Investigation of Klotho G395A and C1818T Polymorphisms and Their Association with Serum Glucose Level and Risk of Type 2 Diabetes Mellitus.

Objective: The objective was to study the association of Klotho gene G395A and C1818T single nucleotide polymorphisms with glycemia, serum, glycosylated hemoglobin (HbA1c) level and the risk of type 2 diabetes mellitus (T2DM) in the Pashtun population of Pakistan. Methods: In this study, 195 normal individuals and 217 T2DM patients were enrolled. All subjects were divided into three groups, namely overall subjects (control + T2DM patients), control individuals and T2DM patients, and their fasting glucose, HbA1c level, lipid profile and C1818T and G395A polymorphisms were determined. Results: The allele frequencies of G395A in overall subjects were 0.568 for A and 0.432 for G. Similarly, allele frequencies for G395A in overall subjects were 0.597 and 0.403 for C and T alleles, respectively. The AA genotype of G395A was observed to be a risk factor for T2DM. In normal individuals, no significant (p > 0.05) association was observed between klotho C1818T and G395A polymorphisms and hyperglycemia. In overall subjects, the C1818T polymorphism was associated (p < 0.05) with high fasting glucose and HbA1c levels in female subjects only. In T2DM patients, both C1818T and G395A polymorphisms were found to be significantly (p < 0.05) associated with high fasting glucose and HbA1c levels both in males and females. Conclusion: The G395A polymorphism was observed to increase the risk of T2DM. Both C1818T and G395 were associated with high fasting glucose and HbA1c levels in T2DM patients.

Folic acid-functionalized graphene quantum dots: Synthesis, characterization, radiolabeling with radium-223 and antiviral effect against Zika virus infection.

The use of graphene quantum dots as biomedical devices and drug delivery systems has been increasing. The nano-platform of pure carbon has shown unique properties and is approved to be safe for human use. In this study, we successfully produced and characterized folic acid-functionalized graphene quantum dots (GQD-FA) to evaluate their antiviral activity against Zika virus (ZIKV) infection in vitro, and for radiolabeling with the alpha-particle emitting radionuclide radium-223. The in vitro results exhibited the low cytotoxicity of the nanoprobe GQD-FA in Vero E6 cells and the antiviral effect against replication of the ZIKV infection. In addition, our findings demonstrated that functionalization with folic acid doesn't improve the antiviral effect of graphene quantum dots against ZIVK replication in vitro. On the other hand, the radiolabeled nanoprobe 223Ra@GQD-FA was also produced as confirmed by the Energy Dispersive X-Ray Spectroscopy analysis. 223Ra@GQD-FA might expand the application of alpha targeted therapy using radium-223 in folate receptor-overexpressing tumors.

Synthesis and preclinical investigation of 99mTc-p-SCN-Bzl-DTPA-cetuximab for targeting EGFR using head and neck squamous cell carcinoma (HNSCC) xenografts.

To assess the preclinical potential of technetium-99m labelled conjugated para-isothiocyanato-benzyl diethylene triamine penta-acetic acid cetuximab (99mTc-p-SCN-Bzl-DTPA cetuximab) for imaging EGFR in HNSCC mice and rabbits xenografts. Cetuximab, a chimeric monoclonal antibody targeting EGFR, was conjugated with p-SCN-Bzl-DTPA followed by labelling with 99mTc. The labelled conjugate was evaluated for in vitro stability in L-cysteine at 37 °C. The 99mTc-p-SCN-Bzl-DTPA cetuximab was also investigated for immunoreactivity, internationalization kinetics, dose escalation (up to 300 µg) and biodistribution in HNSCC mice xenograft. The suitability of labelled moiety as a specific EGFR radio-tracer was assessed in HNSCC rabbit xenograft. 99mTc-p-SCN-Bzl-DTPA cetuximab exhibited more than 98% radiochemical purity at room temperature. In excess L-cysteine, it showed a stable behaviour at 37 °C up to 4 h p.l. The labelled conjugate was internalized in vitro in FaDu tumor cells up to 19.55%. Significantly higher uptake in tumor (at 10 µg; 34.75 ± 0.38% ID/g: pi) was seen in HNSCC mice xenograft with dose escalation assay from 1 to 300 µg/mouse. Blocking of EGFR with excess cetuximab consequently decreased the uptake of tumor up to 6.80 ± 1.25%. SPECT images of rabbit xenograft confirmed increase in tumor to background ratio after 4 h pi and validated its potential in preclinical trial as a specific FaDu tumor tracer. Our in vitro and in vivo preclinical findings indicate that the 99mTc-p-SCN-Bzl-DTPA cetuximab prepared at optimal dose of cetuximab could become a useful tool for EGFR imaging in HNSCC using SPECT.

