Isomerism and solution dynamics of (90)Y-labeled DTPA--biomolecule conjugates

This report describes the synthesis of two DTPA-conjugated cyclic peptides, cyclo(Arg-Gly-Asp-D-Phe-Lys)DTPA (SQ169) and [cyclo(Arg-Gly-Asp-D-Phe-Lys)](2)DTPA (SQ170), and a chromatographic study of their (90)Y complexes (RP762 and RP763, respectively). The goal is to study the solution structure and the possible isomerism of (90)Y-labeled DTPA-biomolecule conjugates at the tracer level (approximately 10(-10) M). RP762 was prepared in high radiochemical purity (RCP > 95%) by reacting 2 microg of SQ169 with 20 mCi of (90)YCl(3) (corresponding to a SQ169:Y ratio of approximately 4:1) in the 0.5 M ammonium acetate buffer (pH 8.0) at room temperature. RP763 was prepared in a similar fashion using SQ170. In both cases, the (90)Y-chelation was instantaneous. By a reversed-phase HPLC method, it was found that RP762 exists in solution as a mixture of two detectable isomers (most likely cis and trans isomers), which interconvert at room temperature. The interconversion of different isomeric forms of RP762 involves a rapid exchange of "wrapping isomers" via the "wagging" of the diethylenetriamine backbone, "shuffling" of the two NO(2) donor sets, and inversion at the ternimal amine-nitrogen atom. The inversion at a terminal nitrogen atom requires simultaneous dissociation of the NO(2) donor set. For RP763, the interconversion of different isomers is much faster than that for RP762 due to the weak bonding of two carbonyl-oxygen donors. Therefore, RP763 shows only one broad radiometric peak in its HPLC chromatogram. The rapid interconversion of different isomers is intramolecular via a partial dissociative mechanism. The results obtained in this study are consistent with the lack of kinetic inertness of (90)Y- and (111)In-labeled DTPA-biomolecule conjugates. Thus, the design of new BFCs should be focused on those which form lanthanide complexes with high thermodynamic stability and more importantly kinetic inertness.     

Comparison of yttrium and indium complexes of DOTA-BA and DOTA-MBA: models for (90)Y- and (111)In-labeled DOTA-biomolecule conjugates

Yttrium and indium complexes of 1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-1-cyclododecylacetylbenzylamine (DOTA-BA) and 1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-1-cyclododecylacetyl-R-(+)-alpha-methylbenzylamine (DOTA-MBA) were prepared in order to study solution structures of (90)Y- and (111)In-labeled DOTA-biomolecule conjugates. (90)Y and (111)In complexes M(L) (M = (90)Y and (111)In; L = DOTA-BA and DOTA-MBA) were prepared from the reaction of MCl(3) with DOTA-BA and DOTA-MBA, respectively, in ammonium acetate buffer. A reverse phase HPLC method revealed that both (90)Y and (111)In complexes show only one radiometric peak in their radio-HPLC chromatograms. It was also found that (111)In(DOTA-BA) and (111)In(DOTA-MBA) are more hydrophilic than their corresponding (90)Y analogues, suggesting different coordination spheres in (111)In and (90)Y complexes of the same DOTA conjugate. Complexes M(L) (M = Y and In; L = DOTA-BA and DOTA-MBA) were prepared and characterized by HPLC, LC-MS, and NMR ((1)H and (13)C) methods. The HPLC concordance experiments for (90)Y(DOTA-MBA)/Y(DOTA-MBA) and (111)In(DOTA-MBA)/In(DOTA-MBA) show that the same complex is prepared at both tracer and macroscopic levels. The NMR data ((1)H and (13)C) clearly demonstrates that Y(DOTA-BA) and Y(DOTA-MBA) exist in solution as one predominant isomer. VT NMR data ((1)H and (13)C) show that In(DOTA-BA) and In(DOTA-MBA) are fluxional at room temperature while Y(DOTA-BA) and Y(DOTA-MBA) become fluxional only at elevated temperatures. The fluxionality of these complexes is due to rapid rotation of acetate/acetamide chelating arms and inversion of ethylenic groups of the macrocyclic ring.     

