Ankur Kaul

Paclitaxel loaded PEGylated gleceryl monooleate based nanoparticulate carriers in chemotherapy.

A PEGylated drug delivery system of paclitaxel (PTX), based on glyceryl monooleate (GMO) was prepared by optimizing various parameters to explore its potential in anticancer therapy. The prepared system was characterized through polarized light microscopy, TEM, AFM and SAXS to reveal its liquid crystalline nature. As GMO based LCNPs exhibit high hemolytic toxicity and faster release of entrapped drug (66.2 ± 2.5% in 24 h), PEGylation strategy was utilized to increase the hemocompatibility (reduction in hemolysis from 60.3 ± 10.2 to 4.4 ± 1.3%) and control the release of PTX (43.6 ± 3.2% released in 24 h). The cytotoxic potential and cellular uptake was assessed in MCF-7 cell lines. Further, biodistribution studies were carried out in EAT (Ehrlich Ascites tumor) bearing mice using (99m)Tc-(Technetium radionuclide) labeled formulations and an enhanced circulation time and tumor accumulation (14 and 8 times, respectively) were observed with PEGylated carriers over plain ones, at 24 h. Finally, tumor growth inhibition experiment was performed and after 15 days, control group exhibited 15 times enhancement in tumor volume, while plain and PEGylated systems exhibited only 8 and 4 times enhancement, respectively, as compared to initial tumor volume. The results suggest that PEGylation enhances the hemocompatibility and efficacy of GMO based system that may serve as an efficient i.v. delivery vehicle for paclitaxel.

The in vivo behavior and antitumor activity of doxorubicin-loaded poly(γ-benzyl L-glutamate)-block-hyaluronan polymersomes in Ehrlich ascites tumor-bearing BalB/c mice

UNLABELLED The in vivo efficacy of doxorubicin (DOX)-loaded poly(γ-benzyl l-glutamate)-block-hyaluronan (PBLG(23)-b-HYA(10))-based polymersomes (PolyDOX) was evaluated. Samples were efficiently labeled with technetium-99m radionuclide with good stability for in vivo studies. PolyDOX enhanced circulation time compared to free DOX. Biodistribution studies revealed selective accumulation of PolyDOX in the Ehrlich ascites tumor (EAT) as a result of passive accumulation and active targeting (CD44-mediated endocytosis) in EAT-bearing mice. Toxicity studies demonstrated PolyDOX is a safe drug carrier, and no hemolysis was observed with PolyDOX equivalent to 200 μg/mL of free DOX. PolyDOX dominantly controlled tumor growth by delaying doubling time of EATs compared to free DOX over 30 days after treatment. PolyDOX also increased life span six times more than free DOX. Hence, it is reasonable to expect that higher DOX levels attributable to PolyDOX improve the therapeutic index and reduce side effects due to site-specific drug accumulation. FROM THE CLINICAL EDITOR In this preclinical project, doxorubicin loaded polymersomes enhanced intracellular uptake of doxorubicin in a murine model of Ehrlich Ascites Tumor (EAT) through CD44 receptor mediated endocytosis, resulting in prolonged Tumor Doubling Time and increase in life span of mice.

Preliminary evaluation of technetium-99m-labeled ceftriaxone: infection imaging agent for the clinical diagnosis of orthopedic infection

OBJECTIVE In this study we sought to assess the efficacy of a technetium-99m (Tc-99m)-labeled third-generation cephalosporin as an infection imaging agent in the accurate detection of the sites of bacterial infection in vivo. DESIGN Ceftriaxone (CRO) was formulated into a ready-to-use single-vial cold kit with a shelf-life of over 6 months and was successfully labeled with technetium. The radiolabeled drug, Tc-99m-CRO, was subjected to the following preclinical evaluations: radiochemical purity, in vitro and in vivo stability, bacterial binding assay, and pharmacokinetic studies in animals and in human patients. RESULTS The kit formulation exhibited excellent radiolabeling efficiency (∼99%) and high in vitro and in vivo stability. The radiolabeled drug exhibited slow blood clearance (12% at 4 h), and the high protein binding and excretion pattern of the labeled formulation mimics the reported pharmacokinetic profile of the drug alone. In the animal model, scintigraphy scans showed higher uptake of the radiopharmaceutical in infectious lesions, even at 1 h post-administration, in comparison to inflammatory lesions. The clinical evaluation of Tc-99m-labeled CRO showed a diagnostic accuracy of 83.3%, and a sensitivity and specificity of 85.2% and 77.8%, respectively. CONCLUSIONS This kit formulation has the potential for imaging bacterial infections with much higher sensitivity and specificity as compared to other Tc-99m-labeled antibiotics available as convenient ready-to-use kits in routine clinical practice.

