Using the Braak staging for neurofibrillary changes as an objective indicator of the progression of Alzheimer's disease, we have performed a systematic search for global gene expression changes in the prefrontal cortex during the course of Alzheimer's disease. In the prefrontal cortex, senile plaques and neurofibrillary changes start to appear around Braak stage III, allowing for the detection of changes in gene expression before, during and after the onset of Alzheimer's disease neuropathology. Two distinct patterns of tightly co-regulated groups of genes were observed: (i) an increase in expression in early Braak stages, followed by a decline in expression in later stages (the UPDOWN clusters; containing 865 genes) and (ii) a decrease in expression in early Braak stages, followed by an increase in expression in later stages (the DOWNUP clusters; containing 983 genes). The most profound changes in gene expression were detected between Braak stages II and III, just before or at the onset of plaque pathology and neurofibrillary changes in the prefrontal cortex. We also observed an increase in intracellular beta amyloid staining from Braak stages I to III and a clear decrease in Braak stages IV to VI. These data suggest a link between specific gene expression clusters and Alzheimer's disease-associated neuropathology in the prefrontal cortex. Gene ontology over-representation and functional gene network analyses indicate an increase in synaptic activity and changes in plasticity during the very early pre-symptomatic stage of the disease. In later Braak stages, the decreased expression of these genes suggests a reduction in synaptic activity that coincides with the appearance of plaque pathology and neurofibrillary changes and the clinical diagnosis of mild cognitive impairment. The interaction of the ApoE genotype with the expression levels of the genes in the UPDOWN and DOWNUP clusters demonstrates that the accelerating role of ApoE-ε4 in the progression of Alzheimer's disease is reflected in the temporal changes in gene expression presented here. Since the UPDOWN cluster contains several genes involved in amyloid precursor protein processing and beta amyloid clearance that increase in expression in parallel with increased intracellular beta amyloid load, just before the onset of plaque pathology in the prefrontal cortex, we hypothesize that the temporally orchestrated increase in genes involved in synaptic activity represents a coping mechanism against increased soluble beta amyloid levels. As these gene expression changes occur before the appearance of Alzheimer's disease-associated neuropathology, they provide an excellent starting point for the identification of new targets for the development of therapeutic strategies aimed at the prevention of Alzheimer's disease.

Doxorubicin (DOX) is an anticancer agent with a wide spectrum of activity. Cumulative dose-related cardiotoxicity, however, is a major side-effect of DOX, in addition to the acute toxicities, such as myelosuppression, nausea and vomiting. The success of DOX, and its limitations in clinical use, have directed research endeavours for the development of analogues of DOX with an improved therapeutic index. Among these are iododoxorubicin, AD-32 and epidoxorubicin (Weiss, 1992). Iododoxorubicin was noted to be promising in phase I trials, but in phase II trials the response rate was too low to warrant further development. AD-32 has greater anti-tumour activity, and less cardiotoxicity, than DOX, but drug formulation and solubility problems prevented its further clinical development. Of the available analogues, only epidoxorubicin appears to have a reduced cardiotoxicity with retention of antitumour activity and is in use in current cancer chemotherapy.Another way to improve the selectivity and efficacy of chemotherapy is the use of non-toxic prodrugs that are preferentially converted into active anticancer agents at the tumour site (Sinhababu and Thakker, 1996). N-L-leucyl-DOX is a prodrug of DOX, to be activated by tumour peptidases (Deprez-de Campeneere et al, 1982). In human ovarian cancer xenografts, N-L-leucyl-DOX was more effective than DOX . Clinical studies on N-L-leucyl-DOX, however, have indicated premature activation of the prodrug in the circulation, because of which the selectivity of the prodrug would be reduced . Elevated enzyme levels in tumour tissue have been reported for β-glucuronidase (Connors and Whisson, 1966). Bosslet et al (1995) and Schumacher et al (1996) have shown that this enzyme is released in the extracellular space as a result of necrosis in tumours. The enzyme can only be detected in very low concentrations in the circulation (Fishman, 1970). Therefore, β-glucuronidase may be exploited for the specific activation of glucuronide prodrugs in tumour tissue.We have developed glucuronide derivatives of anthracyclines and showed that these prodrugs, such as epirubicin-glucuronide (Haisma et al, 1992) and daunorubicin-glucuronides (Leenders et al, 1995;Houba et al, 1996), are relatively non-toxic in vitro and can be activated by β-glucuronidase to yield the active anthracycline. Treatment with the glucuronide prodrug daunorubicin-GA3 (DNR-GA3) induced a better tumour growth delay than daunorubicin (DNR) when studied at equitoxic doses in 3 human ovarian cancer xenografts which were sensitive to DNR (Houba et al, 1998). Comparison of the distribution and pharmacokinetics of DNR and DNR-GA3 demonstrated that the prodrug DNR-GA3 was selectively activated by human β-glucuronidase present in the tumours and resulted in a higher DNR AUC in tumours and lower A novel doxorubicin-glucuronide prodrug DOX-GA3 for tumour-selective chemotherapy: distribution and efficacy in experimental human ovarian cancer Summary The doxorubicin (DOX) prodrug N-[4-doxorubicin-N-carbonyl (oxymethyl) phenyl] O-β-glucuro...

