Tripartate poly(ethylene glycol) prodrugs of the open lactone form of camptothecin

Two PEG prodrugs utilizing conjugation of PEG through the C-21 acid functionality as well as the C-17 OH group of CPT hydroxy-amide open forms were synthesized and characterized. Both of these open lactone tripartate prodrugs were shown to be water soluble and highly effective in MX-1 mouse xenograph studies. Indirect evidence implies that the initial ester or carbonate bond breaking is esterase mediated in the first step of the cascade of CPT release.     

Maleimide-oligo(ethylene glycol) derivatives of camptothecin as albumin-binding prodrugs: synthesis and antitumor efficacy

In situ binding of thiol-reactive prodrugs to the cysteine-34 position of circulating albumin is a new approach in drug delivery. Therefore, five maleimide-bearing derivatives of the anticancer drug camptothecin (CPT) were developed as albumin-binding prodrugs. These compounds were synthesized by reacting heterobifunctional cross-linkers based on oligo(ethylene glycols) [3-6 (O-CH(2)-CH(2)) units] bearing a maleimide group on one end and a carboxylic acid group on the other with camptothecin 20-O-glycinate. Incorporating oligo(ethylene glycol) chains into the prodrugs enhanced their water-solubility when compared to the parent compound (up to 27-fold). HPLC studies showed that the prodrugs react almost quantitatively with the cysteine-34 position of endogenous albumin within a few minutes after incubation of the CPT derivatives with human blood plasma. The therapeutic potential of two of the prodrugs was assessed in nude mice bearing a colon xenograft (HT-29). Both albumin-binding derivatives of camptothecin were well-tolerated and showed enhanced antitumor efficacy when compared to CPT.     

Albumin-binding prodrugs of camptothecin and doxorubicin with an Ala-Leu-Ala-Leu-linker that are cleaved by cathepsin B: synthesis and antitumor efficacy

We have recently validated a macromolecular prodrug strategy for improved cancer chemotherapy based on two features: (a) rapid and selective binding of thiol-reactive prodrugs to the cysteine-34 position of endogenous albumin and (b) acid-sensitive promoted or enzymatic release of the drug at the tumor site [Kratz, F., Warnecke, A., Scheuemann, K., Stockmar, C., Schwab, J., Lazar, P., Druckes, P., Esser, N., Drevs, J., Rognan, D., Bissantz, C., Hinderling, C., Folkers, G., Fichtner, I., and Unger, C. (2002) J. Med. Chem. 45, 5523-33]. In the present work, we developed water-soluble camptothecin (CPT) and doxorubicin (DOXO) prodrugs that incorporate the peptide linker Ala-Leu-Ala-Leu that serves as a substrate for the tumor-associated protease, cathepsin B, which is overexpressed in several solid tumors. Consequently, two albumin-binding prodrugs were synthesized [EMC-Arg-Arg-Ala-Leu-Ala-Leu-Ala-CPT (1) and EMC-Arg-Arg-Ala-Leu-Ala-Leu-DOXO (2) (EMC = 6-maleimidocaproic acid)]. Both prodrugs exhibited excellent water-solubility and bound rapidly and selectively to the cysteine-34 position of endogenous albumin. Further in vitro studies showed that the albumin-bound form of the prodrugs was cleaved specifically by cathepsin B as well as in human tumor homogenates. Major cleavage products were CPT-peptide derivatives and CPT for the CPT prodrug and H-Leu-Ala-Leu-DOXO, H-Leu-DOXO, and DOXO for the doxorubicin prodrug. In vivo, 1 was superior to free camptothecin in an HT-29 human colon xenograft model; the antitumor efficacy of prodrug 2 was comparable to that of free doxorubicin in the M-3366 mamma carcinoma xenograft model at equimolar doses.     

Synthesis of linear, beta-cyclodextrin-based polymers and their camptothecin conjugates

