Cytotoxicity and probable mechanism of action of sulphimidazole

Mario Castellia,*, Monica Malagolia, Lucia Lupoa, Sergio Roffiab, Francesco Paoluccib, Claudio Cermellic, Andrea Zancad and Giosuè Baggioa

a Department of Biomedical Sciences, Section of Pharmacology, University of Modena and Reggio Emilia, Via G. Campi 287, I-41100 Modena, Italy; b Department of Chemistry ‘G. Ciamician’, University of Bologna; c Department of Hygiene, Microbiology and Biostatistics, University of Modena and Reggio Emilia; d Section of Dermatology, ‘C. Poma’ Hospital, Mantova, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sulphimidazole (1-methyl-2((4-aminophenyl)-sulphonyl)-amino-5-nitroimidazole) is a new compound in which a p-aminobenzenesulphonamide radical has been attached at position 2 of the 5-nitroimidazole ring. It possesses a useful spectrum of activity in vitro against various anaerobic microorganisms and its action against aerobic and facultative bacteria is synergically enhanced in association with trimethoprim. In the present study, we determined the cytotoxicity in vitro of sulphimidazole and trimethoprim, both alone and in combination, and analysed the viability of Vero cells and the protein content of their cell lysate in the presence of increasing concentrations of these drugs. Also, in order to verify the hypothesis that the action of sulphimidazole against aerobic and facultative bacteria is mediated by the sulphonamide component of the molecule, while that against anaerobic bacteria depends on the action of the nitro group of the 5-nitroimidazole ring, we studied the mechanism of action of the new compound both indirectly, by means of microbiological techniques, and directly, by determining its oxidoreduction potential with respect to that of metronidazole. The results show that sulphimidazole is only slightly toxic in vitro for Vero cells, either alone or in association with trimethoprim, and that the combination of the two functional groups in a single molecule not only maintains its structure–activity relationship intact but also broadens its antibacterial spectrum.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sulphimidazole (1-methyl-2((4-aminophenyl)-sulphonyl)-amino-5-nitroimidazole) (FigureGo), is a new molecule with a p-aminobenzenesulphonamide radical in position 2 of the 5-nitroimidazole ring.1 Preliminary studies showed that the molecule had interesting antimicrobial and pharmacokinetic characteristics. Sulphimidazole was found to possess a broader spectrum of activity than sulphadiazine, sulphamethoxazole, metronidazole and erythromycin and proved to be more active against Gram-positive and Gram-negative aerobic and facultative bacteria than sulphamethoxazole, though less so than sulphadiazine. Unlike the two known sulphonamides, it demonstrated activity against anaerobic bacteria that, though useful, was less potent than that of erythromycin and metronidazole. Finally, its pharmacokinetics were found to be comparable to those of low-absorption intestinal sulphonamides.1



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Figure. Structures of the compounds discussed in this article.

 
The broad antibacterial spectrum and low intestinal absorption of sulphimidazole led us to study its in vitro antibacterial activity against microorganisms responsible for toxin-mediated intestinal infections such as cholera and enteric fever.2 However, since clinical experience teaches us that in these cases the poorly absorbed chemotherapeutic agent needs to be administered in association with a drug that is easily absorbed by the intestine, we evaluated the effectiveness of sulphimidazole in combination with trimethoprim.3 The results showed that the two drugs exhibited synergic or additive activity against the Gram-negative aerobic and facultative bacteria tested, synergy being seen with 85% of the strains and additive activity with the remaining 15%. Good results were also obtained with anaerobic bacteria; in particular, sulphimidazole– trimethoprim was more effective than cotrimoxazole against all the Clostridium spp. tested, including Clostridium perfringens. Calculation of the fractional inhibitory concentrations (FICs) mostly demonstrated synergy with enhancement of potency.

