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Keywords:

  • capreomycin;
  • drug-palladium complexes;
  • intracellular antitubercular activity;
  • kanamycin;
  • ofloxacin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Declarations
  9. References

Objectives

The aim of this work was to characterize novel palladium (Pd) complexes with second-line antitubercular drugs, namely capreomycin (C), kanamycin (K) and ofloxacin (Ofx), and to address the in vitro extracellular and intracellular activity against Mycobacterium tuberculosis infection.

Methods

Synthesis reaction kinetics and complex properties were assessed. Kf was calculated from the transition state quasi-equilibrium approximation and Arrhenius plot. The complexes were characterized for qualitative solubility, stoichiometry, powder size and morphology, element analysis, and thermal behaviour. Structural analyses were performed by Fourier transform infrared spectroscopy and nuclear magnetic resonance. Activity was evaluated against H37Ra M. tuberculosis strain and in infected THP-1 cells, and compared with that of the parent drugs.

Key findings

The complexes showed log Kf of 6 for CPd and OfxPd, and 10 for KPd indicating good stability. Stoichiometry of 1 : 1, 2 : 3 and 1 : 3 resulted for OfxPd, KPd, and CPd. OfxPd structure matched that in literature, while K and C had more complex structures with possible multiple coexisting species. The complexes had extracellular activity comparable with drugs and an improved efficacy against intracellular infection of M. tuberculosis.

Conclusions

The novel anti-tuberculosis (TB) complexes had promising properties, and extracellular and intracellular activity, which makes them potential tools for intracellular targeting of pulmonary TB.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Declarations
  9. References

The lungs are a well-recognized site for drug administration.[1] Properly tailored drug delivery systems can improve the treatment of lung infections by achieving higher local drug concentrations and reducing systemic exposure.[2, 3] Among other treatments, tuberculosis (TB) therapy may benefit from this strategy.

The efficient clearance of exogenous substances from the lungs complicates the successful development of suitable pulmonary anti-TB drug delivery systems. Various approaches to this problem have been reported, such as the use of polymeric microparticles,[4-6] large porous particles,[7] liposomes[8-12] and polymeric nanoparticles.[13, 14]

These approaches are all aimed at enhancing infected alveolar macrophage targeting. In fact, the Mycobacterium tuberculosis can survive within cells inhibiting lysosome fusion, and this peculiarity makes the alveolar macrophages therapeutic targets.[15] However, the conventional TB therapy is not able to ensure adequate intracellular delivery.[16, 17] In this regard, inhalation may help overcome some of the well-known drawbacks of TB therapy, such as high dose and toxicity, by enhancing local drug accumulation. To do so, rapid drug absorption into the blood stream should be avoided.

Beyond the aforementioned microencapsulation approaches, indeed modulation of drug water solubility is commonly used. Sparingly or highly water-soluble active molecules show often a high burst of drug release from pharmaceutical formulations as they solubilize fast in body fluids. This is the case of injectable second-line antitubercular drugs (sl-ATDs), such as capreomycin (C) and kanamycin (K), and fluoroquinolones like ofloxacin (Ofx).

As a result, they are promptly available for systemic absorption, and in addition, unfavourable physicochemical properties can prevent them from being extensively internalized into cells and from diffusing into tissues.

This is an important aspect as to treat complex forms of TB, like latent and multidrug-resistant TB (MDR-TB), drugs are required to access highly hydrophobic environments where dormant and persistent bacilli find shelter.[18] Moreover, hypoxia, low-nutrient exchange and acidic pH conditions, characterizing the persistent bacilli environment, together with the lack of relevant models of human MDR and latent TB infection limit the successful development of adequate therapies and prevention.[19, 20] These issues represent a tremendous obstacle to the complete eradication of TB infection and explain, together with socio-economic aspects, the widespread latent TB incidence.

Attempts have been made by formulating C as inhalable dry powder;[21-23] nevertheless, such approach is not likely to solve the problem of low tissue penetration and high absorption in the blood stream. Other likely more effective approaches include ionic coupling of C with long-chain fatty acids, such as oleic acid.[24]

Complexation with metals, such as palladium (Pd), may be an alternative. This approach was previously reported for fluoroquinolones, such as Ofx,[25] that in addition has been used to formulate an insoluble dry powder for lung delivery.[26] In spite of the concerns regarding the use of heavy metals in pharmaceutical formulations,[27-32] Pd has been employed to produce a number of active compounds.[33-37] Of course, such concerns limit the development of Pd-bearing medicines; however, data on Pd toxicity are controversial, and as yet fate, adverse reactions as well as accumulation of Pd in the body are insufficiently described in literature.[30] In particular, the effect of the metal when inhaled is unclear, and formulation, therapeutic regime and administration strategy are likely to strongly influence Pd fate and toxicity profile.

