HPLC methods for the determination of bound and free doxorubicin, and of bound and free galactosamine, in methacrylamide polymer-drug conjugates.

Quantitative acid hydrolysis followed by HPLC separation has been established as an analytical procedure for the determination of polymer-bound doxorubicin (an anti-cancer drug) and D-galactosamine (a liver-targetting moiety) in the polymer-drug conjugates FCE 28068 and FCE 28069. Optimal conditions of hydrolysis were determined in both cases: 1 N HCl, 50 degrees C, 1.5 h for doxorubicin, and 6 N HCl, 60 degrees C, 5 h for galactosamine. Appropriate HPLC quantitation of galactosamine required pre-treatment with sodium borohydride and pre-column derivatization with o-phthalalaldehyde and beta-mercaptoethanol. Independent determination of free doxorubicin and glactosamine in untreated polymer samples was also achieved with the same HPLC method up to detection limits of 0.01% and 0.02% respectively. The methods were validated for linearity, precision and repeatability. Validation for accuracy before and after acid hydrolysis was achieved by testing hydrolysis on model compounds and by assessing recovery in polymer solutions spiked with free doxorubicin or galactosamine.

"Hybridization analysis for the quantitative determination of residual DNA in some recombinant proteins expressed in E. Coli cells".

The recent advances in the area of pharmaceutical recombinant DNA products have led to an impressive increase in the number of clinically used therapeutic proteins, for which stringent purity requirements are established by authorities. Nucleic acids are host cell derived contaminants and their allowed level in the final product is in the low pg range, because of the health risk to the patient. Even if fast non-radioactive methods have been recently developed as an alternative to the traditional hybridization assay, the latter is still widely used being a specific and sensitive method. However hybridization quantitative aspects are only partially reviewed in the literature as well as the procedures utilised to cope with the possible interference of the sample protein on the assay results. These topics are described in the present paper by detailing and comparing the methods set up for three recombinant proteins: human pro-Urokinase (r-h-proUK), human basic fibroblast growth factor (r-h-bFGF) and human granulocyte macrophage colony stimulating factor (r-h-GMCSF).

RP-HPLC peptide mapping methods for the analysis of recombinant human pro-urokinase.

Among the techniques available for the detection of protein structure variants such as single point mutations, RP-HPLC peptide mapping plays a key role owing to the high reproducibility of peptide retention times, determined as identity indexes. Because of the possible co-elution of some proteolytic fragments, an improvement of the array of information given by the technique can be achieved by setting up a series of experiments under hydrolytic conditions with different enzymes, followed by appropriate RP-HPLC gradient elutions. Such an experimental approach appears to be particularly useful in the examination of proteins with a high molecular weight, where the resulting RP-HPLC maps are complex. Therefore different RP-HPLC peptide mapping methods have been studied for recombinant human pro-urokinase (r-h-proUK), a thrombolytic agent of apparent molecular weight of 46 kD. The RP-HPLC maps indicate that the methods developed are not only suitable for the qualitative control of the amino acid sequence and arrangement of disulphide bonds but also represent the first demonstration of the identity of the primary structure of the recombinant and of the native species, within the limits of the technique.

High-performance liquid chromatographic analysis of FCE 24304 (6-methylenandrosta-1,4-diene-3,17-dione) and FCE 24928 (4-aminoandrosta-1,4,6-triene-3,17-dione), two new aromatase inhibitors.

The cytochrome P-450-dependent aromatase enzyme plays an important role in hormone-dependent diseases. Many products that inhibit this type of enzyme were obtained: FCE 24304 (I) and FCE 24928 (II) proved to possess remarkable activity and are presently under development. Compounds I and II and their synthetic intermediates are analyzed by means of a high-performance liquid chromatographic method, affording rapid and efficient separation, good resolution and identification of all the examined compounds. The linearity, specificity, sensitivity, precision and accuracy for the method are also provided.

Thermal behaviour and binary phase diagram of (S)-(+)-4,4'-(1-methyl-1,2-ethandiyl)-bis-(2,6-piperazinedione) (dexrazoxane), a cardioprotective agent, and of its (R)-(-)-enantiomer.

