Laura Delprino

In vitro inhibition of animal and higher plants 2,3-oxidosqualene-sterol cyclases by 2-aza-2,3- dihydrosqualene and derivatives, and by other ammonium-containing molecules

2-Aza-2,3-dihydrosqualene and related molecules, a series of new compounds designed as analogues of the transient carbocationic high energy intermediate, occurring in the oxirane ring opening during the cyclization of 2,3-oxidosqualene, were tested in vitro as inhibitors of the microsomal 2,3-oxidosqualene cyclase of animals (rat liver) and of higher plants (maize, pea). These molecules proved to be good and specific inhibitors for the cyclases of both phyla. The inhibition is due to positively charged species and is sensitive to the steric hindrance around the nitrogen-atom. 4,4,10 beta-Trimethyl-trans-decal-3 beta-ol and 4,10 beta-dimethyl-trans-decal-3 beta-ol, which have previously been described (J.A. Nelson et al., J. Am. chem. Soc. 100, 4900 (1978] as inhibitors of the 2,3-oxidosqualene cyclase of chinese hamster ovary cells, were found to be non-competitive inhibitors of the rat liver microsomal enzyme and presented no activity towards the higher plants cyclases. Aza derivatives of these decalines (A. Rahier et al., Phytochemistry, in press), which were aimed to mimic the C-8 carbocationic intermediate occurring during later steps of the 2,3-oxidosqualene cyclization did not inhibit the cyclases. This result underlines the theoretical limitations of the high energy analogues concept in designing enzyme inhibitors. Amongst other molecules tested, 2,3-epiminosqualene was found to be a reversible, non-competitive inhibitor of the cyclases; similarly U18666A was a very potent inhibitor of the microsomal cyclases. In contrast AMO 1618, a known anticholesterolemic agent reported previously to act at the level of the 2,3-oxidosqualene cyclization step, was not found per se to act on the cyclases.

The squalene-2,3-epoxide cyclase as a model for the development of new drugs

The 2,3-oxido squalene (SO) cyclases represent a group of enzymes which convert SO into polycyclic triterpenoids such as lanosterol, cycloartenol, cucurbitadienol and β-amyrin. Taking into account the postulated model of the enzymatic cyclization of SO, we have investigated the possibility of designing compounds that would be selective and potent inhibitors of SO cyclases. Due to the fundamental role of sterols in animal, higher plant and fungal tissues, these inhibitors might behave as very selective (ipocholesterolemic, antifungal or phytotoxic) drugs.Our first approach was the synthesis and biological evaluation of 2-aza-2,3-dihydrosqualene and its derivatives which, being protonated at physiological pH, would present some similarities to the C-2 carbon ion generated by the opening of the oxirane ring of SO. Microsomes from different sources (germinated pea cotyledons, maize seedlings, rat liver and yeasts) were utilized to determine the inhibition values (I50: concentration of inhibitor producing 50% inhibition at a given substrate concentration).From the results obtained so far we conclude that 2-aza-2-dihydrosqualene and its derivatives strongly inhibited the cyclases, the site of the enzyme responsible for binding to the inhibitor is quite sensitive to the steric hindrance, and the degree of the inhibitory activity is greater in higher plants than in rat liver or fungi.

The transformation of 10α-cucurbita-5,24-dien-3β-ol into cucurbitacin C by seedlings of Cucumis sativus

Abstract Labelled 10α-cucurbita-5,24-dien-3β-ol, the simplest tetracyclic triterpene with a cucurbitane skeleton, was transformed into cucurbitacin C in Cucumis sativus seedlings. This transformation has been previously postulated, but this is the first time it has been demonstrated to operate in plant tissues. Two other potential precursors of cucurbitacins, cycloartenol and parkeol, were incubated under the same conditions. Cycloartenol gave only the expected phytosterols whereas parkeol was recovered unchanged.

Comparison of blocked and non-blocked ricin-antibody immunotoxins against human gastric carcinoma and colorectal adenocarcinoma cell lines

SummaryTo avoid non-specific binding of intact ricin-antibody conjugates, we prepared a new blocked thioether-linkedricin-antibody IT, in which the galactosebinding site of ricin had lost the ability to bind to galactosidic residues of Sepharose 6B gel. As carrier agent, the monoclonal antibody AR-3, which defines the CAR-3 tumour-associated antigenic determinant expressed selectively on different human carcinoma cell lines, was used. Purification of the new conjugate was performed in three sequential steps: (1) by HPLC gel filtration on TSK G3000SW to remove the unconjugated ricin: (2) by affinity chromatography on Affi-Gel Blue to separate the free antibody from the conjugate and (3) by affinity chromatography on Sepharose 6B to separate the galactose-binding IT from the non-binding moiety. The cytotoxicity of the blocked and non-blocked thioether-linked IT was compared with that of classical ricin-antibody IT conjugated via SPDP and that of ricin A chain IT. The comparison was made on two different target cell lines (KATO III human gastric carcinoma and HT-29 human colorectal carcinoma) versus two control cell lines (HL-60 promyelocytic pre-leukaemic and COLO38 melanoma). The results showed that the blocked thioether IT displayed a more selective toxicity to target cells than the non-blocked IT and was much more potent than the ricin A chain conjugate.

Side chain degradation and microbial reduction of different steroids by Aspergillus aureogulgens

The Aspergillus aureofulgens ability to cleave the side chain of progesterone (I) and the related C-21 steroids was studied. The enzymic system responsible for the progesterone side chain degradation was demonstrated to be adaptative and to operate by a Baeyer-Villiger mechanism. The cleavage of the side chain of the progesterone and of the related compounds was followed by the stereospecific reduction of the formed androst-4-ene-3,17-dione(II) to the 5 beta-androstan derivatives. Both the oxygenase and reductase activities seemed to be influenced by the growth conditions. Several steroids bearing different skeleton functions and different side chains were also tested in order to correlate the chemical structure with the microbial activity.

