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.

Inhibition of sterol biosynthesis in Saccharomyces cerevisiae by N,N-diethylazasqualene and derivatives.

The ability of some azasqualene derivatives to inhibit yeast cell growth was compared with their inhibition activity on squalene-2,3-oxide cyclase (EC 5.4.99.7) both in living cells and in microsome preparations. Among the compounds tested, N,N-diethylazasqualene showed the best correlation between the activity on squalene-2,3-oxide cyclase and its inhibition of yeast growth. The N-oxide derivative, N,N-diethylazasqualene N-oxide, which was as active as the amine in microsomes, was much less active in living cells, probably because it could not easily penetrate the cell wall. Kinetic analysis of the inhibitory activity of compounds on squalene-2,3-oxide cyclase revealed a sharp difference between N,N-diethylazasqualene and its N-oxide; the former showed a non-competitive-type inhibition, whereas the latter behaved as a competitive inhibitor.

Differential inhibition of fungal oxidosqualene cyclase by 6E and 6Z isomers of 2,3-epoxy-10-aza-10,11-dihydrosqualene.

Inhibitory properties of 6E (compound 1) and 6Z (compound 2) isomers of 2,3-epoxy-10-aza-10,11-dihydrosqualene against oxidosqualene-lanosterol cyclase were assayed on microsomes and whole cells of Saccharomyces cerevisiae and Candida albicans. Only the 6E isomer (compound 1), bearing a correct substrate-like configuration, strongly inhibited the enzyme both in microsomes and cell cultures. The difference between compounds 1 and 2 (which had an unfavorable geometry) was especially evident when measuring [14C]acetate incorporation into non-saponifiable lipids extracted from treated cells. While isomer Z was totally ineffective at up to 30 microM, in cells treated with 5 microM isomer E, labelled oxidosqualene, the level of which was negligible in the control, rose to over 60% of the non-saponifiable lipids.

2,3-Epoxy-10-aza-10,11-dihydrosqualene, a high-energy intermediate analogue inhibitor of 2,3-oxidosqualene cyclase.

2,3-Epoxy-10-aza-10,11-dihydrosqualene, a high-energy intermediate analogue inhibitor of 2,3-oxidosqualene (SO) cyclase was obtained by total synthesis. This involved the preparation of three main building blocks: (1) C17 squalenoid N-methylamine, (2) 3-(diphenylphosphinoyl)propanal, and (3) 5,6-epoxy-6-methylheptan-2-one. The final stages of the reconstruction of the 6E double bond were obtained by a Wittig-Horner reaction which was modified for poorly reactive systems. This compound was designed to mimic the C-8 carbonium ion formed during SO cyclization. Its inhibitory activity on various SO cyclases was evaluated and compared with the 6 Z isomer which has an unfavorable geometry. Only isomer 6 E, the carbocation analogue, was active on SO cyclases from rat liver, pig liver, S. cerevisiae, and C. albicans microsomes, with an I50 varying from 3 to 5 microM. Both E and Z isomers were inactive on squalene epoxidase at the higher concentrations tested.

29-Methylidene-2,3-oxidosqualene derivatives as stereospecific mechanism-based inhibitors of liver and yeast oxidosqualene cyclase.

Two pairs of isomers (18Z)- (8), (18E)-29-methylidene-2,3-oxidohexanorsqualene (21), and (18Z)- (31), (18E)-29-methylidene-2,3-oxidosqualene (34), have been obtained in a fully stereospecific manner, as inhibitors of rat and yeast oxidosqualene cyclase. A new method for the synthesis of C22 squalene aldehyde 2,3-epoxide is reported, as well as that of other 19-modified 2,3-oxidosqualene analogues. We found that the activity is the opposite in the two series: the (E)-hexanormethylidene 21 and the (Z)-methylidene 31 are potent and irreversible inhibitors of oxidosqualene cyclase, while (Z)-hexanormethylidene 8 and (E)-methylidene 34 are almost completely inactive. Reduction of the 18,19-double bond, such as in 39, eliminates the activity, while removal of both of the 19-linked groups such as in heptanor derivative 40 greatly reduces inhibition of the enzyme. (E)-Hexanormethylidene 21 results the first irreversible inhibitor of the series toward the yeast enzyme.

