Formulation of ‘ready-to-use’ human clinical doses of 177Lu-labeled bisphosphonate amide of DOTA using moderate specific activity 177Lu and its preliminary evaluation in human patient

2020 ◽  
Vol 108 (8) ◽  
pp. 661-672
Author(s):  
Sudipta Chakraborty ◽  
Priyalata Shetty ◽  
Rubel Chakravarty ◽  
K. V. Vimalnath ◽  
Chandan Kumar ◽  
...  
Keyword(s):  
Specific Activity ◽  
Specific Binding ◽  
Preliminary Investigation ◽  
Skeletal Metastases ◽  
Preliminary Evaluation ◽  
Binding Studies ◽  
Human Patient ◽  
Biodistribution Studies ◽  
Target Organs

AbstractRadiolabeled macrocyclic bisphosphonate ligands have recently been demonstrated to be highly efficacious in treatment of patients with painful bone metastases. Herein, we report a robust protocol for formulation of therapeutically relevant doses of 177Lu-labeled bisphosphonate amide of DOTA (BPAMD) using moderate specific activity 177Lu produced by direct (n,γ) route and its preliminary investigation in human patients. Doses (2.8 ± 0.2 GBq) were formulated with high radiochemical purity (98.3 ± 0.4 %) using a protocol optimized after extensive radiochemical studies. In vitro binding studies with mineralized osteosarcoma cells demonstrated specific binding of the radiotracer. Biodistribution studies in healthy Wistar rats demonstrated rapid skeletal accumulation with fast clearance from the non-target organs. In a patient administered with 555 MBq dose of 177Lu-BPAMD, intense radiotracer uptake was observed in the metastatic skeletal lesions with insignificant uptake in any other major non-targeted organs. Preliminary clinical investigations carried out after administration of 2.6 GBq of 177Lu-BPAMD revealed significant reduction in pain after 1 week without any adverse effects. The developed protocol for formulation of 177Lu-BPAMD doses using moderate specific activity carrier added 177Lu has been found to be effective and warrants wider investigations in patients with painful skeletal metastases.

High non-specific binding of the β1-selective radioligand 2-125I-ICI-H

2003 ◽  
Vol 42 (04) ◽  
pp. 173-180 ◽  
Author(s):  
M. P. Law ◽  
K. Kopka ◽  
St. Wagner ◽  
S. Luthra ◽  
V. W. Pike ◽  
...  
Keyword(s):  
Specific Activity ◽  
Single Photon ◽  
Specific Binding ◽  
Photon Emission ◽  
Radiochemical Yield ◽  
Emission Computed Tomography ◽  
Biodistribution Studies ◽  
Positron Emission

Summary: Aim: As results of cardiac biopsies suggest, myocardial β1-adrenoceptor density is reduced in patients with chronic heart failure. However, changes in cardiac β2-adrenoceptors vary. With suitable radiopharmaceuticals single photon emission computed tomography (SPECT) and positron emission tomography (PET) offer the opportunity to assess β-adrenoceptors non-invasively. Among the novel racemic analogues of the established β1-selective adrenoceptor antagonist ICI 89.406 the iodinated 2-I-ICI-H showed high affinity and selectivity to β1-adrenoceptors in murine ventricular membranes. The aim of this study was its evaluation as a putative sub-type selective β1-adrenergic radioligand in cardiac imaging. Methods: Competition studies in vitro and in vivo were used to investigate the kinetics of 2-I-ICI-H binding to cardiac β-adrenoceptors in mice and rats. In addition, the radiosynthesis of 2-125I-ICI-H from the silylated precursor 2-SiMe3-ICI-H was established. The specific activity was 80 GBq/µmol, the radiochemical yield ranged from 70 to 80%. Results: The unlabelled compound 2-I-ICI-H showed high β1-selectivity and -affinity in the in vitro competition studies. In vivo biodistribution studies apparently showed low affinity to cardiac β-adrenoceptors. The radiolabelled counterpart 2-125I-ICI-H showed a high degree of non-specific binding in vitro and no specific binding to cardiac β1-adrenoceptors in vivo. Conclusion: Because of its high non-specific binding 2-125I-ICI-H is no suitable radiotracer for imaging in vivo.

