Sustained intra-cartilage delivery of low dose dexamethasone using a cationic carrier for treatment of post traumatic osteoarthritis.

Disease-modifying osteoarthritis drugs (DMOADs) should reach their intra-tissue target sites at optimal doses for clinical efficacy. The dense, negatively charged matrix of cartilage poses a major hindrance to the transport of potential therapeutics. In this work, electrostatic interactions were utilised to overcome this challenge and enable higher uptake, full-thickness penetration and enhanced retention of dexamethasone (Dex) inside rabbit cartilage. This was accomplished by using the positively charged glycoprotein avidin as nanocarrier, conjugated to Dex by releasable linkers. Therapeutic effects of a single intra-articular injection of low dose avidin-Dex (0.5 mg Dex) were evaluated in rabbits 3 weeks after anterior cruciate ligament transection (ACLT). Immunostaining confirmed that avidin penetrated the full cartilage thickness and was retained for at least 3 weeks. Avidin-Dex suppressed injury-induced joint swelling and catabolic gene expression to a greater extent than free Dex. It also significantly improved the histological score of cell infiltration and morphogenesis within the periarticular synovium. Micro-computed tomography confirmed the reduced incidence and volume of osteophytes following avidin-Dex treatment. However, neither treatment restored the loss of cartilage stiffness following ACLT, suggesting the need for a combinational therapy with a pro-anabolic factor for enhancing matrix biosynthesis. The avidin dose used caused significant glycosaminoglycan (GAG) loss, suggesting the use of higher Dex : avidin ratios in future formulations, such that the delivered avidin dose could be much less than that shown to affect GAGs. This charge-based delivery system converted cartilage into a drug depot that could also be employed for delivery to nearby synovium, menisci and ligaments, enabling clinical translation of a variety of DMOADs.

Neutralization of EP217609, a new dual-action FIIa/FXa anticoagulant, by its specific antidote avidin: a phase I study.

INTRODUCTION: EP217609 is a representative of a new class of synthetic parenteral anticoagulants with a dual mechanism of action. It combines in a single molecule a direct thrombin inhibitor and an indirect factor Xa inhibitor. EP217609 can be neutralized by a specific antidote avidin, which binds to the biotin moiety of EP217609. PURPOSE: The primary objective was to assess the neutralization of EP217609 by avidin in healthy subjects. Secondary objectives were to define the optimal avidin monomer/EP217609 molar ratio to achieve an adequate neutralization of EP217609 and to assess the safety and tolerability of EP217609 and avidin. METHODS: Healthy subjects (n = 36) were randomized to a 3 by 3 replicated Latin square design between 3 EP217609 doses (4, 8, 12 mg) and 3 avidin monomer/EP217609 molar ratios (1:1; 2:1; 3:1). EP217609 was administered as a single intravenous bolus, and avidin as a 30-min intravenous infusion, starting 90 min after EP217609 administration. RESULTS: Overall, EP217609 and avidin were well tolerated. One subject experienced a benign and transient typical pseudo-allergic reaction. The administration of EP217609 resulted in dose-dependent increases in pharmacodynamic markers. Avidin triggered a rapid and irreversible neutralization of EP217609 without rebound effect. Adequate neutralization of the anticoagulant activity was achieved with both 2:1 and 3:1 avidin monomer/EP217609 molar ratios. All safety parameters did not show any treatment-emergent clinically relevant changes or abnormalities in any dose group. CONCLUSIONS: These results will allow further investigation in patients requiring a neutralizable anticoagulant as those undergoing cardiac surgery. STUDY REGISTRATION: EudraCT number 2010-020216-10.

A rabbit model demonstrates the influence of cartilage thickness on intra-articular drug delivery and retention within cartilage.

For evaluation of new approaches to drug delivery into cartilage, the choice of an animal model is critically important. Since cartilage thickness varies with animal size, different levels of drug uptake, transport and retention should be expected. Simple intra-articular injection can require very high drug doses to achieve a concentration gradient high enough for drug diffusion into cartilage. New approaches involve nanoparticle delivery of functionalized drugs directly into cartilage; however, diffusion-binding kinetics proceeds as the square of cartilage thickness. In this study, we demonstrate the necessity of using larger animals for sustained intra-cartilage delivery and retention, exemplified by intra-articular injection of Avidin (drug-carrier) into rabbits and compared to rats in vivo. Penetration and retention of Avidin within cartilage is greatly enhanced by electrostatic interactions. Medial tibial cartilage was the thickest of rabbit cartilages, which generated the longest intra-cartilage half-life of Avidin (τ1/2 = 154 h). In contrast, Avidin half-life in thinner rat cartilage was 5-6 times shorter (τ1/2 ∼ 29 h). While a weak correlation (R(2) = 0.43) was found between Avidin half-lives and rabbit tissue GAG concentrations, this correlation improved dramatically (R(2) = 0.96) when normalized to the square of cartilage thickness, consistent with the importance of cartilage thickness to evaluation of drug delivery and retention.

