Zidovudine and Ursodeoxycholic Acid Conjugation: Design of a

Article pubs.acs.org/molecularpharmaceutics

Zidovudine and Ursodeoxycholic Acid Conjugation: Design of a New Prodrug Potentially Able To Bypass the Active Efflux Transport Systems of the Central Nervous System Alessandro Dalpiaz,† Guglielmo Paganetto,‡ Barbara Pavan,*,‡ Marco Fogagnolo,§ Alessandro Medici,‡ Sarah Beggiato,∥ and Daniela Perrone‡ †

Department of Pharmaceutical Sciences, ‡Department of Biology, §Department of Chemistry, and ∥Department of Clinical and Experimental Medicine, Pharmacology Section, University of Ferrara, Ferrara, Italy ABSTRACT: We have synthesized a new prodrug obtained by the 5′-ester conjugation of zidovudine (AZT), an antiviral agent substrate of active efflux transport systems (AET), with ursodeoxycholic acid (UDCA), a bile acid able to permeate into the central nervous system (CNS). We have demonstrated, by HPLC analysis, that UDCA−AZT is quickly hydrolyzed in rat plasma and whole blood (half-life 0.998, P < 0,0001). The solubility values of AZT and UDCA−AZT in water were 27.4 ± 0.9 mg/mL (0.103 ± 0.003 M) and 0.0030 ± 0.0001 mg/mL (4.75 ± 0.19 × 10−6 M), respectively, whereas the UDCA−AZT solubility in the mixture of water and methanol (70:30 v/v) was 0.058 ± 0.002 mg/mL (9.0 ± 0.3 × 10−5 M). The average recoveries ± SD of the compounds from human whole blood and plasma or rat brain and liver homogenates ranged between 71.4 ± 3.1% and 84 ± 3.9%. The concentrations of the prodrug UDCA−AZT and AZT were therefore referred to as peak area ratio with respect to their internal standard 7-n-PX. The precision of the method based on peak area ratio was represented by RSD values ranging between 1.1% and 1.4% for AZT and UDCA−AZT extracted from the different incubation media. The calibration curves referred to these compounds incubated in human whole blood and plasma or rat brain and liver homogenates were linear over the range 0.5−50 μM (n = 8, r > 0.985, P < 0.0001). AZT and its prodrug UDCA−AZT were not degraded in phosphate buffer (pH 7.4) or its mixture with 30% methanol, respectively, during their incubation at 37 °C for eight hours. Taking into account the poor solubility in water of UDCA− AZT, its incubation was performed in the mixture of buffer and methanol, the same solvents employed for the mobile phase of HPLC apparatus. The kinetic studies in plasma and whole blood or tissue homogenates were instead performed by adding the stock solutions (0.3% v/v AZT or UDCA−AZT 10−2 M in DMSO) in the incubation media, taking into account that the protein contents in physiologic fluids allowed solubilization of the lipophilic prodrug. Indeed, problems caused by the poor solubility of these compounds also arisen in transport studies. As a consequence, we have verified that the addition of 0.3% v/ v stock solutions to PBS containing 10 mg/mL BSA allowed 30 μM AZT and UDCA−AZT solutions to be obtained. AZT incubated in rat and human plasma or whole blood, or rat brain and liver homogenates, was not degraded within eight hours. However, the prodrug UDCA−AZT appeared degraded in all these incubation media, showing great rate differences between rat and human species. In particular, Figure 2 reports the degradation profiles of UDCA−AZT in rat plasma and whole blood, evidencing a very fast hydrolysis of UDCA−AZT, whose half-life was estimated lower than 10 s in both incubation media. Figure 3 reports the degradation profiles of UDCA−AZT in human plasma and whole blood, whose halflives were 7.53 ± 0.44 and 3.71 ± 0.16 h, respectively. The degradations followed a pseudo first-order kinetics, confirmed by the linear patterns of corresponding semilogarithmic plots (n = 10, r > 0.981, p < 0.0001) and suggesting, therefore, the prodrug degradation governed by hydrolysis processes. The hydrolysis of UDCA−AZT was confirmed by the AZT appearance in incubation media with amounts increasing during time, as reported in Figure 3. In particular, after sixteen hours of prodrug incubation in plasma the amounts of UDCA− AZT and AZT were 19.69 ± 1.5% and 80.45 ± 4.6%, respectively, of the starting prodrug concentration, whereas after eight hours in human whole blood the amounts of UDCA−AZT and AZT were 20.0 ± 1.3% and 78.97 ± 4.1%, respectively. Similarly, Figure 4 reports the degradation profiles of UDCA−AZT in rat brain and liver homogenates, whose halflives were 7.24 ± 0.45 and 2.70 ± 0.14 min, respectively. Also in this case the pseudo first-order kinetics were confirmed by

