Liposomes as a potential ocular delivery system of distamycin A.

Liposomes containing Distamycin A (DA) may be clinically useful in the treatment of ocular HSV infections, especially in acyclovir-resistant HSV keratitis. This study evaluated the in vitro and in vivo performance of a topical controlled release liposomal formulation containing DA (DA-Lipo) aimed at reducing the toxicity of the encapsulated active agent and improving drug uptake by ocular tissues. The bioavailability of DA in the tear fluid and the DA uptake into the cornea were increased after instillation of DA-Lipo in rabbits, reaching the DA corneal concentration corresponding to IC50 values against HSV without any sign of transcorneal permeation of drug. DA-Lipo was definitely less cytotoxic then plain DA in rabbit corneal epithelial cells. These results provide new insights into the correlation between the in vitro data and the drug kinetics following ocular applications of liposomal vesicles.

Characterization of distamycin A binding to damaged DNA.

We previously reported that distamycin A, a natural antibiotic known as a minor groove binder, could bind to DNA duplexes containing the (6-4) photoproduct formed at its target site, whereas the binding was not observed for duplexes containing the cis-syn cyclobutane pyrimidine dimer in the same sequence context. In this study, we have further analyzed the binding of this drug to lesion-containing duplexes to elucidate its damaged-DNA recognition mechanism. Surface plasmon resonance measurements using various types of DNA showed that distamycin A could bind to several types of lesion-containing DNA. Curve fitting of the CD titration data revealed that the complex formation occurred with K(d) values around 10(-6) and a stoichiometry of 1:1. The results obtained in this study suggested that distamycin A binds to damaged DNA in the same way as to the normal target site, by recognizing the chemical structure of the minor groove.

Binding of f-PIP, a pyrrole- and imidazole-containing triamide, to the inverted CCAAT box-2 of the topoisomerase IIalpha promoter and modulation of gene expression in cells.

An N-formamido pyrrole- and imidazole-containing triamide (f-PIP) has been shown by DNase I footprinting, SPR, and CD studies to bind as a stacked dimer to its cognate sequences: 5'-TACGAT-3' (5'-flank of the inverted CCAAT box-2 of the human topoisomerase IIalpha promoter) and 5'-ATCGAT-3'. A gel shift experiment provided evidence for f-PIP to inhibit protein-DNA interaction at the ICB2 site. Western blot studies showed that expression of the topoisomerase IIalpha gene in confluent NIH 3T3 cells was induced by treatment with f-PIP. The results suggested that the triamide was able to enter the nucleus, interacted with the target site within ICB2, inhibited NF-Y binding, and activated gene expression.

Brostallicin: a new concept in minor groove DNA binder development.

Brostallicin is a bromoacryloyl derivative of distamycin A, which has shown very promising preclinical activity against a variety of human tumors both in vitro and in vivo. The drug has a limited toxicity towards bone marrow precursor cells in vitro resulting in a therapeutic index much higher than those achieved with other distamycin A derivatives. It retains activity against cancer cells resistant to alkylating agents, topoisomerase I inhibitors and cells with mismatch repair deficiency. Brostallicin has a peculiar mechanism of action involving activation upon binding to glutathione (GSH) catalyzed by glutathione-S-transferase (GST). As a consequence, cells expressing relatively high GST/GSH levels are more susceptible to treatment with brostallicin. Considering that increased levels of GST/GSH are often found in human tumors, this could represent an advantage for the drug in the clinic. Initial clinical studies indicate the tolerability of the drug and allow the determination of the optimal dose for subsequent studies. Some partial response were obtained in these initial phase I studies. Altogether, the results suggest brostallicin to be a new promising anticancer agent with a new mechanism of action. It also raises the possibility to use it in combination with other anticancer drugs currently used.

Nature of Interaction between basic fibroblast growth factor and the antiangiogenic drug 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino])-bis-(1,3-naphtalene disulfonate). II. Removal of polar interactions affects protein folding.