68Ga-labeled HBED-CC Variant of uPAR Targeting Peptide AE105 Compared with 68Ga-NODAGA-AE105

2019 ◽  
Vol 18 (9) ◽  
pp. 1289-1294 ◽  
Author(s):  
Kusum Vats ◽  
Rohit Sharma ◽  
Haladhar D. Sarma ◽  
Drishty Satpati ◽  
Ashutosh Dash
Keyword(s):  
Solid Phase ◽  
Evaluation Studies ◽  
Radiochemical Yield ◽  
In Vivo Evaluation ◽  
Peptide Conjugates ◽  
Biodistribution Studies ◽  
Target Organs ◽  
U87mg Tumor

Aims: The urokinase Plasminogen Activator Receptors (uPAR) over-expressed on tumor cells and their invasive microenvironment are clinically significant molecular targets for cancer research. uPARexpressing cancerous lesions can be suitably identified and their progression can be monitored with radiolabeled uPAR targeted imaging probes. Hence this study aimed at preparing and evaluating two 68Ga-labeled AE105 peptide conjugates, 68Ga-NODAGA-AE105 and 68Ga-HBED-CC-AE105 as uPAR PET-probes. Method: The peptide conjugates, HBED-CC-AE105-NH2 and NODAGA-AE105-NH2 were manually synthesized by standard Fmoc solid phase strategy and subsequently radiolabeled with 68Ga eluted from a commercial 68Ge/68Ga generator. In vitro cell studies for the two radiotracers were performed with uPAR positive U87MG cells. Biodistribution studies were carried out in mouse xenografts with the subcutaneously induced U87MG tumor. Results: The two radiotracers, 68Ga-NODAGA-AE105 and 68Ga-HBED-CC-AE105 that were prepared in >95% radiochemical yield and >96% radiochemical purity, exhibited excellent in vitro stability. In vivo evaluation studies revealed higher uptake of 68Ga-HBED-CC-AE105 in U87MG tumor as compared to 68Ga-NODAGAAE105; however, increased lipophilicity of 68Ga-HBED-CC-AE105 resulted in slower clearance from blood and other non-target organs. The uPAR specificity of the two radiotracers was ascertained by significant (p<0.05) reduction in the tumor uptake with a co-injected blocking dose of unlabeled AE-105 peptide. Conclusion: Amongst the two radiotracers studied, the neutral 68Ga-NODAGA-AE105 with more hydrophilic chelator exhibited faster clearance from non-target organs. The conjugation of HBED-CC chelator (less hydrophilic) resulted in negatively charged 68Ga-HBED-CC-AE105 which was observed to have high retention in blood that decreased target to non-target ratios.

scholarly journals Rationale design and synthesis of some novel imidazole linked thiazolidinone hybrid molecules as DNA minor groove binders

2020 ◽  
Vol 11 (2) ◽  
pp. 120-132
Author(s):  
Javeed Ahmad War ◽  
Santosh Kumar Srivastava
Keyword(s):  
Molecular Docking ◽  
Binding Affinity ◽  
Specific Binding ◽  
Minor Groove ◽  
Stable Complex ◽  
Binding Studies ◽  
In Vitro Susceptibility ◽  
Reference Drug ◽  
Hybrid Molecules

A new series of imidazole linked thiazolidinone hybrid molecules was designed and subsequently synthesized through a feasible, three step reaction protocol. The structures of these molecules were established using FT-IR, 1H NMR, 13C NMR and HRMS techniques. In vitro susceptibility tests against some Gram positive (Staphylococcus aureus and Bacillus subtilis) and Gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa) exhibited broad spectrum potency of the molecules. The most potent molecule (S2A7) amongst the screened molecules, showed minimum inhibitory concentration (MIC) value not less than 2.0 µg/mL which was at par with the reference drug Streptomycin. Structure activity relationships revealed nitro and chloro groups being crucial for bioactivity when present at meta position of arylidene ring in 3-(3-(imidazol-1-yl)propyl)-5-(benzylidene)-2-(phenylimino)thiazolidin-4-one. Deoxyribonucleic acid (DNA)and bovine serum albumin (BSA) binding studies for S2A7 under simulated physiological pH were probed using UV-Visible, fluorescence quenching, gel electrophoresis and molecular docking techniques. These studies established that S2A7 has strong binding affinity towards DNA and binds at the minor groove of DNA with binding constant (Kb) of 0.1287×102 L/mol. Molecular docking simulations of S2A7 with DNA and BSA predicted binding affinity of -9.2 and -7.2 kcal/mol, respectively. Van der Waals forces and hydrogen bonding interactions were predicted as the main forces of interaction. With DNA, S2A7 exhibited specific binding affinity towards adenine-thiamine base pairs. The compound S2A7 forms a stable complex with BSA by binding at subdomain IIIA implying high bio-distribution of the compound.

