects because of unwanted accumulation in organs outside of their targeted site of actions. Several studies showed an unintended accumulation of various nanocarriers in female sex organs, especially in the ovaries. Some led to inflammation, fibrosis, or decreasing follicle numbers. However, none of these studies investigated ovarian accumulation in context to both reproductive aging and particle size. Besides the influences of particle size, the biodistribution profile may be altered as well by reproductive aging because of reduced capacities of the reticuloendothelial system (RES), changes in sex steroid hormone levels as well as altering ovarian stromal blood flow. This systematic investigation of the biodistribution of intravenously (i.v) injected nanoemulsions revealed significant dependencies on the two parameters particle size and age starting from juvenile prepubescent to senescent mice. Using fluorescent in vivo and ex vivo imaging, prepubescent mice showed nearly no accumulation of nanoemulsion in their uteri and ovaries, but high accumulations in the organs of the RES liver and spleen independently of the particle size. In fertile adult mice, the accumulation increased significantly in the ovaries with an increased particle size of the nanoemulsions by nearly doubling the portion of the average radiant efficiency (PARE) to ~10% of the total measured signal of all excised organs. With reproductive aging and hence loss of fertility in senescent mice, the accumulation decreased again to moderate levels, again independently of the particle size. In conclusion, the ovarian accumulation of these nanocarriers depended on both the age plus the particle size during maturity.     

Biodistribution of Tc-C60(OH)x in Sprague-Dawley rats after intratracheal instillation

An increasing number of studies have been devoted to studying the respiratory toxicity of carbon nanomaterials, but little information is known, thus far, on the biodistribution of these nanomaterials after inhalation or intratracheal instillation. We synthesized and labeled a polyhydroxylated derivative of fullerene C₆₀(OH)_x (x = 22, 24) with ^99mTc. With single photon emission computed tomography imaging and 〈gamma〉-ray counting techniques, the biodistribution of ^99mTc-C₆₀(OH)_x in Sprague-Dawley rats after intratracheal instillation was studied. It was found that, besides the highest retention in the lung, ^99mTc-C₆₀(OH)_x is distributed mainly in the liver, bone, and spleen, with no uptake found in brain. The long-term retention in the lung and the very fast clearance from the blood revealed a transient characteristic of penetrating the alveolar-capillary barrier. The dimensions and size distribution, as well as in vivo aggregation of ^99mTc-C₆₀(OH)_x, may affect the capability and kinetic process of penetrating the alveolar-capillary barrier. The results provide guidance for further study of the respiratory toxicity of carbon nanomaterials.     

Comparative in vivo toxicity,organ biodistribution and immune response of pristine,carboxylated and PEGylated few-layer graphene sheets in Swiss albino mice: A three month study

We present a comprehensive 3 month report on the acute and chronic toxicity of intravenously administered (20 mg kg⁻¹) few-layer graphene (FLG) and, its carboxylated (FLG-COOH) and PEGylated (FLG-PEG) derivatives in Swiss albino mice. Whole-animal in vivo tracking studies revealed that irrespective of surface modifications, graphene predominantly accumulated in lungs over a period of 24 h. Histological assessment and ex vivo confocal Raman spectral mapping revealed highest uptake and retention in lung tissue, followed by spleen, liver and kidney, with no accumulation in brain, heart or testis. FLG and FLG-COOH accumulated within organs induced significant cellular and structural damages to lungs, liver, spleen, and kidney, ranging from mild congestion to necrosis, fibrosis and glomerular filtration dysfunction, without appreciable clearance. Serum biochemistry analysis revealed that both FLG and FLG-COOH induced elevated levels of hepatic and renal injury markers. Quantitative RT-PCR studies conducted on 23 critical inflammation and immune response markers showed major alterations in gene expression profile by FLG, FLG-COOH and FLG-PEG treated animals. FLG-PEG in spite of its persistance within liver and spleen tissue for 3 months, did not induce any noticeable toxicity or organ damage, and displayed significant changes in Raman spectra, indicative of their biodegradation potential.     

