Single amino acid substitution in the Fc region of chimeric TNT-3 antibody accelerates clearance and improves immunoscintigraphy of solid tumors.

Recent studies in antibody catabolism have identified residues at the CH2-CH3 interface of the IgG heavy chain critical for serum persistence of immunoglobulins. Amino acid substitutions in the Fc region of murine IgG1 were shown to drastically accelerate antibody clearance in mice. Our laboratory has previously described a human-mouse chimeric TNT-3 (chTNT-3) monoclonal antibody directed against a universal nuclear antigen that has potential for the radioimmunotherapy of many solid tumors. In the current study, we engineered a chTNT-3 mutant containing a single amino acid substitution, to determine whether a more rapid clearance profile would make the antibody suitable for diagnostic imaging. METHODS: A single amino acid substitution in the CH2 domain of the human gamma1 constant region was made by polymerase chain reaction mutagenesis. High-level expression was achieved using the Glutamine Synthetase Gene Amplification System, and the chTNT-3 mutant was purified by protein A affinity and ion-exchange chromatography. A radioimmunoassay was performed to examine antigen binding, and in vivo studies were undertaken to evaluate clearance and tumor targeting in human tumor xenograft models. RESULTS: The chTNT-3 mutant retained the high affinity of chTNT-3, with a binding constant of 1.5 x 10(-9) mol/L. The mutant was eliminated rapidly from BALB/c mice, with a beta-phase half-life of 33.8 h, compared to 134.2 h for chTNT-3. Moreover, biodistribution studies in human colon tumor-bearing nude mice reflected this accelerated clearance. Tumor levels of the mutant were, respectively, 65%, 39%, and 36% of the tumor levels achieved with the parental chTNT-3 6, 12, and 24 h postinjection. The rapid clearance of the chTNT-3 mutant from the blood resulted in higher tumor-to-normal organ ratios for many normal tissues. Imaging of tumor-bearing mice with 99mTc-labeled chTNT-3 mutant demonstrated early visualization of tumors in 3 different solid tumor xenograft models. CONCLUSION: The accelerated clearance produced by a single amino acid substitution in the Fc region of chTNT-3 leads to improved imaging in tumor-bearing mice. These studies suggest that a rapidly clearing antibody generated by this approach may be useful for the immunoscintigraphy of human tumors.     

Genital fistula--our experience

Genital fistulas cause immense physical and psychosocial problem in women's life. The present study was conducted to note the varieties of genital fistula as well as their causative factors and the results of the operative corrections. Altogether 42 patients with different varieties of genital fistula were enrolled in the study. The causative factors of the genital fistulas, specially, that of vesicovaginal fistulas were thoroughly enquired. After confirming the diagnosis, the operative corrections were undertaken. Among the varieties of genital fistula, 76.19% were vesicovaginal fistula, 11.90% were rectovaginal fistula and 4.76%, 4.76% and 2.38% cases of ureterovaginal, urethrovaginal and vesicocervical fistulas respectively. The primipara women were the major sufferers of genital fistulas due to obstetric grounds. Regarding aetiologies of vesicovaginal fistulas, 71.87% patients had obstetric reasons, after prolonged labour, instrumental delivery and after caesarean section due to obstructed labour. Abdominal hysterectomy (44.44%) topped the list of the gynaecological causes of vesicovaginal fistulas. The cases of ureterovaginal fistulas were after abdominal hysterectomy. One case of urethrovaginal fistula was due to some chemical application for correction of genital prolapses. The rectovaginal fistulas were mostly due to obstetric reasons. The success rate after the first attempt of repair of vesicovaginal fistula was 82.75% and overall failure was 10.34%. The other varieties of fistulas were repaired with 100% success rate. The incidence of genital fistulas can be reduced by vigilant obstetric care and meticulous surgery.     