The pulmonary administration of the complexes in the form of dry powders may provide sl-ATD with an improved capacity of tissue penetration and an enhanced localized action. According to that, lower dosages and shorter treatments may be conceivable. In this case, the potential benefit of TB therapy improvement may counterbalance the risk of the administration of Pd compounds, especially considering the high sl-ATD systemic toxicity.

In light of these considerations, the present work focused on the synthesis and characterization of new sl-ATD-Pd complexes potentially useful for pulmonary administration in MDR-TB. Based on the previously published complexation strategy for OfxPd, novel sl-ATD-Pd complexes of C (CPd) and K (KPd) with Pd were synthesized and structurally characterized to determine the reaction kinetics and the properties of the complexes. Being OfxPd already reported in literature, the data obtained in this work were compared with those published. The products were then compared with the parent drugs for activity against an M. tuberculosis strain, and in particular, an infected macrophage model was used to test the intracellular activity of such compounds.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Declarations
  9. References

Synthesis of second-line antitubercular drug–palladium complexes

Ofx, K (>90% form A) and C (Sigma Aldrich, Milan, Italy) complexes with Pd were prepared by dropping drug aqueous solutions into a K2PdCl4 (Sigma Aldrich) solution under mixing at room temperature. In the case of Ofx, a dispersion rather than a solution was obtained. Immediately after the addition of the drug, a dark red-brown precipitate started to settle. The reaction mixture was maintained under stirring at room temperature for up to 3 h. The suspension obtained was centrifuged for 10 min at 4000 rpm and 20°C using a Universal 32R Hettich zentrifugen (Tuttlingen, Germany). The solid obtained was carefully washed with water until a clear uncoloured supernatant was obtained, centrifuged and dried for 15 h under vacuum by Edwards high vacuum pump EDM2.

Reaction kinetics study

The reaction kinetics was investigated by measuring complex formation using an Agilent 8453 UV-vis spectrophotometer (Milan, Italy). The analysis was performed by reading the scattering effect elicited by complex precipitation at 800 nm. The kinetic order was determined by assessing the dependence of the reaction rate on each reagent. The initial rate method was used by measuring the difference in absorbance units (ΔAU) between the initial values and those acquired every 30 s over 500 s reaction run. The solutions were allowed to equilibrate at 25°C before each run. All experiments were run in triplicate.

The formation constant Kf was calculated considering a quasi-equilibrium condition approximated by the transition state theory. The Arrhenius plot was used to extrapolate the standard Gibb's free energy at the transition state. Rate constants were determined at 25, 30, 35, 40 and 45°C. Kf was obtained from the standard Gibb's free energy relation at 25°C.

The reaction yield was also used to build a Job's plot[38] to assess the reaction stoichiometry and correlate it with the thermogravimetric differential thermal analysis (TG-DTA) study. At the obtained stoichiometry, the reaction yield was then followed over time to obtain the reaction completion time profile.

Background and calculations

The initial rate method consists in the measurement of the rate of reaction at short times before significant changes in reagent concentrations occur.[38] The general rate equation for the formation of the metal complexes can be written in the following form:

  • display math(1)

where k is the rate constant, [D] and [M] the concentrations of the drug and the metal, respectively, and d and m the exponents indicating the order with respect to the two reagents.

The method was applied by performing rate measurements at increasing reagent concentrations and by comparing initial rates as follows:

  • display math(2)

From equation (2), it is possible to calculate d and m by varying one concentration at a time.

The overall order of reaction is thereby calculated as sum of the two single exponents.

Quasi-equilibrium complex formation constants, Kf, were calculated by applying the transition state theory and the Arrhenius relation (equation (3)).

  • display math(3)

where k is the rate constant, A the pre-exponential factor, E the activation energy of the reaction, R the universal gas constant and T the absolute temperature.

Applying the quasi-equilibrium assumption,[39] the reaction in study can be written as

  • display math

where D is the drug, M the metal ligand, [D-M] the complex at the transition state and DM the reaction product.

The transition state theory assumes that even when reagents and products are not in equilibrium, the complex at the transition state is in quasi-equilibrium with the reagents, and therefore, an equilibrium formation constant Kf can be written (equation (4)).

  • display math(4)

The transition state theory allows approximation of the Arrhenius equation in the following form:[40]

  • display math(5)

where ΔG0 is the standard Gibb's free energy of the transition state, kB is the Boltzmann's constant and h the Planck's constant, and T the absolute temperature.

Equation (5) was used to build an Arrhenius plot and to extrapolate ΔG0, and the general expression for the standard Gibb's free energy in the quasi-equilibrium assumption (equation (6)) was thereby used to calculate Kf at 25°C.