Evaluation of ISCOM matrices clearance from rabbit nasal cavity by gamma scintigraphy

Immune stimulating complexes and/or ISCOM matrices (adjuvant nanoparticles without antigen as a structural component) found potential applications as nasal vaccine adjuvant/delivery system owing to virus like particulate structure and saponin as potent Th1 adjuvant. One of important limiting factor for nasal vaccine delivery is the limited time available for absorption within the nasal cavity due to mucociliary clearance. In this report the clearance rate of ISCOM matrices from nasal cavity of rabbit was determined by gamma scintigraphy. ISCOM matrices were radiolabelled with (99m)Tc by direct labelling method using stannous chloride as a reducing agent. (99m)Tc labelled ISCOM matrices were administered into the nostril of female New Zealand rabbits and 1 min static views were repeated each 15 min until 4h. Clearance rate of ISCOM matrices from nasal cavity was calculated after applying the physical decay corrections. The mean labelling efficiency for ISCOM matrices were calculated as approximately 58.4%. ISCOM matrices showed slower clearance rate compared to sodium pertechnetate control solution (p<0.005) from nasal cavity that may be due to particulate and hydrophobic characters of ISCOM particles even though it was also cleared within 4h from nasal cavity. Mucoadhesive ISCOM formulations that retain in nasal cavity for longer duration of time may reduce the dose/frequency of vaccine for nasal immunization.

Synthesis, conjugation and relaxation studies of gadolinium(III)-4-benzothiazol-2-yl-phenylamine as a potential brain specific MR contrast agent

Magnetic resonance (MR) imaging is widely used in clinical research to map the structural and functional organization of the brain. We have designed and synthesized a Gd-based specific MR contrast agent that binds to regions in the brain. The presented compound {4-[(4-benzothiazol-2-yl-phenylcarbamoyl)-methyl]-7,10-bis-carboxymethyl-1,4,7,10-tetraazacyclododec-1-yl} acetic acid (DO3A-BT) was synthesized by conjugating the chloroacetylated product of 4-benzothiazol-2-yl-phenylamine with a trisubstituted cyclen. The lanthanide complex (Ln-DO3A-BT) was evaluated in vitro for both MR (Gd-DO3A-BT) and optical (Eu-DO3A-BT) imaging applications. The complex Gd-DO3A-BT displays a relaxivity of r1 = 4.18 mM(-1) s(-1) at 4.7 T which is 1.2 times greater than Dotarem and significantly higher than the brain specific MR contrast agent Luxol Fast Blue (LFB). The protonation constant of the ligand (pKa1 = 9.91, pKa2 = 8.22, pKa3 = 5.01) and the stability constant of the complex formed between Gd(III), Eu(III) and Ca(II) and ligand DO3A-BT (log βGdL = 18.4, log βEuL = 18.3, log βZn2L = 7.1, log βCa2L = 6.3) were recorded by potentiometric titration. The constants reflect the high stability of the ligand with lanthanides compared with endogenous metal ions. The transmetalation stability of Gd-DO3A-BT toward Zn proved to be excellent with a rate constant of 3.07 × 10(-5) s(-1) which is in line with other tetraazatetraacetic acid (DOTA)-monoamide complexes. The hydration number (q) was found to be 0.92, and is calculated from the difference in the luminescence lifetime of Eu-DO3A-BT in H2O and D2O solutions to determine the coordination state of this complex. The in vivo biodistribution of (99m)Tc-DO3A-BT in BALB/c mice showed a brain uptake of 1.2% ID g(-1) at 2 min post injection when injected with mannitol which disrupts the blood-brain-barrier (BBB) due to osmotic shock. In vitro binding on the brain homogenate revealed a high uptake by the neuronal/glial cells for in vivo applications.

