Biodistribution In Rats Research Articles

Phase-0/phase-I study of dye-loaded lipid nanoparticles for near-infrared fluorescence imaging in healthy dogs

Near-infrared (NIR) fluorescence imaging using FDA-approved indocyanine green (ICG) has been the subject of numerous studies during the past few years. It could constitute a potentially exciting new paradigm shift in veterinary oncology, especially to develop in vivo fluorescence imaging diagnostics and surgery guidance methods. The objective of this study was to evaluate the pharmacologic and toxicological characteristics in healthy beagle dogs of LipImage™ 815, a formulation made of NIR-dye-loaded lipid nanoparticles. The initial dosage for the evaluation of biodistribution was extrapolated from data in mice and then adapted to define the more adapted dose (MAD) according to the fluorescence results obtained in 5 dogs using a Fluobeam® 800 imaging device (phase 0 study). A single dose acute toxicity study was then performed (3 dogs, phase I study). Before the systemic administration of LipImage™ 815, the dogs presented a very mild residual fluorescence, particularly in the liver and kidneys. After injection, the plasma fluorescence continuously decreased, and the signal was relatively homogeneously distributed throughout the different organs, though more pronounced in the liver and to a lesser extent in the steroid-rich organs (adrenal, ovaries), intestines, lymph nodes and kidneys. A MAD of 2.0μg/kg was found. No evidence of acute or delayed general, hepatic, renal or hematologic toxicity was observed at 1-fold, 5-fold or 10-fold MAD. The results of this phase-0/phase-I study showed that an optimal dosage of LipImage™ 815 of 2.0μg/kg allowed the achievement of a fluorescence signal suitable for surgery guidance application without any acute side effects.

Radiopharmacological characterization of ⁶⁴Cu-labeled α-MSH analogs for potential use in imaging of malignant melanoma.

The melanocortin-1 receptor (MC1R) plays an important role in melanoma growth, angiogenesis and metastasis, and is overexpressed in melanoma cells. α-Melanocyte stimulating hormone (α-MSH) and derivatives are known to bind with high affinity at this receptor that provides the potential for selective targeting of melanoma. In this study, one linear α-MSH-derived peptide Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 (NAP-NS1) without linker and with εAhx-β-Ala linker, and a cyclic α-MSH derivative, [Lys-Glu-His-D-Phe-Arg-Trp-Glu]-Arg-Pro-Val-NH2 (NAP-NS2) with εAhx-β-Ala linker were conjugated with p-SCN-Bn-NOTA and labeled with (64)Cu. Radiochemical and radiopharmacological investigations were performed with regard to transchelation, stability, lipophilicity and in vitro binding assays as well as biodistribution in healthy rats. No transchelation reactions, but high metabolic stability and water solubility were demonstrated. The linear derivatives showed higher affinity than the cyclic one. [(64)Cu]Cu-NOTA-εAhx-β-Ala-NAP-NS1 ([(64)Cu]Cu-2) displayed rapid cellular association and dissociation in murine B16F10 cell homogenate. All [(64)Cu]Cu-labeled conjugates exhibited affinities in the low nanomolar range in B16F10. [(64)Cu]Cu-2 showed also high affinity in human MeWo and TXM13 cell homogenate. In vivo studies suggested that [(64)Cu]Cu-2 was stable, with about 85 % of intact peptide in rat plasma at 2 h p.i. Biodistribution confirmed the renal pathway as the major elimination route. The uptake of [(64)Cu]Cu-2 in the kidney was 5.9 % ID/g at 5 min p.i. and decreased to 2.0 % ID/g at 60 min p.i. Due to the prospective radiochemical and radiopharmacological properties of the linear α-MSH derivative [(64)Cu]Cu-2, this conjugate is a promising candidate for tracer development in human melanoma imaging.

In Vivo Quantification of Inflammation in Experimental Autoimmune Encephalomyelitis Rats Using Fluorine-19 Magnetic Resonance Imaging Reveals Immune Cell Recruitment outside the Nervous System.