  • display math(6)

Then, equation (6) can be written as:

  • display math(7)

Characterization of the complexes

A preliminary qualitative solubility check was performed in water, 1 m HCl, 0.1 m NaOH, 1 m acetic acid and different organic solvents (i.e. dimethylsulfoxide (DMSO), acetone, dichloromethane, chloroform, ethyl acetate, dimethylformamide, acetonitrile, methanol, tetrahydrofuran; JT Baker, analytical grade) by suspending the obtained complexes upon sonication for 1 min and incubation at 25°C for 24 h under magnetic stirring.

The Pd content in the complex was determined by using a Varian 700-Es series inductively coupled plasma optical emission spectroscopy (ICP-OES) system.

Samples were prepared by breaking down the complex in 1 m HNO3 solution by sonication at 60°C for 1 h and orbital rotation for 24 h. The solutions obtained were diluted to fit the 1–12 μg/ml concentration range that was used to build the standard curve.

TG-DTA were performed with a Netzsch STA 449C apparatus, in air flow and heating rate of 10°C/min.

Particle size analysis was performed by a PSS Accusizer C770 (Particle Sizing Systems, Santa Barbara, CA, USA). Samples were suspended in 1% Tween 80 water solution. Mean size was expressed as volume mean diameter (VMD). Morphology was evaluated using a FEG LEO 1525 and elemental composition was determined by Bruker energy dispersive x-ray spectroscopy (EDX) (Milan, Italy). The acceleration potential voltage was maintained at 10 keV. Samples were suspended in water and placed onto carbon tape-coated aluminum stubs. After complete water evaporation, the stubs were sputter-coated with chromium before imaging and analysed. Coating was performed at 20 mA for 30 s.

X-ray powder diffraction patterns were taken with a Philips X'PERT PRO MPD diffractometer operating at 40 kV and 40 mA, with a step size 0.0170 2θ degree and step scan 20 s, using Cu Kα radiation and an X'Celerator detector.

Thermal behaviour was evaluated by differential scanning calorimetry (DSC) using a Mettler Toledo DSC 821 differential calorimeter (Greifensee, Switzerland) equipped with a liquid nitrogen cooling system. The system was calibrated using an indium standard. The complexes were compared with the drugs and the Pd salt profiles. Samples were hermetically sealed in a pin-holed lid 40 μl aluminum pans and scanned at a 5°C/min heating rate between 25 and 350°C. DSC data were treated with STARe software.

Structural characterization

Fourier transform infrared spectroscopy (FTIR)

FTIR spectroscopy analysis was carried out using a Bruker IFS28 spectrometer equipped with a deuterium triglycine sulfate detector. Transmission spectra of drugs and complexes were collected on potassium bromide pellets at room temperature. All spectra were analysed using the KnowItAll® academic edition v.9.5 software.

Nuclear magnetic resonance (NMR)

1H- and two-dimensional correlation spectroscopy (COSY) were recorded at 400 MHz on a Bruker Avance-DRX 400 instrument using standard sequences for the acquisitions. All the chemical shifts are referred to the frequency of tetramethylsilane. As preliminarily established, the complexes and the free drugs were dissolved as follows: C, K, CPd and KPd in a 50 : 50 0.1 M HCl/d6-DMSO, and Ofx and OfxPd in 50 : 50 water/d6-DMSO. These conditions granted stability of the compounds. The strong signal of water was checked for interference with the main diagnostic peaks of drugs and complexes at 296 K.

In vitro activity against Mycobacterium tuberculosis

M. tuberculosis H37Ra strain (American Type Culture Collection 25177) was grown in complete 7H9 broth (supplemented with Middlebrooks ADC enrichment, Difco Laboratories, Detroit, MI, USA) at 37°C, and harvested at mid-exponential phase after 10 days at a density of 2–5 × 107 bacilli/100 μl until use.

The bacteria were separated by centrifugation of 1 ml of suspension in 7H9 broth, resuspended in RPMI-1640, vortexed for 2 min and bath-sonicated for 5 min. The obtained dispersion was rested for 5 min. The drugs, Pd salt and complexes were added to the 7H10 medium according to the scheme in Table 1. Five millilitres of each were seeded into labelled quadrants of sterile Petri dishes corresponding to the four groups in Table 2, that is, control (free of any antimycobacterial agent or salt), drug, Pd salt and complex. After gelling, the Petri plates were inoculated with 100 μl of 100, 10−2 and 10−3 dilutions of a suspension of M. tuberculosis H37Ra strain equivalent to a McFarland 1 standard.[41] Each test was performed in triplicate.