Mesoporous Aerogel Microparticles Injected into the Abdominal Cavity of Mice Accumulate in Parathymic Lymph Nodes

Mesoporous aerogel microparticles are promising drug delivery systems. However, their in vivo biodistribution pathways and health effects are unknown. Suspensions of fluorescein-labeled silica–gelatin hybrid aerogel microparticles were injected into the peritoneum (abdominal cavity) of healthy mice in concentrations of 52 and 104 mg kg⁻¹ in a 3-week-long acute toxicity experiment. No physiological dysfunctions were detected, and all mice were healthy. An autopsy revealed that the aerogel microparticles were not present at the site of injection in the abdominal cavity at the end of the experiment. The histological study of the liver, spleen, kidneys, thymus and lymphatic tissues showed no signs of toxicity. The localization of the aerogel microparticles in the organs was studied by fluorescence microscopy. Aerogel microparticles were not detected in any of the abdominal organs, but they were clearly visible in the cortical part of the parathymic lymph nodes, where they accumulated. The accumulation of aerogel microparticles in parathymic lymph nodes in combination with their absence in the reticuloendothelial system organs, such as the liver or spleen, suggests that the microparticles entered the lymphatic circulation. This biodistribution pathway could be exploited to design passive targeting drug delivery systems for flooding metastatic pathways of abdominal cancers that spread via the lymphatic circulation.     

腺病毒载体介导gag基因在小鼠体内的生物分布

目的研究腺病毒载体介导gag基因在C57BL/6小鼠体内的生物分布。方法经C57BL/6小鼠左后肢股四头肌部位单次注射0.05ml Ad-gag,于给药后不同时间摘取组织脏器,采用Taqman探针实时荧光定量PCR法,以小鼠β-actin基因作为内参,外标绝对定量法分别定量样本中gag和β-actin基因拷贝数。计算每104个小鼠细胞中gag基因的拷贝数,比较各脏器的分布数量。结果外标绝对定量法精密性良好。gag基因主要分布在注射位点、淋巴结、脾脏和肝脏,其他脏器没有分布。给药后3d,gag基因在靶器官分布达峰值,注射位点分布最多,伴随时间延长逐渐减少。结论Ad-gag疫苗在体内不会蓄积并产生全身的特别是生殖系统毒性。本研究可作为重要的安全性试验数据,支持药物在临床应用。     

Pulmonary exposure to single-walled carbon nanotubes does not affect the early immune response against Toxoplasma gondii

ABSTRACT: BACKGROUND: Single-walled carbon nanotubes (SWCNT) trigger pronounced inflammation and fibrosis in the lungs of mice following administration via pharyngeal aspiration or inhalation. Human exposure to SWCNT in an occupational setting may occur in conjunction with infections and this could yield enhanced or suppressed responses to the offending agent. Here, we studied whether the sequential exposure to SWCNT via pharyngeal aspiration and infection of mice with the ubiquitous intracellular parasite Toxoplasma gondii would impact on the immune response of the host against the parasite. METHODS: C57BL/6 mice were pre-exposed by pharyngeal administration of SWCNT (80 + 80 mug/mouse) for two consecutive days followed by intravenous injection with either 1x103 or 1x104 green fluorescence protein and luciferase-expressing T. gondii tachyzoites. The dissemination of T. gondii was monitored by in vivo bioluminescence imaging in real time for 7 days and by plaque formation. The inflammatory response was analysed in bronchoalveolar lavage (BAL) fluid, and by assessment of morphological changes and immune responses in lung and spleen. RESULTS: There were no differences in parasite distribution between mice only inoculated with T. gondii or those mice pre-exposed for 2 days to SWCNT before parasite inoculum. Lung and spleen histology and inflammation markers in BAL fluid reflected the effects of SWCNT exposure and T. gondii injection, respectively. We also noted that CD11c positive dendritic cells but not F4/80 positive macrophages retained SWCNT in the lungs 9 days after pharyngeal aspiration. However, co-localization of T. gondii with CD11c or F4/80 positive cells could not be observed in lungs or spleen. Pre-exposure to SWCNT did not affect the splenocyte response to T. gondii. CONCLUSIONS: Taken together, our data indicate that pre-exposure to SWCNT does not enhance or suppress the early immune response to T. gondii in mice.     