The binary phase diagram of (S)-(+)-4,4'-(1-methyl-1,2-ethandiyl)-bis-(2,6-piperazinedione), 1 (dexrazoxane), a cardioprotective agent, and of its (R)-(-)-enantiomer, 2, has been investigated by differential scanning calorimetry (DSC); the equimolecular mixture of 1 and 2 corresponds to a racemic compound (racemate) whose melting point is higher than that of the enantiomers. Thermal behaviour (DSC) is examined and discussed in comparison with the data obtained by other physical methods (IR spectroscopy and X-ray powder diffraction).

Influence of lipophilicity on cytotoxicity of anthracyclines in LoVo and LoVo/Dx human cell lines.

Quantitative structure-activity relationship studies aimed at improving drug activity profiles require the determination of the physicochemical properties possibly involved in biological action. The lipophilic character of selected anthracyclines has been measured by means of reverse-phase high performance liquid chromatography, selecting appropriate experimental conditions. The capacity coefficients at zero percentage of the organic phase (log K0), which are retention indexes, have been used as lipophilicity descriptors in a QSAR study, involving as biological data the cytotoxicity of anthracyclines in a doxorubicin-sensitive (LoVo) and in a doxorubicin-resistant (LoVo/Dx) human cell lines. The results obtained in these in vitro models indicate that lipophilicity plays a role in anthracycline activity, influencing drug availability at the site of action.

Determination of phenolic ionization constants of anthracyclines with modified substitution pattern of anthraquinone chromophore.

The phenolic ionization constants in aqueous solution of doxorubicin, daunorubicin and nine daunorubicin analogues with different substitution patterns at the anthraquinone moiety have been determined spectrophotometrically upon taking into account aggregation effects with extrapolation to infinite dilution. In contrast with an early literature report [Sturgeon, R.J. & Schulman, S.G. J. Pharm. Sci. 66, 958-961; 1977] the perturbation from amino group ionization on daunosamine sugar was found to be negligible in our spectrophotometric titrations. Accordingly, only phenol hydroxyl ionization constants could be determined in these experiments, but a crude estimate of daunosamine pKa was obtained in a fluorometric titration. From a comparison of suitable analogues it is concluded that the phenolic function at C11 in doxorubicin and daunorubicin is the most acidic one. The higher pKa of the C6-OH group is ascribed in part to electronic effects leading to higher strength of the corresponding hydrogen bond with quinone oxygen, and in part to a steric effect from the bulky sugar group at C7. Ionization constants of nitro or amino substituted derivatives follow the expected trend. In the cases of carminomycin and 6-deoxycarminomycin, which both have another phenolic group at C4, two phenolic ionization processes can be detected in the experimentally accessible pH range (5-12): these are ascribed to C4-OH and C11-OH. An application of the compiled parameters to studies of chemical reactivity and biological activity of anthracyclines is foreseen.

Association of anthracyclines and synthetic hexanucleotides. Structural factors influencing sequence specificity.

The equilibrium and kinetic aspects of the interaction between four anthracyclines and two synthetic self-complementary hexanucleotides was investigated by fluorescence detection. Two of the studied anthracyclines are widely used antitumor drugs: doxorubicin (1, formerly adriamycin) and daunorubicin (2, formerly daunomycin). The other two, 9-deoxydoxorubicin (3) and 3'-deamino-3'-hydroxy-4'-epidoxorubicin (4), are doxorubicin analogues with modifications of the chemical groups that have been proposed as responsible for sequence specificity (Chen, K.-X., Gresh, N. and Pullman, B. (1985). J. Biomol. Struct. Dyn. 3, 445-466). One of the oligonucleotides, d(CGTACG), is identical to that used in the high resolution x-ray structure determination of the daunorubicin intercalative complex (Wang, A. H.-J., Ughetto, G., Quigley, G. J. & Rich, A. (1987). Biochemistry 26, 1152-1163). Binding to this hexanucleotide is compared with intercalation into the d(CGCGCG) duplex, revealing sequence preferences of the four anthracyclines. Taking into account the anthracycline aggregation and the dissociation of the hexanucleotide double standard form, results can be interpreted with a model that assumes complete fluorescence quenching at intercalative sites containing the CG base pair, and a large residual fluorescence after intercalation within the TpA fragment. All four anthracyclines show preferential intercalation at sites near the ends of both hexanucleotide duplexes, partly as a result of positive cooperativity in the formation of di-intercalated species at these sites. Within the limits of experimental error, complete site specificity for the CpG fragment is found in the intercalation of 1 and 2 into d(CGTACG) duplex, whereas analogues 3 and 4 give increasing evidence of intercalation at other sites including the fluorescence-preserving TpA fragment. Site specificity is less pronounced in the association with d(CGCGCG), when cooperativity is taken into account. Kinetic data corroborate the results of equilibrium studies and are interpreted with a mechanism that includes formation of an intermediate bound species followed by drug redistribution to preferential sites. Finally, from a comparison of pertinent site binding constants, approximate free energy contributions to sequence specific DNA interaction, due to C9-OH on the aglycone and -NH3+ on daunosamine, are estimated not to exceed 2 kcal/mol.