Inhibition of cholesterol biosynthesis in 3T3 fibroblasts by 2-aza-2,3-dihydrosqualene, a rationally designed 2,3-oxidosqualene cyclase inhibitor.

The effect of 2-aza-2,3-dihydrosqualene, a new compound designed to inhibit the 2,3-oxidosqualene-lanosterol cyclase [A. Duriatti et al., Biochem. Pharmac. 34, 2765 (1985)] was studied as inhibitor of cholesterol biosynthesis in Swiss 3T3 fibroblasts. Treatment with the drug of cells which were grown for 2 days in a delipidated medium resulted in a marked decrease of [14C]acetate incorporation into the C27-sterol fraction. An IC50 = 0.3 microM was calculated when the cells were preincubated for a period of 4 hr with 2-aza-2,3-dihydrosqualene. This inhibition was correlated with an intracellular accumulation of 2,3-[14C]oxidosqualene and of 2,3:22,23-[14C]dioxidosqualene, indicating that the cyclase was indeed an intracellular target of the drug. A precursor-product relationship of the accumulated [14C]squalene oxide(s) and the [14C]sterols was demonstrated in chase experiments in the absence of drug. Sterols more polar than cholesterol were also detected in treated fibroblasts and in the cells which underwent chase experiments; they were mainly composed of 24,25-epoxycholesterol. The C27-[14C]sterols of [14C]acetate pulse labeled cells consisted in a mixture of desmosterol and cholesterol; treatment of the cells with 2-aza-2,3-dihydrosqualene resulted in a decreased conversion of desmosterol into cholesterol indicating that the delta 24-sterol reductase might be another target of the drug. 2-Aza-2,3-dihydrosqualene at 1 microM affected normal growth of 3T3 fibroblasts, this effect could be prevented by addition of exogeneous cholesterol (50 microM). Growth arrest of the treated cells was correlated with a decrease in cellular sterol content to less than 40% of controls. About 30% of the C27-sterol fraction, of the treated cells, was desmosterol. Our work demonstrates that 2-aza-2,3-dihydrosqualene is a valuable new inhibitor of cholesterol biosynthesis in mammalian cells.

In vitro metabolism of azasqualene derivatives and their effects on aminopyrine N-demethylase activity in rat liver microsomes.

The metabolism of squalene dimethylamine (I), a potent inhibitor of 2,3-oxidosqualene (SO) cyclase, and of sixteen other squalene derivatives was investigated in rat liver microsomes. N-oxidation was the only metabolic pathway observed, squalene dimethylamine N-oxide being the only metabolite isolated from incubation of I. The azasqualane and quaternary ammonium derivatives did not form N-oxides during their metabolism. The inhibition of aminopyrine N-demethylase activity was also studied and the IC50, for compound I, which shows weak competitive inhibition, was determined. At 1 mM concentration the other squalene derivatives showed a range of inhibition activity possibly due to their different lipophilicity.

Inhibition of 2,3-oxidosqualene cyclase and sterol biosynthesis by 10- and 19-azasqualene derivatives.