6(A),6(D)-Bis-(2-amino-2-carboxylethylthio)-6(A),6(D)-dideoxy-beta-cyclodextrin 1, a diamino acid derivative of beta-cyclodextrin, is synthesized and condensed with difunctionalized PEG comonomers to give linear, high molecular weight (M(w) over 50 kDa) beta-cyclodextrin-based polymers (2-4) with pendant functionality (carboxylate). 2-4 are all highly soluble in aqueous solutions (over 200 mg/mL). 20-O-trifluoroglycinylcamptothecin, 5a, and 20-O-trifluoroglycinylglycinylglycinylcamptothecin, 5b, are synthesized and conjugated to 2 to give polymer-camptothecin (CPT) prodrugs. The solubility of CPT is increased by more than three orders of magnitude when it is conjugated to 2. The rates of CPT release from the conjugates HGGG6 (high molecular weight polymer (M(w) 97 kDa), glyglygly linker and 6 wt % CPT loading) and HG6 (high MW polymer (M(w) 97 kDa), gly linker and 6 wt % CPT loading) in either mouse or human plasma are dramatically accelerated over the rates of pure hydrolysis at pH = 7.4, indicating the presence of enzymatic cleavage as a rate-determining step at this pH in the release of the CPT. The pH of aqueous solution has a large effect on hydrolysis rate of CPT from HGGG6 and HG6; the lower the pH, the slower the rate in the range at 4.1 相似文献   

Inter-tissue and inter-species characteristics of the mitochondrial carnitine palmitoyltransferase enzyme system

Properties of the carnitine palmitoyltransferase (EC 2.3.1.21) (CPT) enzyme system were compared in isolated mitochondria from a range of tissues in rodents, monkey, and man. Common features were as follows: (a) while membrane-bound, CPT I, but not CPT II, was inhibited reversibly by malonyl-coenzyme A (CoA) and irreversibly by CoA esters of certain oxirane carboxylic acids; (b) the detergent, Tween-20, readily solubilized CPT II in active form while leaving CPT I membrane associated and catalytically functional; (c) octyl glucoside and Triton X-100 released active CPT II but caused essentially complete loss of CPT I activity. Use of [3H]tetradecylglycidyl-CoA, a covalent ligand for CPT I, yielded estimates of the enzyme's monomeric molecular size: approximately 86 kDa in non-hepatic tissues and approximately 90-94 kDa in liver, depending upon species. A polyclonal antibody to purified rat liver CPT II recognized a single protein in each tissue; its apparent molecular mass was approximately 70 kDa in all rat tissues and approximately 68 kDa in all mouse tissues as well as monkey and human liver. On Northern blot analysis a rat liver CPT II cDNA probe detected a single approximately 2.5-kilobase mRNA in all rat and mouse tissues examined. The following points are emphasized. First, CPT I and II are different proteins. Second, within a species CPT II, but not CPT I, is probably conserved across tissue lines. Third, slight variations in size of both enzymes were found in different species, although, at least in the case of CPT II, significant amino acid identity exists among the various isoforms. Fourth, CPT I, unlike CPT II, requires membrane integrity for catalytic function. Finally, the strategic use of detergents provides a simple means of discriminating between the two enzyme activities.     

Development of albumin-binding camptothecin prodrugs using a Peptide positional scanning library

Designing truly tumor-specific prodrugs remains a challenge in the field of cancer chemotherapy. As a new strategy, we incubated homogenates of a spectrum of human colon tumor xenografts with a fluorogenic positional scanning tetrapeptide library in order to identify peptide sequences that are preferentially cleaved by colon tumors. Our screening experiments at pH 7.4 revealed that Met, Leu, and Lys were preferred amino acids in the position P(1) and Tyr, Phe, and Met in P(2), whereas in P(3) and P(4), the cleavage profiles were less characteristic. However, similar results were obtained when testing breast tumor material and homogenates from healthy murine organs. On the basis of these results, we developed albumin-binding camptothecin (CPT) prodrugs of the general formula EMC-Arg-P(4)-P(3)-P(2)-P(1)-Ala-CPT (EMC = 6-maleimidocaproic acid) that incorporated two peptide linkers: H-Arg-Ala-Phe-Met-OH and H-Arg-Phe-Tyr-Met-OH (P(4)-P(3)-P(2)-P(1)). The incorporation of two arginine residues rendered the prodrugs water-soluble (>7 mg/mL), while the use of alanine as an amino acid spacer proved to be beneficial for the release of the active agent. Incubation studies with homogenates of HT-29 colon tumor tissue and murine spleen, liver, and kidneys demonstrated cleavage of the peptide linker with CPT-peptide derivatives and CPT being the major cleavage products. Although the peptide sequence is not selectively cleaved in colon tumors, an in vivo study in a HT-29 xenograft model showed that the prodrug EMC-Arg-Arg-Ala-Phe-Met-Ala-CPT demonstrated enhanced antitumor efficacy when compared to CPT [( T/ C max: 17% for the prodrug (2 x 12.5 mg/kg CPT equivalents) and 40% for CPT (3 x 12.5 mg/kg)].     

Structural model of the catalytic core of carnitine palmitoyltransferase I and carnitine octanoyltransferase (COT): mutation of CPT I histidine 473 and alanine 381 and COT alanine 238 impairs the catalytic activity.