Progress in identifying new therapies for multiple sclerosis (MS) can be accelerated by using imaging biomarkers of disease progression or abatement in model systems. In this study, we evaluate the ability to noninvasively image and quantitate disease pathology using emerging “hot-spot” 19F MRI methods in an experimental autoimmune encephalomyelitis (EAE) rat, a model of MS. Rats with clinical symptoms of EAE were compared to control rats without EAE, as well as to EAE rats that received daily prophylactic treatments with cyclophosphamide. Perfluorocarbon (PFC) nanoemulsion was injected intravenously, which labels predominately monocytes and macrophages in situ. Analysis of the spin-density weighted 19F MRI data enabled quantification of the apparent macrophage burden in the central nervous system and other tissues. The in vivo MRI results were confirmed by extremely high-resolution 19F/1H magnetic resonance microscopy in excised tissue samples and histopathologic analyses. Additionally, 19F nuclear magnetic resonance spectroscopy of intact tissue samples was used to assay the PFC biodistribution in EAE and control rats. In vivo hot-spot 19F signals were detected predominantly in the EAE spinal cord, consistent with the presence of inflammatory infiltrates. Surprising, prominent 19F hot-spots were observed in bone-marrow cavities adjacent to spinal cord lesions; these were not observed in control animals. Quantitative evaluation of cohorts receiving cyclophosphamide treatment displayed significant reduction in 19F signal within the spinal cord and bone marrow of EAE rats. Overall, 19F MRI can be used to quantitatively monitored EAE disease burden, discover unexpected sites of inflammatory activity, and may serve as a sensitive biomarker for the discovery and preclinical assessment of novel MS therapeutic interventions.

Biomarkers of oxidative stress for in vivo assessment of toxicological effects of iron oxide nanoparticles.

Iron oxide nanoparticles (Fe2O3-IONPs) have revolutionized the industry by significant economic and scientific impacts. Enormous increase in the usage of IONPs has raised concerns about their unseen adverse effects. In the current study, we investigated the effects of IONPs and its bulk on oxidative stress biomarkers, histopathology and biodistribution in rats after 28 days repeated oral treatment at 30, 300 and 1000 mg/kg body weight (b.w.). IONPs size in dry, wet forms and crystallinity was determined using TEM, DLS and XRD. The investigation of oxidative stress biomarkers demonstrated significant increase in lipid peroxidation and decrease in reduced glutathione content in the liver, kidney and the brain of the treated groups in a dose dependant manner. Further, antioxidant enzymes catalase, glutathione S transferase, glutathione peroxidase and glutathione reductase activities were significantly elevated along with significant decrease in superoxide dismutase activity in treated rat organs. ICP-OES analysis revealed dose and size dependant accumulation of IONPs in the liver followed by kidney and the brain than bulk. Moreover, accumulation of IONPs at high dose brought pathological changes only in liver. A large fraction of IONPs was eliminated in urine. Bulk material was substantially excreted in faeces than IONPs suggesting increased absorption of IONPs. In conclusion accumulated IONPs and bulk in organs trigger free radical generation, leading to the induction of oxidative stress condition in rats. The results obtained highlight the importance of toxicity assessments in evaluating the efficiency of IONPs for the safe implementation for diversified applications.

Evaluation of Biodistribution of Functionalized Magnetic Core/Carbon-Shell Nanoparticles in Systemic Method of Administration.

We studied new magnetic nanocomposites consisting of a core(Fe) and carbon-shell inert to biological media. Iron-carbon nanoparticles circulate in the bloodstream for several minutes and are primarily accumulated in the liver; less intensive accumulation was found in the spleen and minimum in the lungs, kidneys, and heart. Accumulation of nanoparticles in the liver leads to the development of destructive processes and is accompanied by activation of compensatory-adaptive mechanisms. In the liver and spleen, structural changes are mild and mainly relate to changes in the microvasculature. In 6 months, the total content of nanoparticles in all tissues decreased due to their elimination from the body and the structure of the organs returned to normal.

