Synthesis, solution conformation, and antibody recognition of oligotuftsin-based conjugates containing a beta-amyloid(4-10) plaque-specific epitope

One possible therapeutic approach to treat or prevent Alzheimer's disease (AD) is immunotherapy. On the basis of the identification of Abeta(4-10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, conjugates with defined structures containing the epitope peptide attached to a tetratuftsin derivative as an oligopeptide carrier were synthesized and their structure characterized. To produce immunogenic constructs, the Abeta(4-10) epitope alone or flanked by alpha- or beta-alanine residues was attached through an amide bond to the tetratuftsin derivative (Ac-[TKPKG]4-NH2) or to a carrier peptide elongated by a promiscuous T-helper cell epitope (Ac-FFLLTRILTIPQSLD-[TKPKG]4-NH2). The conformational preferences of the carrier and conjugates were examined by CD spectroscopy in water and in 1:1 and 9:1 TFE:water mixtures (v/v). We found that the presence of flanking dimers in the conjugates had no effects on the generally unordered solution conformation of the conjugates. However, conjugates with an elongated peptide backbone exhibited CD spectra indicative for a partially ordered secondary structure in the presence of TFE. Comparative ELISA binding studies, using monoclonal antibody raised against the beta-amyloid (1-17) peptide, showed that conjugates with T-helper cell epitope in the carrier backbone exhibited decreased monoclonal antibody recognition. However, we found that this effect was compensated in conjugates comprising the Abeta(4-10) B-cell epitope with the beta-alanine dimer flanking regions at both N- and C-termini. Results suggest that modification of the B-cell epitope peptide from Abeta with rational combination of structural elements (e.g. conjugation to carrier, introduction of flanking dimers) can result in synthetic antigen with preserved antibody recognition.     

Nanoparticulate platinum(II) anticancer drug: synthesis and characterization of amphiphilic cyclotriphosphazene-platinum(II) conjugates

Novel cyclotriphosphazene-platinum(II) conjugates were prepared by hydrolysis and platination of the amphiphilic cyclotriphosphazenes grafted with equimolar hydrophilic methoxy-poly(ethylene glycol) (MPEG) and hydrophobic oligopeptide. These macromolecular conjugates were found to form stable nanoparticles with a mean diameter of approximately 90-200 nm depending on the hydrophobicity of the conjugated (diamine)platinum moieties. The nanoparticulate platinum(II) conjugates have shown temperature and concentration dependent particle sizes. However, the particle sizes of the conjugates were found to decrease to a certain size as the solution concentration was decreased but remained stable even at 10 microM, which is enough for systemic delivery by injection. The conjugates exhibited lower in vitro cytotoxicity than cisplatin but reasonably good activity against selected human tumor cell lines.     

Preparation and characterization of a soluble dextran alpha(1) proteinase inhibitor complex

Purified human alpha(1) proteinase inhibitor, a plasma glycoprotein with a molecular weight of 5.3 x 10(4) daltons and a major serine protease inhibitor has been covalently coupled to dextrans with molecular weights of 1.77 x 10(4) and 1.03 x 10(4) daltons. The coupled conjugates were soluble in aqueous medium and stable up to 6 months at +5 degrees C. Increased moles of dextran/mole protein ratio during coupling resulted in progressive decreases of inhibitory capacity, immunogenicity, and the association constant (k(assoc)) between the enzyme and the inhibitor. Compared to the native protein, the soluble conjugates showed improved stability at pH 3.0 and heat stability at 60 degrees C. At 60 degrees C, no loss of inhibitory capacity has been seen up to 60 min for the conjugates during which time the native protein lost greater than 90% of its inhibitory capacity. The presence of antioxidant catalase was needed to prevent oxidative degradation by hydrogen peroxide.     