Gemcitabine and oxaliplatin in patients with metastatic breast cancer resistant to or pretreated with both anthracyclines and taxanes: clinical and pharmacokinetic data.

Objectives:To evaluate the efficacy, the toxicity and the pharmacokinetics of a gemcitabine (GEM) and oxaliplatin (OXA) combination in metastatic breast cancer patients (MBC), previously treated with anthracycline and taxanes. Methods:A total of 40 women were enrolled; 37 patients had visceral metastases as dominant site of disease, including 20 patients with liver metastases and 14 with multiple visceral metastases. Three patients received neo-adjuvant chemotherapy, 13 patients adjuvant chemotherapy alone, 24 patients chemotherapy for MBC alone. Gemcitabine was given at 1000 mg/m2 on days 1, 8 followed by oxaliplatin at 100 mg/m2 iv on day 2 every 2 weeks (GEM-OXA sequence). Possible pharmacokinetic interactions were studied in 10 patients, by administering on cycle 1 gemcitabine d1/oxaliplatin d2 (GEM-OXA) and on cycle 2 oxaliplatin d1/gemcitabine d2 (OXA-GEM). Results:After a median of 8 cycles, 10 partial response (PR) (25%), 16 stable disease (SD) (40%), and 14 progressive disease (PD) (35%) were obtained. The median duration of response was 6 months (3–9) for responding patients and 4.9 months (2–7.5) for patients with stable disease. For PR, SD and PD patients, median survival was 18 (10–23+), 13 (8–18), and 6 months (4–13), respectively. G3-4 neutropenia occurred in 20% of patients (febrile G3 neutropenia in 2.5%), G3-4 thrombocytopenia and anemia in 15% and 7.5%. The most frequent G3-4 nonhematologic toxicity was represented by peripheral neuropathy (20%), always reversible. The GEM-OXA and OXA-GEM schedules showed a similar pharmacokinetic behavior, with no sequence-dependent interaction. Conclusions:The combination gemcitabine plus oxaliplatin has moderate activity in anthracycline and taxanes resistant/relapsed heavily treated patients, mild toxicity and no administration sequence-dependent pharmacokinetic interactions.

N-oxide as a potential function in the design of enzyme inhibitors. Application to 2,3-epoxysqualene-sterol cyclases

The N-oxides of several tertiary amines possessing an aliphatic long chain are more potent inhibitors of 2,3-epoxysqualene-β-amyrin and -lanosterol cyclases than their parent amines.

Cyclization of squalene-2,3-epoxide to 10α-cucurbita-5,24-dien-3β-ol by microsomes from Cucurbita maxima seedlings

Abstract Labelled 10α-cucurbita-5,24-dien-3β-ol was obtained from [3-3H]squalene-2,3-epoxide incubated with microsomes of Cucurbita maxima seedlings. By contrast the lanostane triterpenoids [2-3H]cycloartenol, [2-3H]parkeol and their corresponding derivatives [2,12-3H]11-ketocycloartenol and [2-3H]24,25-dihydro-90α,11a-epoxyparkeol, incubated under the same conditions, gave no rearranged products with a cucurbitane skeleton. These results suggest that biosynthesis of cucurbitane triterpenoids from squalene-2,3-epoxide occurs through direct cyclization without the intermediacy of lanostane-type triterpenoids such as cycloartenol or parkeol. Labelled cycloartenol, α-amyrin, β-amyrin and 24-methylenecycloartanol were also obtained from [3-3H]squalene-2,3-epoxide in the same enzymatic system yielding labelled 10α-cucurbitadienol.

Conversion of cyclolaudenol to 24α- and 24β-ethylsterols in the cucurbitaceae

Abstract[2-3H]Cyclolaudenol was converted into α-spinasterol, 24β-ethylcholesta-7,25-dien-3β-ol and 24β-ethylcholesta-7,22,25-trien-3β-ol by seedlings ofCucurbita maxima. As 24-methylenecycloartanol is the obligatory precursor of 24-ethylsterols, it can be assumed that the transformation of cyclolaudenol to 24-methylenecycloartanol must have occurred. These results lead us to postulate the existence, in the Cucurbitaceae family, of an enzymatic system capable of isomerizing Δ25 alkylsterols into Δ25(28) sterols.

Antitumour activity of a sterically blocked ricin immunotoxin on a human colorectal adenocarcinoma grafted subcutaneously in nude mice

SummaryWe prepared a ricin-antibody conjugate, lacking the ability to bind the galactosidic residues of Sepharose 6B, a so-called blocked immunotoxin. The monoclonal antibody AR-3 was cross-linked to ricin through a thioether bond. Further studies showed that the immunoconjugate suppressed the tumour growth of HT-29 cells in intraperitoneally grafted nude mice, without showing any undesirable ricin toxicity.In this work, to demonstrate the therapeutic activity of the AR-3—ricin conjugate injected into mice bearing subcutaneous tumour, we first evaluated its pharmacokinetic behaviour and biodistribution. The behaviour of the immunoconjugate injected intravenously was almost intermediate between that of the antibody and ricin. Moreover, when the immunotoxin was intravenously administered to nude mice bearing subcutaneous tumour, no therapeutic effects appeared, in accordance with the relatively low permeability of the immunotoxin from the blood to the skin. In contrast, peritumoral treatment produced a strong reduction of the neoplastic nodules without substantial regrowth of the malignant cells. This result was also achieved when the immunotoxin treatment was performed on a well-established tumour. This finding was strictly related to the specificity of the immunoconjugate, since the analogous treatment with an irrelevant immunotoxin showed therapeutic failure.