The inhibition of 2,3-oxidosqualene-lanosterol cyclase (EC 5.4.99.7) (OSC) by new azasqualene derivatives, mimicking the proC-8 and proC-20 carbocationic high-energy intermediates of the cyclization of 2,3-oxidosqualene to lanosterol, was studied using pig liver microsomes, partially purified preparations of OSC, and yeast microsomes. The azasqualene derivatives tested were: 6E- and 6Z-10aza-10,11-dihydrosqualene-2,3-epoxide 17 and 18, 19-aza-18,19,22,23-tetrahydrosqualene-2,3-epoxide 19 and its corresponding N-oxide 20, and 19-aza-18,19,22,23-tetrahydrosqualene 21. The compounds 17 and 19 (i.e. the derivatives bearing the 2,3-epoxide ring and the same geometrical configuration as the OSC substrate) were effective inhibitors, as shown by the Ki obtained using partially purified OSC: 2.67 microM and 2.14 microM, respectively. Compound 18, having an incorrect configuration and the 19-aza derivative 21, lacking the 2,3-epoxide ring, were poor inhibitors, with IC50 of 44 microM and 70 microM, respectively. Compound 21 was a competitive inhibitor of OSC, whereas 17 and 19 were noncompetitive inhibitors, and showed a biphasic time-dependent inactivation of OSC, their apparent binding constants being 250 microM and 213 microM, respectively. The inhibition of sterol biosynthesis was studied using human hepatoma HepG2 cells. The incorporation of [14C] acetate in the C27 sterols was reduced by 50% by 0.55 microM 17, 0.22 microM 19, and 0.45 microM 21, whereas 2 microM 18 did not affect sterol biosynthesis. In the presence of 17, 19 and 21, only the intermediate metabolites 2,3-oxidosqualene and 2,3,22,23-dioxidosqualene accumulated, demonstrating a very specific inhibition of OSC.

Enhancement of ricin toxin A chain immunotoxin activity: synthesis, ionophoretic ability, and in vitro activity of monensin derivatives.

Site-selective toxin delivery was achieved by coupling monoclonal antibody to the A chain subunit of ricin (RTA-IT). The cell-killing potency of RTA-IT can be drastically increased in vitro by using ionophores such as monensin. To reduce the intrinsic toxicity of monensin and to enhance its in vitro and in vivo activity, we synthesized 7 derivatives characterized by different lipophilicities. These derivatives were also analyzed for ionophoretic activity on intact cells, toxicity, and RTA-IT-enhancing activity. Two different RTA-IT were assayed on a human leukemia cell line. A correlation between lipophilicity, ionophoretic activity, and RTA-IT enhancement was observed. The compounds with the highest polar charge showed low intrinsic toxicity, revealed moderate ionophoretic activity, and were able to enhance RTA-IT only at high concentrations, whereas more lipophilic compounds (with a C28 tail or a phenyl group) showed significant ionophoretic activity and good enhancing properties.

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.

Poly(ethylene glycol)-human serum albumin-paclitaxel conjugates: preparation, characterization and pharmacokinetics.

Paclitaxel has been found to be very effective against several human cancers, such as ovarian, breast and non-small cell lung cancer and has received marketing approval for metastatic cancers. One of main problems with its use is its poor solubility, which makes irritant solubilitazion agents necessary. In previous research we demonstrated that linkage to human serum albumin (HSA) was useful to increase the in vivo performance of paclitaxel. In this article, in order to improve stability and solubility of paclitaxel conjugate, we linked covalently a monomethoxy poly(ethylene glycol) (mPEG) chain to HSA. New thioimidate mPEG derivatives, highly reactive and stable, were used and two different conjugates (with PEG of molecular mass 2 or 5 kDa) were prepared, purified and characterized. The antitumor activity of the free drug and conjugates was tested on three different tumor cell lines. The PEG grafted conjugates maintained high cytotoxicity, similar to that of ungrafted conjugates, with efficient cell binding and internalization followed by release of the drug inside the cell. The changes in pharmacokinetics and distribution of radio-labelled conjugates were evaluated by i.v. administration to mice and compared with those of the free drug and ungrafted conjugates. The total clearance was reduced (from 3.6 ml/h for free drug to 2.9, 1.97 and 1.41 for ungrafted, 2 and 5 kDa PEG conjugates, respectively). Organ uptake was reduced, in particular by liver and spleen.

Preparation, characterization and properties of sterically stabilized paclitaxel-containing liposomes.

