Indium-111-DTPA-folate as a potential folate-receptor-targeted radiopharmaceutical

Indium-111-labeled diethylenetriamine pentaacetic acid (DTPA)-folate was evaluated as a radiopharmaceutical for targeting tumor-associated folate receptors. METHODS: Athymic mice were subcutaneously inoculated with approximately 1.8 x 10(6) folate receptor-positive KB (human nasopharyngeal carcinoma) cells, yielding 0.2- to 0.6-g tumors in 15 days, at which time (111)In-DTPA-folate, (111)In-DTPA or (111)In-citrate was administered by intravenous injection. RESULTS: The (111)In-DTPA-folate conjugate afforded marked tumor-specific (111)In deposition in vivo using this mouse model. The involvement of the folate receptor in mediating tumor uptake of (111)In-DTPA-folate was demonstrated by the blocking of tumor uptake by coadministration of free folic acid (intravenous). The (111)In-DTPA-folate also shows folate receptor-mediated uptake and retention in the kidneys, presumably reflecting radiotracer binding to folate receptors of the proximal tubules. In control experiments, the (111)In-citrate radiopharmaceutical precursor was also shown to afford significant tumor uptake of (111)In, but with much poorer tumor-to-background tissue contrast than that obtained with (111)In-DTPA-folate. Unconjugated (111)In-DTPA showed no tumor affinity. CONCLUSION: Indium-111-DTPA-folate appears suitable as a radiopharmaceutical for targeting tumor-associated folate receptors.     

Design and synthesis of [111In]DTPA-folate for use as a tumor-targeted radiopharmaceutical

The diagnosis of early cutaneous T-cell lymphoma (CTCL) is a difficult point in dermatology. Recently, Southern blot analysis (SBA) and polymerase chain reaction (PCR) have been used to detect clonality in initial lesions in which clinical and histological findings are unspecific. Forty-one samples from 25 patients with CTCL were investigated for the presence of T-cell receptor-gamma gene rearrangement using a nested PCR technique and analysed by polyacrylamide gel electrophoresis (PAGE). Conventional SBA was also performed on 28 samples from 20 of these patients. In addition, 20 samples corresponding to patients with large plaque parapsoriasis (LPP), cutaneous B-cell lymphoma (CBCL) and eczema were analysed by PCR in the same way as were the CTCL specimens. Most of the CTCL specimens (81%) showed clonality on PCR analysis. Among patients with mycosis fungoides, 71% of initial patch lesions and 100% of plaques and tumours showed clonal disease. Clonality could be detected in three of four histologically negative post-treatment lesions. Clonal rearrangement was detected in one of three patients with LPP and in three of 10 patients with CBCL. None of the samples corresponding to patients with eczema showed positive results. SBA was significantly less sensitive than PCR in detecting clonality in CTCL patients (42% among early disease and 60% among advanced cases). The results indicate that this PCR/PAGE technique is a reliable and useful method for the detection of clonality in early skin lesions of CTCL patients and probably in the identification of silent extracutaneous involvement.     

The bioequivalence of a new allopurinol tablet formulation in comparison to a reference formulation]

Recent studies have shown that there are distinct genetic pathways leading to the most malignant astrocytic neoplasm, the glioblastoma. Primary (de novo) glioblastomas are characterized by amplification/overexpression of the EGF receptor (EGFR) and, less frequently, of the MDM2 gene. Another pathway, operative in the progression of low-grade or anaplastic astrocytomas to secondary glioblastomas, is characterized by the frequent occurrence of p53 mutations. In this study, we assessed p53 mutations and EGFR expression in the giant cell glioblastoma. This rare variant is characterized by unusually large, multinucleated giant cells, but tends to be more confined and has been reported to carry a somewhat more favorable prognosis. We analyzed biopsies from 16 patients (mean age at clinical manifestation, 40 years). DNA sequencing revealed that 12 of 16 (75%) giant cell glioblastomas contained a p53 mutation. In 7 patients with two or more surgical interventions, the p53 mutation was already detected in the first biopsy. Focal EGFR overexpression, including multinucleated giant cells, was observed immunohistochemically in 9 of 16 (56%) tumors. However, most tumor areas lacked immunoreactivity, indicating that EGFR overexpression does not play a significant role in the evolution of this glioblastoma variant. These results suggest that giant cell glioblastomas develop de novo with a short preoperative history (mean, 47 +/- 40 days), but contain genetic alterations similar to those observed in secondary glioblastomas.     