Electrostatic interactions enable rapid penetration, enhanced uptake and retention of intra-articular injected avidin in rat knee joints.

Intra-articular (i.a.) drug delivery for local treatment of osteoarthritis remains inadequate due to rapid clearance by the vasculature or lymphatics. Local therapy targeting articular cartilage is further complicated by its dense meshwork of collagen and negatively charged proteoglycans, which can prevent even nano-sized solutes from entering. In a previous in vitro study, we showed that Avidin, due to its size (7 nm diameter) and high positive charge (pI 10.5), penetrated the full thickness of bovine cartilage and was retained for 15 days. With the goal of using Avidin as a nano-carrier for cartilage drug delivery, we investigated its transport properties within rat knee joints. Avidin penetrated the full thickness of articular cartilage within 6 h, with a half-life of 29 h, and stayed inside the joint for 7 days after i.a. injection. The highest concentration of Avidin was found in cartilage, the least in patellar tendon and none in the femoral bone; in contrast, negligible Neutravidin (neutral counterpart of Avidin) was present in cartilage after 24 h. A positive correlation between tissue sGAG content and Avidin uptake (R(2) = 0.83) confirmed the effects of electrostatic interactions. Avidin doses up to at least 1 µM did not affect bovine cartilage explant cell viability, matrix catabolism or biosynthesis.

Avidin as a model for charge driven transport into cartilage and drug delivery for treating early stage post-traumatic osteoarthritis.

Local drug delivery into cartilage remains a challenge due to its dense extracellular matrix of negatively charged proteoglycans enmeshed within a collagen fibril network. The high negative fixed charge density of cartilage offers the unique opportunity to utilize electrostatic interactions to augment transport, binding and retention of drug carriers. With the goal of developing particle-based drug delivery mechanisms for treating post-traumatic osteoarthritis, our objectives were, first, to determine the size range of a variety of solutes that could penetrate and diffuse through normal cartilage and enzymatically treated cartilage to mimic early stages of OA, and second, to investigate the effects of electrostatic interactions on particle partitioning, uptake and binding within cartilage using the highly positively charged protein, Avidin, as a model. Results showed that solutes having a hydrodynamic diameter ≤10 nm can penetrate into the full thickness of cartilage explants while larger sized solutes were trapped in the tissue's superficial zone. Avidin had a 400-fold higher uptake than its neutral same-sized counterpart, NeutrAvidin, and >90% of the absorbed Avidin remained within cartilage explants for at least 15 days. We report reversible, weak binding (K(D) ~ 150 µM) of Avidin to intratissue sites in cartilage. The large effective binding site density (N(T) ~ 2920 µM) within cartilage matrix facilitates Avidin's retention, making its structure suitable for particle based drug delivery into cartilage.

Measurement of local permeability at subcellular level in cell models of agonist- and ventilator-induced lung injury.

Alterations of cell monolayer integrity and increased vascular permeability are key to many pathologies, including atherosclerosis, stroke, lung injury, cancer, digestive disorders and others. Current approaches to probe cell permeability require specific culture conditions and provide an average estimation of trans-monolayer permeability, while analysis of regional monolayer permeability in static and mechanically challenged monolayer at a single-cell scale resolution remains unavailable. We describe a novel method for visualization and rapid quantification of trans-monolayer permeability based on high-affinity interactions between ligand (FITC-conjugated avidin) added in the culture medium, which permeates cell monolayer to reach substrate-bound acceptor (biotinylated gelatin or collagen). This approach was used to simultaneously evaluate general and local permeability responses by endothelial cell (EC) monolayer to a spectrum of barrier protective and barrier disruptive agonists and their combinations. The results revealed the paracellular pathway as the predominant mechanism of agonist-induced mass transport by pulmonary EC. We also detected for the first time, in a direct assay, a synergistic effect of pathologically relevant levels of cyclic stretch (CS) and edemagenic agent thrombin in the development of pulmonary EC hyper-permeability response observed in ventilator-induced lung injury. The reported novel assay provides unique information about local monolayer permeability changes induced by agonists, mechanical factors or molecular perturbations in single cells. However, the spectrum of substrates, assay formats and experimental conditions compatible with this assay suggest its broad application in the areas of endothelial and epithelial biology, cancer research and other fields.