In the present study we set out to evaluate in vitro the cytotoxicity of sulphimidazole both alone and in association with trimethoprim. We then studied the mechanism of action of sulphimidazole to verify our hypothesis that the two components of the new compound (sulphonamide and 5-nitroimidazole) act independently of each other against aerobic, facultative and anaerobic bacteria. MICs were determined using aerobic, facultative and anaerobic bacteria grown in special culture media. This enabled us to establish broadly which part of the molecular structure of sulphimidazole might be involved in the antibacterial activity. However, since sulphimidazole contains a 5-nitroimidazole radical, we studied the mechanism of action electrochemically by determining its oxidoreduction potential using cyclic voltammetry. This allowed us to evaluate whether its action against anaerobes depended mainly on oxidoreduction processes, as in the case of metronidazole and other known 5-nitroimidazoles, whose biological properties and redox potentials are interdependent characteristics.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cytotoxicity tests

The cell line used in the cytotoxicity tests was the Vero line, cultured in minimal essential medium (MEM) (PBI, Milan, Italy) with the addition of 10% fetal bovine serum (FBS) (PBI), in which complement was inactivated by heating at 56°C for 30 min. The medium contained penicillin (100 mg/L) and streptomycin (100 mg/L). After incubation in a controlled environment (37°C, 5% CO2) until confluence was achieved, the cells were cultured in a maintenance medium (MEM containing 5% FBS), under the conditions described above.

Two methods were used: the cell viability test and the determination of the live/dead cells ratio (K), and the determination of the protein content by the Lowry test.4,5 In both cases, using 24-well trays, we seeded 100000 cells in 2 mL of culture medium and left them to grow for 24 h at 37°C in a 5% CO2 atmosphere. The medium was then aspirated and replaced with maintenance medium containing the requisite concentration of the substance being tested. Trays were then incubated for 48 h at 37°C in a 5% CO2 atmosphere.

For the cell viability test, the cells were incubated for 48 h with dilutions of the various compounds, washed with phosphate-buffered saline (PBS) pH 7.4, trypsinized and then counted using 0.5% trypan blue stain in PBS. The live cell count was carried out in a Bürker chamber under an optical microscope (magnification x400).

Lowry's test was used to determine the cell protein content. The cells were incubated for 48 h in contact with the compounds being examined and after removal of the culture medium, and then washed with PBS three times to remove serum. The cells were then treated with a series of salt-based reagents (sodium potassium tartrate and copper sulphate) to obtain a cell lysate containing protein complexed with copper. The lysate was stained with an aqueous solution of the Folin–Ciocalteau reagent containing phenol, and readings of the absorbance of the resultant colour were taken (wavelength 550 nm) and compared with those of standard solutions of bovine serum albumin at known concentrations and of the complete culture medium.

Sensitivity tests

Thirty-five isolates, comprising strains of Gram-positive and Gram-negative facultative aerobic and anaerobic bacteria were tested. The organisms studied comprised both clinical isolates from the laboratory of the Microbiology Service of the Polyclinic of Modena, and type strains including: Escherichia coli ATCC 13762, Klebsiella pneumoniae ATCC 10030, Proteus mirabilis ATCC 10005, Pseudomonas aeruginosa ATCC 14502, Staphylococcus aureus ATCC 25923, Clostridium botulinum ATCC 19397, C. botulinum ISM 70/91 and C. perfringens ATCC 12915. All other bacteria (E. coli, Klebsiella spp., P. aeruginosa, Proteus spp., Shigella spp. and Clostridium spp.) had been recently isolated from pathological material.

The facultative aerobes and the clostridia were cultured in tryptic soy agar and broth (Difco, Milan, Italy), the former at 37 ± 2°C for about 24 h in air, the latter at 37°C for about 48 h in oxygen-free bell jars (Gas-Pack, Oxoid, Milan, Italy).

For antimicrobial sensitivity testing of facultative aerobes, we used Mueller–Hinton agar (Oxoid) in comparison with sulphonamide–trimethoprim agar (Oxoid), to eliminate the antagonistic action against sulphonamides of thymidine in Mueller–Hinton agar, thus highlighting the activity of the p-aminobenzenesulphonamide moiety of sulphimidazole. With anaerobes, we determined the in vitro antibacterial activity of sulphimidazole alone and in combination with trimethoprim, using brain–heart infusion broth containing 0.05 g/L of p-aminobenzoic acid (pABA) (Oxoid), which acts as an antagonist of the sulphonamide component of the new compound, in comparison with Wilkins–Chalgren (Oxoid), to study the activity of the 5-nitroimidazole ring of sulphimidazole.