Table 1. Groups and treatments used in the activity assay against H37Ra Mycobacterium tuberculosis strain
GroupsTreatmentDrugConcentration (μg/ml)
C1C2C3C4
  1. K, kanamycin; Ofx, ofloxacin; Pd, palladium

1Bacteria 
2Bacteria + drugC2.557.510
K1.252.5510
Ofx0.250.512
3Bacteria + PdPdEquivalent to CPdEquivalent to CPdEquivalent to CPdEquivalent to CPd
PdEquivalent to KPdEquivalent to KPdEquivalent to KPdEquivalent to KPd
PdEquivalent to OfxPdEquivalent to OfxPdEquivalent to OfxPdEquivalent to OfxPd
4Bacteria + complexCPdEquivalent to CEquivalent to CEquivalent to CEquivalent to C
KPdEquivalent to KEquivalent to KEquivalent to KEquivalent to K
OfxPdEquivalent to OfxEquivalent to OfxEquivalent to OfxEquivalent to Ofx
Table 2. Reaction stoichiometry, kinetics and Kf for the complexes at 25°C
Balanced reaction stoichiometryOrder of reactionlog Kf
PdDrugOverall
  1. k, kanamycin; K, potassium; Pd, palladium.

C + 3K2PdCl4 = C(PdCl2)3 + 6KCl0.411.46.06
2 k + 3K2PdCl4 = k2(PdCl2)3 + 6KCl0.533.510.10
Ofx + K2PdCl4 = Ofx(PdCl2) + 2KCl0.21.21.46.11

The inoculated plates were then incubated at 37°C, 5% CO2 for 21 days, and colony counting was performed. The results were expressed as the mean of viable counts, namely colony forming units (CFUs), ±standard error compared with the growth of bacteria in the control. A treatment was considered bactericidal if it reduced the 90% of the bacterial viable counts compared with that in the control.[41]

In vitro intracellular activity in infected macrophages

The human leukaemic macrophage cell line (THP-1, human acute monocytic leukaemia, BS TCL 138) was routinely maintained as suspended cells in RPMI-1640 media (Lonza Biowhittaker, Basel, Switzerland) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Lonza Biowhittaker) at 37°C in a 5% CO2 humidified incubator. Cells were grown to a density of 0.8–1 × 106 cells/ml and passaged every 3 days. Before infection with mycobacteria, THP-1 cells at 106 cells/ml were stimulated overnight in a 24-well plates (Falcon, BD Bioscience, Le Pont de Claix cedex, France) with 20 ng/ml phorbol 12-myristate 13-acetate (Microtech, Naples, Italy) to stop proliferation and to allow the cells to adhere and express a macrophage-like phenotype. Non-adherent cells were removed by washing twice with RPMI-1640 media, without serum. Before infection with bacteria, toxicity of drugs, complexes and Pd at the maximum concentrations employed in this experiment (10 μg/ml for C and K, and 2 μg/ml for Ofx) was ruled out by incubation of THP-1 cells up to 72 h. Three-millimetre-diameter glass beads were added to 3 ml of M. tuberculosis H37Ra cultured in 7H9 broth and vigorously vortexed for 5 min to homogenize the suspension. An incubation of 30 min was performed to allow the large particles to settle, and the supernatant was adjusted to a turbidity equivalent to a McFarland standard of 0.5 (1.5 × 108 mycobacteria/ml), and the work dilution of 10−3 was performed to obtain the best final results. This procedure should afford an infection of 5–10 bacilli/cell.

One hundred microlitres of the bacteria suspension was added to each well in plates previously inoculated with THP-1 cells. The cells were infected in triplicate.

After 4 h of incubation, cells were washed with RPMI-1640 medium without serum, and 1 ml/well of each treatment solutions/suspensions were added. Drugs and complexes were added at the maximum concentration used in the previous susceptibility study, which corresponded to 10 μg/ml for C and K, and 2 μg/ml for Ofx.

The growth of bacteria in macrophages incubated with the different solutions/suspensions, and the controls without drugs were evaluated at 3, 7 and 10 days after treatment. At the end of the period of incubation, the supernatant of each well was centrifuged at 300 g for 10 min to pellet the detached cells. These and the adherent cells in the wells were treated with 100 μl of sodium dodecyl sulfate 1% for 30 min at 37°C, followed by 100 μl of bovine serum albumin 1% for neutralization. Then, 30 μl of the lysates were plated in triplicate on complete solid Middlebrooks 7H10 medium (supplemented with Middlebrooks OADC enrichment, Difco Laboratories). The colony growth was counted after an incubation of 21 days at 37°C in a humidified 5% CO2 incubator.

