Pharmacokinetic evaluation of gemcitabine and 2',2'-difluorodeoxycytidine-5'-triphosphate after prolonged infusion in patients affected by different solid tumors.

BACKGROUND: The study determined pharmacokinetic parameters, toxicity profile and preliminary clinical activity of gemcitabine administered i.v. at different infusion rates in patients with a range of solid tumors. PATIENTS AND METHODS: Twenty patients were enrolled for both pharmacokinetic and clinical studies. Gemcitabine 300 mg/m(2) was administered during 1 h, 2 h or 3 h, and as a conventional dose of 1000 mg/m(2) during 30 min infusion. Administration was on days 1, 8 and 15 every 4 weeks. RESULTS: Patients were randomly assigned to one of the four arms. After 30 min infusion of 1000 mg/m(2) gemcitabine the plasma concentration remained above the saturation level of 10-20 microM, whereas after 1, 2 or 3 h infusion 300 mg/m(2) gemcitabine it remained below the saturation level for most of the time (being in the range 2.5-10 microM). Gemcitabine triphosphate was determined in the four arms in white blood cells; for infusion times from 0.5 to 3 h there was a progressive enhancement of gemcitabine triphosphate levels. In all evaluable patients the toxicity was mild, myelosuppression being the main toxicity. No grade 3 or 4 toxicities occurred. Clinical response was similar in patients receiving 300 mg/m(2) gemcitabine in 2 and 3 h and in the 1000 mg/m(2) arm. CONCLUSIONS: 300 mg/m(2) gemcitabine during 3 h infusion produced the highest accumulation of gemcitabine triphosphate. Thus, to achieve the highest possible gemcitabine triphosphate level, prolonged infusion time would appear to be more important than a high dose administered as a short infusion. However, there was no substantial difference in toxicity or antitumoral activity in the all different patient groups.

A preliminary pharmacokinetic study of docetaxel, carboplatin and concurrent radiotherapy for regionally advanced squamous cell carcinoma of the head and neck.

This work investigates the pharmacokinetics and toxicity resulting from the concomitant use of low dose carboplatin (CBCA)/docetaxel (DTX) plus concurrent radiotherapy in patients with head and neck cancer. The study comprised 11 patients with stage III-IV head and neck cancer. All patients received 2 Gy radiotherapy daily, 5 fractions per week, up to a planned total of 70 Gy over 7 weeks. CBCA (AUC 0.4 mg/ml, min/day) was also administrated as 20 min i.v. infusion, starting 1 day before the first radiotherapy fraction. CBCA was administered for 5 consecutive days every 2 weeks (weeks 1, 3, 5 and 7). DTX 30 mg/m2 (1 h i.v. infusion) was given as a single dose on days 10, 24 and 38. CBCA on day 1 and DTX on day 10 were analysed to determine the concentration-time curves during the first 24 h. CBCA Cmax and Cmin in 2-5 days and on day 15 and 29, as well as total plasma platinum on days 2, 3, 4, 5, 29 and 43 were also assayed. By calculating the non-compartmental pharmacokinetic parameters of the two drugs from the available plasma concentrations we found in the first week values similar to those reported in the literature as single agents. In contrast, during subsequent weeks (weeks 3 and 5), a significant and progressive increase of platinum levels was observed. So, it could be assumed that after 2 weeks of CBCA and DTX treatment a bias in dose calculation occurred because the linear relationship between creatinine clearance (used to calculate the expected AUC through the Calvert formula) and CBCA clearance was no longer observed.

2,3,4,5-tetrahydroxy-5-(4-hydroxyphenyl)valeric acid: a new cleavable monofunctional reagent for monoclonal antibody labeling.