Management of thyroid carcinoma-an experience in Bangladesh

All patients (n=154) of thyroid malignancy admitted in the Otoluryngology Department of Bangabandhu Sheikh Mujib Medical University (former IPGMR) between 1986 and 2000 were retrospectively analyzed to find out the extent and result of surgery used for thyroid carcinoma. The other objectives were to find out the incidence of differentiated thyroid carcinoma among the thyroid malignancy and also to find out the age, sex and clinical presentation of papillary and follicular carcinoma. Among all the thyroid malignancy (n-154), Differentiated Thyroid Carcinoma (DTC) was seen in 130 (84.41%) cases, where as papillary carcinoma occurred in 98(63.64%), and follicular carcinoma in 32(20.77%)cases. On the basis of risk factors, the DTC were designated as low and high risk. The year-wise incidence of DTC revealed increasing trend from 1986 (3 cases) to 2000 (23 cases). Among the 98 papillary thyroid carcinoma highest number of cases (35.71%) were seen in 31-40 year age group. The male to female ratio was 1: 1.64. In follicular carcinoma, highest number (35.25%) of cases were also seen in 31-40 year age group. The male to female ratio was 1:1.66. The commonest presentation in papillary carcinoma was thyroid swelling (96.93%). The other presentations were occult thyroid (3.06%), Cervical lymph node metastasis (38.77%) and distant metastasis (2.04%). In Follicular carcinoma, the presentations were thyroid swelling (100%), Cervical lymph node metastasis (6.25%) and Distant metastasis (21.87%). In this series, low risk DTC were treated by Lobectomy & Isthmusectomy plus Thyroxin. In low risk group the rate of recurrence was 6.89% and the mortality was nil in five years follow-up. Except two inoperable cases, all high risk patients were managed by Total thyroidectomy (with or without neck dissection, plus removal of metastatic lesion when required) with Radioiodine ablation plus Thyroxin. . The rate of recurrence was 7.81% and mortality was 1.56% in high risk group in similar period of time. Vocal cord palsy were noted in 5 (3.84%) unilateral, and inane (0.76%) bilateral cases. Hypoparathyroidism was found in 4.61%.     

Brain manganese concentrations in human aging and Alzheimer's disease

Manganese levels have been measured in various brain regions in Alzheimer's disease (AD) and aging using instrumental neutron activation analysis. Mn grand mean for all regions was 0.261 micrograms/g for adult controls and 0.245 micrograms/g for AD and the differences were not statistically significant (p less than 0.05). Highest Mn levels were found in the basal ganglia in controls and AD. No significant alterations in Mn were found with advancing age suggesting that the brain has an efficient homeostatic mechanism regulating Mn concentrations. Mn exhibited a significant positive correlation with Fe. Infants had a significantly lower brain Mn level compared to adults.     

Synthesis and Ex Vivo Autoradiographic Evaluation of Ethyl-��-d-galactopyranosyl-(1,4��)-2��-deoxy-2��-[18F]fluoro-��-d-glucopyranoside��A Novel Radioligand for Lactose-Binding Protein: Implications for Early Detection of Pancreatic Carcinomas with PET

Introduction

Previous studies demonstrated that the lactose-binding protein (hepatocellular carcinoma?Cintestine?Cpancreas and pancreatitis-associated proteins (HIP/PAP)) is upregulated >130 times in peritumoral pancreatic tissue as compared to normal pancreatic tissue. Therefore, we developed a new radiolabeled ligand of HIP/PAP, the ethyl-??-d-galactopyranosyl-(1,4??)-2??-deoxy-2??-[¹⁸F]fluoro-??-d-glucopyranoside (Et-[¹⁸F]FDL) for noninvasive imaging of pancreatic carcinoma using positron emission tomography and computerized tomography (PET/CT).

Methods

The novel precursor and radiolabeling methods for synthesis of Et-[¹⁸F]FDL produced no isomers; the average decay-corrected radiochemical yield was 68%, radiochemical purity >99%, and specific activity >74 GBq/µmol. The radioligand properties of Et-[¹⁸F]FDL were evaluated using an ex vivo autoradiography and immunohistochemistry in pancreatic tissue sections obtained from mice-bearing orthotopic pancreatic tumor xenografts.