Urinary metabolites of rifabutin, a new antimycobacterial agent, in human volunteers.

1. Metabolites of the antimycobacterial agent 4-deoxo-3,4-[2-spiro-(N-isobutyl-4-piperidyl)]-(1H)-imidazo-(2,5-dihydro )- rifamycin S (rifabutin) were isolated from human urine after administration of a single oral dose of the drug. Some of these metabolites were identified by direct inlet mass spectrometry, 1H-n.m.r. spectrometry and, in two cases, by chromatographic comparison with reference compounds. 2. Unchanged drug, 25-O-deacetyl rifabutin and four other metabolites were identified in human urine. 25-O-Deacetyl rifabutin was the main urinary metabolite, other metabolites were characterized as oxidized, and oxidized-deacetylated derivatives. 3. Routes of metabolic transformation were: (a) deacetylation at position 25, (b) oxidation of methyl groups 31 or 32 or at the piperidine nitrogen, and (c) combination of these.

Studies of anthracycline--DNA complexes by circular dichroism.

A series of doxorubicin and daunorubicin analogues have been investigated in aqueous solution and as DNA-bound forms by means of circular dichroism (c.d.) spectroscopy. The structural variants comprise modifications on the amino sugar, on the aliphatic ring and the side chain of the aglycone moiety, and of the substitution pattern of the anthraquinone chromophore. Results with compounds having conformational constraints interfering with optimal fitting to DNA indicate that stereochemistry and conformation of the aliphatic ring predominantly affect c.d. spectra of anthracyclines in DNA-bound as well as in free form. Conformational correspondence with the known structure of the daunorubicin-oligonucleotide complex is inferred from the spectra of derivatives with modifications at position 6 or 11 in the anthraquinone chromophore. On the other hand, a different binding geometry is postulated for compounds either lacking the 4-methoxy group of daunorubicin (idarubicin and derivatives) or having a phenolic function in its place (carminomycin and derivatives). A possible relation with cytotoxic activity is discussed at a speculative level.

Novel rifamycins. III. Synthesis and antibacterial activity of 3-amidino- and of 4-aminoimidazolo[4,5-c]rifamycin derivatives.

A number of semisynthetic rifamycin derivatives modified at position 3 and/or 4, belonging to general structures 2 and 4 (see Scheme 1), have been obtained. The synthesis and the biological activities of the new compounds are described. Compounds 4p and 4q display very good antimycobacterial activity in mice.

Novel rifamycins. I-Synthesis and antibacterial activity of 3-hydrazinorifamycin derivatives.

A number of semisynthetic rifamycin derivatives, modified at position 3, belonging to general structures (II), (III), (IV), (V), (VI), (VII) and (VIII) (see Scheme), have been prepared. The synthesis, structure, and antimicrobial evaluation of the new compounds are described. All the derivatives have in vitro antibacterial activities comparable with that of rifampicin.

o-Methylation on anthracyclines and anthracyclinones. New o-methyl ethers of daunorubicin and its analogues.

The synthesis of four new ethers of daunorubicin, namely 6-, 9-, 11-monomethyl and 6,9-dimethyl, along with the 4'-methyl ethers of 11-deoxydaunorubicin and 11-deoxydoxorubicin is reported. While the methylation of any of the mentioned hydroxyl groups of the aglycone moiety of daunorubicin resulted in a practically complete loss of bioactivity, the methylation of the aminosugar hydroxyl group of 11-deoxydaunorubicin and 11-deoxydoxorubicin increased their effectiveness on P 388 leukemia in mice.