(Invited) Thermal Carbonization of Porous Silicon: The Current Status and Recent Applications

Porous silicon (PSi) is a versatile material for a wide range of applications. Its large surface area combined with easily adjustable pore morphology and surface chemistry offers an excellent platform for many different applications. For example, the refractive index of PSi is strictly depending on the porosity and can be controlled by the current density of electrochemical etching. This enables precise control of the refractive index and allows the fabrication of highly complex optical structures for many interesting applications. However, for decades, the biggest stumbling block for PSi applications has been the chemical instability of PSi. As-anodized PSi is hydrogen terminated and it slowly oxidizes even at room temperature. This instability of as-anodized PSi is unacceptable in most applications. On the other hand, intentional oxidation of PSi, in order to improve the stability, usually leads to too high resistivity for electrical applications and changes the optical properties of PSi too drastically. Moreover, the oxidized PSi is not chemically stable enough for certain type of applications, especially in aqueous conditions. It may react adversely with drugs and its functionalization needs to be done through silanization chemistry which shares the silicon oxide bonding related instability, e.g., for targeted drug delivery applications. Thermal carbonization by acetylene to stabilize PSi was introduced in 2000 [1]. It produces a non-stoichiometric silicon carbide layer on the PSi, which is very stable in harsh environments, like in NaOH, KOH, HF etc. solutions with extreme pH values. It also increases the conductivity of the PSi, which is beneficial for electrical sensor and energy storage applications and it has been found to be nontoxic both in in vitro and in vivo applications. Therefore, the thermally carbonized PSi (TCPSi) has been used in various different applications ranging from gas sensors and biosensors to targeted drug delivery, bioimaging and electrochemical supercapacitors since its introduction [2–9]. In the presentation, the current status and novel applications, in which thermally carbonized PSi has been used, will be discussed. In addition, some new information about the physical and chemical properties of TCPSi will be introduced. REFERENCES: [1] J. Salonen, V.-P. Lehto, M. Björkqvist, E. Laine, L. Niinistö, Studies of Thermally-Carbonized Porous Silicon Surfaces, Physica Status Solidi (a), 182 (2000) 123-126. [2] V. Torres-Costa, R.J. Martin-Palma, J.M. Martinez-Duart, J. Salonen, V.P. Lehto, Effective passivation of porous silicon optical devices by thermal carbonization, Journal of Applied Physics, 103 (2008) 4. [3] J. Tuura, M. Bjorkqvist, J. Salonen, V.P. Lehto, Electrically isolated thermally carbonized porous silicon layer for humidity sensing purposes, Sensors and Actuators B - Chemical, 131 (2008) 627-632. [4] T. Jalkanen, E. Mäkilä, Y.I. Suzuki, T. Urata, K. Fukami, K. Sakka, J. Salonen, Y.H. Ogata, Studies on Chemical Modification of Porous Silicon-Based Graded-Index Optical Microcavities for Improved Stability Under Alkaline Conditions, Advanced Functional Materials, 22 (2012) 3890-3898. [5] B. Sciacca, S.D. Alvarez, F. Geobaldo, M.J. Sailor, Bioconjugate functionalization of thermally carbonized porous silicon using a radical coupling reaction, Dalton Transactions, 39 (2010) 10847–10853. [6] P.J. Kinnari, M.L.K. Hyvönen, E.M. Mäkilä, M.H. Kaasalainen, A. Rivinoja, J.J. Salonen, J.T. Hirvonen, P.M. Laakkonen, H.A. Santos, Tumour homing peptide-functionalized porous silicon nanovectors for cancer therapy, Biomaterials. 34 (2013) 9134–9141. [7] L.M. Bimbo, M. Sarparanta, H.A. Santos, A.J. Airaksinen, E. Mäkilä, T. Laaksonen, L. Peltonen, V.-P. Lehto, J. Hirvonen, J. Salonen, Biocompatibility of thermally hydrocarbonized porous silicon nanoparticles and their biodistribution in rats, ACS Nano. 4 (2010) 3023–3032. [8] J. Salonen, Drug Delivery with Porous Silicon, in: L. Canham (Ed.) Handbook of Porous Silicon, Springer International Publishing, 2014, Ch. 91, pp. 909-919. [9] S. Chatterjee, R. Carter, L. Oakes, W.R. Erwin, R. Bardhan, C.L. Pint, Electrochemical and Corrosion Stability of Nanostructured Silicon by Graphene Coatings: Toward High Power Porous Silicon Supercapacitors, Journal of Physical Chemistry C. 118 (2014) 10893–10902.

