Peripheral benzodiazepine receptor ligand-melphalan conjugates for potential selective drug delivery to brain tumors

To gain insight into the strategy to target PBR ligand-drug conjugates to brain tumors, novel N-imidazopyridinacetyl-melphalan conjugates and the corresponding ethyl esters have been prepared and evaluated for their cytotoxicity in melphalan-sensitive human (SF126, SF188) and rat (RG-2) glioma cell lines. These conjugates exhibited PBR binding affinity with IC(50) values ranging from 57 and 2614 nM. By a computational approach it can be predicted that these conjugates possess significant brain penetration. The stability of the conjugates in 0.05 M phosphate buffer at pH 7.4 and, in some cases, in dilute human serum solution was determined. All the ethyl ester derivatives were stable in 0.05 M phosphate buffer at pH 7.4 and their half-lives exceeded 28 h. Conversely, under the same conditions, the corresponding acids were found to undergo a fast cleavage within a few minutes. HPLC-MS analysis of the mixture from degradation in buffer and physiological medium of the representative cases allowed the identification of their main degradation products. A plausible degradation pathway accounting for the available experimental data is presented.     

BR96 conjugates of highly potent anthracyclines

The 6-maleimidocaproylhydrazone derivatives of highly potent antitumor agents 5-Diacetoxypentyldoxorubicin and Morpholinodoxorubicin were synthesized and conjugated to monoclonal antibody BR96 and control IgG. Immunoconjugate molar ratios were generally 7.5-8.5, and dimer aggregate levels were low. The linkers released parent drug at lysosomal pH 5, while they remained stable at neutral pH. BR96 conjugates were highly potent and antigen specific in vitro. The BR96-DAPDOX conjugate demonstrated an IC(50) of 0.03 micrometer and was at least 300-fold more potent than a non-binding IgG-DAPDOX control conjugate.     

Polymer conjugates of acridine-type anticancer drugs with pH-controlled activation

Acridines are potent DNA-intercalating anticancer agents with high in vivo anticancer effectiveness, but also severe side effects. We synthesized five 9-anilinoacridine-type drugs and their conjugates with biocompatible water-soluble hydrazide polymer carrier. All of the synthesized acridine drugs retained their in vitro antiproliferative properties. Their polymer conjugates were sufficiently stable at pH 7.4 (model of pH in blood plasma) while releasing free drugs at pH 5.0 (model of pH in endosomes). After internalization of the conjugates, the free drugs were released and are visible in cell nuclei by fluorescence microscopy. Their intercalation ability was proven using a competitive ethidium bromide displacement assay.     

"SMART" drug delivery systems: double-targeted pH-responsive pharmaceutical nanocarriers

To develop targeted pharmaceutical carriers additionally capable of responding to certain local stimuli, such as decreased pH values in tumors or infarcts, targeted long-circulating PEGylated liposomes and PEG-phosphatidylethanolamine (PEG-PE)-based micelles have been prepared with several functions. First, they are capable of targeting a specific cell or organ by attaching the monoclonal antimyosin antibody 2G4 to their surface via pNP-PEG-PE moieties. Second, these liposomes and micelles were additionally modified with biotin or TAT peptide (TATp) moieties attached to the surface of the nanocarrier by using biotin-PE or TATp-PE or TATp-short PEG-PE derivatives. PEG-PE used for liposome surface modification or for micelle preparation was made degradable by inserting the pH-sensitive hydrazone bond between PEG and PE (PEG-Hz-PE). Under normal pH values, biotin and TATp functions on the surface of nanocarriers were "shielded" by long protecting PEG chains (pH-degradable PEG(2000)-PE or PEG(5000)-PE) or by even longer pNP-PEG-PE moieties used to attach antibodies to the nanocarrier (non-pH-degradable PEG(3400)-PE or PEG(5000)-PE). At pH 7.4-8.0, both liposomes and micelles demonstrated high specific binding with 2G4 antibody substrate, myosin, but very limited binding on an avidin column (biotin-containing nanocarriers) or internalization by NIH/3T3 or U-87 cells (TATp-containing nanocarriers). However, upon brief incubation (15-30 min) at lower pH values (pH 5.0-6.0), nanocarriers lost their protective PEG shell because of acidic hydrolysis of PEG-Hz-PE and acquired the ability to become strongly retained on an avidin column (biotin-containing nanocarriers) or effectively internalized by cells via TATp moieties (TATp-containing nanocarriers). We consider this result as the first step in the development of multifunctional stimuli-sensitive pharmaceutical nanocarriers.     

