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ABSTRACT: The voltage-dependent M-type potassium current (M-current) plays a major role in controlling brain excitability by stabilizing the membrane potential and acting as a brake for neuronal firing. The KCNQ2/Q3 heteromeric channel complex was identified as the molecular correlate of the M-current. Furthermore, the KCNQ2 and KCNQ3 channel alpha subunits are mutated in families with benign familial neonatal convulsions, a neonatal form of epilepsy. Enhancement of KCNQ2/Q3 potassium currents may provide an important target for antiepileptic drug development. Here, we show that meclofenamic acid (meclofenamate) and diclofenac, two related molecules previously used as anti-inflammatory drugs, act as novel KCNQ2/Q3 channel openers. Extracellular application of meclofenamate (EC(50) = 25 microM) and diclofenac (EC(50) = 2.6 microM) resulted in the activation of KCNQ2/Q3 K(+) currents, heterologously expressed in Chinese hamster ovary cells. Both openers activated KCNQ2/Q3 channels by causing a hyperpolarizing shift of the voltage activation curve (-23 and -15 mV, respectively) and by markedly slowing the deactivation kinetics. The effects of the drugs were stronger on KCNQ2 than on KCNQ3 channel alpha subunits. In contrast, they did not enhance KCNQ1 K(+) currents. Both openers increased KCNQ2/Q3 current amplitude at physiologically relevant potentials and led to hyperpolarization of the resting membrane potential. In cultured cortical neurons, meclofenamate and diclofenac enhanced the M-current and reduced evoked and spontaneous action potentials, whereas in vivo diclofenac exhibited an anticonvulsant activity (ED(50) = 43 mg/kg). These compounds potentially constitute novel drug templates for the treatment of neuronal hyperexcitability including epilepsy, migraine, or neuropathic pain.
Molecular Pharmacology 05/2005; 67(4):1053-66. · 4.88 Impact Factor
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Angewandte Chemie International Edition 02/2005; 44(5):716-20. · 13.45 Impact Factor
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ABSTRACT: Dendrimers are characterized by special features that make them promising candidates for many applications. Here we focus on two such applications: dendrimers as light harvesting antennae, and dendrimers as molecular amplifiers, which may serve as novel platforms for drug delivery. Both applications stem from the unique structure of dendrimers. We present a theoretical framework based on the master equation within which we describe these applications. The quantities of interest are the first passage time (FPT) probability density function (PDF), and its moments. We examine how the FPT PDF and its characteristics depend on the geometric and energetic structures of the dendrimeric system. In particular, we investigate the dependence of the FPT properties on the number of generations (dendrimer size), and the system bias. We present analytical expressions for the FPT PDF for very efficient dendrimeric antennae and for dendrimeric amplifiers. For these cases the mean first passage time scales linearly with the system length, and fluctuations around the mean first passage time are negligible for large systems. Relationships of the FPT to light harvesting process for other types of system-bias are discussed. Comment: ~7 journal pages, 10 figrues, J. Lumi., in press
10/2004;
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ABSTRACT: New self-immolative dendritic molecules have been designed and synthesized. The dendrons are built with a multi-enzymatic triggering mechanism, which initiates their biodegradation through a self-immolative chain fragmentation to release a reporter group from the focal point. The dendritic backbone is constructed from polycarbamate linkages, which are stable to hydrolysis and enhance the dendrons' solubility in water. The degradation can readily take place under physiological conditions on enzymatic triggering.