90Y and 111In complexes of a DOTA-conjugated integrin alpha v beta 3 receptor antagonist: different but biologically equivalent

90Y-TA138 is a (90)Y-labeled nonpeptide integrin alpha(v)beta(3) receptor antagonist that binds with high affinity and specificity to integrin alpha(v)beta(3) receptors overexpressed on both endothelial and tumor cells. (90)Y-TA138 has demonstrated significant therapeutic effects in several preclinical tumor-bearing animal models. Since (90)Y is a pure beta-emitter, (111)In-TA138 has been chosen as the imaging surrogate for dosimetry determination of (90)Y-TA138. This report describes the synthesis of (111)In-TA138 and biological evaluations of both (111)In-TA138 and (90)Y-TA138 in the c-neu Oncomouse model. The HPLC data shows that (111)In-TA138 is more hydrophilic with the retention time approximately 4.5 min shorter than that of (90)Y-TA138 under identical chromatographic conditions. Since the only difference between (111)In-TA138 and (90)Y-TA138 is the metal ion, the HPLC retention time difference strongly suggests that indium and yttrium chelates do not share the same coordination sphere in solution even though they are coordinated by the same DOTA conjugate. Despite their differences in lipophilicity and solution structure, biodistribution data in the c-neu Oncomouse model clearly showed that (111)In-TA138 and (90)Y-TA138 are biologically equivalent with respect to their uptake in tumors and other major organs. Therefore, (111)In-TA138 is useful as an imaging surrogate for (90)Y-TA138 and should be able to predict the radiation dosimetry of (90)Y-TA138, a therapeutic radiopharmaceutical for treatment of rapidly growing tumors.     

Side chain contributions to the interconversion of the topological isomers of guanylin-like peptides.

The peptide hormones guanylin and uroguanylin are ligands of the intestinal guanylyl cyclase-C (GC-C) that is involved in the regulation of epithelial water and electrolyte transport. The small peptides contain 15 and 16 amino acids, respectively, and two disulfide bonds with a 1-3/2-4 connectivity. This structural feature causes the unique existence of two topological isoforms for each peptide in an approximate 3:2 ratio, with only one of the isoforms exhibiting GC-C-activating potential. The two uroguanylin isomers can be separated by HPLC and are of sufficient stability to be studied separately at ambient temperatures while the two guanylin isomers are rapidly interconverting even at low temperatures. Both isomers show clearly distinguishable (1)H chemical shifts. To investigate the influence of certain amino acid side chains on this isomerism and interconversion kinetics, derivatives of guanylin and uroguanylin (L-alanine scan and chimeric peptides) were designed and synthesized by Fmoc solid-phase chemistry and compared by HPLC and 2D (1)H NMR spectroscopy. Amino acid residues with the most significant effects on the interconversion kinetics were predominantly identified in the COOH-terminal part of both peptides, whereas amino acids in the central part of the peptides only moderately affected the interconversion. Thus, the conformational conversion among the isomers of both peptides is under the control of a COOH-terminal sterical hindrance, providing a detailed model for this dynamic isomerism. Our results demonstrate that kinetic control of the interconversion process can be achieved by the introduction of side chains with a defined sterical profile at suitable sequence positions. This is of potential impact for the future development of GC-C peptide agonists and antagonists.     

Therapeutic Efficacy of C-Kit-Targeted Radioimmunotherapy Using 90Y-Labeled Anti-C-Kit Antibodies in a Mouse Model of Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive tumor and prognosis remains poor. Therefore, the development of more effective therapy is needed. We previously reported that high levels of an anti-c-kit antibody (12A8) accumulated in SCLC xenografts. In the present study, we evaluated the efficacy of two antibodies (12A8 and 67A2) for radioimmunotherapy (RIT) of an SCLC mouse model by labeling with the ⁹⁰Y isotope.

Methods

¹¹¹In- or ¹²⁵I-labeled antibodies were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays in c-kit-expressing SY cells and in vivo by biodistribution in SY-bearing mice. Therapeutic efficacy of ⁹⁰Y-labeled antibodies was evaluated in SY-bearing mice upto day 28 and histological analysis was conducted at day 7.