scholarly journals Gold Nanoparticle-Based Fluorescent Theranostics for Real-Time Image-Guided Assessment of DNA Damage and Repair

2019 ◽  
Vol 20 (3) ◽  
pp. 471 ◽  
Author(s):  
Shriya S. Srinivasan ◽  
Rajesh Seenivasan ◽  
Allison Condie ◽  
Stanton L. Gerson ◽  
Yanming Wang ◽  
...  
Keyword(s):  
Molecular Imaging ◽  
Real Time ◽  
Targeted Delivery ◽  
Specific Binding ◽  
Imaging Techniques ◽  
Cancer Cell Line ◽  
Molecular Probe ◽  
Biodistribution Studies

Chemotherapeutic dosing, is largely based on the tolerance levels of toxicity today. Molecular imaging strategies can be leveraged to quantify DNA cytotoxicity and thereby serve as a theranostic tool to improve the efficacy of treatments. Methoxyamine-modified cyanine-7 (Cy7MX) is a molecular probe which binds to apurinic/apyrimidinic (AP)-sites, inhibiting DNA-repair mechanisms implicated by cytotoxic chemotherapies. Herein, we loaded (Cy7MX) onto polyethylene glycol-coated gold nanoparticles (AuNP) to selectively and stably deliver the molecular probe intravenously to tumors. We optimized the properties of Cy7MX-loaded AuNPs using optical spectroscopy and tested the delivery mechanism and binding affinity using the DLD1 colon cancer cell line in vitro. A 10:1 ratio of Cy7MX-AuNPs demonstrated a strong AP site-specific binding and the cumulative release profile demonstrated 97% release within 12 min from a polar to a nonpolar environment. We further demonstrated targeted delivery using imaging and biodistribution studies in vivo in an xenografted mouse model. This work lays a foundation for the development of real-time molecular imaging techniques that are poised to yield quantitative measures of the efficacy and temporal profile of cytotoxic chemotherapies.

scholarly journals Incorporation of a Hydrophilic Spacer Reduces Hepatic Uptake of HER2-Targeting Affibody–DM1 Drug Conjugates

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1168
Author(s):  
Haozhong Ding ◽  
Mohamed Altai ◽  
Sara S. Rinne ◽  
Anzhelika Vorobyeva ◽  
Vladimir Tolmachev ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Histopathological Examination ◽  
Specific Binding ◽  
Hepatic Uptake ◽  
In Vitro Cytotoxicity ◽  
Drug Conjugates ◽  
Biodistribution Studies ◽  
Development Approach

Affibody molecules are small affinity-engineered scaffold proteins which can be engineered to bind to desired targets. The therapeutic potential of using an affibody molecule targeting HER2, fused to an albumin-binding domain (ABD) and conjugated with the cytotoxic maytansine derivate MC-DM1 (AffiDC), has been validated. Biodistribution studies in mice revealed an elevated hepatic uptake of the AffiDC, but histopathological examination of livers showed no major signs of toxicity. However, previous clinical experience with antibody drug conjugates have revealed a moderate- to high-grade hepatotoxicity in treated patients, which merits efforts to also minimize hepatic uptake of the AffiDCs. In this study, the aim was to reduce the hepatic uptake of AffiDCs and optimize their in vivo targeting properties. We have investigated if incorporation of hydrophilic glutamate-based spacers adjacent to MC-DM1 in the AffiDC, (ZHER2:2891)2–ABD–MC-DM1, would counteract the hydrophobic nature of MC-DM1 and, hence, reduce hepatic uptake. Two new AffiDCs including either a triglutamate–spacer–, (ZHER2:2891)2–ABD–E3–MC-DM1, or a hexaglutamate–spacer–, (ZHER2:2891)2–ABD–E6–MC-DM1 next to the site of MC-DM1 conjugation were designed. We radiolabeled the hydrophilized AffiDCs and compared them, both in vitro and in vivo, with the previously investigated (ZHER2:2891)2–ABD–MC-DM1 drug conjugate containing no glutamate spacer. All three AffiDCs demonstrated specific binding to HER2 and comparable in vitro cytotoxicity. A comparative biodistribution study of the three radiolabeled AffiDCs showed that the addition of glutamates reduced drug accumulation in the liver while preserving the tumor uptake. These results confirmed the relation between DM1 hydrophobicity and liver accumulation. We believe that the drug development approach described here may also be useful for other affinity protein-based drug conjugates to further improve their in vivo properties and facilitate their clinical translatability.