Localization of neuroendocrine tumours with [111In] DTPA-octreotide scintigraphy (Octreoscan): a comparative study with CT and MR imaging

Hyperoxia has deleterious effects on lung form and function; however, the molecular events initiated by oxygen exposure remain unclear. We hypothesized that macrophages function as important intermediaries in the protective response of lung tissues after exposure to hyperoxia. This hypothesis was tested by exposing cultured macrophages (RAW 264.7 cells) to hyperoxia for 24 h and then applying the conditioned medium from these cells to cultured pulmonary epithelial cells or to pulmonary microvascular endothelial cells. We observed that the expression of manganese superoxide dismutase mRNA increased in both target cell lines. Therefore, we next hypothesized that exposure of these macrophages to hyperoxia results in a change in gene expression which could be detected by differential display PCR (ddPCR). This hypothesis was tested by exposing RAW 264.7 cells to > or = 95% oxygen (or normoxia) for 24 h, harvesting RNA, and performing ddPCR. A cDNA fragment upregulated by hyperoxia was identified and reamplified. Verification of differential expression of mRNA was done by Northern analysis. A mRNA which was reproducibly upregulated by hyperoxia, as well as by lipopolysaccharide and interferon gamma, was identified. The differentially expressed PCR product was cloned and sequenced, revealing a product with 99% identity to mouse urokinase mRNA. We speculate that one function of pulmonary macrophages following a hyperoxic exposure is to secrete urokinase.     

Bifunctional NHS-BAT ester for antibody conjugation and stable technetium-99m labeling: conjugation chemistry, immunoreactivity and kit formulation

Conjugation chemistry and kit formulated binding of the NHS ester of 6-(4'-(4"-carboxyphenoxy)butyl)-2, 10-dimercapto-2,10-dimethyl-4,8-diazaundecane (NHS-BAT ester) to monoclonal antibodies (MAbs) was investigated. The functionalities of the resulting BAT conjugated and 99mTc-labeled MAbs BW 431/26, MAb 425 and bispecific MDX210 (fragment construct) were tested by immunoreactivity and immunoscintigraphy. METHODS: The kinetics and chemistry of the conjugation reaction were monitored by high-performance liquid chromatography, size-exclusion chromatography and positive fast-atom-bombardment mass spectra (FAB-MS). The 99mTc BAT-MAbs were tested with various immunoreactivity assays. The biodistribution of 99mTc-BAT-BW 431/26 in rats was compared with directly labeled BW 431/26. RESULTS: At pH 8.5 and 25 degrees C, the reactivity of the NHS-BAT ester was high with 90% completion after 30 min. The conjugation yield of 19 microM MAb and 228 microM NHS-BAT ester amounted to 30%. Higher NHS-BAT ester concentrations afforded higher BAT-to-MAb ratios. According to FAB-MS, the conjugation competing hydrolysis surprisingly occurred at the NHS ring. Almost quantitative 99mTc labeling was achieved after 5 min at 25 degrees C. Immunoreactivity of the 99mTc-BAT antibodies showed > 90% recovery and proved to be insensitive to BAT-to-MAb ratios of up to 10. The 99mTc-BAT-BW 431/26 showed similar organ distribution but revealed less urinary excretion compared with the directly labeled BW 431/26. Immunoscintigraphy with 99mTc-labeled and BAT-BW 431/26 and BAT-MAb 425 showed the respective biological function in vivo. CONCLUSION: According to straightforward conjugation chemistry, the ease of 99mTc labeling and the application of a simple ultrafiltration technique, the NHS-BAT ester represents a nondestructive, universally applicable biofunctional ligand to introduce stable 99mTc protein binding sites. Kit formulated conjugation/labeling can be performed with little time requirements and laboratory experience.     