Chemical Communications 08/2004; · 6.17 Impact Factor
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ABSTRACT: "Chemical adaptor systems" are molecules used to link different functionalities, based on unique reactivity that allows controlled fragmentation. Two different mechanistic reactivities were used to prepare chemical adaptor systems. The first is based on a spontaneous intra-cyclization reaction to form a stable ring molecule. Cleavage of the trigger generates a free nucleophile, for example, an amine group, which undergoes intra-cyclization to release the target molecule from the handle part (e.g., a targeting antibody or a solid support for synthesis). The second applied reactivity is an elimination reaction, which is usually based on a quinone-methide-type rearrangement. Similarly, cleavage of the trigger generates a free phenol functionality, which can undergo a self-elimination reaction through a quinone-methide rearrangement to release the target molecule. The adaptor molecules have been applied in the field of drug delivery to release a drug from a targeting device and in the field of solid-phase synthesis to release a synthetic molecule from the solid support. A chemical adaptor molecule has also been used as a building unit to construct dendrimers with a triggered fragmentation.
Chemistry 07/2004; 10(11):2626-34. · 5.93 Impact Factor
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ABSTRACT: A new controlled drug delivery system for selective chemotherapy was developed. It is based on a chemical adaptor unit, that releases a drug by a spontaneous cyclization mechanism after cleavage of an enzymatic substrate. It also provides a generic linkage of a drug with a targeting device in a manner set to be triggered by defined enzymatic activity. The system is generic and allows using a variety of drugs, targeting devices, and enzymes by introducing the corresponding substrate as a trigger for drug release in the chemical adaptor.
Bioorganic & Medicinal Chemistry 05/2004; 12(8):1853-8. · 2.92 Impact Factor
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ABSTRACT: Two new prodrugs of CPT were synthesized, based on carbamate linkages between the 20-hydroxy group of CPT and a linker designed to be enzymatically removed by either Penicillin-G-Amidase or catalytic antibody 38C2. Cell growth inhibition assays showed an up-to-2250-fold difference in toxicity between the prodrugs and the active drug. A significant increase in toxicity was observed upon incubation of the enzyme or the catalytic antibody with the corresponding prodrug. The described derivatives of CPT further our knowledge in the design of prodrugs for use in selective approaches for targeted chemotherapy.
Bioorganic & Medicinal Chemistry 05/2004; 12(8):1859-66. · 2.92 Impact Factor
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ABSTRACT: Self-immolative dendrimers have recently been developed and introduced as a potential platform for a multi-prodrug. These unique structural dendrimers can release all of their tail units, through a self-immolative chain fragmentation, which is initiated by a single cleavage at the dendrimer's core. Incorporation of drug molecules as the tail units and an enzyme substrate as the trigger can generate a multi-prodrug unit that will be activated with a single enzymatic cleavage. We have synthesized the first generation of dendritic prodrugs with doxorubicin and camptothecin as tail units and a retro-aldol retro-Michael focal trigger, which can be cleaved by catalytic antibody 38C2. The bioactivation of the dendritic prodrugs was evaluated in cell-growth inhibition assay with the Molt-3 leukemia cell line in the presence and the absence of antibody 38C2. The dendritic unit was applied as a platform for a heterodimeric prodrug, which achieved a remarkable increase in toxicity with its bioactivation.
Journal of the American Chemical Society 03/2004; 126(6):1726-31. · 9.91 Impact Factor
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Angewandte Chemie International Edition 10/2003; 42(37):4494-9. · 13.45 Impact Factor
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ABSTRACT: Mouse monoclonal antibody 38C2 is the prototype of a new class of catalytic antibodies that were generated by reactive immunization. Through a reactive lysine, 38C2 catalyzes aldol and retro-aldol reactions using the enamine mechanism of natural aldolases. In addition to its remarkable versatility and efficacy in synthetic organic chemistry, 38C2 has been used for the selective activation of prodrugs in vitro and in vivo and thereby emerged as a promising tool for selective chemotherapy. Adding another application with relevance for cancer therapy, designated adaptor immunotherapy, we have recently shown that 38C2 can be chemically programmed to target tumors by formation of a covalent bond of defined stoichiometry with a beta-diketone derivative of an integrin alpha(v)beta(3) targeting RGD peptidomimetic. However, a major limitation for the transition from preclinical to clinical evaluation is the human anti-mouse antibody immune response that mouse 38C2 is likely to elicit in a majority of patients after single administration. Here, we report the humanization of mouse 38C2 based on rational design guided by molecular modeling. In essence, the catalytic center of mouse 38C2, which encompasses a deep hydrophobic pocket with a reactive lysine residue at the bottom, was grafted into a human antibody framework. Humanized 38C2 IgG1 was found to bind to beta-diketone haptens with conserved affinities and revealed strong catalytic activity with identical k(cat) and slightly higher K(M) values compared to the parental mouse antibody. Furthermore, humanized 38C2 IgG1 revealed efficiency in prodrug activation and chemical programming comparable to the parental mouse antibody.