Biodistribution evaluation of 166Ho–DTPA–SPION in normal rats

Super paramagnetic iron oxide nanoparticles (SPION) with a cross-linked dextran coating can be used as a negative contrast agent in magnetic resonance imaging agent. This study describes the preparation, biodistribution of 166Ho–DTPA–SPIONs after intravenous injection in normal Wistar rats. The biodistribution of 166Ho–DTPA–SPIONs showed dramatic uptake in reticuloendothelial system (i.e., more than 60 and 15 % of injected dose was in liver and spleen at 30 min post-injection, respectively) and the clearance of the radiotracer is so fast in other organs. These results suggest that 166Ho–DTPA–SPIONs could be good candidate for theranostic purposes in for liver and spleen malignancies.

Development of bacteriochlorophyll a-based near-infrared photosensitizers conjugated to gold nanoparticles for photodynamic therapy of cancer.

We report the synthesis and characterization of a new sulfur-containing derivative of bacteriochlorophyll a. The latter was isolated from biomass of the nonsulfur purple bacterium Rhodobacter capsulatus strain B10. The developed photosensitizer is N-aminobacteriopurpurinimide with an exocyclic amino group acylated with a lipoic acid moiety, which is a biogenic substance that acts as a cofactor of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes in the body. The disulfide moiety of lipoic acid confers the compound aurophilicity, thus allowing its conjugation with gold nanoparticles (NP-Au) via S-Au bonds. The shape and the size of the resulting nanoconjugate with immobilized photosensitizer (PS-Au) were assessed by dynamic light scattering and transmission electron microscopy. The conjugated nanoparticles are spherical with hydrodynamic diameter of 100-110nm. The PS-Au conjugate absorbs light at 824nm and emits strong fluorescence at 830nm, which allowed in vivo study of its dynamic biodistribution in rats bearing sarcomaM-1. Compared to the free photosensitizer, PS loaded on the gold nanoparticles (PS-Au) showed extended circulation time in the blood and enhanced tumor uptake due to nonspecific passive targeting when the drug accumulates in tumor sites through the leaky tumor neovasculature and does not return to the circulation.

546. Identification of Optimal AAV Vector and Route of Administration for Neonatal Treatment Using the Murine MPS IIIB Model

Mucopolysaccharidoses IIIB (MPS IIIB), is an autosomal recessive lysosomal storage disease caused by defective production of the enzyme α-N-acetylglucosaminidase and is characterized by retention and accumulation of heparan sulfate. It is defined by severe and complex central nervous system (CNS) pathology. No cure exists. Treatment options are limited by the need to overcome the blood-brain barrier and gain successful entry to the CNS. Research suggests that the optimal therapeutic window occurs prior to toddlerhood. Based on this knowledge, using the murine neonatal MPS IIIB model, we evaluate and describe for the first time, therapeutic assessment of two capsid mutated Adeno-associated virus serotype 8 variants for the treatment of MPS IIIB. Our goal is to assess biodistribution and transduction profiles of GFP expressing AAV8(double Y-F) and AAV8(double Y-F+T-V) variants, compared to normal AAV8, administered through four intracranial routes: six site (IC6), thalamic (TH), intracerebroventricular (ICV) and ventral tegmental area (VTA). The serotype exhibiting the best treatment potential is defined by broadest distribution and transduction profiles. Comparative evaluation of vector biodistribution of each of the four administration methods revealed that IC6>ICV>TH>VTA. Surprisingly, evaluation of transduction profiles revealed that AAV8(double Y-F+T-V)>AAV8>AAV8(double Y-F). Based on functional significance, the cortex, hippocampus, thalamus and cerebellum were assessed in each animal. In IC6 administered AAV8(double Y-F+T-V) treated animals, cortical layers II/III, IV and V/VI; as well as, all hippocampal layers were robustly transduced, while the thalamus and cerebellum were modestly transduced. The ICV method yielded heavy localization of vector in the hippocampus and proximal thalamic areas with modest cortical deposition of vector along needle track. The TH and VTA methods proved least desirable to facilitate vector biodistribution. Phenotypic assessment of transduced cells suggest primary robust transduction of neurons compared to astrocytes. For the first time, we have provided proof of concept for the utility of IC6 administration of AAV8(double Y-F+T-V) as a valid therapeutic approach for the early treatment of MPS IIIB with further implications for other monogenic diseases.