Evaluation of 99mTc-Labeled Bevacizumab-N-HYNIC Conjugate in Human Ovarian Tumor Xenografts.

AIM: The aim of the present investigation was to examine the suitability of 99mTc-N-HYNIC-BZMB as a specific vascular endothelial growth factor (VEGF)-targeting agent. Bevacizumab is a recombinant humanized monoclonal antibody that inhibits VEGF. METHODS: N-hydroxysuccinimide-2-hydrazinonicotinic acid (N-HYNIC) was conjugated to BZMB, followed by labeling with 99mTc using N-[Tris(hydroxymethyl)methyl] glycine (tricine), ethylenediamine-N,N'-diacetic acid (EDDA), and nicotinic acid as coligands. 99mTc-labeled BZMB was characterized in terms of 99mTcO4, radiocolloids, and labeled N-HYNIC-BZMB using thin-layer chromatography and HPLC. Poor metastatic SKOV-3 and high metastatic SKOV-3.ip1 human ovarian cancer cell lines were used for in vitro binding uptake of 99mTc-N-HYNIC-BZMB. Biodistribution and scintigraphy accuracy were examined in human ovarian tumor xenografts in rats and rabbits. RESULTS: 99mTc-N-HYNIC-BZMB prepared by using a mixture of tricine and EDDA demonstrated relatively high radiochemical purity (more than 98%). In L-cysteine and serum, it exhibited a stable behavior up to 16 hours. In vitro binding uptake indicated that it targets high metastatic SKOV-3.ip1 tumors. Biodistribution in human ovarian tumor xenografts in rats confirmed a significant uptake in SKOV-3.ip1 tumors (5.69% ± 1.86%, 4 hours). Scintigraphic accuracy in human ovarian tumor xenografts in rabbits validated its suitability as a high metastatic SKOV-3.ip1 radiotracer. CONCLUSION: High radiochemical purity, stability in saline and serum, biodistribution, and scintigraphy of 99mTc-N-HYNIC-BZMB in human ovarian tumor xenografts in rats and rabbits confirmed its suitability as a potential radiotracer for imaging high metastatic SKOV-3.ip1 sites.

Imaging prostate cancer (PCa) with [99m Tc(CO)3 ]finasteride dithiocarbamate.

This investigation aimed to modify finasteride (1) to finasteride dithiocarbamate (2) for subsequent synthesis of the rhenium analogue (3) and [99m Tc]tricarbonyl complexes (4), to assess its prostate cancer (PCa) targeting potential in a rat model. To validate the identity of (4), reference (3) has been synthesized by using fac-[Net4 ]2 [ReBr3 (CO)3 ] precursor and characterized by 1 H-NMR, 13 C-NMR, ESI-MS, and elemental analysis. The analogue (4) was synthesized by using fac-[99m Tc(H2 O)3 (CO)3 ]+ precursor, and its structure was confirmed by comparative HPLC by using (3) as a reference. Further, the suitability of (4) as a PCa imaging agent was investigated in vitro and in vivo. At room temperature, (4) had ≥99% radiochemical purity and remained ≥84% stable in serum. In preclinical studies, biodistribution of (4) in histopathologically established rat model showed adequately high in vivo uptake in the prostate attracting the possibility of using it for noninvasive imaging of PCa.

(99m)Tc-phytate trans-splenic portal scintigraphy in the diagnosis of cirrhotic portal hypertension and compensatory circulation.