Results

[¹¹¹In]12A8 and [¹¹¹In]67A2 specifically bound to SY cells with high affinity (8.0 and 1.9 nM, respectively). 67A2 was internalized similar to 12A8. High levels of [¹¹¹In]12A8 and [¹¹¹In]67A2 accumulated in tumors, but not in major organs. [¹¹¹In]67A2 uptake by the tumor was 1.7 times higher than for [¹¹¹In]12A8. [⁹⁰Y]12A8, but not [⁹⁰Y]67A2, suppressed tumor growth in a dose-dependent manner. Tumors treated with 3.7 MBq of [⁹⁰Y]12A8, and 1.85 and 3.7 MBq of [⁹⁰Y]67A2 (absorbed doses were 21.0, 18.0 and 35.9 Gy, respectively) almost completely disappeared approximately 2 weeks after injection, and regrowth was not observed except for in one mouse treated with 1.85 MBq [⁹⁰Y]67A2. The area of necrosis and fibrosis increased depending on the RIT effect. Apoptotic cell numbers increased with increased doses of [⁹⁰Y]12A8, whereas no dose-dependent increase was observed following [⁹⁰Y]67A2 treatment. Body weight was temporarily reduced but all mice tolerated the RIT experiments well.

Conclusion

Treatment with [⁹⁰Y]12A8 and [⁹⁰Y]67A2 achieved a complete therapeutic response when SY tumors received an absorbed dose greater than 18 Gy and thus are promising RIT agents for metastatic SCLC cells at distant sites.     

Radiometal-labeled peptide-PNA conjugates for targeting bcl-2 expression: preparation,characterization, and in vitro mRNA binding

A new antisense peptide-peptide nucleic acid (peptide-PNA) conjugate, designed for targeting bcl-2 expression, has been radiolabeled, characterized, and evaluated for bcl-2 mRNA binding in a cell-free system. A PNA complementary to the first six codons of the bcl-2 gene was synthesized by standard solid-phase Fmoc chemistry and conjugated to a new derivative of 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA) that allows macrocyclic radiometal chelates to be incorporated into any sequence position of a peptide-PNA conjugate. The DOTA-PNA conjugate was then coupled to a membrane-permeating transduction peptide, PTD-4, designed for intracellular delivery of the radiolabeled PNA. The conjugate was characterized by HPLC and ESI-MS and labeled with (111)In and (90)Y to high specific activities (>1000 Ci/mmol) with high radiochemical purity. Northern blot analysis showed that (90)Y-PTD-4-K(DOTA)-anti-bcl-2-PNA bound specifically to as little as 50 fmol of bcl-2 mRNA, a result equivalent to that obtained with the analogous (32)P-labeled DNA antisense oligonucleotide. Thus, the mRNA targeting properties of (111)In- and (90)Y-PTD-4-K(DOTA)-anti-bcl-2-PNA demonstrate potential for diagnostic imaging and targeted radiotherapy applications in bcl-2-positive cancers.     

Synthesis and evaluation of two technetium-99m-labeled peptidic 2-nitroimidazoles for imaging hypoxia.

The presence of hypoxic cells in solid tumors is a marker for therapy-resistant, aggressive disease. The noninvasive detection of hypoxic cells in tumors by radiolabeled 2-nitroimidazoles is a diagnostic technique under current evaluation. Two peptidic agents, dimethylglycyl-L-seryl-L-cysteinyl-lysyl{N(epsilon)-[1-(2-nitro-1H -im idazolyl)acetamido]}glycine (RP435) and dimethylglycyl-tert-butylglycyl-L-cysteinyl-glycine-[2-(2-ni tro-1H-im idazolyl)ethyl]amide (RP535) have been synthesized. Both agents contain an N(3)S class chelator for (99m)Tc and Re and a 2-nitroimidazole group which can be enzymatically reduced and selectively trapped in cells under hypoxic conditions. Two isomers of (99m)TcO-RP435, which are assumed to be syn and anti conformations, were observed on HPLC analysis. The interconversion of the two isomers in aqueous solution was investigated. In contrast, RP535 chelated (99m)Tc to form a single isomer and no conversion to its counterpart has been observed on HPLC analysis. The tert-butyl group on the chelator may inhibit the formation and interconversion of the syn and anti isomers of (99m)TcO-RP535. Both tracers showed a significant degree of hypoxia-specific accumulation in an in vitro assay, with (99m)TcO-RP535 showing higher selectivity for hypoxic cells than (99m)TcO-RP435. These results suggest that (99m)TcO-RP535 represents a lead compound worthy of further investigation as an agent for imaging hypoxia in tumors.     