Poly(ethylene glycol)-poly(ester-carbonate) block copolymers carrying PEG-peptidyl-doxorubicin pendant side chains: synthesis and evaluation as anticancer conjugates

Water soluble polymer anticancer conjugates can improve the pharmacokinetics of covalently bound drugs by limiting cellular uptake to the endocytic route, thus prolonging plasma circulation time and consequently facilitating tumor targeting by the enhanced permeability and retention (EPR) effect. Many of the first generation antitumor polymer conjugates used nonbiodegradable polymeric carriers which limits the molecular weight that can be safely used to <40,000 g/mol. The aim of this ambitious study was to synthesize and evaluate a novel, prototype biodegradable polymeric system based on high molecular weight, water-soluble functionalized polyesters. The main polymeric platform was prepared from bis(4-hydroxy)butyl maleate (DBM) and poly(ethylene glycol) (PEG4000) blocks to give the polymer DBM2-PEG4000 containing biodegradable carbonate bonds and having a M(w) of 100,000-190,000 g/mol; M(n) of 37,000-53,000 g/mol, and M(w)/M(n) of 3.0-3.7. Using thioether linkages, this polymer was then grafted with HS-PEG3000-Gly-Phe-Lue-Gly doxorubicin (HS-PEG3000-GFLG-Dox) pendant side chains ( approximately 30 per DBM2-PEG chain). The final construct, DBM2-PEG4000-S-PEG3000-GFLG-Dox had a total Dox content of 3-4 wt % and a free Dox content of < or = 0.7% total Dox. During incubation with isolated lysosomal enzymes, the rate of Dox release from the polymer backbone was relatively slow (<5% release over 5 h) compared to that seen for PEG5000-GFLG-Dox alone (>20% over 5 h). The in vitro cytotoxicity was assessed using B16F10 murine melanoma (MTT assay). DBM2-PEG4000-S-PEG3000-GFLG-Dox was 10-20-fold less toxic than free Dox. In vivo antitumor activity of the DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates was assessed using a subcutaneous (s.c.) B16F10 murine melanoma model, and an intraperitoneal (i.p.) L1210 leukaemia model. The increased toxicity (attributed to poor solubility) and low antitumor activity of DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates compared to PEG5000-GFLG-Dox and HPMA copolymer-Dox conjugates was attributed to the slow rate of Dox release. The DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates were considered unfavorable as candidates for further development. However, the successful scale-up synthesis of DBM2-PEG4000-S-PEG3000 constructs suggest that they are worthy of further investigation as carriers for controlled release and targeting of less hydrophobic agents.     

Competitive Immunochemical Determination of Antigens Using Conjugates Containing Co(II) and Ni(II)

A new variant of competitive heterogeneous immunoassay for certain proteinaceous antigens has been developed. The assay is based on the use of the target protein conjugated with Co(II) or Ni(II) ions and immobilized antibodies. The effect of catalytic hydrogen release allows quantitation of the metal ion labels by voltammetry at the final step of the assay. The conjugates have been characterized by spectrophotometry, voltammetry, atomic adsorption spectrometry, and nuclear magnetic relaxation. Based on the use of the conjugate RNase–diethylenetriaminepentaacetic acid–Co(II) (10 : 4 : 4), a competitive immunoassay for RNase has been developed, detecting the target protein in the range 2 × 10^–2–2 × 10^–4 mg/ml.     

Synthesis and evaluation of nanoglobular macrocyclic Mn(II) chelate conjugates as non-gadolinium(III) MRI contrast agents

Because of the recent observation of the toxic side effects of Gd(III) based MRI contrast agents in patients with impaired renal function, there is strong interest on developing alternative contrast agents for MRI. In this study, macrocyclic Mn(II) chelates were conjugated to nanoglobular carriers, lysine dendrimers with a silsesquioxane core, to synthesize non-Gd(III) based MRI contrast agents. A generation 3 nanoglobular conjugate of Mn(II)-1,4,7-triaazacyclononane-1,4,7-triacetate-GA amide (G3-NOTA-Mn) was also synthesized and evaluated. The per ion T(1) and T(2) relaxivities of G2, G3, G4 nanoglobular Mn(II)-DOTA monoamide conjugates decreased with increasing generation of the carriers. The T(1) relaxivities of G2, G3, and G4 nanoglobular Mn(II)-DOTA conjugates were 3.3, 2.8, and 2.4 mM(-1) s(-1) per Mn(II) chelate at 3 T, respectively. The T(1) relaxivity of G3-NOTA-Mn was 3.80 mM(-1) s(-1) per Mn(II) chelate at 3 T. The nanoglobular macrocyclic Mn(II) chelate conjugates showed good in vivo stability and were readily excreted via renal filtration. The conjugates resulted in much less nonspecific liver enhancement than MnCl(2) and were effective for contrast-enhanced tumor imaging in nude mice bearing MDA-MB-231 breast tumor xenografts at a dose of 0.03 mmol Mn/kg. The nanoglobular macrocyclic Mn(II) chelate conjugates are promising nongadolinium based MRI contrast agents.     

