Occurrence of enteric viruses causing clinical diarrhea in small ruminants in northern Indian plains: a reverse transcription PCR based molecular study.

Fowlpox virus (FPV) infects chickens and turkeys giving rise to pock lesions on various body parts like combs, wattles, legs, shanks, eyes, mouth, etc. The birds, affected with FPV, also show anemia and a ruffled appearance which are clinical symptoms of reticuloendotheliosis. Interestingly, the field strains of FPV are integrated with the provirus of reticuloendotheliosis virus (REV). Due to this integration, the infected birds, upon replication of FPV, give rise to free REV virions, causing severe immunosuppression and anemia. Pox scabs, collected from the infected birds, not only show positive PCR results upon performing FPV-specific 4b core protein gene PCR but also show positive results for the PCR of REV-specific env gene and FPV-REV 5'LTR junction. Homogenized suspension of the pock lesions, upon inoculating to the chorio-allantoic membrane (CAM) of 10-day-old specific pathogen-free embryonated chicken eggs, produces characteristic pock lesions in serial passages. However, the lesions also harbor REV mRNA or free virion, which can be identified by performing REV-specific env gene PCR using REV RNA from FPV-infected CAMs. The study suggests successful replication and availability of REV mRNA and free virion alongside the FPV, although the CAM is an ill-suited medium for any retroviral (like REV) growth and replication.

PLGA Nanoparticles for Nose to Brain Delivery of Clonazepam: Formulation, Optimization by 32 Factorial Design, In Vitro and In Vivo Evaluation.

BACKGROUND: Intranasal administration of biodegradable nanoparticles has been extensively studied for targeting the drug directly to CNS through the olfactory or trigeminal route bypassing the blood brain barrier. OBJECTIVE: The objective of the present study was to optimize Clonazepam loaded PLGA nanoparticles (CLO-PNPs) by investigating the effect of process variables on the responses using 32 full factorial design. METHODS: Effect of two independent factors-amount of PLGA and concentration of Poloxamer 188, were studied at low, medium, and high levels on three dependent responses-%Entrapment efficiency, Particle size (nm), and % cumulative drug release at 24hr. RESULTS: %EE, Particle size, and %CDR at 24hr of the optimized batch was 63.7%, 165.1 nm, and 86.96%, respectively. Nanoparticles were radiolabeled with 99mTc and biodistribution was investigated in BALB/c mice after intranasal and intravenous administrations. Significantly higher brain/blood uptake ratios and AUC values in the brain following intranasal administration of CLO-PNPs indicated more effective brain targeting of CLO. Higher brain uptake of intranasal CLO-PNPs was confirmed by rabbit brain scintigraphy imaging. A histopathological study performed on goat nasal mucosa revealed no adverse response of nanoparticles. TEM image exhibited spherical shaped particles in the nano range. DSC and XRD studies suggested Clonazepam encapsulation within the PLGA matrix. The onset of occurrence of PTZ-induced seizures in rats was significantly delayed by intranasal nanoparticles as compared to intranasal and intravenous CLO-SOL. CONCLUSION: This investigation exhibits rapid rate and higher extent of CLO transport in the brain with intranasal CLO-PNPs suggesting a better option as compared to oral and parenteral route in the management of acute status epilepticus.

5-HT1A targeting PARCEST agent DO3AM-MPP with potential for receptor imaging: Synthesis, physico-chemical and MR studies.

Contrast enhancement in MRI using magnetization or saturation transfer techniques promises better sensitivity, and faster acquisition compared to T1 or T2 contrast. This work reports the synthesis and evaluation of 5-HT1A targeted PARACEST MRI contrast agent using 1,4,7,10-tetraazacycloDOdecane-4,7,10-triacetAMide (DO3AM) as the bifunctional chelator, and 5-HT1A-antagonist methoxyphenyl piperazine (MPP) as a targeting unit. The multi-step synthesis led to the MPP conjugated DO3AM with 60% yield. CEST-related physicochemical parameters were evaluated after loading DO3AM-MPP with paramagnetic MRI active lanthanides: Gadolinium (Gd-DO3AM-MPP) and Europium (Eu-DO3AM-MPP). Luminescence lifetime measurements with Eu-DO3AM-MPP and computational DFT studies using Gd-DO3AM-MPP revealed the coordination of one water molecule (q = 1.43) with metal-water distance (rM-H2O) of 2.7 Å and water residence time (τm) of 0.23 ms. The dissociation constant of Kd 62 ± 0.02 pM as evaluated from fluorescence quenching of 5-HT1A (protein) and docking score of -4.81 in theoretical evaluation reflect the binding potential of the complex Gd-DO3AM-MPP with the receptor 5-HT1A. Insights of the docked pose reflect the importance of NH2 (amide) and aromatic ring in Gd-DO3AM-MPP while interacting with Ser 374 and Phe 370 in the antagonist binding pocket of 5-HT1A. Gd-DO3AM-MPP shows longitudinal relaxivity 5.85 mM-1s-1 with a water residence lifetime of 0.93 ms in hippocampal homogenate containing 5-HT1A. The potentiometric titration of DO3AM-MPP showed strong selectivity for Gd3+ over physiological metal ions such as Zn2+ and Cu2+. The in vitro and in vivo studies confirmed the minimal cytotoxicity and presential binding of Gd-DO3AM-MPP with 5-HT1A receptor in the hippocampus region of the mice. Summarizing, the complex Gd-DO3AM-MPP can have a potential for CEST imaging of 5-HT1A receptors.