The bioequivalence of a new amitriptyline tablet formulation in comparison with a reference preparation

An investigation of the bioequivalence of a new tablet formulation (amitriptylin 25 von ct) with 28.3 mg amitriptyline hydrochloride (CAS 549-18-8) was performed in a two-way cross-over study with 18 subjects. The relative bioavailability with respect to a reference preparation for AUC related to amitriptyline (CAS 50-48-6) was 99.3% and for Cmax 100.4%. A positive decision for bioequivalence derived from the usual confidence intervals for both parameters related to amitriptyline and the metabolite nortriptyline (CAS 72-69-5), respectively tmax showed no difference. The new formulation was bioequivalent to the reference.     

Synthesis and antimicrobial activities of 2-[(alpha-methylbenzylidene)-hydrazino]benzoxazoles

New 2-[(alpha-methylbenzylidene)hydrazino]benzoxazole derivatives have been synthesized by reacting ortho or para substituted acetophenones with 2-hydrazinobenzoxazole in ethanol. The structures of the synthesized compounds were confirmed by microanalysis, IR and NMR spectral data. Antimicrobial activities of the compounds were investigated by the microdilution susceptibility test in Mueller-Hinton broth and Sabouraud liquid medium. Test organisms: Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 as Gram (+) bacteria, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa, ATCC 27853 as Gram (-) bacteria, and Candida albicans, Candida stellatoidea, Candida parapsilosis and Candida pseudotropicalis as yeasts. Among the compounds tested 2-[(alpha-methyl-4-chlorobenzylidene)hydrazino]benzoxazole (compounds 4) and 2-[(alpha-methyl-4-nitrobenzylidene)hydrazino]benzoxazole (compound 8) showed the most favorable activity.     

Immunochemical and biochemical characteristics of a preventive preparation against cholera, cholerogen-toxoid]

Cholerogen-toxoid served as a complex vaccine preparation composed of the main pathogenicity factors of cholera vibrio--cholerogen (toxoid), endotoxin and a number of exoenzymes. The preparation contains 65 +/- 7.5% of protein, 12 +/- 1.2% of reducing sugars, 7 +/- 1.2% of lipids, and 2 +/- 0.3% of nucleic acids. Analytic disc-electrophoresis in polyacrylamide gel and immune disc-electrophoresis revealed at least seven individual proteins with the serological activity in the preparation. About 70% of these constituted toxoid proper; the content of O-antigen was 22%. In the cholerogen-toxoid there were revealed seven exoenzymes of cholera vibrio; proteinase, lecithinase, lipase, DNA-ase, RNA-ase, hyaluronidase, amylase. Antibodies against proteinase, lecithinase, amylase and RNA-ase of cholera vibrio were found in the serum of rabbits immunized with cholerogen-toxoid.     

Synthesis and anticonvulsant activity of a new class of 2-[(arylalky)amino]alkanamide derivatives

Although most epilepsies are adequately treated by conventional antiepileptic therapy, there remains an unfulfilled need for safer and more effective anticonvulsant agents. Starting from milacemide, a weak anticonvulsant, and trying to elucidate its mechanism of action, we discovered a structurally novel class of potent and preclinically safe anticonvulsants. Here we report the structure-activity relationship (SAR) study within this series of compounds. Different parts of the structural lead 2-[[4-(3-chlorobenzoxy)benzyl]amino]acetamide (6) were thus varied (Figure 1), and many potent anticonvulsants were found. As an outcome of this study, 57 ((S)-2-[[4-(3-fluorobenzoxy)benzyl]amino]propanamide methanesulfonate, PNU-151774E) emerged as a promising candidate for further development for its potent anticonvulsant activity and outstanding therapeutic indexes (TIs) in different animal tests.     

Evaluation of a new immunologic test kit for rapid detection of group A streptococci, the Abbott Testpack Strep A plus

We compared the Testpack Strep A plus (TPSAP) enzyme-linked immunosorbent assay test for group A beta-hemolytic streptococcal (GAS) antigen rapid detection with blood agar culture in 454 pediatric patients with clinical pharyngitis. Of the 454 patients, 118 (25.9%) had positive oropharyngeal cultures for GAS. TPSAP sensitivity was 89.9% (106 of 118) and specificity was 95.8% (322 of 336). We conclude that the TPSAP is specific enough to indicate treatment for a patient with a positive test but that a negative test should be confirmed by culture.     