Evaluation of biodistribution and transduction profiles of novel AAV8 capsid mutated variants as a therapeutic candidate for the treatment of MPS IIIB

Evaluation of biodistribution and transduction profiles of novel AAV8 capsid mutated variants as a therapeutic candidate for the treatment of MPS IIIB

Radiochemistry, pre-clinical studies and first clinical investigation of 90Y-labeled hydroxyapatite (HA) particles prepared utilizing 90Y produced by (n,γ) route

IntroductionThe scope of using no carrier added (NCA) 90Y [T1/2=64.1h, Eβ(max)=2.28MeV] obtained from 90Sr/90Y generator in radiation synovectomy (RSV) is widely accepted. In the present study, the prospect of using 90Y produced by (n,γ) route in a medium flux research reactor for use in RSV was explored. MethodsYttrium-90 was produced by thermal neutron irradiation of Y2O3 target at a neutron flux of ~1×1014 n/cm2.s for 14 d. The influence of various experimental parameters were systematically investigated and optimized to arrive at the most favorable conditions for the formulation of 90Y labeled hydroxyapatite (HA) using HA particles of 1–10μm size range. An optimized kit formulation strategy was developed for convenient one-step compounding of 90Y-HA, which is easily adaptable at hospital radiopharmacy. The pre-clinical biological evaluation of 90Y-HA particles was studied by carrying out biodistribution and bioluminiscence imaging studies in Wistar rats. The first clinical investigation using the radiolabeled preparation was performed on a patient suffering from chronic arthritis in knee joint by administering 185MBq 90Y-HA formulated at the hospital radiopharmacy deploying the proposed strategy. ResultsYttrium-90 was produced with a specific activity of 851±111MBq/mg and radionuclidic purity of 99.95±0.02%. 90Y-labeled HA particles (185±10MBq doses) were formulated in high radiochemical purity (>99%) and excellent in vitro stability. The preparation showed promising results in pre-clinical studies carried out in Wistar rats. The preliminary results of the first clinical investigation of 90Y-HA preparation in a patient with rheumatoid arthritis in knee joints demonstrated the effectiveness of the formulation prepared using 90Y produced via (n,γ) route in the management of the disease. ConclusionThe studies revealed that effective utilization of 90Y produced via (n,γ) route in a medium flux research reactor coupled with the developed strategy of using HA kits for convenient formulation of 90Y-HA at the hospital radiopharmacy can contribute to sustainable growth in the clinical utilization of 90Y in RSV in the foreseeable future.

Visualisation of dual radiolabelled poly(lactide-co-glycolide) nanoparticle degradation in vivo using energy-discriminant SPECT.

The determination of nanoparticle (NP) stability and degradation in vivo is essential for the accurate evaluation of NP biodistribution in medical applications and for understanding their toxicological effects. Such determination is particularly challenging because NPs are extremely difficult to detect and quantify once distributed in a biological system. Radiolabelling with positron or gamma emitters and subsequent imaging studies using positron emission tomography (PET) or single-photon emission computerised tomography (SPECT) are some of the few valid alternatives. However, NPs that degrade or radionuclides that detach or are released from the NPs can cause artefact. Here, submicron-sized poly(lactide-co-glycolide) nanoparticles (PLGA-NPs) stabilised with bovine serum albumin (BSA) were dual radiolabelled using gamma emitters with different energy spectra incorporated into the core and coating. To label the core, 111In-doped iron oxide NPs were encapsulated inside PLGA-NPs during NP preparation, and the BSA coating was labelled by electrophilic substitution using 125I. After intravenous administration into rats, energy-discriminant SPECT resolved each radioisotope independently. Imaging revealed different fates for the core and coating, with a fraction of the two radionuclides co-localising in the liver and lungs for long periods of time after administration, suggesting that NPs are stable in these organs. Organ harvesting followed by gamma counting corroborated the SPECT results. The general methodology reported here represents an excellent alternative for visualising the degradation process of multi-labelled NPs in vivo and can be extended to a wide range of engineered NPs.