Multidrug resistance-associated protein2 (MRP2) plays an important role in the biliary excretion of glutathione conjugates of 4-hydroxynonenal

Glutathione (GSH) conjugates of 4-hydroxy-trans-2,3-nonenal (HNE) are the final products of lipid peroxidation. In the present study, the role of multidrug resistance-associated protein 2 (MRP2) in biliary excretion of GSH conjugates of HNE (HNE-SG) was studied in vitro by using Madin-Darby canine kidney II (MDCK II) cells expressing human MRP2 and in vivo using a mutant rat strain whose MRP2 expression is defective (Eisai-hyperbilirubinemic rats [EHBR]). A high-performance liquid chromatography method was developed to assay HNE-SG conjugates. Four diastereomeric HNE-SG conjugates could be separated with this method. Three of four HNE-SG conjugates were detectable after incubation of the cell monolayers with HNE. Expression of human MRP2 resulted in a 10-fold increase in HNE-SG conjugates excretion across the apical membrane of MDCK II cells. The four HNE-SG conjugates appeared swiftly in bile from Sprague Dawley rats after intravenous administration of HNE, whereas no detectable HNE-SG conjugates were observed in the bile of EHBR. These results demonstrate the role of MRP2 in the biliary excretion of HNE-SG conjugates.     

N-(2-hydroxypropyl)methacrylamide copolymer-6-(3-aminopropyl)-ellipticine conjugates. Synthesis, in vitro, and preliminary in vivo evaluation

Ellipticine derivatives have potential as anticancer drugs. Their clinical use has been limited, however, by poor solubility and host toxicity. As N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-anticancer conjugates are showing promise in early clinical trials, a series of novel HPMA copolymer conjugates have been prepared containing the 6-(3-aminopropyl)-ellipticine derivative (APE, NSC176328). Drug was linked to the polymer via GFLG or GG peptide side chains. To optimize biological behavior, HPMA copolymer-GFLG-APE conjugates with different drug loading (total APE: 2.3-7% w/w; free APE: <0.1% w/w) were synthesized. Conjugation of APE to HPMA copolymers considerably increased its aqueous solubility (>10-fold). HPMA copolymer-GG-APE did not liberate drug in the presence of isolated lysosomal enzymes (tritosomes), but HPMA copolymer-GFLG-APE released APE to a maximum of 60% after 5 h. The rate of drug release was influenced by drug loading; lower loading led to greater release. Whereas free APE (35 microg/mL) caused significant hemolysis (50% after 1 h), HPMA copolymer-APE conjugates were not hemolytic up to 300 microg/mL (APE-equiv). As would be expected from its cellular pharmacokinetics, HPMA copolymer-GFLG-APE was >75 times less cytotoxic than free drug (IC(50) approximately 0.4 microg/mL) against B16F10 melanoma in vitro. However, in vivo when tested in mice bearing s.c. B16F10 melanoma, HPMA copolymer-GFLG-APE (1-10 mg/kg single dose, APE-equiv) given i.p. was somewhat more active (highest T/C value of 143%) than free APE (1 mg/kg) (T/C =127%). HPMA copolymer-APE conjugates warrant further evaluation as potential anticancer agents.     

Pharmacokinetic, biodistribution, and biophysical profiles of TNF nanobodies conjugated to linear or branched poly(ethylene glycol)

Covalent attachment of poly(ethylene glycol) (PEG) to therapeutic proteins has been used to prolong in vivo exposure of therapeutic proteins. We have examined pharmacokinetic, biodistribution, and biophysical profiles of three different tumor necrosis factor alpha (TNF) Nanobody-40 kDa PEG conjugates: linear 1 × 40 KDa, branched 2 × 20 kDa, and 4 × 10 kDa conjugates. In accord with earlier reports, the superior PK profile was observed for the branched versus linear PEG conjugates, while all three conjugates had similar potency in a cell-based assay. Our results also indicate that (i) a superior PK profile of branched versus linear PEGs is likely to hold across species, (ii) for a given PEG size, the extent of PEG branching affects the PK profile, and (iii) tissue penetration may differ between linear and branched PEG conjugates in a tissue-specific manner. Biophysical analysis (R(g)/R(h) ratio) demonstrated that among the three protein-PEG conjugates the linear PEG conjugate had the most extended time-average conformation and the most exposed surface charges. We hypothesized that these biophysical characteristics of the linear PEG conjugate accounts for relatively less optimal masking of sites involved in elimination of the PEGylated Nanobodies (e.g., intracellular uptake and proteolysis), leading to lower in vivo exposure compared to the branched PEG conjugates. However, additional studies are needed to test this hypothesis.     