An 86Y-labeled mirror-image oligonucleotide: influence of Y-DOTA isomers on the biodistribution in rats

A mirror-image oligonucleotide (L-RNA) was radiolabeled with the positron emitting radionuclide (86)Y (t(1/2) = 14.7 h) via the bifunctional chelator approach. DOTA-modification of the L-RNA (sequence: 5'-aminohexyl UGA CUG ACU GAC-3'; MW 3975) was performed using (S)-p-SCN-Bn-DOTA. (86)Y radiolabeling of the DOTA-L-RNA produced more than one species as evidenced by HPLC radiometric detection. For the identification of the (86)Y-labeled L-RNA, the structural analogue nonradioactive precursor [Y((S)-p-NH2-Bn-DOTA)](-) was synthesized. Two coordination isomers were separated via HPLC adopting the square antiprismatic (SAP) and the twisted square antiprismatic (TSAP) geometry, respectively. Their stereochemical configuration in the solution state was assessed by NMR and circular dichroism spectroscopy. Both [Y((S)-p-NH2-Bn-DOTA)](-) isomers were converted into isothiocyanate derivatives [Y((S)-p-SCN-Bn-DOTA)](-) and conjugated to the L-RNA. The identity of the [(86)Y-DOTA]-L-RNA species was finally established by comparison of the radiometric ((86)Y) and UV-visible chromatographic profiles. Biodistribution studies in Wistar rats showed minor changes in the biodistribution profile of the [(86)Y((S)-p-NH2-Bn-DOTA)](-) complex isomers, while no significant differences were observed for the [(86)Y-DOTA]-L-RNA isomers. High renal excretions were found for the [(86)Y((S)-p-NH 2-Bn-DOTA)](-) complex isomers as well as for the L-RNA isomers.     

融合表达氨基转移酶DsaD和异构酶DsaE合成L-别异亮氨酸

【背景】L-异亮氨酸(L-isoleucine,L-Ile)和L-别异亮氨酸(L-allo-isoleucine,L-allo-Ile)是自然界中广泛存在的一对同分异构体。抗感染抗生素Desotamides结构中含L-别异亮氨酸结构单元,其生物合成途径中的氨基转移酶DsaD和异构酶DsaE可以协作催化L-异亮氨酸和L-别异亮氨酸相互转化。【目的】通过理性设计,使氨基转移酶DsaD和异构酶DsaE融合表达,研究融合蛋白DsaDE催化异亮氨酸和别异亮氨酸相互转化的功能。【方法】利用PCR分别扩增dsaE基因编码区DNA片段、以及含dsaD基因编码区和114个碱基接头序列的DNA片段dsaD-linker,利用酶切位点KpnI将dsaE和dsaD-linker相连,形成das DE重组序列,并克隆至pET28a(+)中,将重组质粒pET28a-dsaDE转化至Escherichia coli BL21(DE3)中进行融合表达,利用Ni-NTA亲和层析法纯化融合蛋白DsaDE;分别以L-异亮氨酸和L-别异亮氨酸为底物进行融合蛋白的体外酶活性检测,利用高效液相色谱对酶反应产物进行分析。【结果】PCR验证、酶切验证以及测序结果证明pET28a-dsaDE重组载体具有正确序列;N-末端和C-末端融合6个组氨酸标签的融合蛋白DsaDE在E. coli BL21(DE3)中获得可溶性表达,经Ni-NTA亲和层析法一步纯化获得纯度约95%的融合蛋白,纯化的融合蛋白DsaDE具有较好的活性,能够催化L-isoleucine和L-allo-isoleucine间的相互转化。【结论】氨基转移酶DsaD和异构酶DsaE成功融合表达,经一步Ni-NTA亲和层析法纯化即可获得纯度较高的融合蛋白,融合蛋白同时具有氨基转移酶和异构酶的活性,为进一步研究L-别异亮氨酸的工业化生产奠定了基础。     

Studies on the racemization of a stereolabile 5-aryl-thiazolidinedione

The enantiomers of the stereolabile peroxisome proliferator-activated receptor (PPAR) agonist, 1, were isolated by preparative chiral chromatography and their absolute configuration established using a combination of chromatographic and NMR methods. Enantiomer interconversion was investigated under a variety of conditions, with rapid racemization being observed in most solvents, including all aqueous systems studied, irrespective of pH. Rapid racemization in both dog and human plasma was confirmed by chiral HPLC with MS detection.     