Apparent permeability coefficients (Papp) of AZT or UDCA− AZT were calculated according to the following equation:37−39

Papp =

dc /dt ·Vr SA·C0

(1)

where Papp is the apparent permeability coefficient in cm/min; dc/dt is the flux of drug across the filters, calculated as the linearly regressed slope through linear data; Vr is the volume in the receiving compartment (A = 0.4 mL; B = 2 mL); SA is the diffusion area (1.13 cm2); and C0 is the initial compound concentration in the donor chamber at t = 0. The permeabilities were determined for the filters alone (Pf) and for the filters covered by cells (Pt). The apparent permeability coefficients PE referred to the cellular monolayer were then calculated as follows:38,40

1 1 1 = − PE Pt Pf

(2)

Statistical Analysis. Statistical comparisons of permeability coefficients or cumulative concentrations obtained from the transport studies were made by one way ANOVA or Student’s t test (GraphPad Prism). P < 0.05 was considered statistically significant. GraphPad Prism was employed for the linear regression of the cumulative amounts of the compounds in the receiving compartments of the Millicell systems. The quality of fit was determined by evaluating the correlation coefficients (r) and P values.

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RESULTS Synthesis of UDCA−AZT Prodrug. The UDCA−AZT prodrug 1 was obtained as white powder in 70% yield and ≥95% purity as determined by their 1H NMR analysis. The molecular weight, 1H NMR, 13C NMR and IR spectra of prodrug were in agreement with those required by its structure. Hydrolysis Studies of UDCA−AZT. A first step of our work was the evaluation of the potential hydrolysis pattern of the UDCA−AZT prodrug in different media such as phosphate buffer, human whole blood or plasma, or rat brain and liver homogenates. In this aim it was necessary to detect and quantify in all incubation media not only the prodrug but also its potential hydrolysis product AZT. In order to do so, an efficacious analytical method was developed based on the employment of a reverse phase C-18 HPLC column and a mobile phase constituted by a mixture of water and methanol following a gradient profile. In particular, the mixture water− methanol 80:20 (v/v), programmed for the first 10 min, allowed the detection of the hydrophilic compounds 7-n-PX (employed as internal standard for the kinetic studies in blood, plasma or brain and liver homogenates) and AZT, whereas the mixture water−methanol (25:75), programmed for the following 10 min, allowed the detection of the hydrophobic prodrug UDCA−AZT. No interferences were observed from whole blood or plasma and brain or liver homogenate extract components. Therefore, this gradient profile allowed us to quantify both the prodrug UDCA−AZT and its hydrolysis product AZT in the same HPLC chromatogram in all incubation media investigated. The chromatographic precision for AZT, UDCA−AZT and 7-n-PX dissolved in aqueous phase and UDCA−AZT dissolved in a water−methanol mixture 70:30 (v/v) were represented by the relative standard deviation (RSD) values ranging, among the different molecules, between 0.84% and 0.98%. The 961