BACKGROUND AND AIM: The trans-splenic portal scintigraphy (TPS) was evaluated as a diagnostic tool in the post viral hepatitis cirrhotic patients of various classes of Child Pugh's (CP) classification. The main aim was to determine the portosystemic shunt index (PSSI) and to compare the results with various clinical grades of disease severity in liver cirrhosis. METHODS: TPS was performed on 72 patients and 10 controls and PSSI was derived. All the 72 patients were categorized according to CP classification into three classes. The cirrhotic patients were categorized as CP A (n = 24),CP B (n = 22), and CP C (n = 26)according to CP criteria. PSSI was compared with different CP classes. RESULTS: In the controlled population, the splenic vein was normal in shape and the mean PSSI was calculated to be 0.178 ± 0.031 (n = 10). For CP classes A, B, and C, the mean PSSI was 0.36 ± 0.04, 0.45 ± 0.05, and 0.54 ± 0.04 (n = 26), respectively. There was statistical significance among groups (p ≤ 0.01).The collateral vessels were mostly uphill or complex. The PSSI index increased as the CP score worsened from A to C. CONCLUSION: PSSI is a useful and minimally invasive tool to detect and quantify the shunt severity, which correlates well with different clinical grades of disease severity.

Synthesis of (99m)TcN-clinafloxacin Dithiocarbamate Complex and Comparative Radiobiological Evaluation in Staphylococcus aureus Infected Mice.

Clinafloxacin dithiocarbamate (CNND) preparation and radiolabeling through [(99m)Tc ≡ N](2+) core with the gamma (γ) emitter ((99m)Tc) was assessed. The potentiality of the (99m)Tc(V) ≡ N-CNND complex was investigated as perspective a Staphylococcus aureus (S.a.) in vivo infection radiotracer in terms of radiochemical stability in normal saline (n.s.), human serum (h.s.), binding efficacy with live and heat killed S.a. and biodistribution in female nude mice model (FNMD). More than 90% stability was observed in n.s. for 4 h with the highest yield of 98.70 ± 0.26% at 30 min after reconstitution. In h.s., the (99m)Tc(V) ≡ N-CNND complex was found stable up to 16 h with 15.35% side products. Maximum in vitro binding (68.75 ± 0.80%, 90 min) with S.a. was observed after 90 min of incubation. In FNMD, (infected with live strain) approximately six-fold higher uptakes was noted in the infected to inflamed and normal muscles. The higher stability in n.s., h.s., higher S.a. (live) up take with specific and targeted in vivo distribution confirmed potentiality of the (99m)Tc(V) ≡ N-CNND complex as perspective S.a. in vivo infection radiotracer.

Phytochemical and antitrypanosomal investigation of the fractions and compounds isolated from Artemisia elegantissima.

CONTEXT: Trypanosoma brucei brucei (T.b. brucei) infection causes death in cattle, while the current treatments have serious toxicity problems. However, natural products can be used to overcome the problems associated with parasitic diseases including T.b. brucei. OBJECTIVE: Artemisia elegantissima Pamp (Asteraceae) was evaluated phytochemically for its constituents and antitrypanosomal potential against T.b. brucei for the first time. Scopoletin isolated from A. elegantissima has shown better potential then the standard drug suramin, used against T.b. brucei. MATERIALS AND METHODS: The ethanol extract of the aerial parts of A. elegantissima was fractionated by column and preparative thin-layer chromatography into six fractions (A-F) yielding 13 compounds, these were evaluated for their antitrypanosomal activity against T.b. brucei at different concentrations. RESULTS: Thirteen compounds were isolated from A. elegantissima: (Z)-p-hydroxy cinnamic acid, stigmasterol, ß-sitosterol, betulinic acid, bis-dracunculin, dracunculin, scopoletin, apigenin, dihydroluteolin, scoparol, nepetin, bonanzin, and 3',4'-dihydroxy bonanzin. The fractions D-F were found to be active at the concentration of 20 µg/ml and three compounds isolated from these fractions, scopoletin (MIC ≤0.19 µg/ml), 3',4'-dihydroxy bonanzin (MIC = 6.25 µg/ml) and bonanzin (MIC = 20 µg/ml), were found to be highly active. DISCUSSION AND CONCLUSION: Artemisia elegantissima was phytochemically and biologically explored for its antitrypanosomal potential against T.b. brucei. The number and orientation of phenolic hydroxyl groups play an important role in the antitrypanosomal potential of coumarins and flavonoids. The compounds 3',4'-dihydroxy bonanzin and scopoletin with low MIC values, hold potential for use as antitrypanosomal drug leads.