Design,characterization, and in vitro antiproliferative efficacy of gemcitabine conjugates based on carboxymethyl glucan

Gemcitabine (GEM) is widely used in clinical practice in the treatment of cancer and several other solid tumors. Nevertheless, the antitumor effect of GEM is partially prevented by some limitations including short half life, and lack of tumor localizing. Carboxymethyl glucan (CMG), a carboxymethylated derivative of β-(1-3)-glucan, shows biocompatibility and biodegradability as well as a potential anticarcinogenic effect. To enhance the antiproliferative activity of GEM, four water soluble conjugates of GEM bound to CMG via diverse amino acid linkers were designed and synthesized. ¹H NMR, FT IR, elementary analysis and RP-HPLC chromatography were employed to verify the correct achievement of the conjugates. In vitro release study indicated that conjugates presented slower release in physiological buffer (pH 7.4) than acidic buffer (pH 5.5) mimicking the acidic tumor microenvironment. Moreover, A549, HeLa and Caco-2 cancer cell lines were used to evaluate the in vitro cytotoxicity of conjugates and the results showed that binding GEM to CMG significantly enhanced antiproliferative activity of GEM on A549 cells. Therefore, these conjugates may be potentially useful as a delivery vehicle in cancer therapy and worthy of further study on structure-activity relationship and antiproliferative activity in vitro and in vivo, especially for lung tumor.     

Peptide substrate specificity of the alpha-amidating enzyme isolated from rat medullary thyroid CA-77 cells

The kinetic parameters were obtained for enzymatic alpha-amidation of peptides of the form N-dansyl-(Gly)4-X-Gly-OH, in which the amino acid at position X was substituted with each of the 20 natural amino acids. The enzyme used in these studies was a highly enriched preparation of alpha-amidating enzyme secreted by a clonal (CA-77) cell line which actively expresses mature alpha-amidated peptides. A 130-fold and 11-fold variation respectively in apparent Km and Vmax values was observed. The effect of the amino acid side chain at position X in stabilization of the enzyme-substrate complex decreased through the series X = planar aromatic or sulfur containing greater than neutral aliphatic greater than polar and basic greater than cyclic aliphatic or acidic.     

Effects of redox buffer properties on the folding of a disulfide-containing protein: dependence upon pH, thiol pKa, and thiol concentration

Aliphatic thiols are effective as redox buffers for folding non-native disulfide-containing proteins into their native state at high pH values (8.0-8.5) but not at neutral pH values (6-7.5). In developing more efficient and flexible redox buffers, a series of aromatic thiols was analyzed for its ability to fold scrambled ribonuclease A (sRNase A). At equivalent pH values, the aromatic thiols folded sRNase A 10-23 times faster at pH 6.0, 7-12 times faster at pH 7.0, and 5-8 times faster at pH 7.7 than the standard aliphatic thiol glutathione. Similar correlations between thiol pK(a) values and folding rates at each pH value suggest that the apparent folding rate constants (k(app)) are a function of the redox buffer properties (pH, thiol pK(a) and [RSH]). Fitting the observed data to a three-variable model (logk(app)=-4.216(+/-0.030)+0.5816(+/-0.0036)pH-0.233(+/-0.004)pK(a)+log(1-e(-0.98(+/-0.02)[RSH]))) gave good statistics: r2=0.915, s=0.10.     