Paclitaxel (Taxol) is a diterpenoid isolated from Taxus brevifolia, approved by the FDA for the treatment of ovarian and breast cancers. Due to its low solubility in water, it is clinically administered dissolved in Cremophor EL, (polyethoxylated castor oil) and ethanol, which cause serious side effects. Inclusion of paclitaxel in liposomal formulations has proved to be a good approach to eliminating this vehicle and improving the drug's antitumor efficacy. We prepared different conventional and PEGylated liposomes containing paclitaxel and determined encapsulation efficiency, physical stability and drug leakage in human plasma. The best conventional liposome formulation was composed of ePC/PG 9:1, while for PEGylated liposomes the best composition was ePC/PG/CHOL/PEG(5000)-DPPE 9:1:2:0.7. PEGylated liposomes were found to be less stable during storage than the corresponding conventional liposomes and to have lower drug release in human plasma at 37 degrees C. In vitro cytotoxic activities were evaluated on HT-29 human colon adenocarcinoma and MeWo melanoma cell lines. After 2 and 48 h, conventional liposomes had the same cytotoxicity as free paclitaxel, while PEGylated liposomes were as active as free drug, only after 48 h. Pharmacokinetics and biodistribution were evaluated in Balb/c mice after i.v. injection of paclitaxel, formulated in Cremophor EL or in conventional or in PEGylated liposomes. Encapsulation of paclitaxel in conventional liposomes produced marked differences over the free drug pharmacokinetics. PEGylated liposomes were long-circulating liposomes, with an increased t(1/2) beta 48.6 h, against t(1/2) beta 9.27 h of conventional liposomes. Biodistribution studies showed a considerable decrease in drug uptake in MPS-containing organs (liver and spleen) at 0.5 and 3 h after injection with PEGylated compared to conventional liposomes.

Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing water-soluble prodrugs of paclitaxel.

Paclitaxel (Taxol) is a diterpenoid isolated from Taxus brevifolia, used clinically for the treatment of ovarian and breast cancer. Due to its aqueous insolubility it is administered dissolved in ethanol and Cremophor EL (polyethoxylated castor oil), which has serious side effects. In order to eliminate this vehicle, in previous work we entrapped paclitaxel in conventional and in polyethylene glycol coated liposomes. However, in neither formulation did we obtain satisfactory entrapment efficiency. In this study we increased the paclitaxel concentration entrapped in liposomes by incorporating different water-soluble prodrugs, such as the 2'-succinyl, 2'-methylpyridinium acetate and 2'-mPEG ester paclitaxel derivatives, in the lipid vesicles. Liposomes containing 2'-mPEG (5000)-paclitaxel showed the best performance in terms of stability, entrapment efficiency and drug concentration (6.5 mgml(-1)). The in vitro cytotoxic activity of this liposomal prodrug was similar to that of the parent drug. The pharmacokinetic parameters for the free and for the liposomal prodrugs fitted a bi-exponential plasma disposition. The most important change in pharmacokinetic values of the prodrug vs. the free drug liposomal formulations was t(1/2)beta, plasma lifetime, which was longer in liposomes containing 2'-mPEG (5000)-paclitaxel.

Drug design based on biosynthetic studies: synthesis, biological activity, and kinetics of new inhibitors of 2,3-oxidosqualene cyclase and squalene epoxidase.

Various classes of inhibitor of 2,3-oxido squalene cyclase have been synthesized and tested on rat liver and Saccharomyces cerevisiae microsomes, 3T3 fibroblast cultures, and various bacteria, fungi, and yeasts. The compounds include azasqualenes, azasqualanes, bis-azasqualenes, bis-azasqualanes, and N-oxide and ammonium derivatives of squalene. In order to better mimic the transition state involved in the SN2-like opening of 2,3-oxidosqualene, we synthesized squalene N-methyloxaziridine. Other derivatives tested were N-methylimine, aminalic hydroperoxide, and N-methylamide. We also attempted to produce new "suicide" inhibitors of SO cyclase, such as a squalenoid epoxide vinyl ether. Many of the products described inhibited the various cyclases, the best having an IC50 of 0.3 microM on plants and 1.5 microM on rat liver microsomes, and good antibacterial and antifungal activity. In a search for inhibitors of squalene epoxidase, a series of mono- and bifunctional squalenoid acetylenes and allenes were synthesized. Some of them proved to be inhibitors of squalene epoxidase.

Melphalan monitoring during hyperthermic perfusion of isolated limb for melanoma: pharmacokinetic study and 99mTc-albumin microcolloid technique.