Journal of Molecular Biology 10/2003; 332(4):889-99. · 4.00 Impact Factor
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Ulrike Schroeder,
Kathrin M Bernt,
Björn Lange,
Jens Wenkel,
Jiang Jikai, Doron Shabat,
Roey Amir,
Nicole Huebener,
Andreas G Niethammer,
Christian Hagemeier,
Lüder Wiebusch,
Gerhard Gaedicke,
Wolfgang Wrasidlo,
Ralph A Reisfeld,
Holger N Lode
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ABSTRACT: Effective therapy of high-risk leukemia with established cytotoxic drugs may be limited by poor antitumor efficacy, systemic toxicity, and the induction of drug resistance. Here, we provide the first evidence that hydrolytically activated prodrugs may overcome these problems. For this purpose, VP16 was functionally blocked by hydrolytically cleavable carbonate linkers with unique characteristics to generate 2 novel prodrugs of VP16. First, we established a more than 3-log higher efficacy of the 2 prodrugs compared with VP16 on a panel of naturally drug-resistant tumor cell lines. Second, the prodrugs did overcome VP16-induced multidrug resistance-1 gene (MDR-1)-mediated multidrug resistance in vitro in a newly established VP16-resistant T-cell leukemia cell line MOVP-3 by functionally blocking MDR-1-mediated efflux. Third, in vivo studies showed a maximum tolerated dose of ProVP16-II (> 45mg/kg), which was at least 3-fold higher than that of VP16 (15 mg/kg). Finally, tests of ProVP16-II in a multidrug-resistant xenograft model of T-cell leukemia expressing MDR-1 indicated that only the mice treated with this prodrug revealed a complete and long-lasting regression of established, drug-resistant leukemia. In summary, the hydrolytically activated etoposide prodrugs proved effective against multidrug-resistant T-cell leukemia in vitro and in vivo and provide proof of concept for a highly promising new strategy for the treatment of MDR-1 drug-resistant malignancies.
Blood 07/2003; 102(1):246-53. · 9.90 Impact Factor
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Angewandte Chemie International Edition 02/2003; 42(3):327-32. · 13.45 Impact Factor
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ABSTRACT: Selective chemotherapy remains a key issue for successful treatment in cancer therapy. The use of targeting approaches like the enhanced permeability and retention (EPR) effect of macromolecules, is consequently needed. Here, we report the preparation of a novel catalytic antibody-polymer conjugate for selective prodrug activation. HPMA copolymer was conjugated to catalytic antibody 38C2 through an amide bond formation between epsilon-amino group of lysine residue from the antibody molecule and a p-nitrophenyl ester of the polymer. The conjugate was purified over a size exclusion column using FPLC. In the isolated fraction, one or two molecules of polymer were conjugated to one molecule of antibody based on gel analysis. The resulting conjugate retained most of its catalytic activity (75-81%) in comparison to the free antibody. The activity was monitored with a fluorogenic substrate and a prodrug activation assay using HPLC. Furthermore, the conjugate was evaluated in vitro for its ability to activate an etoposide prodrug using two different cancer cell lines. Cells growth inhibition using the prodrug and the conjugate was almost identical to inhibition by the free antibody and the prodrug. For the first time, a catalytic antibody was conjugated to a passive targeting moiety while retaining its catalytic ability to activate a prodrug. The conjugate described in this work can be used for selective activation of prodrug in the PDEPT (polymer directed enzyme prodrug therapy) approach by replacing the enzyme component with catalytic antibody 38C2.