Synthesis, Characterization, Biodistribution and Scintigraphic Evaluation of Technetium-99m (99mTc) Labeled Pazufloxacin (PZN): A Novel Infection Radiotracer

In this study tagging of 99mTc with PZN was examined with regards to different concentration of PZN, stannous chloride, 99mTc and pH ranges from 5.00-6.00. The suitability of the 99mTc labeled PZN was assessed in terms of percent radiochemical purity (%RCP) yield in normal saline at concentration value of the stannous chloride as 120 mg, stability in serum (in vitro) at 37°C at different intervals, in vitro up take by live and heat killed E. coli, biodistribution behavior in artificially E. coli (live and heat killed) infected animal model rat and scintigraphic accuracy in artificially E. coli infected animal model rabbit. Maximum %RCP yields of 98.25 ± 0.26% was observed after 30 m of labeling using 2 mg of PZN, 125 μg stannous chloride, 2.5 m Ci sodium pertechnetate and pH 5.3. The labeled PZN showed stable profile in serum at 37°C with a total decay of 16.40 ± 0.18%. The labeled PZN showed 72.55 ± 0.90% maximum bacterial uptake after 90 min in live and in case of heat killed E. coli, no significant uptake was seen. The labeled PZN showed 14.75 ± 0.40% absorption and accumulation in the infected muscle of the rat infected with live E. coli and infected to normal muscle ratio was 6:1. Scintigraphically, it was observed that after few minutes, the activity accumulated in the infected muscle was more than the others sites. The instant investigation substantiated that labeling PZN through this technique as compared to 99mTcV:N~ and 99mTc(CO)3~ showed almost analogous %RCP yield, stability in serum at 37°C, in vitro binding with E. coli, biodistribution profile in animal modal rats and scintigraphic accuracy in animal model rabbit.

Cannabinoid antagonist in nanostructured lipid carriers (NLCs): design, characterization and in vivo study.

This study describes the preparation, characterization, and in vivo evaluation in rats of nanostructured lipid carriers (NLCs) encapsulating rimonabant (RMN) as prototypical cannabinoid antagonist. A study was conducted in order to optimize NLC production by melt and ultrasonication method. NLCs were prepared by alternatively adding the lipid phase into the aqueous one (direct protocol) or the aqueous phase into the lipid one (reverse protocol). RMN-NLCs have been characterized by cryogenic transmission electron microscopy (cryo-TEM), X-ray, photon correlation spectroscopy (PCS) and sedimentation field flow fractionation (SdFFF). Reverse NLCs were treated with polysorbate 80. RMN release kinetics have been determined in vitro by dialysis method. In vivo RMN biodistribution in rats was evaluated after intranasal (i.n.) administration of reverse RMN-NLC. The reverse protocol enabled to prevent the lost of lipid phase and to achieve higher RMN encapsulation efficacy (EE) with respect to the direct protocol (98% w/w versus 67% w/w). The use of different protocols did not affect NLC morphology and dimensional distribution. An in vitro dissolutive release rate of RMN was calculated. The in vivo data indicate that i.n. administration of RMN by reverse NLC treated with polysorbate 80 increased RMN concentration in the brain with respect to the drug in solution. The nanoencapsulation protocol presented here appears as an optimal strategy to improve the low solubility of cannabinoid compounds in an aqueous system suitable for in vivo administration.

Formulation, preclinical evaluation, and preliminary clinical investigation of an in-house freeze-dried EDTMP kit suitable for the preparation of 177Lu-EDTMP.