Kinetic and sequence-structure-function analysis of known LinA variants with different hexachlorocyclohexane isomers

Background

Here we report specific activities of all seven naturally occurring LinA variants towards three different isomers, α, γ and δ, of a priority persistent pollutant, hexachlorocyclohexane (HCH). Sequence-structure-function differences contributing to the differences in their stereospecificity for α-, γ-, and δ-HCH and enantiospecificity for (+)- and (−)-α -HCH are also discussed.

Methodology/Principal Findings

Enzyme kinetic studies were performed with purified LinA variants. Models of LinA2_B90A A110T, A111C, A110T/A111C and LinA1_B90A were constructed using the FoldX computer algorithm. Turnover rates (min⁻¹) showed that the LinAs exhibited differential substrate affinity amongst the four HCH isomers tested. α-HCH was found to be the most preferred substrate by all LinA''s, followed by the γ and then δ isomer.

Conclusions/Significance

The kinetic observations suggest that LinA-γ1-7 is the best variant for developing an enzyme-based bioremediation technology for HCH. The majority of the sequence variation in the various linA genes that have been isolated is not neutral, but alters the enantio- and stereoselectivity of the encoded proteins.     

Characterization of the ysa Pathogenicity Locus in the Chromosome of Yersinia enterocolitica and Phylogeny Analysis of Type III Secretion Systems

Several Gram negative bacteria use a complex system called "type III secretion system" (TTSS) to engage their host. The archetype of TTSS is the plasmid-encoded "Yop virulon" shared by the three species of pathogenic Yersinia (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica). A second TTSS, called Ysa (for Yersinia secretion apparatus) was recently described in Y. enterocolitica 8081, a strain from serotype O:8. In this study, we describe the ysa locus from A127/90, another strain of serotype O:8, and we extend the sequence to several new genes encoding Ysp proteins which are the substrates of this secretion system, and a putative chaperone SycB. According to the deduced protein sequences, the ysa system from A127/90 is identical to that of 8081. It is different from the chromosome-encoded TTSS of Y. pestis but is instead closely related to the Mxi-Spa TTSS of Shigella and to the SPI-1 encoded TTSS of Salmonella enterica. We further demonstrated that the ysa locus is only present in biotype IB strains of Y. enterocolitica. Including this new Ysa system, a phylogenetic analysis of the 26 known TTSSs was carried out, based on the sequence analysis of three conserved proteins. All the TTSSs fall into five different clusters. The phylogenetic tree of these TTSSs is completely different from the evolutionary tree based on 16S RNA, indicating that TTSSs have been distributed by horizontal transfer.     

Rapid miniaturized chromatography for 111In labeled monoclonal antibodies: Comparison to size exclusion high performance liquid chromatography

Our laboratory investigated the use of a rapid miniaturized chromatography system, ITLC-SG with 0.9% NaCl, to assess the radiochemical purity of ¹¹¹In labeled monoclonal antibodies (MoAbs). Radiochemical analysis was performed on numerous ¹¹¹In labeled antibody preparations with labeling efficiencies ranging from 40 to greater than 95% and the results compared to those obtained with size exclusion high performance liquid chromatography (HPLC). The chromatographic procedure involved challenging radiolabeled antibodies with 0.05 M DTPA to chelate unbound and/or non-specific bound ¹¹¹In, spotting on miniaturized instant thin layer-silica gel chromatography strips, developing in 0.9% NaCl, and counting appropriate segments for radioactivity. Results of the study demonstrated that the miniaturized chromatography procedure was rapid, taking less than 4 min to complete, and accurate in assessing the amount of unbound or non-specific bound ¹¹¹In in ¹¹¹In labeled monoclonal antibodies, when compared to size exclusion HPLC.     

Different Rates of Geometric Isomers of Linoleate Hydroperoxide in Acid-catalyzed Decomposition

Methyl linoleate hydroperoxide was decomposed with 0.1 m HCl in acetone-water (9:1, v/v) at 30°C. The decrease in four isomers of the hydroperoxide was monitored by HPLC without any derivatization. In both isomers having 13- and 9-hydroperoxy groups, those having trans, trans dienes decomposed more rapidly than those having cis, trans dienes. In all the isomers, the rates of decomposition were first order with respect to concentrations of the hydroperoxides. The yields of 2-nonenal and 12-oxo-10-dodecenoate were also measured by GC-MS. 12-Oxo-10-dodecenoate was produced only from the 13-isomers and 2-nonenal from the 9-isomers. The rapid decomposition of the trans, trans isomers didn’t seem to be responsible for the formation of these aldehyde products.     