Conversion of rod-shaped gold nanoparticles to spherical forms and their effect on biodistribution in tumor-bearing mice

Gold nanorods that have an absorption band in the near-infrared region and a photothermal effect have been used as nanodevices for near-infrared imaging and thermal therapy. Choice of the optimal shape of gold nanorods which relates optical properties and in vivo biodistribution is important for their applications. In the present study, to investigate the relationship between the shape of gold nanorods and their biodistribution after intravenous injection, we first prepared two types of gold nanorods that had distinct aspect ratios but had the same volume, zeta potential, and PEG density on the gold surface. Biodistributions of the two types of gold nanorods after intravenous injection into tumor-bearing mice were then compared. Although a slight difference in accumulation in the spleen was observed, no significant difference was observed in the liver, lung, kidney, and tumors. These results suggest that biodistribution of the gold nanorods in the aspect ratio range of 1.7 to 5.0, diameter of 10 to 50 nm, and volume of approximately 4 × 10³ nm³ was dependent mainly on surface characteristics, PEG density, and zeta potential.     

Biodistribution of PEGylated graphene oxide nanoribbons and their application in cancer chemo-photothermal therapy

To use graphene oxide nanoribbons (GONRs) in combination with chemo-photothermal therapy, we modified GONRs with phospholipid-polyethylene glycol (PL-PEG) to prepare PEGylated GONRs (PL-PEG-GONRs), followed by investigation of the short-term in vivo biodistribution of ^99mTc-labeled PL-PEG-GONRs and their excretion in mice. The ^99mTc-labeled PL-PEG-GONRs demonstrated a unique biodistribution pattern of rapid accumulation in and excretion from the liver. Moreover, we determined that the PL-PEG-GNORs were excreted from the body through the renal route in urine, and we used hematological analysis to show that the PL-PEG-GNORs were not toxic in vivo. Furthermore, doxorubicin-loaded PL-PEG-GONRs had IC₅₀ values for chemo-photothermal therapy toward U87 glioma cells that were 6.7-fold lower than the IC₅₀ values in traditional chemotherapy. With these advantages, PL-PEG-GONRs could be used as drug nanocarriers to develop an efficient cancer-therapy strategy that would not only improve the efficacy of the therapy, but would also reduce the risk of side effects of the nanocarrier in the body.     

Effect of MWCNTs on Gastric Emptying in Mice

After making model of gastric functional disorder (FD), part of model mice were injected intravenously (i.v.) with oxide multi-walled carbon nanotubes (oMWCNTs) to investigate effect of carbon nanotubes on gastric emptying. The results showed that NO content in stomach, compared with model group, was decreased significantly and close to normal level post-injection with oMWCNTs (500 and 800 μg/mouse). In contrast to FD or normal groups, the content of acetylcholine (Ach) in stomach was increased obviously in injection group with 500 or 800 μg/mouse of oMWCNTs. The kinetic curve of emptying was fitted to calculate gastric motility factor k; the results showed that the k of injection group was much higher than FD and normal. In other words, the gastric motility of FD mice was enhanced via injection with oMWCNTs. In certain dosage, oMWCNTs could improve gastric emptying and motility.     

Distribution of rhodium in mice submitted to treatment with the adduct of rhodium propionate and sodium isonicotinate

The distribution of rhodium in Balb/c mice following intraperitoneal (ip) administration of a solution of adduct of rhodium propionate and sodium isonicotinate has been investigated. The metal concentration was determined in blood and in the following organ tissues: brain, heart, lung, liver, spleen, kidney, testes, and uterus/ovary, and the rhodium concentration was obtained by Inductively Coupled Argon Atomic Emission Spectroscopy (ICP-AES). The metal was detected in all organ tissues examined, mainly in spleen, liver, uterus/ovary and heart. Nine days after the injection, traces of rhodium were found in the liver and kidneys and, twenty days after the injection, only in the liver.     