Pretargeting of necrotic tumors with biotinylated hypericin using 123I-labeled avidin: evaluation of a two-step strategy.

As an alternative to directly targeting of necrotic tissue using hypericin, we synthesized a conjugate of hypericin to biotin for use in a pretargeting approach. With this conjugate, we explored the possibility of a two-step pretargeting strategy using (123)I-labeled avidin as effector molecule directed against necrotic RIF-1 tumors. Hypericin was conjugated to biotin-ethylenediamine in a straightforward coupling method using n-hydroxysuccinimide and dicyclohexylcarbodiimide. The necrosis avidity of the conjugate was first confirmed in necrotic liver tissue by means of fluorescence microscopy. Using autoradiography imaging and whole body-biodistribution, the accumulation of (123)I-avidin in necrotic tumor tissue was evaluated 24 h after administration and 48 h after pretargeting with hypericin-biotin. Analysis of autoradiography images show a higher accumulation of (123)I-avidin in pretargeted compared to nontargeted tissue. However, absolute accumulation of (123)I-avidin in necrotic tumors was low as shown by biodistribution experiments. Direct injection of hypericin-biotin or biotin-fluorescein did not substantially improve (123)I-avidin accumulation after pretargeting, pointing towards a poor penetration of avidin in necrotic tissue. Our results show the feasibility of a pretargeting technique using a small molecule as targeting agent. However, for a more efficient accumulation of the effector molecule in necrotic tissue, other pretargeting strategies need to be investigated.

In vivo distribution of avidin-conjugated MX35 and (211)At-labeled, biotinylated poly-L-lysine for pretargeted intraperitoneal α-radioimmunotherapy.

PURPOSE: Avidin-coupled monoclonal antibody MX35 (avidin-MX35) and astatine-211-labeled, biotinylated, succinylated poly-l-lysine ((211)At-B-PL(suc)) were administered in mice to assess potential efficacy as an intraperitoneal (i.p.) therapy for microscopic tumors. We aimed to establish a timeline for pretargeted radioimmunotherapy using these substances, and estimate the maximum tolerable activity. METHODS: (125)I-avidin-MX35 and (211)At-B-PL(suc) were administered i.p. in nude mice. Tissue distributions were studied at various time points and mean absorbed doses were estimated from organ uptake of (211)At-B-PL(suc). Studies of myelotoxicity were performed after administration of different activities of (211)At-B-PL(suc). RESULTS: We observed low blood content of both (125)I-avidin-MX35 and (211)At-B-PL(suc), indicating fast clearance. After sodium perchlorate blocking, the highest (211)At uptake was found in kidneys. Red bone marrow (RBM) accumulated some (211)At activity. Mean absorbed doses of special interest were 2.3 Gy/MBq for kidneys, 0.4 Gy/MBq for blood, and 0.9 Gy/MBq for RBM. An absorbed dose of 0.9 Gy to the RBM was found to be safe. These values suggested that RBM would be the key dose-limiting organ in the proposed pretargeting scheme, and that blood data alone was not sufficient for predicting its absorbed dose. CONCLUSIONS: To attain a favorable distribution of activity and avoid major toxicities, at least 1.0 MBq of (211)At-B-PL(suc) can be administered 24 hours after an i.p. injection of avidin-MX35. These results provide a basis for future i.p. therapy studies in mice of microscopic ovarian cancer.

Distinct transduction modes of arginine-rich cell-penetrating peptides for cargo delivery into tumor cells.