The objective of the present work was to develop a freeze-dried ethylenediaminetetramethylene phosphonic acid (EDTMP) kit, suitable for the convenient and single-step preparation of (177)Lu-EDTMP, which is currently being evaluated as a promising radiopharmaceutical for providing palliative care to patients suffering from skeletal metastases and to assess the potential of the agent in human patients. Lyophilized EDTMP kits having identical composition with Quadramet(®) were prepared using EDTMP, NaOH, and anhydrous CaCO3. The (177)Lu-EDTMP patient dose was prepared by incubating the kit materials dissolved in 1 mL of water for injection and (177)LuCl3, produced in-house, at room temperature for 15 minutes. Pharmacokinetic behavior of the agent was studied by carrying out biodistribution and scintigraphic imaging studies in normal Wistar rats. Clinical studies were performed by administering the preparation in patients suffering from disseminated skeletal metastases. Five batches of freeze-dried EDTMP kits with 50 kit vials in each batch were prepared. Each kit vial comprised a lyophilized mixture of 35 mg EDTMP, 14.1 mg NaOH, and 5.8 mg of CaCO3. The (177)Lu-EDTMP complex was prepared with excellent radiochemical purity (>99%) and high stability (>98% until 9 days postpreparation) using these kits. Radiochemical studies showed that this kit could be used within a pH range of 6-9 and with (177)Lu having specific activity as low as 925 GBq · g(-1) (25 Ci · g(-1)) for the preparation of up to 3.7 GBq (100 mCi) of (177)Lu-EDTMP. Biodistribution studies in animals revealed selective accumulation of the agent in skeleton (∼ 60% of the injected activity) with major renal clearance. Preliminary clinical studies in 10 patients exhibited selective accumulation of the radiotracer in skeletal lesions and provided significant pain relief thereby improving the quality of life of the patients. Freeze-dried EDTMP kits, suitable for the preparation of patient doses of (177)Lu-EDTMP, have been developed and used in preliminary studies for treating the cancer patients with disseminated skeletal metastases. The kit may also find use for the preparation of other potential bone pain palliation agents, such as (153)Sm-EDTMP.

Biodistribution of a potential chemotherapeutic, dinuclearbisphosphinogold(I) dithiocarbamate, as determined by its 198Au radiolabelled analogue

Dinuclearbisphosphinogold(I) dithiocarbamato, BPDTC, was previously found to have antitumour activity in vitro. 198Au radiolabelled BPDTC (radiochemical yield of 70 ± 6 % and radiochemical purity of >95 %) was used to determine its in vivo biodistribution in Sprague-Dawley rats. Gamma scintigraphs were performed over a period of 48 h and final radioactivity measurements of harvested organs of the test animals after termination was performed at 2, 4 and 48 h. The study successfully showed the biodistribution of the gold complex, with the highest uptake of the compound being observed in the lungs, liver and spleen.

Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial.

Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose–response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2–500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8+ T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX–/– mice 8–10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma FIX activity, 100–500%). These preclinical studies demonstrate a safety:efficacy profile supporting an ongoing phase 1/2 human clinical trial of the scAAV8.FIXR338L vector (designated BAX335).

Biodistribution and in vivo toxicity of aptamer-loaded gold nanostars

This paper reports an in vivo evaluation of toxicology and biodistribution of a highly anisotropic Au nanoconstruct composed of a gold nanostar (AuNS) core and a ligand shell of a G-quadruplex DNA aptamer AS1411 (Apt) supporting both targeting and therapy capabilities. We examined the toxicity of the nanoconstructs (Apt-AuNS) at four different injected concentrations. At the highest dose tested (48 mg/kg), maximal tolerated dose was not reached. Clinical pathology showed no apparent signs of acute toxicity. Interestingly, the nanoconstructs circulated longer in female rats compared to male rats. In two different tumor models, the biodistribution of Apt-AuNS, especially tumor accumulation, was different. Accumulation of Apt-AuNS was 5 times higher in invasive breast cancer tumors compared to fibrosarcoma tumors. These results provide insight on identifying a tumor model and nanoconstruct for in vivo studies, especially when an in vitro therapeutic response is observed in multiple cancer cell lines. From the Clinical EditorThis study investigated the toxicity and distribution of aptamer loaded gold nanostars in a rodent model of invasive breast cancer and fibrosarcoma. Acute toxicity was not identified even in the highest studied doses. Fivefold accumulation was demonstrated in the breast cancer model compared to the fibrosarcoma model. Studies like this are critically important in further clarifying the potential therapeutic use of these nanoconstructs, especially when ex vivo effects are clearly demonstrated.