Nanocarriers with Gentamicin to Treat Intracellular Pathogens

2006 ◽  
Vol 6 (9) ◽  
pp. 3296-3302 ◽  
Author(s):  
C. Lecaroz ◽  
C. Gamazo ◽  
M. J. Blanco-Prieto
Keyword(s):  
Macrophage Activation ◽  
Polymeric Nanoparticles ◽  
Current Treatment ◽  
Delivery Systems ◽  
Oxygen Intermediates ◽  
Water Solvent ◽  
Biodistribution Studies ◽  
Target Organs

Brucellosis is a worldwide zoonosis caused by different species of the genus Brucella. The intracellular localisation of this pathogen, particularly in macrophages, renders treatment difficult since most antibiotics known to be efficient in vitro do not actively pass through cellular membranes. As alternative to current treatment, polymeric drug delivery systems containing gentamicin have been developed. These particulate carriers target the drug into the mononuclear-phagocytic system, where the pathogen resides that will allow intracellular accumulation of the antibiotic after particle degradation. Besides, particle uptake may induce macrophage activation, increasing the production of reactive oxygen intermediates, involved in host defense against the intracellular pathogen. The aim of the present work was to study the suitability of polymeric nanoparticles for gentamicin entrapment in view to treat brucellosis. Different poly(lactide-co-glycolide) PLGA polymers were used to formulate the nanoparticles containing gentamicin by a water-oil-water solvent evaporation method. Furthermore, in vitro macrophage activation upon nanoparticles phagocytosis and in vivo distribution of the nanocarriers in the target organs for Brucella (liver and spleen) were also studied. The nanoparticle sizes were below 350 nm, the gentamicin encapsulation efficiency depended on the polymer type used for their preparation and the in vitro release of the antibiotic exhibited a continuos pattern (PLGA 502H). PLGA 502H nanoparticles were the most suitable due to the highest entrapment and the most sustained release. The nanoparticles were successfully phagocyted by a J774 murine monocytes cell line and biodistribution studies in mice after intravenous administration of the delivery systems revealed that the particles reached the target organs of Brucella (liver and spleen). All together, these results indicate that the nanocarriers described in this work may be suitable as gentamicin delivery system to control brucellosis.

Autoradiographic detection of kinin receptors in the human medulla of control, hypertensive, and diabetic donors

2002 ◽  
Vol 80 (4) ◽  
pp. 249-257 ◽  
Author(s):  
Hudson de Sousa Buck ◽  
Brice Ongali ◽  
Gaétan Thibault ◽  
Charles J Lindsey ◽  
Réjean Couture
Keyword(s):  
Receptor Binding ◽  
Binding Sites ◽  
Specific Binding ◽  
Inferior Olivary Nucleus ◽  
Binding Studies ◽  
Specific Binding Sites ◽  
Kinin Receptors ◽  
Medullary Nuclei ◽  
Inferior Olivary

Kinins have been elected to the status of central neuromediators. Their effects are mediated through the activation of two G-protein-coupled receptors, denoted B1 and B2. Functional and binding studies suggested that B1 and B2 receptors are upregulated in the medulla and spinal cord of hypertensive and diabetic rats. The aim of this study was to localize and quantify kinin receptors in post-mortem human medulla obtained from normotensive, hypertensive, and diabetic subjects, using in vitro receptor autoradiography with the radioligands [125I]HPP-HOE140 (B2 receptor) and [125I]HPP[des-Arg10]-HOE140 (B1 receptor). Data showed specific binding sites for B2 receptor (0.4–1.5 fmol/mg tissue) in 11 medullary nuclei from 4 control specimens (paratrigeminal > ambiguus > cuneate, gelatinous layer of the caudal spinal trigeminal nucleus > caudal and interpolar spinal trigeminal, external cuneate, solitary tract > hypoglossal > gracile > inferior olivary nuclei). Increased density of B2 receptor binding sites was observed in seven medullary nuclei of four hypertensive specimens (paratrigeminal > external cuneate > interpolar and caudal spinal trigeminal, gracile, inferior olivary > hypoglossal nuclei). B2 receptor binding sites were seemingly increased in the same medullary nuclei of two diabetic specimens. Specific binding sites for B1 receptor (1.05 and 1.36 fmol/mg tissue) were seen only in the inferior olivary nucleus in two out of the ten studied specimens. The present results support a putative role for kinins in the regulation of autonomic, nociceptive, and motor functions at the level of the human medulla. Evidence is also provided that B2 receptors are upregulated in medullary cardiovascular centers of subjects afflicted of cardiovascular diseases.Key words: bradykinin, hypertension, diabetes, human brain.