dx.doi.org/10.1021/mp200565g | Mol. Pharmaceutics 2012, 9, 957−968

Molecular Pharmaceutics

Article

case the RSD values were 0.9 and 1.1 for AZT and UDCA− AZT respectively and their calibration curves were linear over the range of 0.04 to 30 μM (n = 10, r > 0.968, P < 0.0001). Transport studies were performed after cell cultures reached the confluence using parallel sets of Millicell well plates with similar TEER values (80 ± 0.9 Ω·cm2). The studies were performed with the employment of PBS containing 1% BSA, useful for transport analysis of lipophilic compounds.36 The permeation profiles of AZT and UDCA−AZT across the Millicell filters alone or coated by monolayers obtained by HRPE cells are reported in Figure 5, where the cumulative concentrations in the receiving compartments are reported (Figures 5A and 5B for AZT; Figures 5C and 5D for UDCA− AZT) . The profiles are referred to the transport from the apical to basolateral compartments (B → A, apical receiving, Figures 5B and 5D) and vice versa (A → B, basolateral receiving, Figures 5A and 5C). The cumulative amounts in the receiving compartments showed a linear profile within 120 min in all cases (r ≥ 0.990, P ≤ 0.001) with the exception of the permeations of AZT and its prodrug UDCA−AZT from the basolateral to apical compartments across the filters not coated by cells. In this case their cumulative amounts showed a hyperbolic pattern, whereas the linearity was maintained within 30 min (dashed lines, r ≥ 0.984, P ≤ 0.002). The resulting slopes of the linear fits were used in eq 1 for calculation of permeability coefficients (Pt and Pf) that were employed to calculate, according to eq 2, the apparent permeability coefficients (PE) of AZT and UDCA−AZT referred to the cellular monolayers, reported in Table 1. The PE values for A → B and B → A transport of AZT were 209 ± 14 × 10−5 and 133 ± 8 × 10−5 cm/min, respectively. The permeation of this drug from the apical to the basolateral compartments appeared therefore higher than its permeation from the basolateral to apical compartments (P < 0.001), suggesting the presence of efflux transport systems active for AZT in the HRPE cellular monolayer. The PE values for A → B and B → A transport of the prodrug UDCA−AZT were 31.3 ± 3.6 and 39.1 ± 1.2, respectively. These values appeared lower than those referred to the AZT permeation across the cellular monolayer (P < 0.001), even if not different between them (P > 0.05). These data suggest the absence of active efflux transport systems for UDCA−AZT in the HRPE monolayer. Figures 5C and 5D evidence also that during permeation of UDCA−AZT across the HRPE cellular monolayer cumulative AZT amounts appeared in the receiving compartments (pointed lines). In particular, during the A → B transport of

Figure 2. Degradation profiles of the prodrug UDCA−AZT and the corresponding appearance profiles of AZT in rat plasma and whole blood. All the values are reported as the percentage of the overall amount of incubated prodrug. Data are reported as the mean ± SD of three independent experiment.

linear patterns of corresponding semilogarithmic plots (n = 7, r > 0.998, P < 0.0001), suggesting the prodrug degradation governed by hydrolysis processes. The hydrolysis of UDCA− AZT was confirmed by the AZT appearance in incubation media with amounts increasing during time, as reported in Figure 4. In particular, UDCA−AZT appeared totally transformed into the drug AZT after 30 and 60 min of incubation in rat liver and brain homogenates, respectively. Millicell Permeation Studies. The second step of our work was constituted by permeability studies to evaluate the bidirectional transport of UDCA−AZT and AZT across the polarized monolayer obtained by HRPE cells. The HPLC analytical method was the same adopted for the quantification of the compounds incubated in the physiologic fluids. In this

Figure 3. Degradation profiles of the prodrug UDCA−AZT and the corresponding appearance profiles of AZT in human plasma and whole blood. All the values are reported as the percentage of the overall amount of incubated prodrug. Data are reported as the mean ± SD of three independent experiment. 962



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Figure 4. Degradation profiles of the prodrug UDCA−AZT and the corresponding appearance profiles of AZT in rat brain and liver homogenates. All the values are reported as the percentage of the overall amount of incubated prodrug. Data are reported as the mean ± SD of three independent experiment.

Figure 5. Permeation kinetics of AZT [A, B] and UDCA−AZT [C, D] across the Millicell filters alone (filters) or coated by monolayers obtained by HRPE cells (cells). The permeations were analyzed from the apical to basolateral compartments (Basolateral receiving; A → B) [A, C] and from the basolateral to apical compartments (Apical receiving; B → A) [B, D]. The cumulative amounts in the receiving basolateral compartment were linear within 120 min (r ≥ 0.990, P ≤ 0.001). The cumulative amounts in the receiving apical compartment were linear within 120 min for permeations across the filters coated by monolayers (r ≥ 0.996, P < 0.0001), whereas they showed a hyperbolic pattern when the permeation was performed across the filters alone. In this case the linearity was maintained within 30 min (dashed lines, r ≥ 0.984, P ≤ 0.002). The resulting slopes of the linear fits were used in eq 1 for calculation of permeability coefficients (Pt and Pf). The pointed lines are related to the AZT amounts accumulated in the receiving compartments in the case of UDCA−AZT permeation across the filters coated by cellular monolayers [C, D]. No AZT amounts were detected in the receiving compartments in the case of UDCA−AZT permeation across filters alone. All data are reported as mean ± SD of three independent experiments.