Concise site-specific synthesis of DTPA-peptide conjugates: application to imaging probes for the chemokine receptor CXCR4

Diethylenetriaminepentaacetic acid (DTPA) is a useful chelating agent for radionuclides such as (68)Ga, (99m)Tc and (111)In, which are applicable to nuclear medicine imaging. In this study, we established a facile synthetic protocol for the production of mono-DTPA-conjugated peptide probes. A novel monoreactive DTPA precursor reagent was synthesized in two steps using the chemistry of the o-nitrobenzenesulfonyl (Ns) protecting group, and under mild conditions this DTPA precursor was incorporated onto an N(ε)-bromoacetylated Lys of a protected peptide resin. The site-specific DTPA conjugation was facilitated by using a highly acid-labile 4-methyltrityl (Mtt) protecting group for the target site of the bioactive peptide during the solid-phase synthesis. A combination of both techniques yielded peptides with disulfide bonds, such as octreotide and polyphemusin II-derived CXCR4 antagonists. DTPA-peptide conjugates were purified in a single step following cleavage from the resin and disulfide bond formation. This site-specific on-resin construction strategy was used for the design and synthesis of a novel In-DTPA-labeled CXCR4 antagonist, which exhibited highly potent inhibitory activity against SDF-1-CXCR4 binding.     

NMR investigations of proline and its derivatives. 4-Proton magnetic resonance parameters and structure of acetyl-proline amide.

The proton magnetic resonance (PMR) spectrum of acetyl-proline amide in D2O solution has been analysed by computer simulation. The spectra of the cis and the trans isomers have been separated and their PMR parameters (chemical shift and coupling constants) are given. Vicinal coupling constants of the pyrrolidine ring are interpreted by means of a Karplus zone relation. The chemical shift effect of the anisotropy of both peptide planes is considered. It follows that both isomers are puckered with Cgamma in an endo position, but the cis isomer is more rigid than the trans isomer, which moreover undergoes a small interconversion of the Cgamma and Cdelta atoms between two extreme spatial positions. The dihedral angle phi has different values in both isomers. Thus, the dihedral angle between the two peptide planes is smaller in the trans isomer than in the cis isomer.     

In vivo therapeutic effect of CDH3/P-cadherin-targeting radioimmunotherapy

Purpose

We examined the possible efficacy of the yttrium-90 (⁹⁰Y)-labeled anti-CDH3/P-cadherin mouse monoclonal antibody (MAb-6) in radioimmunotherapy (RIT) for lung and colorectal cancers that express CDH3/P-cadherin.

Experimental design

MAb-6 was established using genetic immunization. The biodistribution of MAb-6 in nude mice with lung and colorectal cancers was examined by administering indium-111(¹¹¹In)-labeled MAb-6 to mice. The mice were prepared by inoculation of CDH3/P-cadherin-positive (EBC1, H1373, and SW948) and CDH3/P-cadherin-negative (A549 and RKO) tumor cells. Therapeutic effects and toxicity were investigated by administration of ⁹⁰Y-labeled MAb-6 (⁹⁰Y-MAb-6) to EBC, H1373, and SW948-inoculated mice.

Results

Our in vivo results confirmed the specific binding of MAb-6 to tumor cells after intravenous injections of ¹¹¹In-labeled MAb-6 to mice with tumors expressing CDH3/P-cadherin. A single intravenous injection of ⁹⁰Y-MAb-6 (100?μCi) significantly suppressed tumor growth in mice with tumors expressing CDH3/P-cadherin. Furthermore, two injections of ⁹⁰Y-MAb-6 led to complete tumor regression in H1373-inoculated mice without any detectable toxicity.

Conclusions

Our findings demonstrate that CDH3/P-cadherin-targeting RIT with ⁹⁰Y-MAb-6 is a promising strategy for the treatment for cancers expressing CDH3/P-cadherin.     