Preparation of protein conjugates via intermolecular hydrazone linkage

Proteins can be modified at their amino groups under gentle conditions to contain an average of three to six aryl aldehyde or acyl hydrazide groups. These two types of modified proteins at about 10 microM concentration condense with each other at pH approximately 5 to form conjugates linked by hydrazone bonds. Under proper conditions conjugates mainly of dimers and trimers in size or, if desired, higher oligomers can be obtained. The conjugates can be dissociated to their individual protein components by an exchange reaction with an excess of acetyl hydrazide. The reversible hydrazone bonds of conjugates can be reduced with NaCNBH3 to give stable hydrazide bonds. The stability of protein-hydrazone conjugates was found to be significantly greater than that of the model compound, the N-acetylhydrazone of p-carboxybenzaldehyde. This difference is believed to result from the presence of multiple hydrazone linkages in protein conjugates.     

Gallium labeled NOTA-based conjugates for peptide receptor-mediated medical imaging

We report a straightforward and efficient synthetic strategy for the synthesis of three model glycine-arginine-glycine-aspartic acid-glycine (GRGDG) conjugates based on derivatives of NOTA and of their Ga(III) complexes targeted to the integrin α(ν)β(3) receptor. (71)Ga NMR spectroscopy showed that the Ga(III)-labeled conjugates are highly stable in aqueous solution. The (67)Ga-labeled conjugates proved to have high kinetic stability and showed a weak but specific binding to the receptors in a U87MG-glioblastoma cell line.     

Novel cleavable cell‐penetrating peptide–drug conjugates: synthesis and characterization

We report the first drug conjugate with a negatively charged amphipathic cell‐penetrating peptide. Furthermore, we compare two different doxorubicin cell‐penetrating peptide conjugates, which are both unique in their properties, due to their net charge at physiological pH, namely the positively charged octaarginine and the negatively charged proline‐rich amphipathic peptide. These conjugates were prepared exploiting a novel heterobifunctional crosslinker to join the N‐terminal cysteine residue of the peptides with the aliphatic ketone of doxorubicin. This small linker contains an activated thiol as well as aminooxy functionality, capable of generating a stable oxime bond with the C‐13 carbonyl group of doxorubicin. The disulfide bond formed between the peptide and doxorubicin enables the release of the drug in the cytosol, as confirmed by drug‐release studies performed in the presence of glutathione. Additionally, the cytotoxicity as well as the cellular uptake and distribution of this tripartite drug delivery system was investigated in MCF‐7 and HT‐29 cell lines. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of pH-sensitive amphotericin B-poly(ethylene glycol) conjugates and study of their controlled release in vitro

New intravenous conjugates of amphotericin B (AMB) with poly(ethylene glycols) (PEG) (M=5000, 10,000, 20,000) have been synthesized and characterised. The intermediate PEGs possess a 1,4-disubstituted benzene ring with aldehyde group at the end of the chain. The benzene ring is connected with PEG at its 4-position (with respect to the aldehyde group) by various functional groups (ether, amide, ester). Reaction of terminal aldehyde group of the substituted PEGs with AMB gave conjugates containing a pH-sensitive imine linkage, which can be presumed to exhibit antimycotic effect at sites with lowered pH value. All types of the conjugates are relatively stable in phosphate buffer at physiological conditions of pH 7.4 (37 degrees C), less than 5 mol% AMB being split off from them within 24 h. For a model medium of afflicted tissue was used a phosphate buffer (pH 5.5, 37 degrees C), in which controlled release of AMB from the conjugates takes place. The imine linkage is split to give free AMB with half-lives of 2-45 min. The rate of acid catalysed hydrolysis depends upon substitution of the benzene ring; however, it does not depend on molecular weights of the PEGs used. The conjugates with ester linkage undergo enzymatic splitting in human blood plasma and/or blood serum at pH 7.4 (37 degrees C) with half-lives of 2-5 h depending on molecular weights of the PEGs used (M = 5000, 10,000, 20,000). At first, the splitting of ester linkage produces the relatively stable pro-drug, that is, 4-carboxybenzylideniminoamphotericin B, which is decomposed to AMB and 4-formylbenzoic acid in a goal-directed manner only at pH 7 (t1/2 = 2 min, pH 5.5, 37 degrees C). A goal-directed release of AMB is only achieved by acid catalysed hydrolysis of imine linkage, either from the polymeric conjugate or from the pro-drug released thereof. The LD50 values determined in vivo (mouse) are 20.7 mg/kg and 40.5 mg/kg for the conjugates with ester linkage (M = 10,000 and 5000, respectively), which means that they are ca. 6-11 times less toxic than free AMB.     