Carnitine palmitoyltransferase I (CPT I) and carnitine octanoyltransferase (COT) catalyze the conversion of long- and medium-chain acyl-CoA to acylcarnitines in the presence of carnitine. We propose a common three-dimensional structural model for the catalytic domain of both, based on fold identification for 200 amino acids surrounding the active site through a threading approach. The model is based on the three-dimensional structure of the rat enoyl-CoA hydratase, established by x-ray diffraction analysis. The study shows that the structural model of 200 amino acids of the catalytic site is practically identical in CPT I and COT with identical distribution of 4 beta-sheets and 6 alpha-helices. Functional analysis of the model was done by site-directed mutagenesis. When the critical histidine residue 473 in CPT I (327 in COT), localized in the acyl-CoA pocket in the model, was mutated to alanine, the catalytic activity was abolished. Mutation of the conserved alanine residue to aspartic acid, A381D (in CPT I) and A238D (in COT), which are 92/89 amino acids far from the catalytic histidine, respectively (but very close to the acyl-CoA pocket in the structural model), decreased the activity by 86 and 80%, respectively. The K(m) for acyl-CoA increased 6-8-fold, whereas the K(m) for carnitine hardly changed. The inhibition of the mutant CPT I by malonyl-CoA was not altered. The structural model explains the loss of activity reported for the CPT I mutations R451A, W452A, D454G, W391A, del R395, P479L, and L484P, all of which occur in or near the modeled catalytic domain.     

Development and characterization of cholinephosphotransferase antibody

In the present study, we generated antibodies in rabbits against two synthetic peptides, one based on peptide sequence from yeast CPT cDNA (position 86 to 98 of the amino acid sequence) and the other from our guinea pig CPT cDNA (it corresponds to amino acid positions 119 to 130 according to yeast CPT gene). The antibody titers were measured by both dot blot analysis and ELISA using Keyhole limpets hemocyanin coupled CPT peptides. The CPT antibody recognized a single band by Western blot analysis of proteins from guinea pig liver mitochondria and microsomes. The molecular weight of the protein recognized by Western blot analysis is close to the predicted molecular weight (46 kDa) of yeast CPT. Further analysis revealed that the antibody inhibited CPT activity in both subcellular fractions in a dose dependent manner, thus confirming the specificity of the antibody against both subcellular CPT.     

Improving enzymes for cancer gene therapy

New techniques now make it feasible to tailor enzymes for cancer gene therapy. Novel enzymes with desired properties can be created and selected from vast libraries of mutants containing random substitutions within catalytic domains. In this review, we first consider genes for the ablation of tumors, namely, genes that have been mutated (or potentially can be mutated) to afford enhanced activation of prodrugs and increased sensitization of tumors to specific chemotherapeutic agents. We then consider genes that have been mutated to provide better protection of normal host tissues, such as bone marrow, against the toxicity of specific chemotherapeutic agents. Expression of the mutant enzyme could render sensitive tissues, such as bone marrow, more resistant to specific cytotoxic agents.     

聚乙二醇在化疗药物成药性研究中的应用——以阿霉素为例

多数抗肿瘤药物的水溶性差、系统毒性和多药耐药性已成为其临床应用所面临的主要问题,而利用聚乙二醇材料构建前药或合适的 递药系统来克服这些问题,备受广大药学研究者的关注。以具有良好抗肿瘤活性和分子荧光特性的阿霉素为例,综述聚乙二醇在化疗药物 前药和递药系统的构建及制备等成药性研究中的应用,为高效低毒抗肿瘤药品的进一步研究与开发提供参考。     

Functional and structural basis of carnitine palmitoyltransferase 1A deficiency

Carnitine palmitoyltransferase 1A (CPT1A) is the key regulatory enzyme of hepatic long-chain fatty acid beta-oxidation. Human CPT1A deficiency is characterized by recurrent attacks of hypoketotic hypoglycemia. We presently analyzed at both the functional and structural levels five missense mutations identified in three CPT1A-deficient patients, namely A275T, A414V, Y498C, G709E, and G710E. Heterologous expression in Saccharomyces cerevisiae permitted to validate them as disease-causing mutations. To gain further insights into their deleterious effects, we localized these mutated residues into a three-dimensional structure model of the human CPT1A created from the crystal structure of the mouse carnitine acetyltransferase. This study demonstrated for the first time that disease-causing CPT1A mutations can be divided into two categories depending on whether they affect directly (functional determinant) or indirectly the active site of the enzyme (structural determinant). Mutations A275T, A414V, and Y498C, which exhibit decreased catalytic efficiency, clearly belong to the second class. They are located more than 20 A away from the active site and mostly affect the stability of the protein itself and/or of the enzyme-substrate complex. By contrast, mutations G709E and G710E, which abolish CPT1A activity, belong to the first category. They affect Gly residues that are essential not only for the structure of the hydrophobic core in the catalytic site, but also for the chain-length specificity of CPT isoforms. This study provides novel insights into the functionality of CPT1A that may contribute to the design of drugs for the treatment of lipid disorders.     