Gaussian and linear deconvolution of LC-MS/MS chromatograms of the eight aminobutyric acid isomers

Isomeric molecules present a challenge for analytical resolution and quantification, even with MS-based detection. The eight aminobutyric acid (ABA) isomers are of interest for their various biological activities, particularly γ-aminobutyric acid (GABA) and the d- and l-isomers of β-aminoisobutyric acid (β-AIBA; BAIBA). This study aimed to investigate LC-MS/MS-based resolution of these ABA isomers as their Marfey's (Mar) reagent derivatives. HPLC was able to separate three Mar-ABA isomers l-β-ABA (l-BABA), and l- and d-α-ABA (AABA) completely, with three isomers (GABA, and d/l-BAIBA) in one chromatographic cluster, and two isomers (α-AIBA (AAIBA) and d-BABA) in a second cluster. Partially separated cluster components were deconvoluted using Gaussian peak fitting except for GABA and d-BAIBA. MS/MS detection of Marfey's derivatized ABA isomers provided six MS/MS fragments, with substantially different intensity profiles between structural isomers. This allowed linear deconvolution of ABA isomer peaks. Combining HPLC separation with linear and Gaussian deconvolution allowed resolution of all eight ABA isomers. Application to human serum found a substantial level of l-AABA (13 μM), an intermediate level of l-BAIBA (0.8 μM), and low but detectable levels (<0.2 μM) of GABA, l-BABA, AAIBA, d-BAIBA, and d-AABA. This approach should be useful for LC-MS/MS deconvolution of other challenging groups of isomeric molecules.     

The mechanism of oleic acid nitration by *NO(2)

Fatty acid nitration is a recently discovered process that generates biologically active nitro lipids; however, its mechanism has not been fully characterized. For example, some structural details such as vinyl and allyl isomers of the nitro fatty acids have not been established. To characterize lipids that originated from a biomimetic reaction of *NO(2) with oleic acid, we synthesized several isomers of nitro oleic acids and studied their chromatography and mass spectra by various techniques of mass spectrometry. LC/MS analysis performed on a high resolution micro column detected molecular carboxylic anions of various oleic acid nitro isomers (NO(2)OA). Esterification of NO(2)OA with pentafluorobenzyl bromide and diisopropylethylamine as a catalyst produced a unique isoxazole ester derivative exclusively from allyl NO(2)OA isomers via dehydration of the nitro group at ambient temperatures. This new analytical procedure revealed that *NO(2) generated two vinyl and two allyl isomers of NO(2)OA. The vinyl isomers showed high regioselectivity with the 1.8:1 preference for the 10-NO(2)OA isomer that was absent among allylic isomers. The nitration also generated elaidic acid via cis-trans isomerization and diatereoisomers of vicinal nitro hydroxy, nitro keto and alpha-nitro epoxy stearic acids with high stereo and regioselectivity. Nitration of small unilamelar phospholipid vesicles resulted in several phospholipids containing nitro lipids and elaidic acid amenable to hydrolysis by phospholipase A(2).     

Investigations of N-linked macrocycles for 111in and 90Y labeling of proteins

To simplify the synthesis of macrocyclic chelators, commercially available macrocyclic amines were condensed with halogenated acetic acid to prepare the five chelators 12N4 (DOTA), 14N4 (TETA), 15N4, 9N3 and 12N3. Only 12N4 and 9N3 showed efficient labeling of the free chelator with ¹¹¹In and ⁹⁰Y. Serum stability studies at 37 °C with In-labeled DTPA, 12N4 and 9N3 showed no loss of label over 2 days whereas, with ⁹⁰Y, only 12N4 showed stabilities comparable to DTPA. The 12N4 chelator was derivatized by attaching biotin on one N-acetate group to simulate the attachment to protein. The serum stability for both ¹¹¹In and ⁹⁰Y was identical to that of biotin derivatized DTPA and lower than that of the free chelators. Biodistribution studies in normal mice of a model protein (avidin) labeled with ⁹⁰Y via biotinylated 12N4 and biotinylated DTPA showed identical distribution at 1 day except in bone where the %ID/g for the macrocyclic-conjugated protein (3.4 ± 0.5, N = 8) was significantly (P < 0.001) lower than that of the DTPA-conjugated protein (9.4 ± 0.9, N = 7). In conclusion, macrocycles may be readily synthesized from the macrocyclic amines and several show useful stabilities with In and Y. When N-linked to a protein, the Y biodistribution was found to be superior to that of the corresponding DTPA-coupled protein.