The following chemotherapeutic agents were used: metronidazole (Janssen, Rome, Italy), sulphadiazine (Bracco, Milan, Italy), sulphamethoxazole (Roche, Milan, Italy), trimethoprim (Biomedica, Foscama, Italy) and sulphimidazole (Proter, Milan, Italy). All were dissolved in slightly alkaline solution (pH 7.8), and the stock solutions, prepared at the time of use, were filter-sterilized (millipore filters, 0.2 µm), then diluted as required in the various culture media at pH 7.4.

MICs were determined using doubling dilutions in agar as described by Ericsson & Sherris.6 The microorganisms were used in the tests at a concentration of about 104–106 cfu/mL. Growth was assessed after incubation for 18–24 h at 37°C for the facultative aerobes, and after 48 h in an oxygen-free atmosphere at 37°C in the case of the anaerobes.

Electrochemical experiments

The electrochemical experiments were carried out using the method described in Castelli et al.7 Tetraethylammonium tetrafluoroborate (TEATFB; Aldrich, Milan, Italy) was used as support electrolyte as received. Dry vacuum-distilled N,N-dimethylformamide (DMF; Aldrich) was purified using sodium anthracenide to remove any traces of water and oxygen, according to the method of Sagi et al.8 The solvent was distilled via a closed system in an electrochemical cell containing the supporting electrolyte and the species under examination, immediately before performing the experiment. A potassium dihydrogen phosphate buffer (pH 7) (Riedel-de Hahn, Seelz, Germany) was used as solvent in some experiments. All other chemicals were of reagent grade.

Electrochemical experiments were carried out in an airtight, single-compartment cell described elsewhere,9 using platinum as working and counter electrodes and an aqueous saturated calomel electrode (SCE) as reference electrode. In the steady-state experiments, either a mercury-film electrode10 or a platinum ultramicroelectrode (10 µm diameter; Goodfellow, Cambridge, UK) sealed in glass was used. E1p,c is the one-electron peak potential for the first reduction process determined in aprotic medium (DMF) with cyclic voltammetry,11 while E1/2 is the half-wave potential obtained from the steady-state current– potential curves.12,13 All potentials refer to the SCE.

Voltammograms were recorded with an AMEL Model 552 potentiostat controlled by an AMEL Model 568 programmable function generator, an AMEL Model 865 A/D converter (AMEL, Milan, Italy), a Hewlett Packard 7475A digital plotter (Hewlett Packard, Milan, Italy) and a Nicolet Model 3091 digital oscilloscope (Nicolet, Madison, WI, USA). In the experiments with the ultramicroelectrode, a home-made high-gain potentiostat was used.14 The minimization of the uncompensated resistance effect in the voltammetric measurements was achieved by the positive-feedback circuit of the potentiostat.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results relating to the toxicity of sulphimidazole, trimethoprim and sulphimidazole–trimethoprim for Vero cells are reported in Tables I and IIGo. The cell count and the K ratio (live/dead cells) show that sulphimidazole was only slightly toxic at the highest doses employed, the level of toxicity being similar to that of trimethoprim. Sulphimidazole–trimethoprim also proved toxic for Vero cells only at the highest doses. In all the other cases, however, there was not much difference between association and control data. This finding was corroborated by the Lowry test data relating to the protein content of the cell lysate, using the same range of concentrations.


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Table II. In vitro cytotoxicity of sulphimidazole (SIZ) and trimethoprim (TMP) in association (SIZ + TMP) for Vero cells
 
In microbiological tests, we compared the in vitro antimicrobial activity of sulphimidazole with that of sulphadiazine, sulphamethoxazole and metronidazole using sulphonamide–trimethoprim agar rather than Mueller– Hinton agar; this was to avoid the effect on the sulphonamide activity of any thymidine that might be present in the latter culture medium, which would increase the MICs.15 On the other hand, the antibacterial activity of sulphimidazole against some facultative aerobes grown on sulphonamide–trimethoprim agar was slightly greater than that of sulphamethoxazole (Table IIIGo). The same table reports the parallel MICs obtained when using Mueller–Hinton agar, and shows that the composition of the culture medium may influence the antimicrobial activity of the compound by interacting, in this case, with the sulphonamide component of the sulphimidazole molecule.