Rat liver carnitine palmitoyltransferase 1 forms an oligomeric complex within the outer mitochondrial membrane

Carnitine palmitoyltransferase (CPT) 1A catalyzes the rate-limiting step in the transport of long chain acyl-CoAs from cytoplasm to the mitochondrial matrix by converting them to acylcarnitines. Located within the outer mitochondrial membrane, CPT1A activity is inhibited by malonyl-CoA, its allosteric inhibitor. In this study, we investigate for the first time the quaternary structure of rat CPT1A. Chemical cross-linking studies using intact mitochondria isolated from fed rat liver or from Saccharomyces cerevisiae expressing CPT1A show that CPT1A self-assembles into an oligomeric complex. Size exclusion chromatography experiments using solubilized mitochondrial extracts suggest that the fundamental unit of its quaternary structure is a trimer. When studied in blue native-PAGE, the CPT1A hexamer could be observed, however, suggesting that under these native conditions CPT1A trimers might be arranged as dimers. Moreover, the oligomeric state of CPT1A was found unchanged by starvation and by streptozotocin-induced diabetes, conditions characterized by changes in malonyl-CoA sensitivity of CPT1A. Finally, gel filtration analysis of several yeast-expressed chimeric CPTs demonstrates that the first 147 N-terminal residues of CPT1A, encompassing its two transmembrane segments, trigger trimerization independently of its catalytic C-terminal domain. Deletion of residues 1-82, including transmembrane 1, did not abrogate oligomerization, but the latter is limited to a trimer by the presence of the large catalytic C-terminal domain on the cytosolic face of mitochondria. Based on these findings, we proposed that the oligomeric structure of CPT1A would allow the newly formed acylcarnitines to gain direct access into the intermembrane space, hence facilitating substrate channeling.     

The utility of camptothecin as a synergist of Bacillus thuringiensis var. kurstaki and nucleopolyhedroviruses against Trichoplusia ni and Spodoptera exigua

We studied the effect of combining microbial pesticides with camptothecin (CPT) on the mortality of two lepidopteran insects: Trichoplusia ni (Hübner) and Spodoptera exigua (Hübner). CPT is an alkaloid that is often used as an anticancer agent. Here, CPT was evaluated as a microbial pesticide synergist of Bacillus thuringiensis (Bt) and insect baculovirus. The toxicity of CPT and its synergistic effects on two microbial pesticides were studied using the diet overlay method. Bioassay results showed that CPT significantly enhances the toxicity of Bt variety kurstaki to S. exigua and T ni. In addition, CPT strongly enhanced the infectivity of Autographa californica (Speyer) multinucleocapsid nucleopolyhedrovirus (AcMNPV) and S. exigua nucleopolyhedrovirus (SeMNPV). Using light microscopy, we found that CPT disrupts the peritrophic membrane of T. ni larvae and severely affects the structure of the midgut, resulting in an abnormal gut lumen morphology. We speculate that CPT increases toxicity by affecting the permeability of the peritrophic membrane.     

Different sites of inhibition of carnitine palmitoyltransferase by malonyl-CoA, and by acetyl-CoA and CoA, in human skeletal muscle.

