A new radiopharmaceutical compound (131I-PR81) for radioimmunotherapy of breast cancer: labeling of antibody and its quality control.

PR81 is a monoclonal antibody that binds with high affinity to MUC1, which is over expressed on breast and other tumors. The objective of this study was to evaluate the application of this antibody against MUC1 as a radioimmunotherapeutical agent. Monoclonal antibody (PR81) against MUC1 was prepared, characterized, purified, and labeled with 131I. The immunoreactivity of radiolabeled mAb PR81with MUC1 (the native protein), BSA-P20 (a 20 amino acid corresponding the tandem repeat of MUC1) and MCF7 cell line were performed by RIA. In vitro stability of radiolabeled mAb in human serum was determined by thin layer chromatography (TLC). Cell toxicity and in vitro internalization studies were performed with the MCF7 cell line, and the tissue biodistribution of the radioiodinated PR81 was evaluated in normal BALB/c mice at 4, 24 and 48 hrs. The tumor imaging was performed in BALB/c mice with breast xenograft tumors at 24 and 72 hr after the complex injection. The labeling efficiency was found to be 59.9% ± 7.9%. MAb-131I conjugates showed high immunoreactivity towards MUC1 protein, BSA-P20 and MCF7 cell line. In vitro stability of the labeled product in human serum was found to be more than %50 over 24 hr. Cell toxicity and in vitro internalization studies showed that the mAb-131I conjugate inhibited 80% growth of the MCF7 cultured cell lines in vitro in a high concentration and up to %60 of the conjugate internalized after 24 h. Biodistribution studies were performed in normal BALB/c mice at 4, 24 and 48 hrs post-injection and no important accumulation was observed in vital organs. The tumors were visualized with high sensitivity after 24 and 72 hr in radioimmunoscintographical studies. These results show that the new radiopharmaceutical may be considered as a promising candidate for therapy of breast cancer.

Peritoneal retention of liposomes: Effects of lipid composition, PEG coating and liposome charge.

In the treatment of peritoneal carcinomatosis, systemic chemotherapy is not quite effective due to the poor penetration of cytotoxic agents into the peritoneal cavity, whereas intraperitoneal administration of chemotherapeutic agents is generally accompanied by quick absorption of the free drug from the peritoneum. Local delivery of drugs with controlled-release delivery systems like liposomes could provide sustained, elevated drug levels and reduce local and systemic toxicity. In order to achieve an ameliorated liposomal formulation that results in higher peritoneal levels of the drug and retention, vesicles composed of different phospholipid compositions (distearoyl [DSPC]; dipalmitoyl [DPPC]; or dimiristoylphosphatidylcholine [DMPC]) and various charges (neutral; negative, containing distearoylphosphatidylglycerol [DSPG]; or positive, containing dioleyloxy trimethylammonium propane [DOTAP]) were prepared at two sizes of 100 and 1000nm. The effect of surface hydrophilicity was also investigated by incorporating PEG into the DSPC-containing neutral and charged liposomes. Liposomes were labeled with (99m)Tc and injected into mouse peritoneum. Mice were then sacrificed at eight different time points, and the percentage of injected radiolabel in the peritoneal cavity and the tissue distribution in terms of the percent of the injected dose/gram of tissue (%ID/g) were obtained. The ratio of the peritoneal AUC to the free label ranged from a minimum of 4.95 for DMPC/CHOL (cholesterol) 100nm vesicles to a maximum of 24.99 for DSPC/CHOL/DOTAP 1000nm (DOTAP 1000) vesicles. These last positively charged vesicles had the greatest peritoneal level; moreover, their level remained constant at approximately 25% of the injected dose from 2 to 48h. Among the conventional (i.e., without PEG) 100nm liposomes, the positively charged vesicles again showed the greatest retention. Incorporation of PEG at this size into the lipid structures augmented the peritoneal level, particularly for negatively charged liposomes. The positively charged PEGylated vesicles (DOTAP/PEG 100) had the second-greatest peritoneal level after DOTAP 1000; however, their peritoneal-to-blood AUC ratio was low (3.05). Overall, among the different liposomal formulations, the positively charged conventional liposomes (100 and 1000nm) provided greater peritoneal levels and retention. DOTAP/PEG100 may also be a more efficient formulation because this formulation can provide a high level of anticancer drug into the peritoneal cavity and also can passively target the primary tumor.

Effect of liposome size on peritoneal retention and organ distribution after intraperitoneal injection in mice.

Peritoneal carcinomatosis is a serious concern when treating digestive or ovarian tumors. Treatment with systemic chemotherapy suffers from poor penetration of cytotoxic agents into the peritoneal cavity and is not quite effective. Local delivery of drugs, especially as controlled-release delivery systems like liposomes, could provide sustained and higher drug levels and reduce systemic toxicity. In order to investigate the effect of liposome size on peritoneal retention, liposomes composed of distearoylphosphatidylcholine and cholesterol (DSPC/CHOL, molar ratio 2:1) were prepared at four sizes of 100, 400, 1000 and 3000 nm. Subsequently, these liposomes were labeled with (99m)Tc complex of hexamethylpropyleneamineoxime ((99m)Tc-HMPAO) and injected into mouse peritoneum. Then, mice were sacrificed at eight different time points and the percentage of injected radiolabel in the peritoneal cavity and the organ distribution in terms of percentage injected dose/gram tissue (%ID/g) were obtained. Results showed that the free label ((99m)Tc-HMPAO) was cleared very rapidly from the cavity so that after 5 min and 7h only 6.89+/-2.51% and 0.91+/-0.51% of the injected dose was recovered, respectively. However, for the liposomal formulations, this recovery value ranged from 8.47+/-1.62% to 29.99+/-12.06% at 7h. Peritoneal retention of the vesicles was increased with their size, and the highest retention rate was obtained with 1000 nm liposomes with an AUC value 15.51 times that of (99m)Tc-HMPAO. In blood, as expected, 100 nm liposomes showed much higher levels because of their greater stability. Their greater blood concentration also caused increased levels in the heart and kidneys, although their organ to blood AUC ratio was the lowest. Overall, among the different sized neutral liposomes investigated, the 1000 nm vesicles seemed to be the most optimal, achieving a greater peritoneal level and retention.