The application of cell-penetrating peptides (CPPs) for delivering various cargo molecules with biological functions into cells has gained much attention in recent years. However, the internalization mechanisms and delivery properties of CPP-cargo remains controversial. In this study, low- and high-molecular-weight cargoes attached to arginine-rich CPPs were employed: the former was the fluorescein isothiocyanate-labeled nona-arginine (CPP-FITC), and the latter was the fluorescently labeled nona-arginine-avidin complex (CPP-avidin). We measured the intracellular trafficking of CPP-FITC and CPP-avidin in four cancer cell lines in a series of microenvironments altered by the presence or absence of serum, different temperatures and different incubation times. The results revealed that CPP-cargo delivery exhibited no specificity toward any cell line, but the levels were found to be related to cell type and cargo. Furthermore, their endocytic mechanisms were investigated via incubation with related endocytic inhibitors. Two different types of CPP-cargo were required to cross the plasma membrane to bind to cell surface-associated heparan sulfate proteoglycans in a time-dependent manner. CPPs and small cargoes attached to CPP may enter cells rapidly via direct translocation in addition to the endocytic route. Translocation of large components linked to CPP tended to be mediated by macropinocytosis in an energy-dependent manner with slower rates for larger compounds. In contrast, the clathrin-dependent pathway is not essential to the translocation of either type of CPP-cargo.

Preclinical pharmacology and safety of a novel avidin derivative for tissue-targeted delivery of radiolabelled biotin.

We recently described an oxidized avidin variant, named AvidinOX(®) , which is a product that chemically links to tissue proteins while maintaining the capacity to uptake intravenously administered biotin. Such product proved to be successful in targeting radionuclide therapy in a mouse model of inoperable breast cancer. Here, we show that the uptake of a single or multiple doses of biotin (up to five times), by the tissue-bound AvidinOX(®) , is stable for 2 weeks. Taking into account that oxidized avidin is the first chemically reactive protein to be proposed for clinical use, we evaluated its tolerability, immunogenicity and mutagenicity. Present in vitro data indicate that AvidinOX(®) (up to 10 µg/5 × 10(5) cells) does not affect cell viability or proliferation of PC3 human prostate cancer or 3T3 mouse fibroblast cell lines as well as primary mouse spleen cells. Safety pharmacology and toxicology studies were conducted using AvidinOX(®) up to the highest concentration compatible with its solubility (about 12 mg/mL), representing four times the product concentration intended for human use, and in the maximum administrable volume compatible with each study system. The intramuscular administration in rat and monkey induced a moderate to strong inflammatory response particularly after a second administration and consistently with the induction of an immune response. Interestingly, the intramuscular administration of AvidinOX(®) to rodents and monkeys exhibiting very high anti-avidin antibody titres was well tolerated with no systemic symptoms of any kind. Intravenous administration of AvidinOX(®) , performed to mimic an accidental injection of the dose intended for a local administration (15 µL of 3.3 mg/mL solution), showed significant localization of the product into the spleen not associated with uptake of the radiolabelled biotin intravenously injected after 24 hr, thus suggesting rapid inactivation. No mutagenic activity was induced by oxidized avidin in prokaryotic and eukaryotic cells. Overall, the present data indicate that AvidinOX(®) is well tolerated in rodents and non-human primates, thus supporting its clinical use within protocols of radionuclide therapy of inoperable tumour lesions.

Recording intracellular molecular events from the outside: glycosylphosphatidylinositol-anchored avidin as a reporter protein for in vivo imaging.

UNLABELLED: With the emergence of multimodal imaging strategies, genetically encoded reporters that can be flexibly combined with any imaging modality become highly attractive. Here we describe the use of glycosylphosphatidylinositol (GPI)-anchored avidin, an avidin moiety targeted to the extracellular side of cell membranes via a GPI anchor, as a reporter for in vivo imaging. Being present on the outside of cells, avidin can be visualized with any type of biotinylated imaging agent, without the requirement that the probe be membrane-permeable. We used the avidin-GPI system to monitor the activity of hypoxia-inducible factors (HIFs)-oxygen-sensing transcription factors, which play a major role in regulating cancer progression-in a mouse tumor allograft model. METHODS: Mouse C51 cells were stably transfected with pH3SVG, a reporter construct driving the expression of avidin-GPI from an HIF-sensitive promoter. The transfected cells were subcutaneously implanted into BALB/c nude mice. At 10 d after tumor inoculation, mice received an intravenous injection of either alexa-594-biocytin or (67)Ga-DOTA-biotin, and tumor HIF activity was imaged using fluorescence reflectance imaging or SPECT. RESULTS: In vitro cell experiments demonstrated the functionality and HIF-dependent regulation of the avidin-GPI reporter construct. In vivo, avidin-GPI was targeted specifically in allograft tumors with biotinylated imaging probes using both fluorescence imaging and SPECT. Analysis of the reporter expression pattern on ex vivo tumor tissue sections indicated a good overlap, with areas of hypoxia. CONCLUSION: We have demonstrated the utility of avidin-GPI as a reporter for multimodal in vivo imaging using both a fluorescence and a SPECT approach to assess intracellular oxygen signaling in a mouse tumor model.