The results were expressed as CFU ± standard error compared with the growth of bacteria in the untreated cells. The result was considered readable when the count of bacteria was included between 50 and 100 colonies/inoculum of lysate, independently from the initial inoculum of bacteria in the cells.

Statistical analysis

Activity data were analysed for statistical differences by applying two-way ANOVA and all pairwise multiple comparison post-hoc Holm-Sidak's method to determine differences among and within groups at a 0.05 significance level.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Declarations
  9. References

Complex formation kinetics and yield

The reaction kinetics of complex formation was followed by measuring the absorbance at 800 nm upon precipitation of the insoluble complex. The initial rate method was employed to determine the overall order of reaction. In this regard, the rate expressed as ΔAU was determined by keeping alternatively constant the drug and the metal at 25°C. The curves obtained are reported in Figure 1. A close observation of the plots revealed that the kinetics were nearly zero order for the metal in the case of Ofx and always <0.5 in the case of C and K (Table 2). For the drugs, the order was much higher, and it reached 3 for K, whereas it was nearly first order in C and Ofx.

figure

Figure 1. Initial rate plots for the complex formation. The plots were used to determine the reaction order.

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As shown in Table 2, the reaction for C and Ofx resulted following an overall pseudo-first-order kinetics, while for K the reaction order was 3.5. This value indicated a complex behaviour of K with Pd. In this regard, the reaction stoichiometry obtained by ICP-OES determination of the amount of metal bound to the drugs was OfxPd 1 : 1, CPd 1 : 3 and KPd 2 : 3 (Table 2). The 1 : 1 ratio for OfxPd corresponds to what already reported by Vieira et al.[25]

The same result was suggested by TG-DTA measurements (Figure 2). OfxPd showed three phases of mass loss: an initial 5.8% up to 200°C, about 31% between 200 and 400°C, and a final 43% between 400 and 650°C (Figure 2a). The following residual mass loss between 780 and 800°C was assigned to 1 mmol PdO that is the main species because of Pd oxidation around 800°C. The PdO residue was confirmed by XDR at 780°C (International Center for Diffraction Data – PdO, ICDD number 41–1107).

figure

Figure 2. Thermogravimetric differential thermal analysis profiles of (a) ofloxacin with palladium, (b) capreomycin with palladium and (c) kanamycin with palladium.

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Likewise, CPd lost 9.8% up to 200°C, and 54.7% between 200 and 500°C. The 7.5 % loss between 500 and 800°C was ascribed to PdO (Figure 2b).

KPd had a 6.8% loss up to 200°C and two-step decrease of 34.2% and 32.7% in the 200–500°C range and residual 2.3% between 500 and 800°C assigned to PdO (Figure 2c).

These profiles were interpreted as an initial water evaporation, degradation and following combustion of the organic matter up to 800°C, at which Pd is oxidized to PdO. These assumptions were confirmed by the DTA profiles. The total mass losses were 82% for OfxPd, 72% for CPd and 76% for KPd, which confirmed the stoichiometry previously determined by ICP-OES analysis.

Further confirmation was then obtained by plotting reaction yield versus the metal molar fraction displayed by the Job's plot in Figure 3a. The maxima obtained for the three complexes matched perfectly the molar ratios of 1 : 1, 1 : 3 and 2 : 3 for OfxPd, CPd and KPd, respectively, corresponding to those previously found by TG-DTA and ICP-OES.

figure

Figure 3. (a) Job's plot built measuring the yield against palladium molar fraction; (b) Arrhenius plots reporting rate constants determined in the range 25–45°C. The plots were used to extrapolate the transition free energy and to calculate the quasi-equilibrium constants; (c) time profile of reaction yield for the complex formation. Values expressed as mean ± standard deviation, n = 3.

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The final balanced reaction expressions are those thereby reported in Table 2.

Using this information, an Arrhenius plot was built to extrapolate the transition free energy and therefore to calculate Kf (Figure 3b). This procedure is based on the transition state theory, which allows determination of Kf under the hypothesis of quasi-equilibrium conditions between the transition state and the reagents (38). The obtained log Kf values at 25°C are reported in Table 2.

The highest value obtained for KPd matched the higher order kinetics, and this suggest higher stability of the complex compared with OfxPd and CPd, which in turn showed similar values of Kf that somehow correlate with the similar pseudo-first-order kinetics earlier measured.