Bioorganic & Medicinal Chemistry 10/2002; 10(9):3023-9. · 2.92 Impact Factor
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ABSTRACT: Two 4'-propylcarbonoxy derivatives (2,3) of etoposide (1), a topoisomerase II inhibitor, were synthesized and evaluated as potential prodrugs for anticancer therapy. Their activation via hydrolysis mechanisms was determined as a function of pH in buffer solutions, in human serum and in the presence of carboxyl ester hydrolase. Cytotoxicity was determined on various tumor cell lines and compared to the parent compound. On cell lines exhibiting resistance to etoposide we observed an enhanced cytotoxicity of the prodrugs of up to three orders of magnitude.
Bioorganic & Medicinal Chemistry Letters 03/2002; 12(4):557-60. · 2.55 Impact Factor
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ABSTRACT: Aldolase antibody 38C2-catalyzed resolutions of tertiary aldols were studied. Tertiary aldols proved to be very good substrates for antibody catalyzed retro-aldol reactions. The catalytic proficiency, (kcat/KM)/kuncat, of the antibody for these reactions was on the order of 1010 M-1. A fluorogenic tertiary aldol allowed for the quantitative study of enantiomeric excess as a function of reaction conversion, revealing an E value of ca. 160 in this case. Study of a variety of substrates demonstrated that antibody-catalyzed retro-aldolization provides rapid entry to highly enantiomerically enriched tertiary aldols, typically >95% ee, containing structurally varied, heteroatom-substituted quaternary carbon centers. The utility of this approach to natural product syntheses has been demonstrated with the syntheses of (+)-frontalin, the side chain of Saframycin H, and formal syntheses of (+)- and (−)-mevalonolactone.
07/1999;
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ABSTRACT: Hohe Enantiomerenanreicherung nach 50 % Umsatz: Racemate von Aldolen konnten durch die Titelreaktion [Gl. (1)] mit den Aldolase-Antikörpern 38C2 und 33F12 gespalten werden; die ee-Werte der nicht umgesetzten Aldole betrugen in den meisten Fällen >95 %. Weil die Antikörper auch die Aldolreaktion, also die Rückreaktion, katalysieren, ist es möglich, beide Enantiomere mit einem einzigen katalytischen Antikörper zu synthetisieren.
Angewandte Chemie 03/1999; 110(18):2609 - 2612.
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ABSTRACT: High enantiomeric enrichment after 50% conversion: Racemates of aldols can be resolved by the title reaction [Eq.(1)] by use of the aldolase antibody 38C2 or 33F12; the ee values of the unconverted aldols are greater than 95% in most cases. Since the antibodies also catalyze the aldol reaction–that is, the reverse reaction–it is possible to prepare both enantiomers using the same antibody catalysts.
Angewandte Chemie International Edition 12/1998; 37(18):2481 - 2484. · 13.45 Impact Factor
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ABSTRACT: This paper describes the substrate specificity, synthetic scope, and efficiency of aldolase catalytic antibodies 38C2 and 33F12. These antibodies use the enamine mechanism common to the natural Class I aldolase enzymes. Substrates for these catalysts, 23 donors and 16 acceptors, have been identified. The aldol acceptor specificity is expected to be much broader than that defined here since all aldehydes tested, with the exception polyhydroxylated aldehydes, were substrates for the antibodies. 38C2 and 33F12 have been shown to catalyze intermolecular ketone−ketone, ketone−aldehyde, aldehyde−ketone, and aldehyde−aldehyde aldol addition reactions and in some cases to catalyze their subsequent dehydration to yield aldol condensation products. Substrates for intramolecular aldol reactions have also been defined. With acetone as the aldol donor substrate a new stereogenic center is formed by attack on the si-face of the aldehyde with ee's in most cases exceeding 95%. With hydroxyacetone as the donor substrate, attack occurs on the re-face, generating an α,β-dihydroxy ketone with two stereogenic centers of the α-syn configuration in 70 to >98% ee. With fluoroacetone donor reactions, the major product is a syn α-fluoro-β-hydroxy ketone with 95% ee. Studies of retroaldol reactions demonstrate that the antibodies provide up to 108-fold enhanced efficiency relative to simple amine-catalyzed reactions.