Fibroblast growth factor-2 (basic FGF), a potent inducer of angiogenesis, and the naphthalene sulfonic distamycin A derivative, 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino])-bis-(1,3-naphtalene disulfonate) (PNU145156E), which exhibits in vivo antiangiogenic activity, form a tight reversible (1:1) complex. PNU145156E binds to the heparin and the selenate-binding sites on bFGF. The cis bFGF-heparin (2:1) complex, essential for the activation of the angiogenic process, is thus prevented. The nature of the forces involved in bFGF:PNU145156E complex, using the wild-type and the K128Q, K138Q, K134Q, and K128Q-K138Q point mutated bFGFs was sought. Based on thermodynamic analysis of the complexation constants, protein temperature stability profiles by ultraviolet absorption, circular dichroism measurements, fluorescence Förster energy-transfer, and anisotropy studies, in harmony with the published x-ray crystallographic structure, the following molecular interactions are proposed: reduced coulombic interactions, hence loosening of the complex by the removal of charged polar groups from the bFGF-heparin binding cleft resulted in decreased binding constants and in a change in the binding mode from polar to nonpolar. Concomitantly, upon mutation, the protein was rendered more compact, less flexible, and less aqueously exposed compared with the wild type. These were further pronounced with the double mutant: weaker dominantly nonpolar protein-drug interactions were accompanied by conspicuous folding. With heparin, however, wild-type bFGF forms a tighter complex with a more compact structure.

PNU-145156E, a novel angiogenesis inhibitor, in patients with solid tumors: a phase I and pharmacokinetic study.

Our aim was to establish, in patients with solid tumors, the dose-limiting toxicity, maximum tolerated dose (MTD), and pharmacology of PNU-145156E, a new sulfonated distamycin A derivative that blocked circulating angiogenesis-promoting growth factors in animal studies and exhibited an antitumor effect in murine solid tumors. In a Phase I study, PNU-145156E was administered i.v. every 6 weeks. Included were patients with solid tumors; an Eastern Cooperative Oncology Group performance score

A phase I and pharmacokinetic study of tallimustine [PNU 152241 (FCE 24517)] in patients with advanced cancer.

Tallimustine [PNU 152241 (FCE 24517)] is a synthetic derivative of the DNA minor groove binder distamycin A, in which the NH2-terminal formyl group is substituted by benzoyl mustard. In this Phase I clinical trial, patients with advanced solid tumors received i.v. bolus injections of tallimustine daily for 3 consecutive days. Patients were treated at six dosage levels of 33.3 micrograms/m2/day to 250 micrograms/m2/day for 3 consecutive days, with courses of therapy repeated every 28 days. Detailed pharmacokinetic blood sampling was performed during the first 3 days of the first course of tallimustine. The plasma samples were analyzed by high-performance liquid chromatography with UV detection. Forty-eight eligible patients were treated at all six dosage levels. The dominant dose-related toxicity of tallimustine was neutropenia, becoming dose limiting at 250 micrograms/m2/day. At this dosage level, one patient experienced febrile neutropenia, and a second patient died on study of indeterminate cause. Thrombocytopenia was not observed, and only 10 patients developed anemia < 8.0 gm/dl. Sporadic elevation of liver enzymes or bilirubin was observed but was not dose related. Pharmacokinetic analysis gave reliable results for 33 patients. For most patients, analysis of the data best fit a three-exponential model. Dose-related increases in areas under the concentration-time curve and end-of-infusion concentrations were observed. There was no significant plasma accumulation of tallimustine over the 3 days of administration. The terminal half-life of tallimustine in individual patients ranged from 6.83 to 39.02 h following the last dose. In summary, the recommended Phase II dosage for tallimustine is 200 micrograms/m2/day for 3 consecutive days every 28 days. Neutropenia is the principal toxicity of this agent at this dosage and schedule.

Cytotoxic and antitumor activity of MEN 10710, a novel alkylating derivative of distamycin.

MEN 10710 is a new synthetic distamycin derivative possessing four pyrrole rings and a bis-(2-chloroethyl)aminophenyl moiety linked to the oligopyrrole backbone by a flexible butanamido chain. Its biological properties have been investigated in comparison with the structurally related compound, tallimustine (FCE24517), and the classical alkylating agent, melphalan (L-PAM). Cytotoxic potency of MEN 10710 was increased from 10- to 100-fold, as compared to tallimustine or L-PAM in murine L1210, human LoVo and MCF7 tumor cell lines. MEN 10710 was still active against L1210/L-PAM leukemic cells, while a partial cross-resistance was observed in LoVo/DX and in MCF7/DX cells selected for resistance to doxorubicin and expressing a MDR phenotype. Treatment with verapamil (VRP) reduced the resistance to tallimustine, but not to MEN 10710, in MCF7/DX cells. The cytotoxic effects reflect in vivo antitumor potency and toxicity in the treatment of human tumor xenografts. MEN 10710 was more effective in A2780/DDP, an ovarian carcinoma selected for resistance to cisplatin. On the other hand, the IC30 for inhibiting murine granulocyte/macrophage colony formation was 50 times higher for MEN 10710 than for tallimustine, suggesting a lower myelotoxic potential. In conclusion, the particular biological profile of MEN 10710 characterized by a marked cytotoxic potency, an interesting antitumor efficacy and a reduced in vitro myelosuppressive action may represent a further improvement in the rational design of a novel distamycin-related alkylating compound.