CPP-protein constructs induce a population of non-acidic vesicles during trafficking through endo-lysosomal pathway.

The major limitation in the application of bioactive molecules is their low permeation across plasma membrane. Effective transporters - cell-penetrating peptides (CPPs) - are utilized to enhance uptake of various cargo upon attachment to its sequences. Still, information about relevance of different endocytic routes during CPP-cargo internalization is ambiguous and underlying mechanism(s) of intracellular trafficking is even less understood. We first defined involvement of recycling pathway in trafficking of 3 different CPPs - transportan, oligoarginine and Tat - complexed to avidin-TexasRed in Cos-7 cells in relation to trans-Golgi network spatially constraining recycling endosomes. By confocal microscopy, only a negligible fraction of complexes-containing vesicles were found inside trans-Golgi ring suggesting its marginal role in CPP-mediated delivery. Secondly, we characterized engagement of endo-lysosomal pathway to assess acidity of complexes-containing vesicles. CPPs induced 3 different populations of complexes-containing vesicles which size and proportion depended on CPP, time and concentration. In time, more complexes were targeted to low-pH structures. However, a population of complexes-containing vesicles was observed to retain rather neutral pH. Induction of vesicles with non-acidic pH generated i.e. by caveolin-dependent endocytosis or by CPPs themselves during intracellular trafficking could be the key step in inducement of escape of complexes from endosomal structures, a limiting step in effective cargo delivery by CPPs.

OXavidin for tissue targeting biotinylated therapeutics.

Avidin is a glycoprotein from hen egg white that binds biotin with very high affinity. Here we describe OXavidin, a product containing aldehyde groups, obtained by ligand-assisted sugar oxidation of avidin by sodium periodate. OXavidin chemically reacts with cellular and tissue proteins through Schiff's base formation thus residing in tissues for weeks while preserving the biotin binding capacity. The long tissue residence of OXavidin as well as that of OXavidin/biotinylated agent complex occurs in normal and neoplastic tissues and immunohistochemistry shows a strong and homogenous stromal localization. Once localized in tissue/tumor, OXavidin becomes an "artificial receptor" for intravenous injected biotin allowing tumor targeting with biotinylated therapeutics like radioisotopes or toxins. Moreover, present data also suggest that OXavidin might be useful for the homing of biotinylated cells. Overall, OXavidin exhibits a remarkable potential for many different therapeutic applications.

Intraoperative avidination for radionuclide therapy: a prospective new development to accelerate radiotherapy in breast cancer.

PURPOSE: In a continuous effort to seek for anticancer treatments with minimal side effects, we aim at proving the feasibility of the Intraoperative Avidination for Radionuclide Therapy, a new procedure for partial breast irradiation. EXPERIMENTAL DESIGN: To assess doses of 90Y-DOTA-biotin to target (i.e., breast tumor bed) and nontarget organs, we did simulation studies with 111In-DOTA-biotin in 10 candidates for conservative breast surgery. Immediately after quadrantectomy, patients were injected with 100-mg avidin in the tumor bed. On the following day, patients were given 111In-DOTA-biotin (approximately 111 MBq) i.v. after appropriate chase of biotinylated albumin (20 mg) to remove circulating avidin. Biokinetic studies were done by measuring radioactivity in scheduled blood samples, 48-h urine collection, and through scintigraphic images. The medical internal radiation dose formalism (OLINDA code) enabled dosimetry assessment in target and nontarget organs. RESULTS: Images showed early and long-lasting radioactive biotin uptake in the operated breast. Rapid blood clearance (<1% at 12 h) and urine excretion (>75% at 24 h) were observed. Absorbed doses, expressed as mean+/-SD in Gy/GBq, were as low as 0.15+/-0.05 in lungs, 0.10+/-0.02 in heart, 0.06+/-0.02 in red marrow, 1.30+/-0.50 in kidneys, 1.50+/-0.30 in urinary bladder, and 0.06+/-0.02 in total body, whereas in the targeted area, they increased to 5.5+/-1.1 Gy/GBq (50% ISOROI) and 4.8+/-1.0 Gy/GBq (30% ISOROI). CONCLUSION: Our preliminary results suggest that Intraoperative Avidination for Radionuclide Therapy is a simple and feasible procedure that may improve breast cancer patients' postsurgical management by shortening radiotherapy duration.