Influence of structural variation on nuclear localization of DNA-binding polyamide-fluorophore conjugates

A pivotal step forward in chemical approaches to controlling gene expression is the development of sequence-specific DNA-binding molecules that can enter live cells and traffic to nuclei unaided. DNA-binding polyamides are a class of programmable, sequence-specific small molecules that have been shown to influence a wide variety of protein-DNA interactions. We have synthesized over 100 polyamide-fluorophore conjugates and assayed their nuclear uptake profiles in 13 mammalian cell lines. The compiled dataset, comprising 1300 entries, establishes a benchmark for the nuclear localization of polyamide-dye conjugates. Compounds in this series were chosen to provide systematic variation in several structural variables, including dye composition and placement, molecular weight, charge, ordering of the aromatic and aliphatic amino-acid building blocks and overall shape. Nuclear uptake does not appear to be correlated with polyamide molecular weight or with the number of imidazole residues, although the positions of imidazole residues affect nuclear access properties significantly. Generally negative determinants for nuclear access include the presence of a beta-Ala-tail residue and the lack of a cationic alkyl amine moiety, whereas the presence of an acetylated 2,4-diaminobutyric acid-turn is a positive factor for nuclear localization. We discuss implications of these data on the design of polyamide-dye conjugates for use in biological systems.     

Immobilized ligands of high stability: Hydroxyalkyl methacrylate gel conjugates with oligo- and polypeptides

The long-time stability of conjugates prepared from epoxy derivatives of hydroxyalkyl methacrylate gels and peptides or proteins has been investigated at various pH values. These conjugates were found to be extremely stable. The observed slow release of nitrogen is due mainly to a splitting of the peptide bond adjacent to the covalent bond anchoring the peptide to the matrix. This peptide bond is partially labilized by microenvironmental influences of the matrix. Since in all experiments only a fraction of the immobilized ligand became detached, it is suggested that there is a subpopulation of fixed ligands which are susceptible to a matrix-induced bond splitting.     

Acid wash in determining cellular uptake of Fab/cell-permeating peptide conjugates

Successful intracellular delivery of various bioactive molecules has been reported using cell-permeating peptides (CPPs) as delivery vectors. To determine the effects of CPPs on the cellular uptake of immunoglobulin Fab fragment, conjugates of a radio-iodinated Fab fragment with CPPs (CPP-(125)I-Fab) derived from HIV-1 TAT, HIV-1 REV, and Antennapedia (ANP) were prepared. These vectors are rich in basic amino acids, and their strong adsorption on cell surfaces often results in overestimation of internalized peptides. Cell wash with an acidic buffer (0.2M glycine-0.15M NaCl, pH 3.0) was thus employed in this study to remove cell-surface adsorbed CPP-(125)I-Fab conjugates. This procedure enabled clearer understanding of the methods of internalization of CPP-(125)I-Fab conjugates. The kinetics of internalization of REV-(125)I-Fab conjugate was rapid, and a considerable fraction of REV-(125)I-Fab was taken up by HeLa cells as early as 5 min after administration. It was also shown that cellular uptake of these conjugates was significantly inhibited in the presence of endocytosis/ macropinocytosis inhibitors, in the order REV-(125)I-Fab > or = TAT-(125)I-Fab > or = ANP-(125)I-Fab; this order was the same as for effectiveness of intracellular delivery. Simultaneous cell washing with phosphate-buffered saline (PBS) and this acidic buffer effectively separated the internalized conjugates from the cell-surface-adsorbed ones, and considerable differences were observed in these amounts dependent on the employed CPPs.     