A novel minor groove binding reagent designed to serve as a "truck" to carry DNA modifying moieties into the major groove.

A site selective DNA minor groove binding tripyrrole peptide has been synthesized as a "truck" to place chemical functionalities into the major groove which are capable of physically modifying DNA, acting as catalysts to hydrolyze DNA, or effectively protecting DNA from various DNA modifying enzymes. The equilibrium dissociation constants for the binding of this peptide to an A3T3 dsDNA binding site have been determined to be nanomolar, and they are compared to the constants for other minor groove binding agents.

Antitumor activity of FCE 26644 a new growth-factor complexing molecule.

FCE 26644, or 7,7'-(carbonyl-bis[imino-N-methyl-4, 2 pyrrole carbonyl-imino(N-methyl-4,2-pyrrole)carbonyl-imino])-bis-(1,3- naphthalene)disulfonic acid, belongs to the newly synthesized class of sulfonated derivatives of distamycin A. FCE 26644 is a noncytotoxic molecule capable of inhibiting the binding of basic fibreblast growth factor (bFGF), platelet-derived growth factor (PDGF beta) and interleukin 1 (IL-7) to their receptors and to block bFGF-induced vascularization in vivo as well as neovascularization in the chorioallantoic membrane. FCE 26644 and suramin, a compound possessing the same terminal half-life (t1/2) in mice and, presumably, the same mode of action, inhibit the growth of solid murine tumors, M5076 reticulosarcoma, and MXT and S180 fibrosarcoma and are inactive against B16F10 melanoma. The activity of FCE 26644 was constantly observed at nontoxic doses, at variance with suramin. FCE 26644 was also found to maintain activity against M5076 resistant to cyclophosphamide and to be equally active against UV 2237 and UV 2237/ADR fibrosarcoma.

Phase I study of the novel distamycin derivative tallimustine (FCE 24517).

BACKGROUND: Tallimustine, a benzoyl nitrogen mustard derivative of the antiviral agent distamycin A, is a new alkylating agent which binds to A-T rich regions of DNA in the minor groove producing highly sequence-specific alkylations. Its main preclinical features are a significant antitumor activity in animal models and a lack of cross-resistance in vitro and in vivo with L-PAM. Myelotoxicity was dose-limiting in animals, with a more than 100-fold difference in bone marrow sensitivity between mice and dogs. PATIENTS AND METHODS: Forty adult patients (pts) with solid malignancies were entered in the study. The drug was administered as an IV bolus every 4 weeks. CBC was repeated twice a week and serial assessments of renal function were performed in the week following the first cycle. From the starting dose of 50 micrograms/m2, corresponding to 1/3 of the highest non-toxic dose (HNTD) in dogs, there were increases through 10 dose levels, with reliance only on the features of the myelotoxicity observed. RESULTS: The main toxic effect was neutropenia which was dose-limiting, selective and short-lasting. Only previously-untreated pts received doses of 750 micrograms/m2 or more, with grade 4 neutropenia occurring in > or = 75% of the cycles. The maximally tolerable dose (MTD) was defined as 1250 micrograms/m2, with 3 of 3 pts developing febrile neutropenia requiring IV antibiotics. A platelet count of < 100 x 10(3)/microliters was observed in only one pt. Bone marrow aspiration performed in selected pts on days 8 and 15 confirmed a highly selective impairment by tallimustine of the myeloid lineage, with rapid recovery of the proliferative compartment. Pharmacokinetic studies performed at 1000 micrograms/m2 and 1250 micrograms/m2 showed a rapid fall of the plasma levels within the first 2 hours with drug concentrations between 100 ng/ml and 400 ng/ml within the first hour. A partial response of 4 months' duration was reported in one previously-untreated pt with cutaneous recurrences of malignant mesothelioma. CONCLUSIONS: The report of some antitumour efficacy, the high selectivity of neutropenia, the lack of significant non-hematological toxic effects and the occurrence of detectable but still low plasma drug concentrations suggest that further clinical evaluation of higher doses of tallimustine in combination with colony-stimulating factors would be justified.