derived by the hydrolysis processes appeared, also in this case, to be a substrate of efflux transport systems.

the prodrug the AZT amounts detected in the basolateral compartment during this time were the same (P > 0.05) as the permeated UDCA−AZT, whereas during its B → A transport the AZT amounts in the apical compartment were lower than those of the permeated prodrug (P < 0.01). No AZT amounts were detected in the receiving compartments in the case of UDCA−AZT permeation across filters alone. These data indicate that UDCA−AZT was partially hydrolyzed during the permeation across the HRPE cellular monolayers. AZT

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DISCUSSION The CNS appears to be a sanctuary of HIV, the anti-HIV agents being unable to cross the physiological barriers between blood and CNS.10−12 Similarly, intracellular liver infections caused by pathogenic viruses are difficult to eradicate with conventional drug therapy, because the antiviral agents fail to 963



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Table 1. Permeability Coefficients (PE) (×10−5 cm/min) of AZT and Its Prodrug UDCA−AZT Transported by the Monolayer Obtained by HRPE Cells on Millicell Systema compd

A→B

B→A

AZT UDCA−AZT

209 ± 14 31.3 ± 3.6

133 ± 8b 39.1 ± 1.2

amide conjugates of some gelatinase inhibitors are more rapid in whole blood than in plasma.52 It is therefore known that the esterase activity measured in plasma, in vitro, can be likely an underestimation of the true activity in whole blood due to the absence of erythrocyte esterase activity.53 In human blood UDCA−AZT appeared able to control the release of AZT; this phenomenon may contribute to increase its relatively short half-life in vivo and to reduce the risks related to its dose dependent side effects on bone marrow. In rat brain and liver homogenates UDCA−AZT was hydrolyzed with rates higher (half-lives ranging from 2.70 ± 0.14 to 7.24 ± 0.45 min) than those detected in human blood compartments. As a consequence, the prodrug appeared potentially able to target and release AZT in the CNS or in the liver cells in dependence of its permeation pattern in these compartments. In the aim to study the permeation properties of the prodrug across physiological barriers, we have considered the human retinal pigment epithelium (HRPE) cells (whose main function is to form the natural blood−retinal barrier) as good candidates to constitute an in vitro model for transport studies. We have already utilized an established HRPE cell line to investigate on the involvement of the carrier mediated transport (CMT) by SVCT2 and GLUT 1 of vitamin C- and glucose-conjugated prodrugs of several neuroactive agents into the CNS. Our studies were performed in HRPE cells cultured in classical culture dishes and were based on the intracellular uptake analysis of the compounds.51,54−56 Following these studies, it has been recently proposed that the SVCT2 transporter, expressed on neoplastic tissues and choroid ependymal cells, may be exploited as a potential target for drug-loaded pharmaceutical carriers.57 To improve the culture environment for the HRPE epithelial cell line, Millicell filter inserts were applied, feeding the HRPE cell monolayer with media of different composition at the apical and basolateral side.58 This arrangement allowed the cells to acquire directional polarization characterized by epithelial barrier properties, as demonstrated by TEER measurements (about 80 Ω·cm2). After confluence, the HRPE cell layer separated an upper from a lower compartment. The upper compartment represented the apical side of the HRPE cells, corresponding to the retinal- or neural-facing domain of the retinal pigment epithelium monolayer, whereas the lower compartment represented the basolateral side of the HRPE cells, corresponding to the choroid- or blood-facing side of the monolayer.59 This system provided a very useful tool for investigating blood−brain or blood−CSF and blood−retinal barrier transport functions.60 It is indeed known that RPE cells show, both in vivo and when cultured on filter inserts, TEER values (25−100 Ω·cm2) very similar to those obtained by a cell line deriving from the blood−CSF barrier of the choroid plexus epithelium (CPE).61,62 Tight junctional complexes of the CPE form, therefore, a barrier between the CSF and fenestrated capillaries, similar to the organization of the choroid and RPE. Furthermore, RPE exhibits, along with the CPE, a reversed apical polarity of the electrogenic pump Na,K-ATPase, which is located at the basolateral level in most epithelia, but is expressed at the apical side of both RPE and CPE.63 It was also demonstrated that another continuous cell line of human RPE, ARPE-19 cells, when grown on inserts, develops TEER values very similar to those observed in classic in vitro model of blood−brain barrier represented by RBE4 cells, a SV40immortalized cell line of rat brain endothelium,64 whose main limitation, however, appears precisely due to its rodent and not

a

The values were obtained from Pt and Pf coefficients according to eq 2. The coefficients are referred to the transport from the apical compartment (A) to the basolateral compartment (B) and vice versa . Data are reported as the mean ± SD of three independent experiments. bp < 0.01 as compared to PE value of AZT transported from A to B.