Abstract 823: Water-soluble ureidomustine (BO-1055) potently suppresses orthotopic 22Rv/HL2 prostate tumor xenografts: early preclinical studies

Abstract In developed countries, prostate cancer (PCa) is one of the leading causes of death in men. PCa often recurs and metastasizes after an initial response to hormone therapy. Once PCa progresses to the castration-resistant stage, it becomes incurable. New and effective agents against PCa are therefore urgently needed. The objective of this study is to evaluate the antitumor activity of ureidomustine (BO-1055), which is a chemically stable and water-soluble phenyl N-mustard-benzamide conjugate previously synthesized from our laboratory. We previously demonstrated that ureidomustine exhibited potent therapeutic efficacy in nude mice bearing various human tumor xenografts, such as human colon cancer HCT-116 and prostate adenocarcinoma PC-3 xenografts (subcutaneous implantation), inhibiting growth by &amp;gt;95% and &amp;gt;99%, respectively. To broaden the antitumor spectrum of this agent, we evaluated the therapeutic efficacy of ureidomustine in nude mice bearing 22Rv/HL2 human PCa tumors implanted at an orthotopic site. The results showed that ureidomustine was as efficacious as docetaxel (&amp;gt;92% tumor suppression) and more potent than carboplatin (62% suppression) against 22Rv/HL2 PCa tumors in an orthotopic xenograft model. It revealed that ureidomustine exhibited low toxicity in mice, with no apparent pathological changes. We also studied the cytotoxicity of ureidomustine against normal murine cells and human hematopoietic stem cells (cord blood CD34+). The results showed that this agent exhibited low cytotoxicity against human hematopoietic stem cells (cord blood CD34+), primary human endothelial cells, and mesenchymal stem cells (BSC). Moreover, we performed the early preclinical studies including pharmacokinetics in rats, rat plasma protein binding, metabolic stability in rat liver microsomes, the 14 day intravenous acute toxicity in mice, and the biodistribution in rat, to determine whether this agent is a candidate for future clinical studies. The results indicate that ureidomustine is as potent as docetaxel against 22Rv/HL2 PCa cells in orthotopic xenograft models with low toxicity. In addition, this agent has low toxicity to human hematopoietic stem/progenitor cells and stromal cells such as BM-derived endothelial cells (BMEC) and MSC. Furthermore, early preclinical studies (ADME) suggest that ureidomustine has high potential for the treatment of advanced PCa in the clinic. Citation Format: Tsann-Long Su, Yi-Ren Chen, Jae-Hung Shieh, Tung-Hu Tsai, Malcolm A.S. Moore, Te-Chang Lee. Water-soluble ureidomustine (BO-1055) potently suppresses orthotopic 22Rv/HL2 prostate tumor xenografts: early preclinical studies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 823. doi:10.1158/1538-7445.AM2014-823

Evaluation of cancer imaging potential and photodynamic therapy efficacy of copper (II) benzyloxypheophorbide-a

The biological potential of a synthetic copper chlorophyll derivative was investigated via in vivo and in vitro experiments. The Cu-chlorophyll derivative photosensitizer (Cu-PH-A) was labeled with 131I with high efficiency (92.9 ± 4.2%) using the iodogen method. Cell culture studies were performed with the MCF-7 and MDAH-2774 cell lines after radiolabeling. The photosensitizing activity of Cu-PH-A was more effective in MDAH-2774 cells than in MCF-7 cells at a concentration of 50 μM. When the biodistribution in female Albino Wistar rats was examined, uptake of the radiolabeled photosensitizer was maximal in the liver and ovaries after 60 min. It is concluded that radiolabeled Cu-chlorophyll derivative photosensitizer has high uptake in ovaries in normal rats. In addition, the intercellular uptake and PDT efficacy of the Cu-PH-A in MDAH-2774 were good compared with MCF-7 cells. This photosensitizer could be useful for both ovary tumour imaging and PDT.