Polymer-bound camptothecin: initial biodistribution and antitumour activity studies.

Camptothecin (CPT) is a potent, antitumour drug acting mainly through inhibition of topoisomerase I during the S-phase of the cell cycle. Despite its impressive antitumour activity, clinical development was halted for unpredictable toxic events. Two soluble N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers were synthesised to contain CPT (5 wt.% and 10 wt.%). CPT was covalently linked at its alpha-hydroxyl group to the polymers through a Gly-Phe-Leu-Gly- spacer. In-vitro, CPT-conjugates were fairly resistant to hydrolysis in plasma as in buffer at neutral pH (0.2-0. 4% free CPT/h), while elastase and cysteine-proteases were able to release the active drug. Plasma levels in mice after intravenous administration of CPT-conjugates confirmed the modest hydrolysis in plasma. Plasma levels were approximately 5-fold lower than those observed at the highest tolerated dose of CPT administered in classical vehicles. Biodistribution in HT29 human colon carcinoma bearing mice was carried out after i.v. injection of [3H]CPT-conjugate and free [3H]CPT. Radioactivity uptake in tumour was evident only after [3H]CPT-conjugate treatment. Repeated intravenous administration of CPT-conjugates to HT29-bearing mice gave more than 90% tumour inhibition, some complete tumour regressions and no toxic deaths. The improved pharmacological profile on HT29 human colon carcinoma xenografts of the first poly(HPMA)-CPT conjugates might be ascribed to their prolonged intra-tumour retention and sustained release of the active drug.

Mechanism, structure-activity studies, and potential applications of glutathione S-transferase-catalyzed cleavage of sulfonamides.

The mechanism of sulfonamide cleavage of PNU-109112, a potent HIV-1 protease inhibitor, by glutathione-S-transferase (GST) was investigated in the presence of reduced GSH. GST-catalyzed sulfonamide cleavage takes place via the nucleophilic attack of GSH on the pyridine moiety of the substrate with formation of the GS-para-CN-pyridinyl conjugate, the corresponding amine, and sulfur dioxide. Structure activity studies with a variety of sulfonamides indicate that an electrophilic center alpha to the sulfonyl group is required for cleavage. Substituents that withdraw electron density from the carbon atom alpha- to the sulfonyl group facilitate nucleophilic attack by the GS(-) thiolate bound to GST. The rate of sulfonamide cleavage is markedly affected by the nature of the electrophilic group; replacement of para-CN by para-CF(3) on the pyridine ring of PNU-109112 confers stability against sulfonamide cleavage. On the other hand, stability of sulfonamides is less dependent on the nature of the amine moiety. These principles can be applied to the synthesis of sulfonamides, labile toward cellular GST, that may serve as prodrugs for release of bioactive amines. Tumors are particularly attractive targets for these sulfonamide prodrugs as GST expression is significantly up-regulated in many cancer cells. Another potential application could be in organic synthesis, where protection of amines as the corresponding activated sulfonamides can be reversed by GST/GSH under mild conditions.

Ring-B modified anthracyclines.

One of the most investigated classes of antitumor drugs is represented by anthracyclines. Over thirty years since the original discovery of daunorubicin and doxorubicin thousands of anthracycline analogues have been synthesized and tested to identify compounds superior to the parent drugs in terms of increased therapeutic effectiveness, reduced toxicity or both. Previous structure-activity studies had shown that minor modifications of the anthracycline structure can result not only in active agents, but, more importantly, analogues with reduced cardiotoxicity and activity on multi drug resistance. The fact that 4-demethoxydaunorubicin showed higher potency than daunorubicin and a reduced cardiotoxicity, prompted us to explore novel analogues with altered substitution patterns on the anthraquinone system, particularly ring-B. In this review we will describe total synthesis and antitumor activity of three classes of derivatives: whereby one hydroxyl group in ring-B was either removed or replaced with nitro or amino groups. While these modifications yielded anthracyclines with a promising pharmacological activity, they did not modify activity on multidrug resistant (MDR) tumors. On the other hand, introduction of morpholino group in the sugar part of these new molecules, dramatically increased activity on MDR tumors. We conclude that activity on MDR tumors is not bound to modifications in the aglycone moiety of anthracyclines.