Radiochemical investigations of (99m)Tc-N(3)S-X-BBN[7-14]NH(2): an in vitro/in vivo structure-activity relationship study where X = 0-, 3-, 5-, 8-, and 11-carbon tethering moieties

Bombesin (BBN), a 14 amino acid peptide, is an analogue of human gastrin releasing peptide (GRP) that binds to GRP receptors (GRPr) with high affinity and specificity. The GRPr is overexpressed on a variety of human cancer cells, including prostate, breast, lung, and pancreatic cancers. The specific aim of this study was to develop (99m)Tc-radiolabeled BBN analogues that maintain high specificity for the GRPr in vivo. A preselected synthetic sequence via solid-phase peptide synthesis (SPPS) was designed to produce N(3)S-BBN (N(3)S = dimethylglycyl-l-seryl-l-cysteinylglycinamide) conjugates with the following general structure: DMG-S-C-G-X-Q-W-A-V-G-H-L-M-(NH(2)), where the spacer group, X = 0 (no spacer), omega-NH(2)(CH(2))(2)COOH, omega-NH(2)(CH(2))(4)COOH, omega-NH(2)(CH(2))(7)COOH, or omega-NH(2)-(CH(2))(10)COOH. The new BBN constructs were purified by reversed phase-HPLC (RP-HPLC). Electrospray mass spectrometry (ES-MS) was used to characterize the nonmetalated BBN conjugates. Re(V)-BBN conjugates were prepared by the reaction of Re(V)gluconate with N(3)S-X-BBN[7-14]NH(2) (X = 0 carbons, beta-Ala (beta-alanine), 5-Ava (5-aminovaleric acid), 8-Aoc (8-aminooctanoic acid), and 11-Aun (11-aminoundecanoic acid)) with gentle heating. Re-N(3)S-5-Ava-BBN[7-14]NH(2) was also prepared by the reaction of [Re(V)dimethylglycyl-l-seryl-l-cysteinylglycinamide] with 5-Ava-BBN[7-14]NH(2). ES-MS was used to determine the molecular constitution of the new Re(V) conjugates. The (99m)Tc conjugates were prepared at the tracer level by each the prelabeling, post-conjugation and pre-conjugation, postlabeling approaches from the reaction of Na[(99m)TcO(4)] with excess SnCl(2), sodium gluconate, and corresponding ligand. The (99m)Tc and Re(V) conjugates behaved similarly under identical RP-HPLC conditions. In vitro and in vivo models demonstrated biological integrity of the new conjugates.     

Peptide T-araC conjugates: solid-phase synthesis and biological activity of N4-(acylpeptidyl)-araC

Due to the capability of peptidyl derivatives of araC to behave as prodrugs of this antimetabolite, and because of the well known biological properties of peptide T and its analogues (in particular that of targeting CD4+ cells), new peptide T-araC conjugates were prepared and tested in vitro for antiproliferative activity. The aim was that of specifically delivering the antitumor drug to CD4+ cells. N4-(Acylpeptidyl)-derivatives of araC were synthesized by a new general approach involving solid-phase synthesis, which allows mild conditions, avoids the usually required protection of the glycoside portion of nucleosides and affords high yields of the final products. After the demonstration that peptide T-araC conjugates were able to activate chemotaxis by binding CD4 receptor on monocyte membranes, the antiproliferative activity was evaluated against a panel of leukemia lymphoma and carcinoma cell lines derived from human tumors, three CD4+ cell lines included. Title compounds resulted effective as antiproliferative agents at concentrations 4- to 10-fold higher than those of araC alone, did not preferentially inhibit CD4+ cells and proved stable not only in cell culture medium containing 20% FCS, but also in human plasma. All this suggests their potential utility in vivo.     