BACKGROUND: The kinetics of melphalan leakage from extracorporeal fluid to the peripheral blood was studied in ten patients undergoing hyperthermic isolation perfusion of the lower limbs as an adjuvant treatment in high-risk melanoma. MATERIALS AND METHODS: Systemic leakage was monitored by a new technique using 99mTc-albumin microcolloid. Serial samples were drawn from a peripheral vein and from the perfusion circuit during surgical treatment and analysed by HPLC. RESULTS: The leakage measured with 99mTc-albumin microcolloid ranged from 1.5 to 18%/h (mean 8%/h). The average concentrations in the perfusate were 200-300-fold those found in the systemic circulation. A good correlation (R=0.945) was obtained between systemic AUC (0 to 1 hour) and leakage measured through the 99mTc procedure. Negligible toxicity was found and the survival rate yielded 92% of objective response. CONCLUSION: By studying the pharmacokinetic data of melphalan in the circuit and in the systemic circulation, we were able to validate the 99mTc procedure used during clinical perfusion. Moreover, considering the efficiency of the system as well as the minimum toxicity and the high survival rate, a reduction of perfusion time may be considered.

Preparation and biological characterization of conjugates consisting of ricin and a tumor-specific non-internalizing MAb.

MOv18, a non-internalizing monoclonal antibody (MAb) with restricted tumor specificity, was conjugated to ricin toxin (RT). According to their ability to bind to galactose residues of Sepharose 6B, the immunoconjugates were fractionated into Bound and Unbound MOv18-RT. The two conjugates could be distinguished by SDS-PAGE, in vivo toxicity and agglutination capability. When the binding activity of both fractions was compared by solid-phase RIA to that of native MAb, it proved to be similar on the relevant target cells but significantly increased on the non relevant cells. On the latter, galactose totally cancelled the binding of the Unbound immunoconjugate, whereas it could only partially reverse that of the Bound MOv18-RT. By in vitro cytotoxic activity, either in the presence or absence of galactose, only a slight selectivity for relevant versus non-relevant target cells was observed for both conjugates. It seems that in the presence of a MAb which is incapable of internalization, the conjugate cytotoxicity could only be attributed to RT, with a loss of the MAb's specificity.

Antitumoral activity of liposomes and immunoliposomes containing 5-fluorouridine prodrugs.

Liposomes and immunoliposomes containing cytotoxic agents may be highly efficacious in intracavity therapy of malignancies confined principally to the peritoneal cavity. To assess the feasibility of this locoregional treatment, we prepared two derivatives of 5-fluorouridine (5-FUR), a highly cytotoxic metabolite of 5-fluorouracile, and incorporated them into REV liposomes, prepared with the reverse phase evaporation method. Encapsulation efficiency, drug leakage, and stability were determined, and size analysis and differential scanning calorimetry were carried out to evaluate the drug delivery potential of liposomes containing 5'-palmitoyl-5-FUR, 5'-succinyl-5-FUR, or the parent drug 5-FUR. The most suitable drug for encapsulation, in terms of minimum leakage and encapsulation efficiency, was 5'-palmitoyl-5-FUR, which differential scanning calorimetry indicated as being firmly anchored to the lipid bilayer. Thus, 5'-palmitoyl-5-FUR was chosen to prepare a chemotherapeutic liposome-monoclonal antibody conjugate (immunoliposome). The covalent linkage between antibody and liposome was realized by coupling the thiolated monoclonal antibody AR-3 with REV liposomes, containing N-[4-(p-maleimidophenyl)butyryl]phosphatidylethanolamine. The cytotoxic activity of drug-bearing liposomes and immunoliposomes was evaluated on the HT-29 human colon adenocarcinoma cell line; the immunoliposomes had higher cytotoxicity than liposomes or 5-FUR. To explore the potential of these drug formulations in anticancer therapy, we ip injected liposomes or immunoliposomes into athymic mice ip grafted with human HT-29 cell line. In this mouse model, the immunoliposome containing 5'-palmitoyl-5-FUR displayed the best antitumoral activity, since on day 27 postgraft only 5% of residual tumor mass was present, compared to control mice; there was a close relationship between exposure time of tumor tissue to the drug and antitumor potency.

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. 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.