Modified dipeptide-based nanoparticles: vehicles for targeted tumor drug delivery.

AIM Different nanoparticles have been investigated to deliver chemotherapeutic agents, but complex synthesis procedures and biocompatibility issues raise concerns in developing them for safe human usage. The aim of this work is to develop α,β-dehydrophenylalanine-containing, self-assembled, amphipathic dipeptide nanoparticles for tumor-targeted drug delivery and therapy. MATERIAL & METHODS Solution-phase peptide synthesis was used to synthesize dipeptides. Nanoparticles were prepared by molecular self-assembly. A tumor distribution study was carried out using a radiolabeling method. Tumor regression studies were carried out in murine ascitic tumors in BALB/c mice and breast tumor xenografts in in nonobese diabetic/severe combined immunodeficiency mice. RESULTS Arg-α,β-dehydrophenylalanine formed self-assembled nanoparticles that could be easily derivatized with folic acid. Folic acid-derivatized nanoparticles showed enhanced cellular uptake and, when loaded with doxorubicin, showed enhanced tumor regression compared with underivatized nanoparticles or native drug, without any adverse side effects, both in vitro and in vivo.

Comparative Evaluation of Glutamate-Sensitive Radiopharmaceuticals: Technetium-99m–Glutamic Acid and Technetium-99m–Diethylenetriaminepentaacetic Acid–bis(Glutamate) Conjugate for Tumor Imaging

Single-photon emission computed tomography has become a significant imaging modality with huge potential to visualize and provide information of anatomic dysfunctions that are predictive of future diseases. This imaging tool is complimented by radiopharmaceuticals/radiosubstrates that help in imaging specific physiological aspects of the human body. The present study was undertaken to explore the utility of technetium-99m (⁹⁹(m)Tc)-labeled glutamate conjugates for tumor scintigraphy. As part of our efforts to further utilize the application of chelating agents, glutamic acid was conjugated with a multidentate ligand, diethylenetriaminepentaacetic acid (DTPA). The DTPA-glutamate conjugate [DTPA-bis(Glu)] was well characterized by IR, NMR, and mass spectroscopy. The biological activity of glutamic acid was compared with its DTPA conjugate by radiocomplexation with ⁹⁹(m)Tc (labeling efficiency ≥98%). In vivo studies of both the radiolabeled complexes ⁹⁹(m)Tc-Glu and ⁹⁹(m)Tc-DTPA-bis(Glu) were then carried out, followed by gamma scintigraphy in New Zealand albino rabbits. Improved serum stability of ⁹⁹(m)Tc-labeled DTPA conjugate indicated that ⁹⁹(m)Tc remained bound to the conjugate up to 24 hours. Blood clearance showed a relatively slow washout of the DTPA conjugate when compared with the labeled glutamate. Biodistribution characteristics of the conjugate in Balb/c mice revealed that DTPA conjugation of glutamic acid favors less accumulation in the liver and bone and rapid renal clearance. Tumor scintigraphy in mice showed increasing tumor accumulation, stable up to 4 hours. These preliminary studies show that ⁹⁹(m)Tc-DTPA-bis(Glu) can be a useful radiopharmaceutical for diagnostic applications in single-photon emission computed tomography imaging.