Innovative, multifunctional sequential oligopeptide carriers Soc(n)-I and SOC(n)-II: functions-technology-perspectives

An innovative type of multifunctional helicoid artificial carriers, formed by the repetitive Lys-Aib-Gly (SOC(n)-I) or by the Aib-Lys-Aib-Gly (SOC(n)-II), with structural rigidity and regularity were successfully applied in our laboratory for anchoring antigenic/immunogenic peptides. The carriers, designed to display a predetermined 3D structure, adopt the 3(10) helical conformation, while the attached peptides preserve their original "active" conformation. The constructed conjugates were used as substrates in solid phase immunoassays, as well as for generating potent and specific immune responses.     

Fibroblast aggregation by suspension with conjugates of poly(ethylene glycol) and RGD

Cell aggregates may be useful components of artificial organs and mammalian cell bioreactors, but many cells do not naturally aggregate. In a previous report,(4) we described a method for promoting neural cell aggregation by addition of water-soluble conjugates of cell adhesion peptides, containing the three amino acid sequence Arg-Gly-Asp (RGD), and poly(ethylene glycol) (PEG). Here, we examined the mechanism of conjugate-induced aggregation using fibroblasts and a variety PEG-peptide conjugates. Aggregation was monitored during rotation culture of fibroblasts in the presence of unconjugated GRGDY and PEG; monofunctional (PEG-GRGDY) and bifunctional (GRGDY-PEG-GRGDY) conjugates; and bifunctional conjugates produced with a similar, but non-cell-binding, peptide (GRGEY-PEG-GRGEY). GRGDY-PEG-GRGDY conjugates induced rapid and pronounced fibroblast aggregation that was dose-dependent; at the highest concentration tested (5 mg/mL GRGDY-PEG-GRGDY), cell aggregates were produced more quickly ( approximately 1 h) and were significantly larger at 24 h (mean radius approximately 66 mum) than at slightly lower concentrations (1.7 and 3.3 mg/mL). Aggregation with GRGDY-PEG-GRGDY was completely inhibited by dissolved GRGDY (1.7 mg/mL). Neither unmodified GRGDY, unmodified PEG, PEG-GRGDY, nor GRGEY-PEG-GRGEY conjugates led to significant aggregation. The extent of aggregation depended on PEG molecular weight: conjugates with 3400 M(w) PEG produced aggregates with significantly larger mean radius than conjugates with 20,000 M(w) PEG. When 1N-8A fibroblasts, genetically engineered to produce recombinant nerve growth factor (NGF), were aggregated with GRGDY-PEG-GRGDY, aggregated cells produced more NGF per cell than nonaggregated cells. Aggregation of cells may lead to improved cell function, such as the increase in NGF production observed here, which could be useful in large-scale cell culture and construction of artificial organs or tissue transplants for tissue engineering. (c) 1996 John Wiley & Sons, Inc.     

A spectrophotometric method for determination of fluorophore-to-protein ratios in conjugates of the blue fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA)

7-Amino-4-methylcoumarin-3-acetic acid (AMCA) has been found to be a useful fluorophore for immunofluorescence. The present study describes a spectrophotometric method for determining the ratio of moles AMCA to moles protein (or the f/p ratio) in an AMCA-conjugated IgG. The concentration of a substance absorbing light can be determined spectrophotometrically using Beer's Law: Absorbance = Concentration x Extinction coefficient. From Beer's law, one can derive the following formula for determining the f/p ratio of AMCA-IgG conjugates: f/p = (epsilon 280IgG).A350 - (epsilon 350IgG).A280/(epsilon 350AMCA).A280 - (epsilon 280AMCA).A350 where A is the optical density of the conjugate at the given wavelength and epsilon is the extinction coefficient of a substance at the wavelength specified. Using conjugates of model proteins, it was found that the extinction coefficients of the AMCA moiety of AMCA-conjugated protein were 1.90 x 10(4) at 350 nm and 8.29 x 10(3) at 280 nm. Similarly, it was found that the extinction coefficients of swine IgG were 1.56 x 10(3) at 350 nm and 1.26 x 10(5) at 280 nm. Thus, for AMCA-conjugated swine IgG: f/p = (1.26 x 10(5)).A350 - (1.56 x 10(3)).A280/(1.47 x 10(4)).A280 - (6.42 x 10(3)).A350 [corrected]. Based on this formula, the f/p ratios of some AMCA-IgG conjugates useful for immunohistochemistry have been found to range between 6 and 24.     