In spite of the higher Kf for KPd, the yield was much lower compared with those obtained for CPd and OfxPd. Maximum yield was >60% w/w for OfxPd, about 40% w/w for CPd and barely 16% w/w for KPd (Figure 3a). Even completion times were different, being faster the formation of OfxPd (1 h) compared with the 3 h required by CPd and KPd (Figure 3c). This is consistent with the lower hindrance and steric repulsion for Ofx that binds Pd just in a 1 : 1 ratio. Moreover, the reaction between Ofx and Pd occurs in different conditions as the drug is much less soluble in water and is mainly present as a solid dispersion. This makes the appearance of the precipitate much faster, while in the other cases, the product precipitation occurs through a slower flocculation process that indeed retards precipitate formation.

Size, morphology and thermal behaviour

The precipitates resulted in a completely amorphous product as observed by XRD analysis (data not shown). OfxPd showed the most irregular granules of about 10 μm in VMD with a rather rough surface and clustered to give larger aggregates (Figure 4a). CPd was characterized by small bean-like-shaped particles with a regular and homogeneous surface and VMD <5 μm (Figure 4b). KPd particles were even smaller (< 1 μm) more irregular in shape and surface, and easily clumped to form clusters of about 10 μm (Figure 4c).

figure

Figure 4. Scanning electron microscopy pictures of (a) ofloxacin with palladium, (b) kanamycin with palladium and (c) capreomycin with palladium powders, aside corresponding particle size distributions of the dried powders of the three complexes.

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EDX analysis of the powders confirmed the presence of Pd together with Cl with intense peaks at about 2.8 and 2.6 keV, respectively, which supports the hypothesis of metal binding to drugs in the form of PdCl2 as already reported in Table 2 (Figure 5).

figure

Figure 5. Energy dispersive x-ray spectroscopy spectrum of the ofloxacin with palladium complex showing the peaks for palladium and Cl. Equivalent spectra were obtained for capreomycin with palladium and kanamycin with palladium complexes.

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The DSC profiles of the complex powders overlap with the DTA profiles previously shown (Figure 6). In fact, all three complexes showed the presence of water with a broad band up to 120–130°C. Moreover, they showed large bands in the range 220–240°C for OfxPd, 250–270°C for KPd and 260–280°C for CPd assigned to thermal disruption of the complexes. These bands match those ascribable to the thermal degradation of the corresponding drugs, which were recorded at 275°C for Ofx, 285°C for K and around 260°C for C, thereby indicating that complex disruption correlates with a degradation process involving the drugs as well. These data suggest a stability rank with temperature: OfxPd < CPd ≈ KPd.

figure

Figure 6. Differential scanning calorimetry profiles of the complexes compared with the drugs and palladium salt.

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Structural characterization of the complexes

All the main FTIR bands were modified by the binding with the metal (Figure 7). In detail, for Ofx, the C = O stretching at 1700/cm was suppressed, while the carbonyl C = O stretching at 1600/cm shifted to 1580/cm. This indicates a specific interaction of both groups with the metal. Moreover, being the difference between the antisymmetric and symmetric COO stretching (1600 e 1400/cm) larger than 200/cm, a monodentate chelation of PdCl2 on the ketone and carboxyl groups was theorized.[42] These results match those already reported by Vieira et al.[25]

figure

Figure 7. Fourier transform infrared spectroscopy profiles of the complexes and the drugs.

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More difficult is the interpretation of FTIR signals in the case of CPd and KPd. Nevertheless, CPd shows a shift of the Amide II band from 1400/cm to about 1550/cm, indicating a likely interaction of the peptide amide groups with the metal. To support this hypothesis, the wide N-H stretching band at 3300/cm became sharper upon binding with the metal, probably as a consequence of a reduce capacity of H-bonding with water.

In addition, some bending signals in the 800–1400/cm region disappeared in the complex, symptom of a higher rigidity of the molecule when bound by the metal.

In the case of KPd, the amine C-N stretching at 1126/cm lost intensity and shifted to 1146/cm. The glycosidic bond C-O-C stretching at 1033/cm showed a slight shift to 1038/cm. This may suggest that Pd binds mainly to the amine groups, as also the N-H bending at 1600/cm shifted down to 1580/cm, with a small possibility of involvement of the oxygen on the glycosidic bond. A further evidence is the split of the O-H and N-H stretching bands at 3348/cm into three distinct bands at 3419, 3219 and 3130/cm; meanwhile, the alkyl C-H stretching at 2926 cm−1remained unchanged.

Unlike for OfxPd, for CPd and KPd from FTIR data it was not possible to establish whether Pd binds in a monodentate way.

NMR was performed by dissolving drugs and complexes in 50 : 50 0.1 m HCl/d6-DMSO and 50 : 50 water/d6-DMSO. The strong signal of water did not impair the analysis of the main diagnostic peaks of the drugs and the complexes when the spectra were recorded at 296 k.