The synthesis and preliminary biological assays of a new monofunctional reagent to reversible derivatize monoclonal antibodies is described. This compound, comprising a 1,4-polyiol moiety, is cleavable by means of sodium periodate in mild conditions; moreover it also contains a phenolic residue suitable for 125I labelling and a carboxylic group for reaction with epsilon-lysyl amino group of antibodies. These features are suitable to study the monoclonal antibodies cell-internalization process and antigen expression on cell surface. The 125I labelled reagent has been coupled to the monoclonal antibody AR-3, an IgG1 directed toward the CAR-3 antigen widely expressed on human ovarian and colorectal adenocarcinomas. The cleavage capability is tested in different conditions both on reagent and on derivatized MAb. Cell labelling experiments are performed both on target and untarget cell lines.

Pharmacokinetics of an antibody-ricin conjugate administered intraperitoneally to mice.

Immunotoxins have been extensively studied for the treatment of neoplasias; their intracavitary administration could be useful for the therapy of tumors confined to the pleural or peritoneum spaces. To study the feasibility of this "locoregional" treatment, a pharmacokinetic study of immunotoxins delivery is necessary. Ricin, a plant toxin extracted from the seeds of Ricinus communis, has often been used in immunoconjugates for its high activity; nevertheless, appropriate strategies have been necessary to limit the aspecific toxicity. We previously prepared a AR-3-ricin immunotoxin lacking the ability to bind galactosidic cell surface residues, a so-called sterically blocked immunotoxin. The monoclonal antibody AR-3, an IgG1 specific to the CAR-3 antigen, was able to recognize human colorectal adenocarcinomas. Preclinical trials in nude mice, intraperitoneally grafted with the target neoplasia, showed that this immunotoxin suppressed tumor growth without showing any undesirable ricin toxicity. In the present work we report the pharmacokinetic properties of this immunotoxin, showing the in vivo stability and a relatively long blood survival. With a biodistribution study in tumor-bearing mice, we demonstrate that in tumor-invaded tissues, the concentration of the specific AR-3-ricin immunotoxin was higher and progressively increased in a multiple-dose regimen. In contrast, an irrelevant immunotoxin behaved differently because it did not show specific tumor uptake. Moreover the pharmacokinetic data reported in this work improve the potential for "locoregional" treatment of malignancy with blocked immunotoxins.

A new approach in the synthesis of immunotoxins: ribosome inactivating protein noncovalently bound to monoclonal antibody.

This study describes the synthesis of a new generation of immunotoxins made by a noncovalent interaction between a monoclonal antibody derivatized with a dichlorotriazinic dye and the ribosomal inhibitor protein gelonin. The scheme of preparation has several advantages with respect to the traditional methods, which used heterobifunctional cross-linkers, such as a higher overall yield of production and the homogeneity of the obtained conjugate. Moreover, because no chemical derivatization of the gelonin was required, the unconjugated ribosome inactivating protein was recovered unaltered and therefore can be reused in other synthetic processes. This immunoconjugate was stable when tested in mouse serum and showed an interesting slow elimination rate when administered intravenously in mice. Although a high dye derivatization degree induced a modification of the specificity of the monoclonal antibody, the native specificity was restored after conjugation with gelonin. Furthermore the noncovalent linkage did not affect the gelonin inhibitory activity; in fact, the specific cytotoxic activity seemed to be similar to that of other disulfide-linked immunotoxins previously prepared in our laboratories.

Toxin-targeted design for anticancer therapy. II: Preparation and biological comparison of different chemically linked gelonin-antibody conjugates.

To obtain more potent immunotoxins for anticancer therapy a gelonin-AR3 antibody immunoconjugate was prepared with different new linkers and coupling procedures. The gelonin was derivatized with the heterobifunctional thioimidate linkers ethyl-acetyl-3-mercaptopropionthioimidate (AMPT) and 3-(4-carboxamidophenyldithio)propionthioimidate (CDPT), and with the succinimidyl type reagents N-succinimidyl-3-(4-carboxamidophenyldithio)propionate (SCDP) and N-succinimidyl-S-acetyl thiolacetate (SATA). The biological activity of gelonin modified with different linkers (AMPT, CDPT, SCDP, SATA) was determined by a rabbit reticulocyte assay. We found that AMPT was the molecule of choice to derivatize the toxin, confirming the preferability of thioimidate linkers. The monoclonal antibody Mab was derivatized with CDPT and SCDP. Then the following immunoconjugates were prepared with different procedures: Mab-CDPT with gelonin-AMPT; Mab-CDPT with gelonin-CDPT; Mab-SCDP with gelonin-SATA. To verify whether selection of the most suitable coupling procedure could affect the antitumoral activity of the gelonin-AR3 immunoconjugate, the three immunotoxins were tested on target HT-29 human colon carcinoma cells versus nontarget MeWo cells. The gelonin immunoconjugate linked via the AMPT-CDPT thioimidate reagents showed highest antitumoral activity as well as best selectivity for the target cells.