Organometallic 99mTc(III) '4 + 1' bombesin(7-14) conjugates: synthesis, radiolabeling, and in vitro/in vivo studies

Bombesin (BBN) peptide exhibits high selectivity and affinity for the gastrin-releasing peptide receptor (GRPr). The GRPr is overexpressed on many human cancer cell types, thus making BBN a potent delivery vehicle for radionuclide targeting. In this study, the biologically active minimal sequence BBN(7-14) was labeled using the novel Tc '4 + 1' mixed-ligand system, [Tc(NS3)(CN-R)], in which Tc(III) is coordinated by a monodentate isocyanide linker bearing the peptide and the tetradentate, tripodal chelator, 2,2',2'-nitrilotriethanethiol (NS3). BBN(7-14) was N-terminally modified with Gly-Gly-Gly, betaAla, and Ser-Ser-Ser spacer groups (X) and functionalized with 4-(isocyanomethyl)benzoic acid (L1) or 4-isocyanobutanoic acid (L2), resulting in a series of [M(NS3)(L-X-BBN(7-14))] conjugates (M = 99mTc, Re). The isocyanide ligand frameworks were introduced using novel bifunctional coupling agents. The spacer groups (X), the monodentate isocyanide units, and a tetradentate NS3 chelator bearing a pendant carboxylic acid (NS3COOH) were proposed as pharmacological modifiers. 99mTc-labeling was performed in a two-step procedure by first preparing 99mTc-EDTA/mannitol followed by reactions with the isocyanides and NS3 or NS3COOH ligand frameworks. The 99mTc complexes were obtained with a radiochemical yield of 30-80% depending on the amount of the isocyanide (20-100 nmol) used. These new conjugates were purified by reversed-phased high-performance liquid chromatography (RP-HPLC) to give a radiochemical purity of >or=95%. The 99mTc conjugates exhibited high in vitro stability (>90%, 24 h). Analogous nonradioactive Re conjugates were synthesized and characterized by electrospray ionization mass spectrometry (ESI-MS). RP-HPLC analyses of the Re conjugates indicated that they exhibited identical retention times to the corresponding 99mTc conjugates under identical HPLC conditions, demonstrating structural similarity between the two metalated species. The [Re(NS3)(L-X-BBN(7-14))] conjugates exhibited GRPr affinity in the nanomolar range as demonstrated by in vitro competitive binding assays using PC-3 human prostate cancer cells. In vitro internalization/externalization assays indicated that approximately 65% of [99mTc(NS3)(L2-betaAla-BBN(7-14))] conjugate was either surface-bound or internalized in PC-3 cells. Cell-associated activity for all other 99mTc conjugates was below 20%. Biodistribution studies of [99mTc(NS3)(L-betaAla-BBN(7-14))], L = L1 or L2, in normal, CF-1 mice showed minimal accumulation in normal pancreas (a tissue expressing the GRPr in high density in rodent models) and rapid hepatobiliary elimination. Introduction of a carboxyl group onto the NS3 ligand framework had only minimal effects to increase renal excretion. Activity distribution and accumulation was highly dominated by the relatively lipophilic '4 + 1' complex unit.     

Detection of the low density lipoprotein (LDL) receptor on nitrocellulose paper with colloidal gold-LDL conjugates

Gold-low density lipoprotein (LDL) conjugates were used to detect the LDL receptor on nitrocellulose paper. Solubilized rat liver membrane proteins were subjected to electrophoresis and electroblotted onto nitrocellulose paper. The receptor was then detected as a red band (within 10 min) by overlaying with the LDL conjugates. The coloration was prevented by unlabeled LDL, EDTA, and suramin but not by unlabeled HDL3. In the dot blot assay, detection with the colloidal gold-LDL conjugates was as sensitive as both the autoradiographic method with 125I-labeled LDL and the biotinylated LDL method; the estimated limit of detection by scanning densitometry was 1.6 femtomoles of receptor protein. When the coloration obtained with the colloidal gold-LDL conjugates was intensified by photochemical silver staining, down to 200 attomoles of the LDL receptor could be detected. In this assay, the EDTA-sensitive binding of colloidal gold-LDL to solubilized hepatic membrane proteins was 12 times higher for rats treated with 17 alpha-EE than for normal rats. The use of colloidal gold-LDL conjugates is therefore a very easy, safe, inexpensive, fast and sensitive method for the detection of the LDL receptor on nitrocellulose paper. Furthermore, with silver staining and scanning densitometry, the colloidal gold-LDL conjugates could be used in a dot blot assay to quantify tissue and cell LDL receptors down to attomolar levels.     

