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Supramolecular drug inclusion complex constructed from cucurbit[7]uril and the hepatitis B drug Adefovir

ABSTRACT The interaction between cucuribit[7]uril (Q[7]) and Adefovir (ADV) has been studied in aqueous solution by 1H NMR spectroscopy, electronic absorption spectroscopy, Isothermal Titration Calorimetry and mass spectrometry. The results revealed that an inclusion complex was formed via encapsulation of the purine rings of the guest ADV, while the phosphonomethoxyethyl group was prevented from entering the cavity. ITC data revealed that the formation of this 1:1 inclusion complex is mainly driven by favourable enthalpy changes. Studies investigating the release of ADV from the inclusion complex revealed enhanced rates under acidic conditions, although the rates were slower than observed for the free guest under the same conditions. Thermal stability studies indicated that the included form of ADV was more stable that the free form. GRAPHICAL ABSTRACT


Introduction
Adefovir (ADV) was originally of interest in the 1990s for the treatment for HIV, but complications with dosage size versus kidney problems led to its withdrawal by the FDA. [1] However, the use of lower dosages proved fruitful for the treatment of hepatitis B, and in the early 2000s, Adefovir was approved for use. [2] The medicinal potential of compounds such as Adefovir can be broadened and/or improved if delivery to specific targets in the body is achieved without degradation. With this in mind, we are interested in the host-guest properties of cucurbit[n]urils, Q[n]s, which given their enhanced solubility, recognition properties, ability to cross cell membranes and favorable toxicity profiles are attractive as containers/scaffolds for drug delivery.
[3] This is exemplified by the work of Isaacs et al who have made use of cucurbit [7]uril to deliver oxaliplatin to cancer cells, [4] whilst Wang et al reported reduced toxicity but preservation of anticancer activity for Q [7] encapsulated camptothecin. [5] The host-guest complex formed between Q [7] and oxaliplatin demonstrated enhanced antitumour activity (using colorectal cells) versus only oxaliplatin, which illustrated the potential for supramolecular chemotherapy. [6] There is also potential for such an approach to be employed in the war against neurodegenerative diseases, such as Parkinson's disease. [7] Other complexation studies on Q[n]s and multinuclear platinum complexes suggested that Q-based  [7] can improve the in-vitro and in-vivo uptake of the dye molecule coumarin-6; [9] coumarin forms a 1:1 inclusion complex with Q [7] but a 2:1 complex with Q[8]. [10] Other drugs such as atenolol, glibenclamide, memantine and paracetamol can be stabilized in the solid state by forming inclusion complexes with Q [7], [11] whilst increased stability (2 to 3x versus similar sized β-cyclodextrin) constants are observed for the anaesthetics procaine, tetracaine, procainamide, dibucaine and prilocaine in aqueous solution. [12] The histamine H2-receptor antagonist rantidine has also exhibited increased stability in acidic aqueous solution in the presence of Q [7], [13] as did the antituberculosis drugs pyrazinamide and isoniazid. [14] Furthermore, the problematic cardiotoxicity of the antituberculosis drug clofazimine can be almost completely eliminated by complexation with Q [7]. [15] It is against this background that we now report our findings on the interaction of Q [7] with Adefovir (ADV) (see Scheme 1). Results are compared against out earlier study of the pro-virucide Adefovir bis(L-leucine propyl)ester (PMEA-Leu) -see scheme 1, left. [16] Scheme 1. Schematic molecular structures of ADV, PMEA-Leu and Q [7].

NMR spectroscopy
In order to investigate the complexation of Q [7] with ADV in solution, 1  were all accommodated within the cavity of Q [7], whereas the phosphonomethoxyethyl was prevented from entering the cavity. These results differ from our early observations for the pro-virucide Adefovir bis(L-leucine propyl)ester (PMEA-Leu), [16] where the two ends of the branches (ie. the leucine propyl groups)

UV absorption spectroscopy
The supramolecular interactions of the Q[7]/ADV host-guest inclusion complex were then further investigated by the use of UV spectroscopy. As shown in Figure 2A  absorption spectra of the ADV became weaker. These observations indicate that the interaction between Q [7] and ADV has occurred. Furthermore, in Figure 2C the stoichiometry was confirmed by a Job's plot, and the UV data can be fitted to a 1:1 binding model.