reach the therapeutic concentrations in the cytosol of cells.4 These phenomena have been attributed to the expression of AET systems on the membranes of cells, including those of the BBB and CPs, where they cause the efflux of drugs into the bloodstream.9,15−17,41 An efflux transporter activity was first recognized as “multidrug resistance” during the treatment of tumors with anticancer drugs, and it was attributed to the Pglycoprotein transporter (P-gp) expressed on the cell surfaces.42,43 This efflux transporter was then detected in the brain capillary of several species.44 P-gp is a member of the ATP binding cassette (ABC) superfamily together with multiresistance proteins (MPRs) and breast cancer protein (BCRP).45,46 Other efflux transporters have been recognized to belong to the soluble carriers (SLC) superfamily: these are the organic anion transporters (OATs) and the organic anion transporter polypeptide (OATP).45,46 AZT is a substrate of some of these transporters; as a consequence its ability to reach the cytosol of cells is generally poor and its entry in the CNS is hampered.4,9,15−17 The opportunity to elude these efflux systems should be therefore of great importance in the aim to increase the antiviral effects of AZT both at intracellular level and in the CNS. Interestingly, it has been recently demonstrated that UDCA, a biliary acid known for its antiapoptotic properties,31 is able to permeate the CNS in a dose dependent manner,32 suggesting its ability to elude the AET systems. As a consequence, we have conjugated AZT with UDCA via an ester bond in the aim to evaluate not only its potential ability to be hydrolyzed in physiologic fluids but also its bidirectional transport across a polarized cellular monolayer able to simulate several properties of BBB and CPs. The studies have been performed with the conjugate in the solubilized state, taking into account that, in general, the state of candidates may affect their interaction with enzymes and transporters in the media.47 UDCA−AZT showed high stability in phosphate buffer, whereas it was hydrolyzed in plasma and whole blood, with rates that were greatly different between the rat and human species. In particular, the half-life of UDCA−AZT was lower than 10 s in both plasma and whole blood of rats, whereas the hydrolysis rate was sensibly lower in human whole blood and plasma (half-lives = 3.71 ± 0.16 h and 7.53 ± 0.44 h, respectively) indicating that the plasma of rodents has the strongest carboxylesterase activity, as previously described by other authors.48 In human whole blood the UDCA−AZT degradation was sensibly faster than that in plasma, suggesting that it was governed by enzymatic processes localized in both the plasmatic and cellular components of the blood, as previously found by us for several other ester prodrugs.49−51 Similarly, it has been recently reported that hydrolyses of the 964