Real time observation of diffusion and bioaffinity binding processes in single polyelectrolyte-coated microcapsules: a fluorescence-based approach.

We report on using fluorescence microscopy to study, visualize and determine the diffusion phenomena into and bioaffinity binding within single microcapsules in real time by using biotin-fluorescein as diffusive species and encapsulated avidin as binding partner. Microcapsules were constructed by entrapment of avidin within an agarose matrix and encapsulated with polyelectrolyte layers by Layer-by-Layer (LbL) polyelectrolyte self assembly. A "ring" of high fluorescence intensity advancing with time towards the capsule centre was observed during incubation of capsules with fluorescent-labeled biotin. Fluorescence intensity was build up in capsule areas where binding to avidin occurred and was visualized in real time. A model for the diffusion process in microcapsules was developed and experimental data was plotted and fitted well with trends predicted by the model. The value of the diffusion coefficient for biotin-fluorescein was determined to be 3.5x10(-8)cm(2)/s, which is comparable to literature values of similar sized molecules.

Avidin bioconjugate with a thermoresponsive polymer for biological and pharmaceutical applications.

A thermoresponsive polymer, N-isopropylacrylamide-co-acrylamide (Mn 6 kDa) with a lower critical solution temperature (LCST) of 37 degrees C, was activated and conjugated to avidin to yield a derivative with 200 kDa molecular weight. Gel permeation analysis demonstrated that the new bioconjugate possessed an apparent size corresponding to a 220 kDa globular protein. Photon correlation spectroscopy and turbidometric studies showed that the bioconjugate underwent temperature dependent phase transitions. The protein-co-polymer bioconjugate displayed the same onset phase transition temperature (LCST) as the original synthetic co-polymer. Nevertheless, the aggregation profile of the bioconjugate shifted at higher temperature as compared to the original polymer. This indicated that the aggregation behaviour coil-to-globule transition of the co-polymer was modified by anchoring to the protein surface. Circular dichroism analysis showed that the co-polymer conjugation did not alter the protein tertiary structure tertiary the aromatic amino acid environment. The bioconjugate maintained 85+/-3% of native avidin affinity for biotin and biotin-Mab, and high affinity was maintained after three heating cycles. Pharmacokinetic studies demonstrated that the co-polymer bioconjugation increased the avidin residence time in the bloodstream. The distribution phase of avidin-co-polymer was longer than the native protein by a factor of 20. The co-polymer conjugation decreased by three-fold the distribution extent of avidin and reduced significantly its up-take to the liver.

A target cell-specific activatable fluorescence probe for in vivo molecular imaging of cancer based on a self-quenched avidin-rhodamine conjugate.

A target cell-specific activation strategy for improved molecular imaging of peritoneal implants has been proposed, in which fluorophores are activated only in living targeted cells. A current example of an activatable fluorophore is one that is normally self-quenched by attachment to a peptide backbone but which can be activated by specific proteases that degrade the peptide resulting in "dequenching." In this study, an alternate fluorescence activation strategy is proposed whereby self-quenching avidin-rhodamine X, which has affinity for lectin on cancer cells, is activated after endocytosis and degradation within the lysosome. Using this approach in a mouse model of peritoneal ovarian metastases, we document target-specific molecular imaging of submillimeter cancer nodules with minimal contamination by background signal. Cellular internalization of receptor-ligand pairs with subsequent activation of fluorescence via dequenching provides a generalizable and highly sensitive method of detecting cancer microfoci in vivo and has practical implications for assisting surgical and endoscopic procedures.

IART: intraoperative avidination for radionuclide treatment. A new way of partial breast irradiation.

A new procedure, known as Intraoperative Avidination for Radionuclide Therapy (IART), is described in breast cancer patients. In this paper, we provide proof of the principle that intraoperative injection of avidin in the tumour bed after quadrantectomy allows homing in of intravenously (IV) administered radioactive biotin to the target site. This approach of targeted therapy consists of two steps: (i) "avidination" of the anatomical area of the tumour with avidin injected by the surgeon, into and around the tumour bed; (ii) targeting the anatomical area of the tumour by IV injection of radiolabelled biotin. The scintigraphic images demonstrated fast and stable uptake of labelled biotin at the site of operated breast. The radiation dose released to the index quadrant was more than 5 Gy/GBq, consistent with a boost of 20 Gy for an activity of 3.7 GBq 90Y-biotin (100mCi). A further large clinical trial facing IART in combination with reduced external-beam radiotherapy is, in our opinion, fully justified.