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Table III. In vitro antibacterial activity on sulphonamide–trimethoprim agar and Mueller–Hinton agar of sulphimidazole (SIZ), sulphadiazine (SDZ), sulphamethoxazole (SMX) and metronidazole (MZ) against some facultative aerobes
 
The antibacterial activity of sulphimidazole against some anaerobes grown in brain–heart infusion agar with the addition of pABA is reported in Table IV. The resultant MICs are in line with those relating to the same antibacterial activity determined using other culture media with the exclusion of pABA (Table VGo). The finding demonstrates that the activity of the sulphonamide component of the molecule had no influence on the compound's anti-anaerobic activity, the MICs of sulphimidazole for anaerobes being practically identical, whether the metabolic antagonist (pABA) is present or not. Hence, the anti-anaerobic activity of sulphimidazole can be attributed to the presence and activation of the nitroimidazole component, as is true of all the known 5-nitroimidazoles.16


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Table V. In vitro antibacterial activity on Wilkins–Chalgren agar of sulphimidazole (SIZ), metronidazole (MZ), sulphamethoxazole (SMX) and trimethoprim (TMP) against some species of the genus Clostridium
 
To verify this claim, we determined the oxidation– reduction potential of sulphimidazole by means of cyclic voltammetry, as in the case of the precursors of sulphimidazole, namely, V1 (1-methyl-2-(methylthio)-5-nitro- 1H-imidazole or sulphuridazole) and V2 (1-methyl-2-(methylsulphonyl)-5-nitro-1H-imidazole or sulphonidazole).7 The results, expressed in E1p,c (cathode peak monoelectronic potential) (Table VI), show that the redox potential of sulphimidazole (–1.40 V) is between that of metronidazole (–1.00 V) and that of V2 (–1.60 V); this demonstrates that the biological activity of these compounds, be it cytotoxic or antimicrobial, can be correlated with their electronic affinity.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Most, if not all, of the biological characteristics of the 5-nitroimidazoles depend on metabolic reactions that lead to the reduction of the nitro group and the formation of a highly reactive intermediate which is responsible not only for the antimicrobial and radio-sensitizing properties of these compounds, but also for their cytotoxic and mutagenic properties. However, both the cell toxicity of the metabolites of the various 5-nitroimidazoles and the possible therapeutic importance of the different substituents in positions 1 and 2 of the imidazole ring require fuller investigation. Metronidazole, for example, is metabolized in the liver by means of oxidation processes mediated by the radicals both in position 1 and in position 2. These metabolites are generally less active as antimicrobial agents, but are probably more toxic for host cells than the starting compound, and accumulation of intermediates can create serious problems in patients suffering from renal and hepatic dysfunction.17

The sulphimidazole precursors V1 and V2 are so cytotoxic for Vero cells as to exclude systemic application.7 On the other hand, our present findings, relating to the cytotoxicity of sulphimidazole, both alone and in combination with trimethoprim, show that the p-aminobenzenesulphonamide radical in position 2 on the nitroimidazole ring (FigureGo) drastically reduces the toxicity of the precursors; only at the highest doses does slight cytotoxicity occur (Tables I and IIGo). Although the nitro group is acknowledged as playing a crucial role in the biological activity of the 5-nitroimidazoles, the other molecular structures also appear to exert an important influence on their therapeutic properties.

The mechanisms by which the different substituents in position 2, either alone or, more probably, in conjunction with those in position 1, influence the structure–activity relationship of the 5-nitroimidazoles are still unknown. Some authors have suggested that the different radicals may modify their osmotic properties, and hence the uptake of the molecules by the cells.18 Others have pointed out that the different structures present can influence the distribution of the highly reactive reduced intermediates inside the cells, thereby conditioning the access to the molecular targets of the 5-nitroimidazoles.19

The formulae of some 5-nitroimidazoles are shown in the FigureGo. The presence of different radicals in position 1 of the 5-nitroimidazole ring does not modify the compound's activity against anaerobes, nor does it appreciably enhance it against aerobic and facultative microorganisms.20 The presence of substituents in position 2, on the other hand, can increase the spectrum of activity, as demonstrated by experiments with Neisseria gonorrhoeae;19 while metronidazole is slightly active against this microorganism, other 5-nitroimidazoles with various radicals in position 2 have very low MICs. Moreover, this activity does not correlate with that against the more familiar targets of this compound, such as Bacteroides fragilis and C. perfringens.19 Sulphimidazole, which exerts an inhibitory effect against anaerobes and facultative aerobes, probably owes the latter activity to the p-aminobenzenesulphonamide radical present in position 2.