Results and Discussion

Et-[¹⁸F]FDL binding to peritumoral pancreatic tissue sections strongly correlated with HIP/PAP expression (r?=?0.81) and could be completely blocked by treatment with 1 mM lactose.

Conclusion

These results suggest that Et-[¹⁸F]FDL is a promising agent which should be evaluated for detection of early pancreatic carcinomas by PET/CT imaging.     

Pharmacokinetics, Metabolism, Biodistribution, Radiation Dosimetry, and Toxicology of 18F-Fluoroacetate (18F-FACE) in Non-human Primates

Introduction

To facilitate the clinical translation of ¹⁸F-fluoroacetate (¹⁸F-FACE), the pharmacokinetics, biodistribution, radiolabeled metabolites, radiation dosimetry, and pharmacological safety of diagnostic doses of ¹⁸F-FACE were determined in non-human primates.

Methods

¹⁸F-FACE was synthesized using a custom-built automated synthesis module. Six rhesus monkeys (three of each sex) were injected intravenously with ¹⁸F-FACE (165.4?±?28.5?MBq), followed by dynamic positron emission tomography (PET) imaging of the thoracoabdominal area during 0?C30?min post-injection and static whole-body PET imaging at 40, 100, and 170?min. Serial blood samples and a urine sample were obtained from each animal to determine the time course of ¹⁸F-FACE and its radiolabeled metabolites. Electrocardiograms and hematology analyses were obtained to evaluate the acute and delayed toxicity of diagnostic dosages of ¹⁸F-FACE. The time-integrated activity coefficients for individual source organs and the whole body after administration of ¹⁸F-FACE were obtained using quantitative analyses of dynamic and static PET images and were extrapolated to humans.

Results

The blood clearance of ¹⁸F-FACE exhibited bi-exponential kinetics with half-times of 4 and 250?min for the fast and slow phases, respectively. A rapid accumulation of ¹⁸F-FACE-derived radioactivity was observed in the liver and kidneys, followed by clearance of the radioactivity into the intestine and the urinary bladder. Radio-HPLC analyses of blood and urine samples demonstrated that ¹⁸F-fluoride was the only detectable radiolabeled metabolite at the level of less than 9% of total radioactivity in blood at 180?min after the ¹⁸F-FACE injection. The uptake of free ¹⁸F-fluoride in the bones was insignificant during the course of the imaging studies. No significant changes in ECG, CBC, liver enzymes, or renal function were observed. The estimated effective dose for an adult human is 3.90?C7.81?mSv from the administration of 185?C370?MBq of ¹⁸F-FACE.

Conclusions

The effective dose and individual organ radiation absorbed doses from administration of a diagnostic dosage of ¹⁸F-FACE are acceptable. From a pharmacologic perspective, diagnostic dosages of ¹⁸F-FACE are non-toxic in primates and, therefore, could be safely administered to human patients for PET imaging.     

Evaluation of 9-[(3-18F-fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]-FHPG) in vitro and in vivo as a probe for PET imaging of gene incorporation and expression in tumors.

Preparation of 9-[(3-18F-fluoro-1-hydroxy-2-propoxy)methyl]-guanine ([18F]-FHPG) for clinical use, and its evaluation as a positron emission tomography (PET) imaging agent for gene incorporation and expression in tumors are reported. In vitro studies in human colon cancer cells, HT-29, transduced with the retroviral vector G1Tk1SvNa and nontransduced (wild type) showed 4, 8, 12, and 15 times higher uptake of the probe in 1, 3, 5, and 7 h, respectively, in transduced cells compared with the controls. In vivo studies in tumor-bearing nude mice demonstrated that the tumor uptake of the radiotracer is three and six-fold higher in 2 and 5 h, respectively, in transduced cells compared with the control cells. These results suggest that [18F]-FHPG is a potential in vivo PET imaging agent for monitoring gene incorporation and expression in gene therapy of cancer.