03/1998;
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ABSTRACT: Two 4 0 -propylcarbonoxy derivatives (2,3) of etoposide (1), a topoisomerase II inhibitor, were synthesized and evaluated as potential prodrugs for anticancer therapy. Their activation via hydrolysis mechanisms was determined as a function of pH in buffer solutions, in human serum and in the presence of carboxyl ester hydrolase. Cytotoxicity was determined on various tumor cell lines and compared to the parent compound. On cell lines exhibiting resistance to etoposide we observed an enhanced cyto-toxicity of the prodrugs of up to three orders of magnitude. # 2002 Elsevier Science Ltd. All rights reserved. Etoposide (1) is a widely used, highly effective anti-cancer drug against a broad spectrum of tumors including pediatric cancers such as acute lymphatic lymphomas, rhabdomyosarcomas and neuroblastomas, as well as in most common adult cancers. 1,2 It is also used in bone marrow transplantation conditioning regi-mens. However, the therapeutic use of etoposide is lim-ited by toxicity involving mainly myelosuppression. 3 A major problem for the use of this drug (and other topoisomerase inhibitors) is the development of multi-drug resistance. 4,5 Efforts are presently made to synthe-size new derivatives of the natural podophyllotoxin to overcome some of these shortcomings. 6À8 An alternative approach to new analogues of therapeutic agents is the synthesis of prodrugs. In previous work 9À12 we synthe-sized prodrugs of various antitumor agents to improve their bio-availability, phamacokinetics and aqueous solubility. For paclitaxel 11,12 we established that pro-drugs incorporating hydrolytically cleavable moieties are effective in lowering the systemic toxicity of the drug in animal models and most importantly also in patients. 13 Furthermore these prodrugs of paclitaxel revealed a dramatically improved pharmacokinetics and could be formulated for intravenous delivery with a minimum of toxic organic vehicles. Based on these findings the hydrolytic activation approach was applied to etoposide, an important drug in pediatrics and adult oncology.
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ABSTRACT: Single-triggered disassemble dendrimers were recently developed and introduced as a potential platform for a multi-prodrug. These unique structural dendrimers can release all of their tail units through a self-immolative chain fragmentation initiated by a single cleavage at the dendrimer's core. There are several examples for the bioactivation of first-generation self-immolative dendritic prodrugs. However, enzymatic activation failed for second-generation self-immolative dendrimers. The hydrophobic large molecular structure of the dendritic prodrugs results in aggregation under aqueous conditions and prevented the enzyme from reaching the triggering substrate. Here we show a simple solution for the enzymatic activation of second-generation self-immolative dendrimers. Poly(ethylene glycol) (PEG) was conjugated to the dendritic platform via click chemistry. The poly(ethylene glycol) tails significantly decreased the hydrophobic properties of the dendrimers and thereby prevented aggregate formation. We designed and synthesized a dendritic prodrug with four molecules of the anticancer agent camptothecin and a trigger that can be activated by penicillin-G-amidase. The PEG5000-conjugated, self-immolative dendritic prodrug was effectively activated by penicillin-G-amidase under physiological conditions and free camptothecin was released to the reaction media. Cell-growth inhibition assays demonstrated increased toxicity of the dendritic prodrug upon incubation with the enzyme.
Bioconjugate Chemistry 17(6):1432-40. · 4.93 Impact Factor