Multivalent poly(ethylene glycol)-containing conjugates for in vivo antibody suppression

Poly(ethylene glycol) (PEG) was incorporated into multivalent conjugates of the N-terminal domain of beta(2)GPI (domain 1). PEG was incorporated to reduce the rate of elimination of the conjugates from plasma and to putatively improve their efficacy as toleragens for the suppression of anti-beta(2)GPI antibodies and the treatment of antiphospholipid syndrome (APS). Three structurally distinct types of multivalent platforms were constructed by incorporating PEG into the platform structures in different ways. The amount of PEG incorporated ranged from about 5000 g per mole to about 30000 g per mole. The platforms were functionalized with either four or eight aminooxy groups. The conjugates were prepared by forming oxime linkages between the aminooxy groups and N-terminally glyoxylated domain 1 polypeptide. The plasma half-life of each conjugate, labeled with (125)I, was measured in both mice and rats. The half-lives of the conjugates ranged from less than 10 min to about 1 h in mice, and from less than 3 h to about 19 h in rats. The ability of five tetravalent conjugates to suppress anti-domain 1 antibodies in immunized rats was also measured. Incorporation of PEG in the conjugates significantly reduced the doses required for suppression, and the amount of reduction correlated with the amount of PEG incorporated.     

Oligonucleotide conjugates of Eu(III) tetraazamacrocycles with pendent alcohol and amide groups promote sequence-specific RNA cleavage

Eu(III) complexes of two neutral bifunctional tetraaaza macrocyclic ligands {1-[1-carboxamido-3-(4-nitrophenyl)propyl]-4,7,10-tris(2-hydroxyethyl)-1,4,7,10-tetraazacyclododecane and 2-(4-nitrobenzyl)-1,4,7,10-tetrakis(2-hydroxyethyl)-1,4,7,10-tetraazacyclododecane} are prepared. Eu(III) complexes of the isothiocyanate derivatives of these macrocycles are treated with oligonucleotides containing 2′-O-propylamine linkers to form conjugates. Hydrolytic cleavage of an oligoribonucleotide is promoted by Eu(III) macrocyclic oligonucleotide conjugates containing complementary (antisense) sequences. Cleavage is not observed in the presence of Eu(III) conjugates containing scrambled sequences nor by free complex. Despite the fact that one of the free macrocyclic complexes is more reactive than the other, the extent of cleavage observed is similar for conjugates containing either Eu(III) macrocyclic complex.     

Bis(acridinylthiourea)platinum(II) complexes: synthesis,DNA affinity,and biological activity in glioblastoma cells

The preparation of two novel bis(acridine)platinum(II) complexes is reported. The 4+ charged conjugates associate strongly with double-stranded native DNA (K(i)>10(6)), possibly through bisintercalation. A cell viability assay was used to demonstrate that both compounds are capable of mediating cytotoxicity at micromolar concentrations in SNB19 brain tumor cells.     

Prostate-cancer-targeted N-(2-hydroxypropyl)methacrylamide copolymer/docetaxel conjugates

Biodistribution, pharmacokinetics, and efficacy of prostate-cancer-targeted HPMA copolymer/DTX conjugates are evaluated in nude mice bearing prostate cancer C4-2 xenografts. PSMA-specific monoclonal antibodies 3F/11 are used as the targeting moiety. Control conjugates tumor accumulation to total background organs (heart, lung, kidney, liver, spleen and blood) accumulation increase substantially with time for the targeted conjugate, and the ratio at 48 h is 7-fold higher than that at 6 h. Preliminary evaluation of the efficacy of the conjugates in vivo show tumor growth inhibition for all HPMA copolymer/DTX conjugates.