Circular dichroism studies on anthracycline antitumor compounds. Relationship between the molecular structure and the spectroscopic data.

The band assignment of the circular dichroism (CD) spectra of anthracyclines can provide us with the tools to study the interaction of these molecules with biomolecules, such as DNA and membranes, and also with metal ions. This paper reports the CD spectra of 17 anthracycline derivatives and the tentative assignment of the bands to specific electronic transitions. The deprotonation of some anthracyclines, such as doxorubicin, daunorubicin, and idarubicin, have been also studied in order to characterize the electronic transitions involved in the acid-base process. Our evidence suggests the following assignment. The position of the band assigned to pi-->pi transition, polarized along the short axis of the molecule ( approximately 290 nm), does not depend on the hydroxyl group at C(11) (presence and/or ionization state), whereas the position of the band assigned to the pi-->pi transition ( approximately 480 nm), polarized along the long axis, is strongly dependent on it. Concerning the n-->pi transitions, the bands at approximately 320 and approximately 350 nm have a strong contribution of the n-->pi C(12)=O transition and the n-->pi C(5)=O transition, respectively.

Effectiveness of anthracycline against experimental prion disease in Syrian hamsters.

Prion diseases are transmissible neurodegenerative conditions characterized by the accumulation of protease-resistant forms of the prion protein (PrP), termed PrPres, in the brain. Insoluble PrPres tends to aggregate into amyloid fibrils. The anthracycline 4'-iodo-4'-deoxy-doxorubicin (IDX) binds to amyloid fibrils and induces amyloid resorption in patients with systemic amyloidosis. To test IDX in an experimental model of prion disease, Syrian hamsters were inoculated intracerebrally either with scrapie-infected brain homogenate or with infected homogenate coincubated with IDX. In IDX-treated hamsters, clinical signs of disease were delayed and survival time was prolonged. Neuropathological examination showed a parallel delay in the appearance of brain changes and in the accumulation of PrPres and PrP amyloid.

Morpholinylanthracyclines: cytotoxicity and antitumor activity of differently modified derivatives.

The relationship between different chemical modifications on morpholinylanthracyclines and their ability to overcome multidrug resistance (MDR) has been evaluated testing all compounds in vitro on LoVo and LoVo/DX human colon adenocarcinoma cells and in vivo disseminated P388 and P388/DX murine leukemias. Results obtained led us to the following conclusions: 1) the insertion of the morpholinyl or the methoxymorpholinyl group on position 3' of the sugar moiety confers the ability to overcome MDR in vitro and in vivo; conversely, 4' morpholinyl compounds are effective on MDR cells only in vitro and result inactive in vivo on DX-resistant leukemia; 2) all chemical modifications performed on 3' morpholinyl or methoxymorpholinyl derivatives, that is substitutions on the aglycone or on position 2 of the morpholino ring, do not interfere with the activity of the compounds: all derivatives present in fact the same efficacy on sensitive and resistant models. It is concluded that position 3' in the sugar moiety plays a crucial role in the ability of morpholinyl-anthracyclines to overcome MDR.

Influence of structural modifications at the 3' and 4' positions of doxorubicin on the drug ability to trap topoisomerase II and to overcome multidrug resistance.