The inhibition of carnitine palmitoyltransferase (CPT, EC 2.3.1.21) by malonyl-CoA, acetyl-CoA and free CoA was studied in sonicated skeletal-muscle homogenates from normal human subjects and from five patients with a mutant CPT [Zierz & Engel (1985) Eur. J. Biochem. 149, 207-214]. (1) Malonyl-CoA, acetyl-CoA and CoA were competitive inhibitors of CPT with palmitoyl-CoA. (2) Acetyl-CoA and CoA inhibited normal and mutant CPT to the same degree, whereas malonyl-CoA inhibited mutant CPT more than normal CPT. (3) Triton X-100 abolished the inhibition of normal CPT by malonyl-CoA, but not by acetyl-CoA or CoA. Triton X-100 by itself caused loss of activity of the mutant CPT. (4) In the concentration range 0.1-0.4 mM, the inhibitory effects of any two of the three inhibitors were synergistic. (5) The inhibitory constants (Ki) for acetyl-CoA and CoA were close to 45 microM. The Ki for malonyl-CoA was 200-fold lower, or 0.22 microM. Addition of 40 microM-acetyl-CoA or CoA resulted in a 3-fold increase in the Ki for acetyl-CoA. Addition of 20 microM-CoA resulted in a 3-fold increase in the Ki for acetyl-CoA. (6) The findings indicate that acetyl-CoA and CoA can inhibit CPT at the catalytic site or a nearby site which is different from that at which malonyl-CoA inhibits CPT. (7) The fact that small changes in the concentration of acetyl-CoA and CoA can antagonize the inhibitory effect of malonyl-CoA suggests that these compounds could modulate the inhibition of CPT by malonyl-CoA.     

Mutation of Gly721 alters DNA topoisomerase I active site architecture and sensitivity to camptothecin

DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA via a concerted mechanism of DNA strand cleavage and religation. Top1p is the cellular target of the anti-cancer drug camptothecin (CPT), which reversibly stabilizes a covalent enzyme-DNA intermediate. Top1p clamps around duplex DNA, wherein the core and C-terminal domains are connected by extended alpha-helices (linker domain), which position the active site Tyr of the C-terminal domain within the catalytic pocket. The physical connection of the linker with the Top1p clamp as well as linker flexibility affect enzyme sensitivity to CPT. Crystallographic data reveal that a conserved Gly residue (located at the juncture between the linker and C-terminal domains) is at one end of a short alpha-helix, which extends to the active site Tyr covalently linked to the DNA. In the presence of drug, the linker is rigid and this alpha-helix extends to include Gly and the preceding Leu. We report that mutation of this conserved Gly in yeast Top1p alters enzyme sensitivity to CPT. Mutating Gly to Asp, Glu, Asn, Gln, Leu, or Ala enhanced enzyme CPT sensitivity, with the acidic residues inducing the greatest increase in drug sensitivity in vivo and in vitro. By contrast, Val or Phe substituents rendered the enzyme CPT-resistant. Mutation-induced alterations in enzyme architecture preceding the active site Tyr suggest these structural transitions modulate enzyme sensitivity to CPT, while enhancing the rate of DNA cleavage. We postulate that this conserved Gly residue provides a flexible hinge within the Top1p catalytic pocket to facilitate linker dynamics and the structural alterations that accompany drug binding of the covalent enzyme-DNA intermediate.     

A humanized aldolase antibody for selective chemotherapy and adaptor immunotherapy

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.     

Protein arginine (N)-methyl transferase 7 (PRMT7) as a potential target for the sensitization of tumor cells to camptothecins

PRMT7 belongs to the protein arginine methyl-transferases family. We show that downregulation of PRMT7alpha and beta isoforms in DC-3F hamster cells was associated with increased sensitivity to the Top1 inhibitor camptothecin (CPT). This effect was not due to a change in Top1 contents or catalytic activity, or to a difference in the reversal of DNA breaks. Overexpression of PRMT7alpha and beta in DC-3F cells had no effect on CPT sensitivity, whereas it conferred a resistance to DC-3F/9-OH-E cells for which both isoforms are reduced by two- to three-fold as compared to DC-3F parental cells. Finally, downregulation of the human PRMT7 could also sensitize HeLa cells to CPT, suggesting that it could be used as a target to potentiate CPT derivatives.     

Synthesis, hydrolysis studies and phamacodynamic profiles of amide prodrugs of dexibuprofen with amino acids

The present investigation deals with the synthesis of novel prodrugs of dexibuprofen with amino acids with an aim to achieve potent anti-inflammatory activity and less gastrointestinal toxicity. Structures of synthesized compounds were confirmed by spectral and elemental analyses. In vitro hydrolytic studies in simulated intestinal fluid, 80% plasma and rat faecal matter showed satisfactory release of dexibuprofen due to enzymatic cleavage. The synthesized prodrugs were evaluated for anti-inflammatory activity, analgesia, ulcerogenicity and histopathology. The anti-inflammatory activity of dexibuprofen was 43.3% whereas an improved value of 73.4, 77.3, 72.8 and 64.5% was observed for the synthesized prodrugs. The percentage analgesia of the prodrugs increased, whereas a decrease in the mean ulcer index values than dexibuprofen was observed. The histopathological studies revealed less ulceration in the gastric region when treated with prodrugs. Thus, the prodrugs were proved to be better in action as compared with the parent drug.