Tissue uptake study and photodynamic therapy of melanoma-bearing mice with a nontoxic, effective chlorin

Chlorins have intense red absorptions and high tumor affinities that make them interesting candidates for photodynamic therapy (PDT) of cancer. This paper reports cytotoxicity, phototoxicity, in vitro cellular uptake, and in vivo biodistribution and PDT efficacy of a synthetic chlorin derivative (TCPCSO₃H) towards Cloudman melanoma cells (S91). No cytotoxic effects were observed in vitro at concentrations up to 20 μm, and no toxicity was observed in vivo in DBA mice with doses up to 2 mg kg^?1. Pharmacokinetics and biodistribution of TCPCSO₃H were evaluated in vivo in DBA mice bearing S91 tumors. TCPCSO₃H demonstrated preferential accumulation in S91 mouse melanoma, with tumor‐to‐normal tissue ratios of 5 and 11 for muscle and skin, respectively, 24 h after intravenous injection of 2 mg kg^?1. Photodynamic therapy performed under these conditions with 70 mW cm^?2 diode laser irradiation at 655 nm for 25 min (total light dose=105 J cm^?2) resulted in scab formation, followed by temporary or permanent (>60 days) tumor remission. According to the Kaplan–Meier analysis, the median survival time of the control group was 9 days, whereas that of the treated group was 38 days.     

乙型脑炎病毒减毒株SA14-14-2在小鼠体内的动态分布

目的研究乙型脑炎病毒减毒株SA14-14-2在小鼠体内的动态分布。方法将SA14-14-2株分别经脑内和皮下途径接种小鼠,采用BHK21细胞病毒蚀斑法检测接种后不同时间病毒在小鼠不同脏器中的分布。同时以不同稀释度的强毒株SA14为对照。结果SA14-14-2株经脑内感染小鼠后,除在脑组织中短时间检出病毒外,在其他组织中均未检出病毒;经皮下感染小鼠后,在任何组织中均未检出病毒。而强毒株SA14无论经脑内还是皮下途径感染小鼠,在各种脏器中均可检出病毒。结论SA14-14-2株经皮下感染小鼠后,病毒不能在体内复制,证明乙型脑炎病毒减毒活疫苗具有较好的安全性。     

In Vivo Evaluation of Sgc8-c Aptamer as a Molecular Imaging Probe for Colon Cancer in a Mouse Xenograft Model

Recent biotechnological applications in the field of clinical oncology led to the identification of new biomarkers as molecular targets of cancer, and to broad developments in the field of personalized medicine. Aptamers are oligonucleotides (ssDNA or RNA) that are selected to specifically recognize a molecular target with high affinity and specificity. Based on this, new horizons for their use as molecular imaging probes are being explored. The objective of this work was to evaluate the Sgc8-c aptamer conjugated with Alexa Fluor 647 fluorophore as an imaging probe in a colon tumor xenograft mouse model, with potential application in molecular imaging. In this study, the LS174T cell line was used to induce colorectal adenocarcinoma in nude mice. After confirmation of PTK7 overexpression by immunohistochemistry, in vivo studies were performed. Pharmacokinetic, in vivo and ex vivo biodistribution imaging, and a competition assay were evaluated by fluorescence imaging. In vivo visualization of the probe in the tumors was assessed two hours after aptamer probe administration, exhibiting excellent tumor-to-background ratios in biodistribution studies and high specificity in the competition test. Our results demonstrated the functionality of Scg8-c as an imaging probe for colon cancer, with potential clinical applications.     

Pharmacokinetics and tissue distribution of vinorelbine delivered in parenteral lipid emulsion