The comparison of 1HNMR spectra of C with those reported in literature[43, 44] allowed us, with the support of the COSY's correlations, to assign the resonance of some characteristic protons confirming that the form I is the major component (>98%) and that it is present as a couple of species (IA and IB) in a ratio of 45 : 55.

In the 1HNMR of CPd, it was observed a general shielding for the NH and NH2 signals that were also affected by a not well-interpretable peak broadness that is probably due to the coexistence of more different Pd complex species. The major shielding effects were recorded for H12 (0.3 ppm) and H9, H10 (up to 0.4 ppm) suggesting an important involvement of the tetrahydropyrimidinic moiety (Figure 8a). Similarly, the shielding of CH2 6’ as unique signal of the β-lysine moiety effected by the presence of Pd may be, in our opinion, correlated to Pd complexation.

figure

Figure 8. Proposed structures and palladium binding of (a) capreomycin, (b) kanamycin and (c) ofloxacin obtained from nuclear magnetic resonance measurements. Numbers indicate the nuclei involved in the interaction with the metal.

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In the case of K, the supplied drug powder was mainly in the form A with a negligible trace of form B. Even in this case, the K 1HNMR profile was compared with that reported in literature.[45]

The 1HNMR spectra of KPd are characterized by a general broadness. Some evident shifts on the resonances of characteristic protons were observed: signals of Ha and Ha’ on C2 position appeared as a collapsed multiplet at 1.5 ppm, slight deshielding was observed for protons in C1 and C3 positions, whereas the C1’ and C1' ’ protons resulted to be shielded at high field frequencies (Figure 8b). On the basis of these evidences, the coordination reported in Figure 8b should be hypothesized, where the two Pd may contemporary or singularly bind the two sites.

The 1HNMR spectra of Ofx[46] and OfxPd suggest, in the latter case, the co-existence of three species, one of which largely more abundant (>95%). The signals relative to the methyl groups on the complex are only slightly shifted (<0.1 ppm) in comparison with those of the native molecule, and this suggest that the piperazine and the morpholine heteroatoms are probably not directly involved in the main interaction with the metal. The =C-H signal at 8.4 ppm was downfield shifted at 8.7 ppm in the complex, and this should be correlated to a different orientation of the anisotropic effects of the carboxylic group blocked by a hydrogen bond in Ofx and by the Pd coordination in the complex (Figure 8c).

In vitro efficacy against Mycobacterium tuberculosis

The analysis of the in vitro activity against M. tuberculosis H37Ra strain demonstrated that the complexes maintain an efficacy comparable with that of the parent drugs (Figure 9). The concentration range was chosen to include the minimal inhibitory concentration (MIC) reported in literature, which against susceptible strains is <4 μg/ml for C and K and <1 μg/ml for Ofx.[47]

figure

Figure 9. Activity against Mycobacterium tuberculosis H37Ra strain of the complexes and drugs. The effect of palladium was also evaluated. The experiment was run at four concentrations according to Table 1. ***, **, * statistically different at 95% significance level (n = 3). Values expressed as mean ± standard deviation.

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Either KPd or OfxPd had identical activity compared with free drugs, with the exception of OfxPd at 0.25 μg/ml that showed a little lower activity compared with the free drug (P < 0.001). A slight but significant activity reduction was instead observed for CPd at 2.5, 5 and 7.5 μg/ml (P < 0.001, P < 0.001, P = 0.067). Overall, the estimated MIC for KPd and OfxPd were practically unchanged compared with the free drugs (4 and 0.5 μg/ml), while it was increased for CPd to about 7.5 μg/ml.

Negligible bactericidal action could be ascribed to Pd, which had some effect only at the highest concentrations in the Ofx and K groups.

More interesting were the findings of the activity in THP-1 infected cells over time (Figure 10). C and CPd in this case had identical killing efficacy at all times investigated. KPd was more effective than K, in particular at 7 and 10 days after treatment with an average of 80% of killing compared with the 55% of the drug (P = 0.002). Ofx and OfxPd were equally 100% effective only after 7 days. Interestingly, at day 3, OfxPd was superior to Ofx by reaching 60% of CFU reduction compared with a bare 40% of the drug (P < 0.001).

figure

Figure 10. Activity against Mycobacterium tuberculosis H37Ra strain in infected THP-1 cells. The experiment was run at the C4 concentration reported in Table 1. **, * statistically different at 95% significance level (n = 3). Values expressed as mean ± standard deviation.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Declarations
  9. References

While OfxPd formation and structure basically matched the work of Vieira et al.[25] with a monodentate binding of PdCl2 on the carboxyl and carbonyl groups of the fluoroquinolone, the kinetics of formation measured, in the case of KPd and perhaps CPd, suggests the possibility of multiple co-existing species.