No abstract

A glucuronide doxorubicin prodrug N‐[4‐doxorubicin‐N‐carbonyl (oxymethyl) phenyl] O‐β‐glucuronyl carbamate (DOX‐GA3) has been developed to improve the antitumor effects of doxorubicin (DOX). The prodrug was originally designed to be activated into drug by human β‐glucuronidase (GUS) released from tumor cells in necrotic areas of tumor lesions. The aim of this study was to further improve the antitumor effects of DOX‐GA3 by means of antibody‐directed enzyme prodrug therapy (ADEPT). We thus investigated if the administration of an enzyme‐immunoconjugate prepared from the pancarcinoma Ep‐CAM specific monoclonal antibody (MAb) 323/A3 and β‐glucuronidase would result in improved antitumor effects because of additional enzyme localization in tumor tissue. In vitro, the prodrug DOX‐GA3 was found to be 12‐times less toxic than the parent drug DOX in a human ovarian cancer cell line. Immunospecific and complete activation of the prodrug took place when the cells were pretreated with 323/A3‐β‐glucuronidase conjugate. In nude mice bearing s.c. human ovarian cancer xenografts (FMa) the maximum tolerated dose (MTD) of DOX‐GA3 (500 mg/kg weekly × 2) was much higher when compared with that of DOX (8 mg/kg weekly × 2). In mice bearing well‐established FMa xenografts, the standard treatment of DOX at the MTD (8 mg/kg weekly × 2) resulted in a tumor growth inhibition of 67%. Treatment with DOX‐GA3 at a single dose of 500 mg/kg resulted in a better tumor growth inhibition of 87%. The combination of DOX‐GA3 (500 mg/kg) with 323/A3‐mGUS conjugate and anti‐GUS MAb 105, to clear circulating conjugate, improved the antitumor effect even further to 98%. At the lower dose of 250 mg/kg DOX‐GA3 tumor growth inhibition (34%) was not better than that of DOX. The combination, however, of DOX‐GA3 at 250 mg/kg and 323/A3‐mGUS conjugate plus MAb 105 again greatly improved the antitumor effect (growth inhibition of 93%). DOX given at 8 mg/kg weekly x 2 did not result in tumor regressions. As a result of ADEPT, the number of regressions of tumors improved from 0 out of 12 to 9 out of 11 at a dose of 250 mg/kg DOX‐GA3. At the higher prodrug dose (500 mg/kg) the number of regressions improved from 2 out of 12 to 9 out of 10 as a result from the addition of enzyme‐immunoconjugate. Our studies show that the efficacy of the widely used anti‐cancer agent DOX may be improved by using the prodrug DOX‐GA3, in combination with the tumor‐specific enzyme‐immunoconjugate 323/A3‐mGUS and a conjugate clearing antibody. © 2001 Wiley‐Liss, Inc.