To better define the role of the amino sugar in the pharmacological and biochemical properties of anthracyclines related to doxorubicin and daunorubicin, we have investigated the effects of various substituents at the 3'- and 4'-positions of the drug on cytotoxic activity and ability to stimulate DNA cleavage mediated by DNA topoisomerase II. The study shows that the nature of the substituent at the 3'-position but not the 4'-position is critical for drug ability to form cleavable complexes. The amino group at the 3'-position is not essential for cytotoxic and topoisomerase II-targeting activities, because it can be replaced by a hydroxyl group without reduction of activity. However, the presence of bulky substituents at this position (i.e., morpholinyl derivatives) totally inhibited the effects on the enzyme, thus supporting previous observations indicating that the cytotoxic potencies of these particular derivatives are not related to topoisomerase II inhibition. This conclusion is also supported by the observation that 3'-morpholinyl and 3'-methoxymorpholinyl derivatives are able to overcome atypical (i.e., topoisomerase II-mediated) multidrug resistance. Because a bulky substituent at the 4'-position did not reduce the ability to stimulate DNA cleavage, these results support a critical role of the 3'-position in the drug interaction with topoisomerase II in the ternary complex. An analysis of patterns of cross-resistance to the studied derivatives in resistant human tumor cell lines expressing different resistance mechanisms indicated that chemical modifications at the 3'-position of the sugar may have a relevant influence on the ability of the drugs to overcome specific mechanisms of resistance.

Growth-inhibitory properties of novel anthracyclines in human leukemic cell lines expressing either Pgp-MDR or at-MDR.

The objective of the experiments reported in this paper was the identification of promising anthracycline analogs on the basis of lack of cross-resistance against tumor cells presenting either P-glycoprotein multidrug resistance (Pgp-MDR) or the altered topoisomerase multidrug resistant (at-MDR) phenotype. Differently modified anthracycline analogs known to be active against MDR cells were assayed in vitro against CEM human leukemic cells, and the sublines CEM/VLB100 and CEM/VM-1 exhibiting respectively the Pgp-MDR and the at-MDR phenotype. Two classes of molecules, in which the -NH2 group in C-3' position is substituted with a morpholino, methoxymorpholino (morpholinyl-anthracycline), or an alkylating moiety, present equivalent efficacy in the drug-sensitive and the two drug-resistant sublines. These results indicate that such molecules may exert their cytotoxic effect through a mode of action different from that of "classical" anthracyclines and is not mediated through topoisomerase II inhibition. Both molecules represent novel concepts in the field of new anthracyclines derivatives.

In vivo anti-tumour activity of FCE 23762, a methoxymorpholinyl derivative of doxorubicin active on doxorubicin-resistant tumour cells.

FCE 23762 is a new doxorubicin derivative obtained by appending a methoxymorpholinyl group at position 3' of the sugar moiety. The compound is greater than 80 times more potent than doxorubicin, it is highly lipophilic, and presents equivalent anti-tumour activity when administered by i.p., i.v. or oral route. The pattern of anti-tumour activity of FCE 23762 differs from that of doxorubicin in maintaining anti-tumour activity against two P388 murine leukaemia sublines resistant to doxorubicin and, although at borderline levels of efficacy, against LoVo human colon adenocarcinoma resistant to doxorubicin. FCE 23762 exhibits remarkable efficacy against MX-1 human mammary carcinoma, with most treated mice being cured both after i.v. and oral treatment. Anti-tumour activity was also observed against L1210 murine leukaemia and two sublines resistant to cis-platinum and melphalan, M5076 murine reticulosarcoma, MTV murine mammary carcinoma and N592 human small cell lung cancer.

Doxorubicin-3'-NH-oestrone-17-oxime-ethyl-carbonyl, a doxorubicin-oestrone conjugate that does not redox cycle in rat liver microsomes.

In summary doxorubicin-3'-NH-oestrone-17-oximethyl-carbonyl (Dox-Oes) is a covalent adduct of the anthracycline antitumor agent doxorubicin and oestrogen. Dox-Oes does not generate free radicals in rat liver microsomes as detected by electron spin resonance spectroscopy or redox cycle as shown by lack of superoxide anion formation and NADPH oxidation. Furthermore Dox-Oes actually inhibits free radical formation by doxorubicin used in equimolar amounts. The lack of free radical formation by doxorubicin when covalently linked to oestrone supports the development of Dox-Oes as a non-cardiotoxic derivative whilst potentially improving its targeting to oestrogen positive breast tumour cells.