Novel indium-111 labeled gastrin peptide analogues (MG-CL1-4): synthesis and quality control.

Radiolabeled neuropeptides are widely investigated to diagnose and therapy of tumors. These peptides get internalization after binding with particular receptors at the surface of cells and finally move to lysosome. Internalization into tumor cells helps in mapping the infected site. Minigastrin peptide analogues (MG-CL1-4) were synthesised and labeled with 111-In radioisotope under different sets of conditions for imaging CCk-2 receptor bearing tumors. Different parameters such as temperature (80-100°C), pH (4-12), incubation time (5-30 minutes) and dilution effect were investigated to get the maximum labeling yield and stability. The results indicated that MG-CL1-4 is successfully labeled with indium-111 at pH 4.5 with heating at 98°C for 15 minute. At these conditions i.e. heating, pH and incubation minimum oxidized and maximum labeling yield, more than 94 %, was obtained. The labeling stability was studied by incubating the radiolabeled complex for predefined time points in PBSA and blood serum. Results show that more than 90% radiolabeled MG-CL1-4 remained intact.

(99m)Tc(CO)(3)-Ibritumomab tiuxetan; a novel radioimmunoimaging (RII) agent of B cell non-Hodgkin's lymphoma (NHL).

To exploit the B-lymphocyte antigen-CD20 binding capacity of the Ibritumomab tiuxetan (IBTN) monoclonal antibody (mAb) for imaging, the over-expression of B cells in non-Hodgkin's lymphoma (NHL) (a myeloproliferative disorder of the lymphatic system) was investigated. In the current investigation, we present the labeling of the IBTN with technetium-99m ((99m)Tc) through [(99m)Tc(CO)(3)](+) precursor for radioimmunoimaging (RII) of the tumor prior to its treatment with (90)Y labeled IBTN. Labeled IBTN was radiobiologically characterized in terms of radiochemical purity, in vitro stability in human plasma, immunoreactivity, binding with Raji and Ramos cells and biodistribution in a female nude mouse (FNM) model. It was observed that the reduced IBTN (rIBTN) showed more promising radiobiologic characteristics than the nonreduced IBTN. Significantly higher transchelation was seen in excess cysteine compared with histidine. The radioconjugate showed higher saturated binding affinity with CD20 antigen. Significantly higher target (tumor) to background ratios were observed 1 h post-injection (p.i.). Based on radiochemical purity, in vitro stability, immunoreactivity, binding and biodistrubtion in the FNM model, we recommend the radiolabeling of the rIBTN using tricarbonyl technique as a potential RII agent.

Radiosynthesis and biological evaluation of the (99m)Tc-tricarbonyl moxifloxacin dithiocarbamate complex as a potential Staphylococcus aureus infection radiotracer.

In the present investigation, radiosynthesis of the (99m)Tc-tricarbonyl moxifloxacin dithiocarbamate complex ((99m)Tc(CO)(3)-MXND) and its biological evaluation in male Wister rats (MWR) artificially infected with Staphylococcus aureus (S. aureus) was assessed. The (99m)Tc(CO)(3)-MXND complex was radiochemically examined in terms of stability in saline and in serum and biologically its in-vitro binding with S. aureus and percent absorption in MWR models. Radiochemically the (99m)Tc(CO)(3)-MXND complex showed more than 90% stability in saline up to 240 min and in serum 14.95% undesirable species was appeared within 16h. In-vitro the (99m)Tc(CO)(3)-MXND complex showed saturated binding with S. aureus. In MWR artificially infected with live S. aureus the complex showed about six fold higher uptakes in the infected muscle as compared to the normal muscle. However, insignificant change in the uptake of (99m)Tc(CO)(3)-MXND complex in the infected and inflamed or normal muscle was observed in the MWR infected with heat killed S. aureus. The (99m)Tc(CO)(3)-MXND complex disappeared from the circulatory system and appeared in the urinary system within 60-90 min followed by excretion through normal route of urinary system. Based on the elevated and stable radiochemical succumb in saline, serum, saturated in-vitro binding with S. aureus and higher accumulation in the target organ of the MWR, we recommend the (99m)Tc(CO)(3)-MXND complex for radio-localization of the infection induced by S. aureus in human.