A method for stabilising technetium-99m exametazime prepared from a commercial kit

Technetium-99m exametazime (99mTc-d,l-HMPAO), prepared by the reconstitution of the Ceretec kit, is widely used in the investigation of regional cerebral blood flow. The radiochemical purity specification of not less than 80% lipophilic complex requires that the kit is used within 30 min of reconstitution. In certain circumstances this imposes restrictions on its clinical availability. A number of approaches to extending the 30-min shelf life have been proposed and these are discussed. A method of stabilising the kit by the addition of 200 micrograms cobalt chloride hexahydrate (CoCl2 x 6H2O) in 2 ml of water is described. The addition of this solution can extend the shelf life of the reconstituted kit to at least 5 h post reconstitution.     

3,5-二溴-4-二甲氨基过溴化物苯基荧光酮与锆络合物的分光光度法研究

用合成的新试剂３，５－二溴－４－二甲氨基过溴化物苯基荧光酮作显色剂，以分光光度法测定微量锆。在阳离子表面活性剂ＣＴＭＡＢ存在下，０．４ｍｏｌ／ＬＨＣｌ介质中，试剂与锆形成１：４的红色络合物，其最大吸收波长在５３０ｎｍ处，锆含量在０～１０μｇ／２５ｍｌ范围内服从比尔定律，表观摩尔吸光系数为１．２５×１０５Ｌ·ｍｏｌ－１·ｃｍ－１．方法简便、快速、灵敏度高，已用于铝合金样品中微量锆的测定，获得满意的结果。     

ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine]: a novel, orally effective antinociceptive agent acting via neuronal nicotinic acetylcholine receptors: II. In vivo characterization

The antinociceptive effects of ABT-594, a novel nicotinic acetylcholine receptor (nAChR) ligand, were examined in rats in models of acute thermal (hot box) and persistent chemical (formalin test) pain. Also, the effects of ABT-594 treatment on motor function and electroencephalogram (EEG) were determined. In the hot box and formalin test (i.e., phase 1 and 2), acute treatment with ABT-594 (0.03, 0.1 and 0.3 mumol/kg i.p.) produced significant dose-dependent antinociceptive effects. In the hot box, the efficacy of ABT-594 was maintained after a repeated dosing paradigm (5 days b.i.d.i.p.). ABT-594 was fully efficacious in the formalin test when administered before formalin, and also retained significant efficacy (0.3 mumol/kg i.p.) when administered after formalin injection. The antinociceptive effects of ABT-594 in the hot box and formalin tests were attenuated by pretreatment with the nAChR antagonist, mecamylamine, and in animals treated with the nAChR antagonist chlorisondamine, given centrally (10 micrograms/rat i.c.v. 5 days before), but not in animals pretreated with the opioid receptor antagonist, naltrexone. Acute treatment with ABT-594 produced an initial decrease in open-field locomotor activity, which was absent in animals dosed repeatedly (5 days b.i.d.) with ABT-594. Also, acute treatment with ABT-594 decreased body temperature and decreased the amount of time the animals could maintain balance in an edge-balance test. These effects were no longer present in animals dosed repeatedly with ABT-594. At antinociceptive doses, ABT-594 produced activation of free running EEG in contrast to the sedative-like effects of morphine. Full antinociceptive efficacy was maintained in both the hot box and formalin tests after oral administration, whereas the effects on motoric performance were attenuated. In conclusion, these data demonstrate that ABT-594 is a potent antinociceptive agent with full efficacy in models of acute and persistent pain and that these effects are mediated predominately by an action at central neuronal nAChRs. In addition, antinociceptive effects were maintained after repeated dosing, whereas effects of ABT-594 on motor and temperature measures were attenuated in animals treated repeatedly with ABT-594. Thus, compounds acting at nAChRs may represent a novel approach for the treatment of a variety of pain states.