scholarly journals Fibrin and fibrinogen degradation products with an intact D-domain C- terminal gamma chain inhibit an early step in accessory cell-dependent lymphocyte mitogenesis

Blood ◽  
1993 ◽  
Vol 81 (11) ◽  
pp. 3006-3014 ◽  
Author(s):  
SC Robson ◽  
R Saunders ◽  
LR Purves ◽  
C de Jager ◽  
A Corrigall ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Degradation Products ◽  
Specific Binding ◽  
Lymphocyte Transformation ◽  
Mononuclear Leukocytes ◽  
Accessory Cell ◽  
Binding Studies ◽  
Gamma Chain

Abstract Although the low molecular weight degradation products of fibrinogen (FgDP) and fibrin (FbDP) are known to inhibit lymphocyte blastogenesis, the effect of purified macro-molecular FgDP and FbDP (molecular weight, 90 to 200 Kd) is unclear. We have examined the effect of these latter FgDP and FbDP and find that products that contain the D domain inhibit lymphocyte proliferation in response to T-cell mitogens, allogeneic mononuclear leukocytes, and anti-CD3 in vitro. Plasmic digestion of D1 in the absence of calcium with removal of the C-terminal end of the gamma chain or disruption of the gamma-gamma C-terminal cross-link site of D-dimer (DD) by puffadder venom (PAV-D) abrogates their inhibitory potential. Prior incubation of monocytes with DD or D1 inhibits subsequent lymphocyte transformation. Binding studies with radiolabeled DD and PAV-D confirm that monocytes interact only with DD. This specific binding may be competitively inhibited by monoclonal antibodies to CD11b/CD18 or by peptide analogues of the C-terminal gamma chain of fibrinogen that mimic the adhesion recognition site of integrins. We postulate that DD and D1 bind to CD11b/CD18 on adherent monocytes and modulate lymphocyte activation. These products are typically present in the plasma of patients with disseminated intravascular coagulation with sepsis and could therefore influence inflammatory processes in vivo.

scholarly journals A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

2020 ◽  
Vol 48 (16) ◽  
pp. 8914-8926
Author(s):  
Erin E Cutts ◽  
J Barry Egan ◽  
Ian B Dodd ◽  
Keith E Shearwin
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Attachment Site ◽  
Binding Energies ◽  
Sequence Specificity ◽  
Binding Studies ◽  
Binding Model ◽  
Specific Binding Sites

Abstract The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

scholarly journals Heterologous Expression of Functional Ptr ToxA

2000 ◽  
Vol 13 (4) ◽  
pp. 456-464 ◽  
Author(s):  
Robert P. Tuori ◽  
Thomas J. Wolpert ◽  
Lynda M. Ciuffetti
Keyword(s):  
Fusion Protein ◽  
Heterologous Expression ◽  
Specific Activity ◽  
Maximal Activity ◽  
Cysteine Residue ◽  
Binding Studies ◽  
Reading Frame ◽  
Pyrenophora Tritici Repentis ◽  
Ptr Toxa

Ptr ToxA, a proteinaceous host-selective toxin (HST) produced by the fungus Pyrenophora tritici-repentis, was expressed in Escherichia coli and purified as a polyhistidine-tagged, fusion protein (NC-FP). NC-FP, consisting of both the N and C domains of the ToxA open reading frame (ORF), is produced as an insoluble protein in E. coli at approximately 10 to 16 mg per liter of culture. Following in vitro refolding, NC-FP elicits cultivar-specific necrosis in wheat, with a specific activity similar to that of native Ptr ToxA. A fusion protein consisting of only the C domain has approximately 10 to 20% of the activity of native Ptr ToxA. These data suggest that (i) the N domain is important for maximal activity of Ptr ToxA, (ii) the N domain does not function to eliminate activity of the protoxin, and (iii) post-translational modifications of Ptr ToxA are not essential for activity. A C domain construct with a cysteine residue mutated to glycine is inactive. This, plus the observation that toxin activity is sensitive to reducing agents, provides evidence that the two cysteine residues in Ptr ToxA are involved in a disulfide bond that is essential for activity. The heterologous expression of Ptr ToxA provides a valuable tool for addressing a number of issues such as receptor binding studies, structure/function studies, and screening wheat cultivars for disease resistance.

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