Preparation and characterization of soluble conjugates of defined molecular sizes prepared by linkage of pepsin and carboxypeptidase A to dextrans

Soluble conjugates of pepsin and carboxypeptidase A were prepared by covalent linkage of the enzymes to an amino derivative of dextran. By fractionating the dextran derivatives before and after enzyme coupling, three conjugates, with median Stokes radii between 4.0 and 11.7 nm and with a range of 25% of the median, were prepared from each enzyme. The pepsin and carboxypeptidase A conjugates contained about 35% and 3% protein, respectively. Both types had specific activities close to those of the native enzymes and were stable at -20 degrees C. The pH-activity curve was unaffected by linkage of either enzyme to dextran. The stabilities at 30 degrees C of pepsin at pH 6-7 and carboxypeptidase A at pH 3.5-9.0 were increased by linkage to dextran. No significant amount of unbound enzyme was released from either type of conjugate in skim milk. The molecular sizes, deduced from the intrinsic viscosities and the diffusion coefficients of all conjugates, were close enough to the Stokes radii to indicate that the molecules were approximately spherical. Physical measurements also indicated that the molecules were dextranlike and highly hydrated.     

Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates

Antibody drug conjugates (ADCs) are highly cytotoxic drugs covalently attached via conditionally stable linkers to monoclonal antibodies (mAbs) and are among the most promising next-generation empowered biologics for cancer treatment. ADCs are more complex than naked mAbs, as the heterogeneity of the conjugates adds to the inherent microvariability of the biomolecules. The development and optimization of ADCs rely on improving their analytical and bioanalytical characterization by assessing several critical quality attributes, namely the distribution and position of the drug, the amount of naked antibody, the average drug to antibody ratio, and the residual drug-linker and related product proportions. Here brentuximab vedotin (Adcetris®) and trastuzumab emtansine (Kadcyla®), the first and gold-standard hinge-cysteine and lysine drug conjugates, respectively, were chosen to develop new mass spectrometry (MS) methods and to improve multiple-level structural assessment protocols.     

Synthesis and hydrolytic evaluation of acid-labile imine-linked cytotoxic isatin model systems

In this study a series of isatin-based, pH-sensitive aryl imine derivatives with differing aromatic substituents and substitution patterns were synthesised and their acid-catalysed hydrolysis evaluated. These derivatives were functionalised at the C3 carbonyl group of a potent N-substituted isatin cytotoxin and were stable at physiological pH but readily cleaved at pH 4.5. Observed rates of hydrolysis for the embedded imine-acid moiety were in the order para-phenylpropionic acid>phenylacetic acid (para>meta)>benzoic acid (meta>para). The ability to fine-tune hydrolysis rates in this way has potential implications for optimising imine linked, tumour targeting cytotoxin-protein conjugates.     

Sequence-specific conjugates of oligo(2'-O-methylribonucleotides) and hairpin oligocarboxamide minor-groove binders: design, synthesis, and binding studies with double-stranded DNA

New conjugates of triplex-forming pyrimidine oligo(2'-O-methylribonucleotides) with one or two 'head-to-head' hairpin oligo(N-methylpyrrole carboxamide) minor-groove binders (MGBs) attached to the terminal phosphate of the oligonucleotides with a oligo(ethylene glycol) linker were synthesized. It was demonstrated that, under appropriate conditions, the conjugates form stable complexes with double-stranded DNA (dsDNA) similarly to triplex-forming oligo(deoxyribonucleotide) (TFO) conjugates containing 5-methylated cytosines. Kinetic and thermodynamic parameters of the complex formation were evaluated by gel-shift assay and thermal denaturation. Higher melting temperatures (Tm), faster complex formation, and lower dissociation constants (Kd) of the triple helices (6-7 nM) were observed for complexes of MGB-oligo(2'-O-methylribonucleotide) conjugates with the target dsDNA compared to the nonconjugated individual components. Interaction of MGB moieties with the HIV proviral DNA fragment was indicated by UV/VIS absorption changes at 320 nm in the melting curves. The introduction of thymidine via a 3',3'-type 'inverted' phosphodiester linkage at the 3'-end of oligo(2'-O-methylribonucleotide) conjugates (3'-protection) had no strong influence on triplex formation, but slightly affected complex stability. At pH 6.0, when one or two hairpin MGBs were attached to the oligonucleotide, both triplex formation and minor-groove binding played important roles in complex formation. When two 'head-to-head' oligo(N-methylpyrrole) ligands were attached to the same terminal phosphate of the oligonucleotide or the linker, binding was observed at pH >7.5 and at high temperatures (up to 74 degrees). However, under these conditions, binding was retained only by the MGB part of the conjugate.     