Direct labelling of octreotide with 99mTc: effect of different concentration of reducing agents and amount of sodium pertechnetate on radiolabelling efficiency.

Octreotide, a synthetic analog of natural hormone somatostatin, was labelled with 99mTc. Labelling was accomplished by reduction of the cysteine bridge, which provided sulfhydryl groups for chelating with 99mTc. Sodium ascorbate and sodium dithionite in different concentrations were used as reducing agents. Different amounts of sodium pertechnetate were used for labelling of peptide. When the mass ratio of peptide and sodium ascorbate was 1:100 and the final concentration of dithionite in the labelling vial was 0.2-0.4 microg/microl with 0.18-1.48 GBq sodium pertechnetate more than 80% radiolabelling efficiency was confirmed by RP-HPLC, ITLC-SG and C18 Cartridge analysis. The stability of the 99mTc-peptide bond was evaluated by human serum challenge and that showed the stability was 90% after 4h.

Polymerase chain reaction of bacterial genomes with single universal primer: application to distinguishing mycobacteria species.

The polymerase chain reaction with a single universal primer (UP-PCR) was applied to bacterial strains and mycobacteria isolates alongside conventional methods. A universal protocol of preparation of PCR samples from cultures representing Escherichia coli, Enterobacter aerogenes, Serratia marcescens, Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, Mycobacterium tuberculosis, Mycobacterium bovis, and several non-tuberculous mycobacteria was found to be reproducible and efficient with these organisms. The bands of UP-PCR products observed in an agarose gel after electrophoresis were species-specific and provided an efficient means of distinguishing bacterial species. The applicability of this approach to mycobacteria identification was assessed by comparing the DNA bands obtained for different strains. Three reference strains and 22 clinical isolates of M. tuberculosis and M. bovis produced very similar DNA banding patterns. They comprised a triplet of prominent and several minor fragments within the 200-500 base pair (bp) size range and were the easiest to interpret. The DNA profiles of unrelated mycobacteria clearly differed from each other when subjected to electrophoretic analysis and correlated well with results of culture method. The method provides a real promise of its application in clinical studies as a simple assay for distinguishing between tubercle bacilli.

Detection and identification of non-tuberculous mycobacterial infections in 6,472 tuberculosis suspected patients.

Between March 1993 and March 1994, 82 patients with infection by non-tuberculous mycobacteria (NTM) and 443 patients with tuberculosis (TB) were registered among 6,472 patients with suspected tuberculosis. Skin-test reactivity to purified protein derivative (PPD) in patients demonstrated indurations of 10-14 mm or more for the majority of patients in both groups. Most patients with NTM infection had abnormal chest roentgenograms showing sporadic infiltrations, nodular abscesses, and cavities resembling TB radiological evidence. The similarity in age range, PPD skin reaction, and radiological evidence in patients infected with NTM or Mycobacterium tuberculosis (MTB) can mislead the physician. Some NTM species were recovered more often than others. Mycobacterium fortuitum from 22 clinical specimens (26.8%); Mycobacterium gastric 19 (23.1%); and Mycobacterium terrae complex 15 (18.3%). The antimicrobial drug susceptibility tests of the isolated organisms showed that 42 (9.5%) isolates of MTB were resistant to isoniazid and 31 (7.0%) to streptomycin. a few strains (1.3%) were identified as being resistant to a combination of 3 primary drugs. These findings suggest that drug-resistant mycobacterial infections are becoming an important problem in the region.

Use of polymerase chain reaction for primary diagnosis of pulmonary tuberculosis in the clinical laboratory.

A nested polymerase chain reaction (PCR) has been used for the rapid detection of tubercule bacilli in respiratory specimens from 287 patients suspected of tuberculosis. The results of PCR testing were compared with isolation methods (conventional culture and Bactec system) in 110 smear-positive and 177 smear-negative patients. There were only 4 false negative results by PCR in the 171 specimens that were M. tuberculosis complex culture-positive. Of 92 PCR-positive samples prepared from the smear-positive specimens 90 (97.8%) were confirmed by culture. However, a poor correlation was obtained between initial 122 PCR-positive results and combined 81 culture recovered organisms in smear-negative patients. After verification of the efficacy of isolation method, retesting PCR-positive culture-negative samples, and studies of patients' clinical histories, only 18 of the cases were found to be associated with the disease. The other 29 results out of the original 47 discrepants were considered PCR false positives, possibly due to contamination. In conclusion, the PCR assay described is suitable for implementation in daily routine work with respiratory specimens, however it should be validated with culture, especially for the smear-negative patients.