Comparison of intracellular drug retention, DNA damage and cytotoxicity of derivatives of doxorubicin and daunorubicin in a human colon adenocarcinoma cell line (LoVo).

Formation of DNA single strand breaks (SSB) was assayed by alkaline elution in LoVo cells treated with doxorubicin, daunorubicin and six derivatives of these drugs modified either in the chromophore or the sugar. Seven compounds showed a biphasic relationship (initial increase and then a decrease) for the formation of DNA-SSB over the concentration range 0.05-10 micrograms/ml. At a drug concentration in the range causing an increase of DNA damage very fast repair of DNA-SSB was observed for 4'-deoxydoxorubicin and 4-demethoxydaunorubicin; the kinetics of DNA-SSB investigated after drug removal at a drug concentration reducing DNA-SSB showed a time dependent increase of DNA damage for both drugs although with different patterns. 4'-Deoxydoxorubicin reduced the effect of radiations on the rate of elution of DNA in a way resembling the formation of DNA interstrand cross links (ISC) at concentrations at which DNA-SSB were reduced. DNA-ISC were not produced by chemical reactions occurring during sample processing for alkaline elution and this derivative was not metabolized by LoVo cells. The IC50 of the anthracyclines were on a several log range, though for most of the derivatives the cytotoxicity curve showed a plateau at growth inhibition of about 15-30% at increasing intracellular drug levels. A relationship between DNA damage and cytotoxicity was observed only in a very small range of DNA-SSB. It is likely that the different effects of these anthracyclines on the formation of DNA-SSB depend on a qualitatively different interaction between drug-DNA and topoisomerase II when the drug concentration is raised.

Chemical and biological characterization of 4'-iodo-4'-deoxydoxorubicin.

4'-Iodo-4'-deoxydoxorubicin is a doxorubicin (DXR) analogue with greater lipophilicity and reduced basicity of the amino group. In vitro 4'-iodo-4'-deoxydoxorubicin is more cytotoxic than DXR against a panel of human and murine cell lines and is characterized by a higher and faster uptake. In vivo, the spectrum of activity of 4'-iodo-4'-deoxydoxorubicin is comparable to that of DXR, but the new compound has higher activity against murine P388 leukemia resistant to DXR and against pulmonary metastases from Lewis lung carcinoma. Moreover, the new analogue exhibits antitumor activity also after p.o. administration and shows no cardiotoxicity in experimental systems.

4-Demethoxy-4-hydroxydaunorubicin 6-methyl ether and 11-methyl ether, two active isomers of daunorubicin.

The preparation of two isomers of daunorubicin, one having a completely chelated dihydroxyanthraquinone chromophor (III) and one have having such a chromophor in a emichelated form (IV), is reported. The former is as active as daunorubicin, the latter shows a considerable activity, however at much higher doses.

4-Demethoxy-4-alkoxydaunorubicins.

The synthesis and the antitumor activities of a group of 4-demethoxy-4-alkoxydaunorubicins (VIII) are reported. The new compounds were found to have an activity equal or superior to that of daunorubicin, at somewhat higher doses.

Doxorubicin 14-ethers.

The synthesis and the antitumor activities of a group of doxorubicin (adriamycin) 14-ethers are reported. The new compounds were found to have an activity similar to that of daunorubicin, at somewhat higher doses, but inferior to that of doxorubicin.

Preparation and biological evaluation of 4-O-demethyldaunorubicin (carminomycin I) and of its 13-dihydro derivative.

A mutant strain of Streptomyces peucetius produced an anthracycline antibiotic whose structure has been established to be 4-O-demethyl-13-dihydrodaunorubicin (4), by application of spectroscopic methods and chemical degradation. A new synthesis of 4-O-demethyl-daunorubicin (carminomycin I, 2) starting from daunomycinone, together with the comparison of the antitumor activity of the anthracycline glycosides 2 and 4 are also reported.