Toxin-targeted design for anticancer therapy. I: Synthesis and biological evaluation of new thioimidate heterobifunctional reagents.

In an effort to obtain a more potent and specific immunotoxin for cancer therapy, we designed a series of heterobifunctional linkers characterized by a thioimidate group linked to a S-acetyl thiol (4, 5) or substituted aryldithio group (6-10). These ligands were synthesized by a Pinner-type process from the corresponding nitrile derivatives obtained by thiol-disulphide exchange reaction, reaction with substituted benzene-sulphenyl chloride, or other known procedures. To check the reagent of choice for immunoconjugate preparation, we studied thioldisulphide exchange kinetics between the intermediate nitrile derivatives and cysteine. Among the tested aryldithio derivatives (6-10), we selected ethyl 3-(4-carboxamido-phenyldithio)propionthioimidate (CDPT, 9) for further studies. By analyzing the rate of incorporation of the linkers 4, 5, and 9 in a model immunoglobulin G protein, we found similar results with CDPT 9 and ethyl S-acetyl 3-mercaptopropionthioimidate ester hydrochloride (AMPT, 5) because both reagents showed a linear correlation between the number of introduced thiol groups and factors such as time and protein and reagent concentrations. Comparison of the two acetylthio-derivative ligands 4 and 5 showed that AMPT 5 was more stable toward deacetylation than ethyl S-acetyl 2-mercaptopropionthioimidate ester hydrochloride (AMAT, 4). By comparing the kinetic and biological parameters of seven new thioimidate linkers, we found that two of these (CDPT and AMPT) could be superior ligands for protein-protein conjugation. They offer advantages over the commercially available compounds, such as minimal perturbation of the protein structure, controlled reactivity, and good stability.

A new 'solid phase' procedure to synthesize immunotoxins (antibody-ribosome inactivating protein conjugates).

A method to produce immunotoxins (conjugates comprising of a monoclonal antibody and toxin) using ribosome inactivating protein anchored on an affinity gel derivatized with triazinic dye is described. The adsorbed toxins were activated with 2-imino-thiolane and then conjugated to monoclonal antibody activated by SPDP. The "heterogeneous phase" system offered several advantages, reducing the usually required purification steps and opening a way to automatize the conjugation procedure.

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

We 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 evalauted 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 specifcity of the immunoconjugate, since the analogous treatment with an irrelevant immunotoxin showed therapeutic failure.

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

To avoid non-specific binding of intact ricin-antibody conjugates, we prepared a new blocked thioether-linked ricin-antibody IT, in which the galactose binding 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.

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 beta-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.

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.

Biosynthesis of Sterols and Triterpenoids in Tissue Cultures of Cucurbita maxima.

The biosynthesis of sterols and triterpenoids in CUCURBITA MAXIMA was studied by analysis of unsaponifiable fraction of tissues from different development stages of the plant (seeds, seedlings, adult plant and tissue culture) and by feeding germinating seeds and tissue cultures with [2- (14)C]-acetate. Synthesis of cucurbitacins does not occur in callus tissues of CUCURBITA MAXIMA, whereas a wide variety of 4,4-dimethylsterols present in these tissues testifies of a high level of squaleneoxide cyclase activity in growing callus. The peculiarity of Cucurbitaceae among the higher plants is also discussed comparing the side chain biosynthesis of sterols in CUCURBITA MAXIMA to that operating in other higher plants.

Side chain degradation and microbial reduction of different steroids by Aspergillus auroefulgens.

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.