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human origin.65 Moreover, some properties, particularly useful for studies of penetration selectivity, were found more appropriate in the case for ARPE-19 cells with respect to RBE4 cells.61,62,66 Furthermore, BBB continuous cell lines have been reported to produce poor correlation between in vivo and in vitro permeability data,65 whereas data based on a wide range of test substances showed that the epithelial blood−retinal barrier largely reflects the pharmacological properties (permeability coefficient and ratio plasma/brain) of the endothelial blood−brain barrier and expresses the same two major efflux pump fundamental efflux systems, such as multidrug resistance protein (P-glycoprotein) and multidrug resistance−associated protein (MRP).67 The above considerations allow us to introduce the established HRPE cells as the startup for obtaining in vitro monolayers showing epithelial barrier characteristics. These monolayers should constitute a valuable model for mechanistic studies of drug transport across the physiologic barriers between blood and CNS. With respect to the monolayers derived by primary culture of CPE cells, the HRPE monolayers were obtained by easier and cheaper ways, allowing avoidance of the problems related to impurity of cell population or the relatively short lifespan.68 Moreover, the human origin of HRPE cells should allow them to share several structural and functional features with the human CPE barrier and several functional features with the human BBB. The apparent permeability coefficient (PE) of AZT across the HRPE monolayers was significantly higher from the apical to basolateral compartments in comparison with the PE value referred to the opposite way, suggesting the presence of active efflux systems that in vivo should transport this drug from the CNS to the bloodstream. These data indicate the conformity of our model not only with the AZT behavior in vivo, where its efflux clearance from CNS has been found to be higher than the influx clearance into CNS,18 but also with cellular models obtained by native or immortalized choroidal epithelial cells, where AZT showed higher efflux than influx apparent permeability coefficients.68,69 The AZT active efflux transporters have yet to be fully characterized at the molecular level. Currently, it is known that AZT is not a P-gp substrate70 and that Bcrp does not play a significant role in limiting the CNS distribution of AZT in rats.71 Moreover, it has been suggested that the AZT efflux can be associated with an Oat system expressed by choroid plexus,69 even if other efflux transporters may be involved.68 We have then employed our HRPE model to study the permeation of UDCA−AZT, whose apparent permeability coefficients appeared slightly lower than that of AZT influx. This phenomenon can be attributed not only to the relatively high molecular weight of the prodrug but also to its partial hydrolysis that we have detected during UDCA−AZT permeation across the cellular monolayer. Also in this case the AZT permeation in the basolateral compartment was higher than that in the apical compartment, confirming the presence of active efflux systems for this drug. On the other hand, the influx and efflux permeability coefficients of UDCA−AZT were not different between them, suggesting that this prodrug is not a substrate of active efflux transporters. On the basis of these data we can hypothesize that UDCA−AZT is not effluxed from body compartments, including the CNS, that normally actively efflux AZT. A similar CNS drug delivery system could potentially be used in the treatment of other neurological impairments such as Alzheimer or Parkinson’s disease.

Taking into account the aspects described above, the prodrug UDCA−AZT appears to be a good candidate for intravenous administration after its encapsulation in long circulating or functionalized nanoparticles able to promote brain targeting.72−75 Moreover, UDCA−AZT seems suitable for nasal administration, a promising way for the brain uptake of neuroactive agents.76,77 We have demonstrated that microparticulate formulations based on chitosan can be useful in promoting the CNS entry of neuroactive drugs via the nasal way.78,79 Our results may help guide and improve future pharmacological treatment against HIV, even if further studies will be necessary to better investigate the in vivo administration and effects of the prodrug.

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CONCLUSIONS

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AUTHOR INFORMATION

We have proposed a new prodrug obtained by the conjugation of AZT with UDCA, in the aim to obviate the poor ability of AZT to permeate in the CNS or intracellular compartments. It is indeed known that AZT is a substrate of active efflux transporters that hamper it to reach the brain and intracellular therapeutic targets. On the other hand, it has been very recently reported that UDCA is able to permeate into the CNS in a dose dependent manner, suggesting its ability to avoid the AET systems. We have demonstrated that the prodrug UDCA−AZT is hydrolyzed in human blood compartments, inducing a controlled release of AZT. The release profile should allow, in vivo, mitigation of its dose dependent unwanted effects on bone marrow. UDCA−AZT was hydrolyzed also in homogenates of rat brain and liver, suggesting the potential ability of this prodrug to release AZT in these compartments, provided that it is able pass through. Taking into account the increasing demands of in vitro models that can allow study of the mechanistic phenomena related to the permeation of drugs across physiological barriers, we have employed an established HRPE cell line to obtain a cellular monolayer showing epithelial barrier characteristics. We analyzed the permeability of AZT and UDCA−AZT for this monolayer evidencing that the influx permeation coefficient of AZT was lower than that of the efflux rate, in conformity with the AZT behavior in vivo. On the other hand, the influx and efflux permeation coefficients of UDCA−AZT were the same, suggesting the ability of the prodrug to avoid the AET systems, and thus, its potential ability, in vivo, to target AZT in the CNS and at the intracellular level. Taking into account that neither UDCA nor UDCA−AZT appears able to interact with AET systems, the UDCA molecular structure may be proposed as a useful reference for the design of drugs or prodrugs characterized by the potential ability to avoid the AET systems, or, in other words, potentially characterized by the absence of multidrug resistance phenomena.

Corresponding Author

*Department of Biology, General Physiology Section, University of Ferrara, via L. Borsari, 46, 44121 Ferrara, Italy. Phone: +39-0532-455476. Fax: +39-0532-455450. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 965



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ACKNOWLEDGMENTS We thank PCA (Prodotti Chimici Alimentari) spa Basaluzzo (AL), Italy, and ICE srl Reggio Emilia, Italy, for financial support.

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