Preliminary investigations into the mechanism of action of sulphimidazole were conducted by means of microbiological techniques. In the case of the aerobes and facultative aerobes, sulphonamide–trimethoprim agar was used, while brain–heart infusion agar containing 0.05 g/L pABA was used with anaerobes. Comparison of the results obtained using the different culture media shows that sulphimidazole had slightly better antibacterial activity against facultative aerobes than sulphamethoxazole. However, a more important observation related to sulphimidazole's mechanism of action in relation to facultative bacteria, which was similar to that of the other sulphonamides. It can therefore be argued that sulphimidazole acts against facultative bacteria by competitive inhibition of dihydropteroate synthetase, the enzyme that facilitates the condensation of pABA with pteridine. The synergy between sulphimidazole and trimethoprim (Table VIIGo) also points to a sulphonamide-like mechanism of action.


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Table VII. In vitro antibacterial activity of sulphimidazole (SIZ), trimethoprim (TMP) alone and in association (SIZ + TMP) against Gram-negative and Gram-positive enteropathogens
 
Table IIIGo, which shows the MICs of metronidazole for facultative aerobes confirms its lack of antibacterial activity against aerobes. The point of assessing its activity on both sulphonamide–trimethoprim and Mueller–Hinton agar was to demonstrate that the 5-nitroimidazole ring has no effect on the antibacterial properties of metronidazole in relation to facultative aerobes, whether in rich or poor culture medium. This observation again leads us to suppose that it is only the sulphonamide component of sulphimidazole and not the 5-nitroimidazole ring that acts against the facultative aerobes.

Similar conclusions can be drawn from an analysis of the results of sulphimidazole in relation to anaerobes. In experiments using culture medium containing 0.05 g/L pABA, the sulphonamide component of the new compound proved inactive. Since the sulphonamide component of sulphimidazole was antagonized by pABA, the marked inhibition of the growth of anaerobes must be due entirely to its 5-nitroimidazole radical, which is clearly not affected by the essential metabolites (such as traces of thymidine or pABA) present in brain–heart infusion medium (Table IV); for the MICs obtained for this culture medium are practically identical to those found against the same type of microorganism using a culture medium without pABA (Table VGo).

The MICs of sulphamethoxazole and trimethoprim were determined in the same way as were those of sulphimidazole–trimethoprim and cotrimoxazole. Using pABA-free culture media, the action of sulphimidazole against anaerobes was more intense than that of sulphamethoxazole or trimethoprim. Finally, while a combination of sulphamethoxazole and trimethoprim showed indifference to clostridia, the combination of sulphimidazole and trimethoprim showed synergic activity against the same bacteria; indeed, when trimethoprim, which has a neglible effect on anaerobes, is combined with sulphimidazole, it potentiates the anti-anaerobic activity (Table IV). The biochemical mechanism involved in the anti-anaerobic synergy between sulphimidazole and trimethoprim is different from that exerted by sulphimidazole–trimethoprim against facultative aerobes. Sulphimidazole has a sulphonamide component as well as a 5-nitroimidazole ring, and thus trimethoprim interacts with both groups but in a different way. The synergic effect on clostridia, due to the nitroimidazole component of sulphimidazole, is probably of a complementary type, similar to that exerted by trimethoprim and oxylinic acid on aerobic microorganisms.2123 In this case, the effect of quinolone on DNA is potentiated by that exerted by trimethoprim on the biosynthesis of the nucleotide bases and of the proteins.

It is therefore possible that, with the interaction between sulphimidazole and trimethoprim, the intracellular effect of sulphimidazole's 5-nitroimidazole radical is potentiated only by a complementary and not sequential type of trimethoprim-mediated synergy, which inhibits the base metabolites for the construction of the double helix of DNA. While trimethoprim exerts its activity on the enzymes responsible for the biosynthesis of the precursors of amino acids and nucleotides, the nitroimidazole component of sulphimidazole would appear to act directly on DNA, probably by means of the mechanism of action typical of 5-nitroimidazoles.16

An important observation emerges from these preliminary microbiological assays which already partly confirms the initial hypotheses: the two different components of sulphimidazole's molecular structure, sulphonamide and nitroimidazole (FigureGo), would appear to act separately on facultative aerobes and on anaerobes, respectively.