Quantitative Structure–Property Relationship (Correlation Analysis) of Phosphonic Acid-Based Chelates in Design of MRI Contrast Agent

Nuclear magnetic resonance imaging is a very useful tool in modern medical diagnostics, especially when gadolinium (III)‐based contrast agents are administered to the patient with the aim of increasing the image contrast between normal and diseased tissues. With the use of soft modelling techniques such as quantitative structure–activity relationship/quantitative structure–property relationship after a suitable description of their molecular structure, we have studied a series of phosphonic acid for designing new MRI contrast agent. Quantitative structure–property relationship studies with multiple linear regression analysis were applied to find correlation between different calculated molecular descriptors of the phosphonic acid‐based chelating agent and their stability constants. The final quantitative structure–property relationship mathematical models were found as – quantitative structure–property relationship Model for phosphonic acid series (Model 1) − log KML = {5.00243(±0.7102)}− MR {0.0263(±0.540)}n = 12 l r l = 0.942 s = 0.183 F = 99.165 quantitative structure–property relationship Model for phosphonic acid series (Model 2) − log KML = {5.06280(±0.3418)}− MR {0.0252(± .198)}n = 12 l r l = 0.956 s = 0.186 F = 99.256.

Targeted theranostic liposomes: rifampicin and ofloxacin loaded pegylated liposomes for theranostic application in mycobacterial infections

Theranostic liposomes are effective drug delivery systems for the management of infections. With added features of targeting and stealth, theranostic liposomes can be made more effective. We report the synthesis and validation of targeted stealth theranostic liposomes for the management of mycobacterial infections. The targeted drug delivery systems for infections are devised to improve the therapeutic window of encapsulated drugs by increasing their delivery to the target area and minimizing the drug-associated toxicity. FR+ targeted pegylated liposomal formulation was developed for in vivo imaging of mycobacterial infections. The encapsulated drugs for mitigation are rifampicin and ofloxacin. The prepared liposomes were characterized for physicochemical properties and stability. In vitro release properties, mycobacterial activity, in vivo blood-kinetics, bio-distribution, and bio-efficacy of the prepared lyophilized liposomes were assessed. The mean particle size of the liposomes was 160.6 nm with considerable colloidal stability observed up to 120 days. The results of in vitro investigations indicate good encapsulation efficiency of 66.89 (±10.9)% and 40.61 (±8.7)% for rifampicin and ofloxacin respectively with excellent anti-mycobacterial activity. The pharmacokinetics data corroborate a slow biphasic pattern with a much longer terminal half-life of 19.13 h. The tissue distribution studies revealed high blood pool activity even up to 24 h post injection (p.i.) with the maximum organ localization in the spleen, liver, and kidneys at one hour p.i. Further, in vivo scintigraphic studies in the murine model of TB infection showed higher uptake at infected lesions at two hours p.i. The blocking imaging experiments showed minimized uptake, which confirms specific targeting. Therapeutic efficacy studies further confirmed that liposomal delivery of the anti-TB drugs is efficacious in the murine model of infection. In conclusion, preliminary studies demonstrated that the formulated liposomes can be an effective theranostic agent against mycobacterial infections in the mouse model.

Design, synthesis and preliminary evaluation of a novel SPECT DTPA-bis-triazaspirodecanone conjugate for D2 receptor imaging

On the basis of pharmacological behaviour, dopamine receptors are divided into D1 and D2 subtype, which are primarily responsible for several neurophysiological anomalies. Assessment and validation with SPECT based conjugates provides a promising and cost effective insight to detect molecular changes in such neurological diseases. Spiperone, a well known “butryophenone” based D2 receptor antagonist, has been explored to design a novel conjugate for SPECT evaluation. The molecular docking pose analysis of the designed molecule was explored with dopamine D2 receptor. The molecule showed high affinity (−51.318 kcal mol−1) due to conserved interactions with Asp114 and Phe389 of D2 receptor. DTPA with triazaspirodecanone moiety of spiperone was synthesized using bifunctional chelation approach with 88% yield and has been characterized using NMR and mass spectroscopy. The radiolabeling efficiency of 99mTc-DTPA-bis-(1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one) was 98%. The complex showed appreciable brain uptake in mice. Receptor binding experiments revealed a Kd of 6.26 nM with maximum localization of the conjugate in the striatum. Thus, these studies could be viewed as novel and informative, initial proof-of-concept approach to the field of 99mTc-labeled radioligand design.