The pharmacokinetics and biodistribution of vinorelbine (VRB) delivered in a parenteral lipid emulsion (VLE) were evaluated, and compared with that of a commercial vinorelbine injectable solution (Navelbine® i.v., VS). After intravenous administration to rats at doses of 2.25, 4.5, and 9 mg/kg, the AUC_{0–24 h} was significantly different (1.15 ± 0.27, 2.65 ± 0.82, and 8.18 ± 1.10 mg/L/h for 2.25, 4.5, and 9 mg/kg, respectively), other pharmacokinetic parameters of VLE, such as the Cmax, CL, and Vss, were also dependent on the dose of VLE. The pharmacokinetics of VLE in rats was dose‐dependent in the dosage range of 2.25–9 mg/kg. VLE exhibited significantly higher VRB concentration than that of VS at the initial time points, it also produced a higher AUC_{0–24 h}, MRT and lower CL in comparison with VS. In beagle dogs, VLE exhibited pharmacokinetic bioequivalence to VS. The tissue distribution in mice of VLE was modified compared to VS, the AUC_{0–48 h} of VLE was grater in the liver, spleen, kidney and small intestine, but a little lower in the heart and lung. Practical applications: To reduce the severe local venous irritation of vinorelbine (VRB) at the aim of improve patient compliance, a parenteral vinorelbine loaded lipid emulsion (VLE) has been developed, and then systematical preclinical evaluations have been done. The pharmacokinetics and tissue distribution study reported here are part of the preclinical evaluation. VLE exhibited non‐linear relationship between dose and AUC_{0–24 h} after single intravenous dose (at doses of 2.25, 4.5, and 9 mg/kg) to rats. Pharmacokinetic bioequivalence between VLE and VS was found after single intravenous infusion administration a dose of 1.33 mg/kg to beagle dogs. Tissue distribution results showed that the AUC_{0–48 h} of VLE was grater in the liver, spleen, kidney, and small intestine, but a little lower in the heart and lung. These results could provide information for the further pharmacodynamics and pharmacology study, and finally for clinical use of VLE.     

On the in vitro and in vivo properties of four locked nucleic acid nucleotides incorporated into an anti-H-Ras antisense oligonucleotide

Locked nucleic acid (beta-D-LNA) monomers are conformationally restricted nucleotides bearing a methylene 2'-O, 4'-C linkage that have an unprecedented high affinity for matching DNA or RNA. In this study, we compared the in vitro and in vivo properties of four different LNAs, beta-D-amino LNA (amino-LNA), beta-D-thio LNA (thio-LNA), beta-D-LNA (LNA), and its stereoisomer alpha-L-LNA in an antisense oligonucleotide (ODN). A well-known antisense ODN design against H-Ras was modified at the 5'- and 3'-ends with the different LNA analogues (LNA-DNA-LNA gapmer design). The resulting gapmers were tested in cancer-cell cultures and in a nude-mouse model bearing prostate tumor xenografts. The efficacy in target knockdown, the biodistribution, and the ability to inhibit tumor growth were measured. All anti H-Ras ODNs were very efficient in H-Ras mRNA knockdown in vitro, reaching maximum effect at concentrations below 5 nM. Moreover, the anti-H-Ras ODN containing alpha-L-LNA had clearly the highest efficacy in H-Ras knockdown. All LNA types displayed a great stability in serum. ODNs containing amino-LNA showed an increased uptake by heart, liver, and lungs as compared to the other LNA types. Both alpha-L-LNA and LNA gapmer ODNs had a high efficacy of tumor-growth inhibition and were nontoxic at the tested dosages. Remarkably, in vivo tumor-growth inhibition could be observed at dosages as low as 0.5 mg kg(-1) per day. These results indicate that alpha-L-LNA is a very promising member of the family of LNA analogues in antisense applications.     

In Vivo Albumin-Binding of a C-Functionalized Cyclam Platform for 64Cu-PET/CT Imaging in Breast Cancer Model

An improved glucose-chelator-albumin bioconjugate (GluCAB) derivative, GluCAB-2^Mal, has been synthesized and studied for in vivo ⁶⁴Cu-PET/CT imaging in breast cancer mice models together with its first-generation analogue GluCAB-1^Mal. The radioligand works on the principle of tumor targeting through the enhanced permeability and retention (EPR) effect with a supportive role played by glucose metabolism. [⁶⁴Cu]Cu-GluCAB-2^Mal (99 % RCP) exhibited high serum stability with immediate binding to serum proteins. In vivo experiments for comparison between tumor targeting of [⁶⁴Cu]Cu-GluCAB-2^Mal and previous-generation [⁶⁴Cu]Cu-GluCAB-1^Mal encompassed microPET/CT imaging and biodistribution analysis in an allograft E0771 breast cancer mouse model. Tumor uptake of [⁶⁴Cu]Cu-GluCAB-2^Mal was clearly evident with twice as much accumulation as compared to its predecessor and a tumor/muscle ratio of up to 5 after 24 h. Further comparison indicated a decrease in liver accumulation for [⁶⁴Cu]Cu-Glu-CAB-2^Mal.