Isothermal Titration Calorimetry
To study the thermodynamics parameter of the complexation between ADV and Q[7], we conducted ITC experiments at 298.15 K in pure water. The titration graphs and the thermodynamic parameters data are shown in Figure 3 and Table 1 respectively, and the experimental results revealed a Ka value of (4.25±0.22)×10 4 M -1 . This Ka value is indicative of effective binding between ADV and Q [7]. Furthermore, the negative enthalpy variation, ∆H°= (-29.05±0.13) kJ·mol -1 and the negative entropy variation, T∆S° = (-2.41±0.26) kJ·mol -1 , indicate that the formation of the inclusion complex between ADV and Q [7] is mainly driven by favourable enthalpy changes, accompanied by small negative (unfavourable) entropy changes.

MALDI-TOF mass spectrometry
Analysis of the inclusion complex by MALDI-TOF mass spectrometry revealed (see Figure 4) an intense signal at m/z=1436.01, which corresponds to ADV/Q [7] (calculated 1435.53), thereby providing direct support for the formation of the 1:1 stoichiometry for the host-guest inclusion complex ADV/Q [7].

Controlled release behaviour
To understand the controlled release performance of this inclusion complex, the ADV release from inclusion complex ADV/Q [7] was investigated in water at 37 ℃ ( Figure 5). The ADV and ADV/Q [7] are released, via the use of a dialysis bag (for full details see experimental section), on an orbital oscillator and the solution curve for drug release is obtained based on the solution absorption of the drug at different times ( Figure 5). When the solution pH was kept at 6.8 using a NaH2PO4/ Na2HPO4 buffer, the ADV was totally released after 25 min. Whereas, in the case of the inclusion complex ADV/Q [7], the ADV was released from the Q[7] over 145 min, which indicated that the release time of ADV from the inclusion complex was longer than that in the case of the free guest. The released amounts of ADV and ADV/Q [7] were respectively 57.8% and 58.2%. When the pH was about 1.2, the ADV was totally released after 35 min., whilst ADV was released from Q [7] showed over 85 min.; the released amounts of ADV and ADV/Q [7] were 48.7% and 45.1% respectively ( Figure 6). In short, these results indicate that the pH of the solvent medium can act as a trigger for release, ie for the free drug, the released amounts of ADV at pH 6.8 was more than at pH 1.2, and similarly for the ADV/Q [7] complex, at pH 6.8, both the release time and the amounts of drug was more than at pH 1.2.

Thermal stability analysis
From the DTA spectra ( Figure 6), it is also evident that ADV and Q [7] interacted with each other. Analysis of the thermal stabilities is via the use of differential scanning calorimetry (DSC) and thermogravimetry (TG). As shown in Figure

Conclusion
We have investigated the interaction between cucuribit [7]uril (Q [7]) and the hepatitis B drug Adefovir by a variety of techniques such as 1 H NMR spectroscopy, electronic absorption spectroscopy, Isothermal Titration Calorimetry and mass spectrometry. At an ADV:Q [7] ratio in excess of 1:1, NMR observations indicate that the guest purine rings are encapsulated by the Q [7], whilst the phosphonomethoxyethyl was prevented from entering the cavity. ITC results indicate that the formation of the 1:1 inclusion complex is mainly driven by favourable enthalpy changes. The controlled release of ADV from this inclusion complex was investigated in water at 37 o C at pHs 6.8 and 1.2 using an orbital oscillator. The release was faster under more acidic conditions, though in both cases the process was slower than that observed for the free guest.
Analysis of the starting materials and the inclusion complex by differential scanning calorimetry (DSC) indicated that the new inclusion compound afforded a form of ADV that was more thermally stable than the free guest. These studies suggest there is potential to use Q [7] to enhance the stability of ADV-type molecules and to control the release of such drug molecules by manipulation of the pH employed. Further studies are on-going in our laboratory to investigate the ability of Q [7] to act as a scaffold for other drug molecules.

Materials and apparatus
The host Q [7] was prepared according to the literature method. [16] Adefovir (ADV) was obtained from Aldrich and was used without further purification. All other

H NMR spectroscopy
To study the host-guest complexation of Q [7] and ADV, all the 1 H NMR spectra,

Isothermal titration calorimetry (ITC) experiments
Microcalorimetric experiments were conducted using an isothermal titration