The difficulty in resolving CPd and KPd structure was due to the molecular complexity of the parent drugs, which are characterized by different forms occurring in different ratios and all participated to the binding with the metal. Therefore, different forms of the complexes can be inferred and the stoichiometry ratios of 1 : 3 for CPd and 2 : 3 for KPd may just be the overall combination of several possible species.

In the case of CPd, the molecule is characterized by four different isoforms: IA, IB, IIA and IIB.[43, 44] As observed from NMR signals, the form I is predominant over the form II that lacks of the β-lysine moiety, while forms A and B occur in a nearly 55-45 ratio, which is consistent with the work of Nomoto et al.[43] From our data, it seems that Pd could give rise not only to intramolecular but also intermolecular binding even in light of the involvement of the lateral β-lysine residue. However, the formation of intermolecular complexes is a remote possibility because of the size of C that produces a considerable hindrance that opposes the vicinal binding of a second C molecule. It is thereby conceivable that the 1 : 3 ratio in CPd could be the result of the intracellular binding of Pd, which can however give rise to multiple species.

This situation may also fit the KPd case, which is almost totally in the form A, where the ratio of 2 : 3 may depict alternation of 1 : 1 and 1 : 2 species. This may exclude the unlikely possibility that Pd could bind two K molecules, which is not consistent with the presence of Cl detected by EDX. Such a hypothesis can be ruled out even considering the high repulsion that would be generated by the proximity of two large K molecules linked to the same centre. This high thermodynamic energy could not conceive the high log Kf value for KPd formation.

Even though we could not determine the nature of binding of Pd in CPd and KPd, the evidence previously obtained indicating the consistent presence of Cl suggests the same situation observed for OfxPd, where the metal binds as PdCl2 in a monodentate way. Of course, a deeper investigation is required to unravel the possible different structures produced by these complicated interactions, but this study is beyond the aims of the present work.

The structural complexity of the complexes may explain the activity reduction observed for CPd compared with the free drug with a significant increase of the MIC value. It must be underlined that all MIC values here reported have been only estimated, as this study was aimed at investigating a comparative activity rather than an absolute efficacy of these compounds. Therefore, all MIC should not be considered as absolute values.

In the case of C-Pd, the presence of Pd may affect the uptake through the bacterial wall. Residues as the β-lysine side chain provide peptide antibiotics with the capacity to interact with bacterial cell walls.[48] The β-lysine residue in C is one of the sites of binding of Pd, and this may partially impair the peptide ability to penetrate bacterial membrane. Another factor affecting uptake may be the insolubility of CPd that, on the one side, may enhance macrophage intake but, on the other side, may reduce the amount of the compound available for bacteria cell uptake over time.

In the case of K, activity was not affected by Pd binding as the amine groups that allow aminoglycoside interaction with bacterial walls are only partially occupied by the metal.[48]

The hydrophobicity provided by Pd binding seemed to partially enhance the intracellular efficacy of the complexes compared with the drugs. In the case of Cpd, such enhanced penetration may have balanced the negative effect previously discussed.

Another important factor to be considered is the stability of Pd binding in the intracellular environment. Even though the Kf values suggest a high binding capacity of the metal, the activity results seem to support a possible liberation of the drug from the complex in the cell milieu. In fact, although in OfxPd the metal binds the quinolone centres recognized as responsible for the killing activity, the complex showed even better performance compared with the free drug. This may infer that the active moiety is freed from the metal inside cells, hypothesis that requires further investigation.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Declarations
  9. References

Novel anti-TB complexes were synthesized reacting in simple and green conditions a Pd salt with sl-ATD. The results for OfxPd confirmed those reported in literature[25]. CPd and KPd showed an expected complexity, which is the result of the complex structural nature of the drugs. Even though not conclusive, a hypothesis of structure was formulated for CPd and KPd on the basis of clear evidence of well-defined metal binding sites and the likely presence of Pd in the chloride form. Further studies will be needed to precisely define the possible existence of multiple forms of the complexes. The complexes showed activity comparable with that of the parent drugs and an improved intracellular efficacy. This outcome may be worth of further investigation to understand whether such compounds may have real potential to enhance intracellular targeting of TB. Moreover, toxicity studies, mandatory in the case of heavy metal-bearing compounds, will be performed to evaluate the real possible impact in TB therapy of these complexes. A step forward will consist in investigating whether such complexes may prevent some of the mechanisms at the origin of resistance development to the drugs employed.

Declarations

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Declarations
  9. References

Conflict of interest

The Author(s) declare(s) that they have no conflicts of interest to disclose.

Acknowledgement

This work was partially performed at the University of Perugia Interdepartmental Laboratories of Nanomaterials (LUNA).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Declarations
  9. References