Radiosynthesis of (99m)Tc(CO)3-Clinafloxacin Dithiocarbamate and Its Biological Evaluation as a Potential Staphylococcus aureus Infection Radiotracer.

BACKGROUND: Clinafloxacin dithiocarbamate (CNND) was radiolabeled with technetium-99m ((99m)Tc) using [(99m)Tc(CO)3(H2O)3](+) and assessed for its radiochemical stability in saline and serum, its in vitro binding with methicillin-resistant Staphylococcus aureus (MRSA) and biodistribution in female nude mice (FNM) artificially infected with live and heat-killed MRSA. METHODS: In normal saline (NS) the (99m)Tc(CO)3-clinafloxacin dithiocarbamate ((99m)Tc(CO)3-CNND) showed radiochemical stability with a maximum value of 99.10 ± 0.20% and remained stable up to 4 h (92.65 ± 0.18%). RESULTS: In human serum at 37°C within 16 h of incubation, 14.85% side products as a result of de-tagging developed. Incubation with MRSA gave saturated binding with a maximum value of 72.75 ± 1.20%. Almost six-fold higher uptake was seen in the infected muscle of the FNM as compared to the inflamed and normal muscle. The (99m)Tc(CO)3-CNND complex showed a normal route of excretion from the body of the FNM model. CONCLUSION: The higher stability in NS, HS, saturated in vitro binding with a live strain of MRSA and six-fold higher uptake in the target organ showed the (99m)Tc(CO)3-CNND complex to be a potential MRSA infection radiotracer.

Radiosynthesis and biodistribution of (99m)Tc-rifampicin: a novel radiotracer for in-vivo infection imaging.

(99m)Tc-rifampicin ((99m)Tc-RMP) a new radioantibiotic complex was synthesized specifically for the infection localization caused by methicillin-resistant Staphylococcus aureus (MRSA). The in-vitro radiochemical purity (RCP) yield, in-vivo biodistribution behavior in artificially infected rats (AIT) and scintigraphic accuracy in artificially infected rabbit (AIB) of the (99m)Tc-RMP complex was investigated using different concentration of the RMP, sodium pertechnetate (Na(99m)TcO(4)), stannous chloride dihydrate (SnCl(2) x 2H(2)O) at different pH ranges 5-6. The best RCP yield observed at 30, 60, 90 and 120 min after labeling was; 98.95+/-0.20, 98.15+/-0.24, 96.50+/-0.27 and 91.55+/-0.22%, respectively, using 1.5 mg RMP, 175 microL of SnCl(2) x 2H(2)O (1 microg/microL in 0.01 N HCl), 3 mCi of Na(99m)TcO(4), at pH 5.6. Initially in the infected muscle (INM) of the AIT the activity was lower but after 90 min it went up to 18.35+/-0.20% from 5.95+/-0.25%. The activity in the inflamed muscle (IMM), normal (NM) muscle, blood, liver and spleen was initially high that decreased with time. The ratios of the INM/NM and IMM/NM were 7.34+/-0.74 and 1.20+/-0.85, respectively. The whole body static (WBS) imaging of the MRSA infected rabbit confirmed the usefulness of the (99m)Tc-RMP as a precise radiotracer for MRSA infection imaging. On the basis of in-vitro RCP, in-vivo biodistribution and scintigraphic precision, we recommend the (99m)Tc-RMP complex prepared aseptically for in-vivo assessment of MRSA infection.