To gain further insight into the role of the nitro group of sulphimidazole in anti-anaerobic activity, we compared sulphimidazole's redox properties with those of metronidazole, determining their E1p,c values by means of cyclic voltammetry. The efficacy of 5-nitroimidazoles correlates with their electro-affinity, depending on the degree of reduction.2426 If the nitro-imidazole derivative has been only partially reduced, the redox potential is greater and the efficacy of the compound is more marked. This occurs, for example, in the case of radio-sensitization of hypoxic cells in mammals, when the radio-sensitization takes place in a few milliseconds. If, however, the nitro-imidazole derivative has been completely reduced, the more negative redox potential, and hence lower electro-affinity, increases the cytotoxicity of the compound. These observations were noted both in the determination of the cytotoxicity of the 5-nitroimidazoles on hypoxic cells in mammals and in studies of their antimicrobial activity, both events that take longer than radio-sensitization.2731 Since the experiments on both the cytotoxicity and the antimicrobial activity of the compounds were conducted over a period of 24–48 h, reduction of the compounds is likely to have been complete, reinforcing the correlation between low redox potential and high cytotoxicity. The E1p,c values for metronidazole, sulphimidazole, V1 and V2 are reported in Table VI. The data show that the redox potential of V1 (–1.16 V) is a little more negative than that of metronidazole (–1.00 V), while that of V2 (–1.60 V) is much more negative7 and that of sulphimidazole (–1.40 V) falls between those of metronidazole and V1 and that of V2.

The above findings give an insight into the mechanism of action of sulphimidazole. However, a distinction needs to be drawn between the behaviour of sulphimidazole against anaerobes and facultative aerobes. In particular, since the microbiological assays indicate that the component active against anaerobes is the 5-nitroimidazole ring, the following premises can be adduced: (i) the redox potential is relative to the mono-electronic reduction of the nitro group; (ii) the E1p,c value found for sulphimidazole (–1.40 V) (Table VI) falls between those of metronidazole (–1.00 V) and V1 (–1.16 V) and that of V2 (–1.60 V); (iii) the experimental conditions are such that reduction of the compound being examined can be said to be complete; (iv) since the cytotoxicity towards these microorganisms by the compound with the highest redox potential (metronidazole) is already high, the activity of sulphimidazole should be substantially the same as that of metronidazole and V1, as is confirmed by the fact that sulphimidazole's anti-aerobic activity correlates with its redox behaviour (Table VI). Bearing in mind that, as demonstrated by the microbiological assays, the sulphonamide component of sulphimidazole is that biologically active against facultative aerobes, while its redox potential reflects the properties of the 5-nitroimidazole ring, the electrochemical behaviour of sulphimidazole is only indicative of the anti-anaerobic characteristics of the molecule. Finally, it should be pointed out that the cytotoxicity values of the compounds under investigation for Vero cells are in agreement with their electrochemical properties.

In the case of sulphimidazole, our findings vindicate the rationale of combining the functional groups of two different classes of chemotherapeutic agents, namely, sulphonamides and 5-nitroimidazoles, in a single molecule. The finding that the spectrum of antimicrobial activity is broadened relative to that of the single compounds, suggests that sulphimidazole may act in the course of mixed infections both as a sulphonamide and as a 5-nitroimidazole.


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Table I. In vitro cytotoxicity of sulphimidazole (SIZ) and trimethoprim (TMP) for Vero cells
 

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Table IV. In vitro antibacterial activity on brain-heart infusion agar containing pABA of sulphimidazole (SIZ), sulphamethoxazole (SMX) and trimethoprim (TMP) and of the associations SIZ + TMP and SMX + TMP against some species of the genus Clostridium
 

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Table VI. Biological properties of the compounds analysed in relation to their redox potential and chemical structure
 

    Acknowledgments
 
We should like to thank Mr Ergisto Angeli, senior technician at the Department of Physics, University of Ferrara, for his expert technical assistance and the Cambridge Centre of English, Modena, for translation and typing services. This work was supported in part by grants from the ‘Ministero dell’Università e della Ricerca Scientifica e Tecnologica' and from the ‘Consiglio Nazionale delle Ricerche’, Rome.


    Notes
 
* Corresponding author. Tel: +39-59-2055366; Fax: +39-59-2055367; E-mail: Castma24{at}mail.unimo.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 4 May 1999; returned 6 July 1999; revised 26 October 1999; accepted 31 January 2000





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