Radiolabeling optimization and characterization of (68)Ga labeled DOTA-polyamido-amine dendrimer conjugate - Animal biodistribution and PET imaging results.

The present study describes the optimization of (68)Ga radiolabeling with PAMAM dendrimer-DOTA conjugate. A conjugate (PAMAM-DOTA) concentration of 11.69µM, provided best radiolabeling efficiency of more than 93.0% at pH 4.0, incubation time of 30.0min and reaction temperature ranging between 90 and 100°C. The decay corrected radiochemical yield was found to be 79.4±0.01%. The radiolabeled preparation ([(68)Ga]-DOTA-PAMAM-D) remained stable (radiolabeling efficiency of 96.0%) at room temperature and in serum for up to 4-h. The plasma protein binding was observed to be 21.0%. After intravenous administration, 50.0% of the tracer cleared from the blood circulation by 30-min and less than 1.0% of the injected activity remained in blood by 1.0h. The animal biodistribution studies demonstrated that the tracer excretes through the kidneys and about 0.33% of the %ID/g accumulated in the tumor at 1h post injection. The animal organ's biodistribution data was supported by animal PET imaging showing good 'non-specific' tracer uptake in tumor and excretion is primarily through kidneys. Additionally, DOTA-PAMAM-D conjugation with αVß3 receptors targeting peptides and drug loading on the dendrimers may improve the specificity of the (68)Ga labeled product for imaging and treating angiogenesis respectively.

Intranasal clobazam delivery in the treatment of status epilepticus.

The aim of the present investigation was to prepare and characterize clobazam mucoadhesive microemulsion (CZMME) to assess brain drug uptake and protection against pentylenetetrazole (PTZ)-induced convulsions in mice. Clobazam microemulsion (CZME) and CZMME were prepared by titration method and characterized. Brain uptake and pharmacokinetic parameters were calculated from drug concentration in mice brain versus time plots following intranasal administration of radiolabeled CZME and CZMME, intravenous and intranasal administration of radiolabeled clobazam solution. Gamma scintigraphy imaging of rabbit brain following intranasal administration was performed. Formulations were investigated for the onset of seizures in PTZ-challenged mice. Brain targeting efficiency and direct nose-to-brain transport percentage for mucoadhesive microemulsion suggested an improved brain uptake following intranasal administration. The findings were supported by gamma scintigraphy images. Delay in onset of PTZ-induced seizures with CZMME compared with positive control and placebo-treated groups confirmed the improved brain uptake. However, extensive animal studies followed by clinical trials are necessary to develop a product suitable for emergencies of acute seizures in status epilepticus and patients suffering from drug tolerance and hepatic impairment on long-term use in treatment of epilepsy, schizophrenia, and anxiety.

Marcaje indirecto de anticuerpos monoclonales empleando la N2-dietilentriamino-pentaacetil lisina amida como agente quelatante del 99mTc / Indirect labeling of monoclonal antibodies with 99mTc, using N2-diethylentriamine-pentaacetil lysine amide as chelating agent

Los anticuerpos monoclonales y sus fragmentos marcados se han empleado ampliamente en el diagnóstico y seguimiento de diferentes tipos de neoplasias. El objetivo del trabajo fue desarrollar una metodología para el marcaje indirecto de anticuerpos con 99mTc, partiendo de la N2-dietilentriamino-pentaacetil lisina amida como agente quelatante. Mediante reacción con un exceso molar de 2-iminotiolano (1:200) y reducción con un exceso de 2-mercaptoetanol (1:2000) se generaron en el anticuerpo 3.5 +/- 0.6 y 5.8 +/- 0.5 grupos –SH, respectivamente, por lo que se continuó el trabajo con la segunda metodología. Se incubó el h-R3 reducido durante 12 h con N6-ciclohexilmaleimido-N2-dietilentriaminopentaacetil lisina amida, obtenida previamente mediante la reacción de la N2-dietilentriamino-pentaacetil lisina amida con sulfosuccinimidil–4(N-maleimido-metil) ciclohexano-1-carboxilato de sodio. La eficiencia de marcaje con 99mTc del anticuerpo monoclonal h-R3 modificado según este método fue de (98.6 +/-1.4) por ciento, manteniendo una estabilidad satisfactoria hasta 24 h frente al exceso de L-cisteína (1:300). Conclusiones: La metodología desarrollada permitió el marcaje indirecto del anticuerpo monoclonal humanizado h-R3 con 99mTc de forma satisfactoria, empleando la N2- dietilentriaminopentaacetil lisina amida como agente quelatante bifuncional.