Synthesis and properties of cell-targeted Zn(II)-phthalocyanine-peptide conjugates

Two Zn-Pc-peptide conjugates bearing either a short linker or a long PEG-linker between the macrocycle and a bifunctional peptide containing the nucleoplasmin and HIV-1 Tat 48-60 sequences have been synthesized in order to increase the Pc cell-targeting ability and to evaluate the effect of the linker. The presence of the peptide chain increased the water solubility of the Pc macrocycle and, consequently, its fluorescence in aqueous solutions. The highest fluorescence quantum yields were observed at low pH (5.0) for both conjugates and were always higher for the conjugate bearing the short linker. Both conjugates were found to have low dark cytotoxicity toward human HEp2 cells (IC50 > 77 microM) but were highly phototoxic (IC50 < 2 microM at 1 J cm-2). The conjugate bearing the long PEG-linker accumulated the most within cells (26 times more than the unconjugated Zn-Pc), followed by the short linker conjugate (17 times more than the unconjugated Zn-Pc). Both conjugates were found to localized preferentially within the cell lysosomes.     

Enzymatic and nonenzymatic synthesis of glutathione conjugates: application to the understanding of a parasite's defense system and alternative to the discovery of potent glutathione S-transferase inhibitors

A primary pathway for metabolism of electrophilic compounds in Schistosoma japonicum involves glutathione S-transferase (SjGST)-catalyzed formation of glutathione (GSH) conjugates. As part of a program aimed at gaining a better understanding of the defense system of parasites, a series of aromatic halides (1-8), aliphatic halides (9, 10), epoxides (11-20), alpha,beta-unsaturated esters (21, 22), and alpha,beta-unsaturated amides (23, 24) were prepared, and their participation in glutathione conjugate formation was evaluated. Products from enzymatic and nonenzymatic reactions of these substances with glutathione were characterized and quantified by using reverse-phase high-performance liquid chromatography (HPLC), NMR, and fast atom bombardment mass spectrometry (FAB-MS) analysis. Mechanisms for formation of specific mono(glutathionyl) or bis(glutathionyl) conjugates are proposed. Although the results of this effort indicate that SjGST does not catalyze addition or substitution reactions of 1, 3, 4, 7-9, 11-13, 15-17, 19-21, and 24, they demonstrate that 2, 5, 6, 14, 18, and 23 undergo efficient enzyme-catalyzed conjugation reactions. The kcat values for SjGST with 23 and 18 are about 886-fold and 14-fold, respectively, larger than that for 5. This observation suggests that 23 is a good substrate in comparison to other electrophiles. Furthermore, the initially formed conjugation product, 23a, is also a substrate for SjGST in a process that forms the bis(glutathionyl) conjugate 23b. Products arising by enzymatic and nonenzymatic pathways are generated under the conditions of SjGST-activated GSH conjugation. Interestingly, production of nonenzymatic GSH conjugates with electrophilic substrates often overwhelms the activity of the enzyme. The nonenzymatic GSH conjugates, 9a-11a, 16a, 21a, and 22a, are inhibitors of SjGST with respective IC50 values of 1.95, 75.5, 0.96, 19.0, 152, and 0.36 microM, and they display moderate inhibitory activities against human GSTA2. Direct evidence has been gained for substrate inhibition by 10 toward SjGST and GSTA2 that is more potent than that of its GSH conjugate 10a. The significance of this work is found in the development of a convenient NMR-based technique that can be used to characterize glutathione conjugates derived from small molecule libraries as part of efforts aimed at uncovering specific potent SjGST and GSTA2 inhibitors. This method has potential in applications to the identification of novel inhibitors of other GST targets that are of chemotherapeutic interest.