Labeled monoclonal antibodies and their fragments are been widely employed for the diagnosis and follow up of different kinds of neoplasm. The aim of the present work was to develop a method for indirect labeling of antibodies with 99mTc, using N2-diethylentriamine-pentaacetil lysine amide as chelating agent. By reactions with a 200-fold molar excess of 2-iminothiolane, and the reduction using a 2000-fold molar excess of 2-mercaptoethanol, 3.5 +/- 0.6 and 5.8 +/- 0.5 sulfhydril groups, respectively, were generated in the antibody. Thus, work was continued using the second procedure. Reduced h-R3 was incubated for 12 h with N6-cyclohexylmaleimide-N2-diethylentriamine-pentaacetil lysine amide, previously obtained by the reaction of N2-diethylentriamine-pentaacetil lysine amide with sodium sulfosuccinimidyl–4(N-maleimidomethyl)cyclohexane-1-carboxylate. Labeling efficiency of h-R3 monoclonal antibody, modified by this method with 99mTc, was (98.6 +/- 1.4) percent. A satisfactory stability of the label was observed up to 24 h in presence of a 300-fold molar excess of L-cysteine. Conclusions: Developed procedure allowed satisfactory indirect labeling of the humanized monoclonal antibody h-R3 with 99mTc, using N2-diethylentriamine-pentaacetil lysine amide as bifunctional chelating agent.

Preparation and pharmacological evaluation of a new radiopharmaceutical, technetium-99m-5-fluorouracil, for tumor scintigraphy.

UNLABELLED: 5-fluorouracil (5-FU) has been used for cancer chemotherapy since more than four decades. There are reports of use of (18)F and (19)F analogues of 5-FU for tumor studies using PET and NMR respectively. However, study pertaining to its use in g-scintigraphy is still lacking. In the present study, we have optimized the methodology to radiolabel it with technetium-99m ((99m)Tc) efficiently and evaluated its physicochemical and biological properties. METHODS: 5-FU was radiolabeled with (99m)Tc and evaluated for physicochemical properties. Blood kinetics were studied in rabbits and biodistribution was carried out in normal as well as tumor bearing mice. In vivo and in vitro tumor uptake of the radiocomplex was evaluated in Ehrlich Ascites Tumor (EAT) bearing mice and human breast cancer cell line (MDA-MB-468). RESULTS: The resultant radiopharmaceutical ((99m)Tc-5-FU) has been found to be stable up to 24 h in both in vitro normal and physiological conditions. The blood clearance of the (99m)Tc-5-FU showed a bi-phasic pattern. High extraction of (99m)Tc-5-FU by the liver (36.41+/-2.79% of injected dose/g tissue) has been observed in mice, along with time dependent increase in the solid tumor to muscle ratio (2:1) measured at 4 h. Incubation of the radiocomplex with human breast cancer cells lines also showed time dependent increase in the uptake of the tracer. CONCLUSION: It can be concluded that the (99m)Tc-5-FU possesses selectivity towards solid tumor tissue.

Synthesis of trans-1,2-cyclohexyldinitrilo tetramethylene phosphonic acid and its radiolabelling with 99mTc for the detection of skeletal metastases.

BACKGROUND AND AIM: The development of bone-seeking radiopharmaceuticals for the detection of malignant bone lesions could further improve the diagnostic accuracy of routine bone scanning. This study aimed to provide a convenient synthesis of trans-1,2-cyclohexylenedinitrilo tetramethylene phosphonic acid (CDTMP) and an improved preparation of its (99m)Tc complex. METHODS: CDTMP was prepared from trans-1,2-cyclohexyldinitrilotetraacetic acid by reaction with phosphorus trichloride and it was labelled with (99m)Tc. Toxicity and biodistribution studies were carried out in BALB/c mice, while blood clearance and bone scintigraphy studies were carried out in rabbits. (99m)Tc-CDTMP was evaluated for the detection of malignant bone lesions in 11 patients. Bone scintigraphy (a methylene diphosphonate scan) was performed to detect metastases at diagnosis and follow-up. RESULTS: The radiolabelling efficiency was found to be >97% and the stability in serum indicated that (99m)Tc remained bound to the chelate, CDTMP, for up to 24 h. Blood clearance showed a quick wash-out from the circulation and the biological half-lives (t12) were 55 min (F) and 8 h 48 min (S). The LD50 was 110 mg.kg(-1) as determined by toxicity studies. The drug was excreted mainly through renal route and the accumulation of (99m)Tc-CDTMP in bone was 7.69+/-0.65%ID/g at 1 h. The mean ratio of bone lesion to soft tissue was 6.8+/-0.69 and of bone lesion to normal bone was 5.67+/-0.82. Visual image analysis of (99m)Tc-CDTMP was clinically comparable to the interpretation of imaging studies with (99m)Tc-MDP. CONCLUSION: These preliminary data support increased bone uptake by the tetraphosphonate complex of (99m)Tc. This suggests that CDTMP complexed with therapeutic radionuclides should be evaluated for therapy of skeletal metastases.