Improvement of Water Solubility of Josamycin by Inclusion Complex with γ-Cyclodextrin

1 Medicinal Chemistry Research Team, Faculty of Medicine and Pharmacy, University Mohammed V, Souissi, BP 6203, Rabat, Morocco 2 Pharmaceutical and Toxicological analysis Research Team, Laboratory of Pharmacology and Toxicology, Faculty of Medicine and Pharmacy, University Mohammed V, Souissi, BP 6203, Rabat, Morocco 3 Centre de Recherche en Epidémiologie Clinique et Essais thérapeutiques, Faculty of Medicine and Pharmacy, University Mohammed V, Souissi, BP 6203, Rabat, Morocco


Introduction
Cyclodextrins (CDs) (Figure 1) are macrocyclic oligosaccharides constituted by 6, 7, or 8 D-glucose units forming α-, βand γ-cyclodextrin, respectively. They have the property of forming inclusion complex with various quest molecules with suitable polarity and dimension because of their special molecular structure/hydrophobic internal cavity and hydrophilic external surface. The most probable binding involves the insertion of the lipophilic portion of the guest molecule into the cavity of the host and displacement of the water molecules located inside the cavity [1][2][3].
The Complexation with cyclodextrins has been widely used to improve the solubility, dissolution rate, stability, bioavailability of poorly water-soluble drugs, and elimination of undesired properties of drug, such as unpleasant odor and taste [4][5][6][7][8][9]. In our knowledge no study has been reported in the literature which interested to improve solubility of Josamycin propionate.
Josamycin propionate (JMP) ( Figure 2) is a macrolide antibiotic, produced by Streptomyces narbonensis vary [10][11][12], characterized by a large lactone ring, a ketone group and a glycosidically linked amino sugar attached to nucleus which explains the basicity of this antibiotic [13]. According to the Biopharmaceutical Classification System (BCS), this compound can be classed in class II, poorly soluble in aqueous medium but permeable [14,15].
The aim of this study was to prepare and to characterize inclusion complexes of Josamycin propionate in γ-CD, to provide a way to increase their aqueous solubility. Phasesolubility diagram was used to evaluate the solubility of this macrolide and to determine its stability constant of the complex. The characterization of the binary mixtures as performed using differential scanning calorimetry (DSC), powder X-ray diffractometry, and Fourier transformationinfrared spectrometry (FTIR). Finally, the shift in the nuclear magnetic resonance spectroscopy ( 1 H NMR) was performed to confirm the existence of the inclusion complex.

Materials.
Josamycin propionate (pharmaceutical grade) was obtained graciously from National Laboratory of Drug Control (Morocco). γ-cyclodextrin was purchased from Sigma; all other reagents were of analytical grade. Distilled water was used throughout the experiment.

Phase Solubility Studies.
Phase solubility diagrams were obtained at room temperature (27 • C) in water. An excess amount of the guest molecule JMP (100 mg) was added to a series of 5 mL flasks each containing increasing quantities of γ-CD (ranging from 0 to 232 mg/mL). The suspension was shaken for 72 h, after the steady state was reached. A portion of the sample was appropriately diluted with methanol and analyzed by an UV spectrophotometric method at 232 nm to assess the concentration of the Josamycin propionate dissolved according to European Pharmacopeia [13]. The phase solubility was constructed by plotting the total dissolved JMP concentration against the total γ-CD concentration. The stability constant (Ks) of the complex was calculated from the slope of phase solubility diagram according to Higuchi and Connors equation: where S0 is the solubility of JMP in absence of γ-CD.

Preparation of Binary
Mixtures. The complex of Josamycin propionate with γ-CD in 1 : 1 molar ratio was prepared by the coprecipitation method, by dissolving an appropriate amount of JMP (x mg) and γ-CD (y mg) in 25 mL of water ethanol (V/V). The solution was stirred for 10 h and the solvent was removed at 45 • C. The physical mixture (noncomplexed drug) was obtained by mixing an exactly weighed (1 : 1 molar ratio) amounts of JMP and γ-CD powders in mortar with continuous stirring for 10 min.

Powder X-Ray Diffraction (XRD)
. XRD was used as another quantitative measure of crystallinity of JMP and developed to characterize the formation of the inclusion complexation. The powder X-ray diffraction patterns of JMP, γ-CD, physical mixture, and corresponding complex were recorded using an automatic powder diffractometer (Philips X Pert PRO). X-ray patterns were obtained in the angular range of 5-50 • 2θ, with 2 s fixed time for each 0.04 • step.

FTIR Spectroscopy.
The IR spectra of pure materials, physical mixture and corresponding complex were measured, using an FTIR spectrophotometer (TANSOR 27 Bruker ATR) using KBr pelleting. The scans were executed at a resolution of 8 cm −1 from 4000 cm −1 to 500 cm −1 .
2.6. 1 H NMR Spectroscopy. The 1 H NMR spectra of the samples were recorded at 25 • C using the NMR spectrometer (AVANCE 300 Bruker) employing DMSO-d 6 as a solvent. The H 1 NMR chemical shift (Δδ) caused upon complexation were measured only for both H 5 and H 3 which are located inside the cyclodextrin cavity, to confirm the inclusion complex of JMP and calculated according to the following formula:

Solubility Studies.
The phase solubility diagram of JMPγ-CD system was reported in (Figure 3).The solubility of JMP increases linearly as a function of γ-CD concentration (0-250 mg/mL). And the solubility curve can be classified as A P type [2], according to the Higuchi and Connors classification. When there is a linear increase in guest molecule solubility with increase in cyclodextrin concentration, a cyclodextrin complex of the guest results from 1 : 2 mol/mol interactions. According with this theory, it is possible to assume that 1 : 2 mol/mol JMP/γ-CD inclusion compound was formed. The solubility calculated for JMP in water was 0.35 mg/mL at 27 • C; this solubility increased linearly giving 4.60 mg/mL in the presence of 250 mg/mL γ-CD. The stability constant value calculated was 3060 M −1 , also the larger constant observed indicate that JMP interact strongly with γ-CD. Furthermore the correlation between the solubility of JMP and the concentration of γ-CD suggested that water soluble complex was formed [14][15][16]. Figure 4 shows the infrared spectra of γ-CD, JMP, physical mixture, and corresponding complex. JMP has a carbonyl band of 1600-1750 cm −1 , may be due to a ring lactone [17,18], in the spectrum of the physical mixture there is no change. Whereas in the IR spectrum of the corresponding complex there is a significance decrease was observed in its intensity. IR spectrum of JMP contains a band with shoulders (3000-3500 cm −1 ) due to hydrate form [19].

IR Spectra Studies.
In the spectrum of γ-CD some bands in the range 1030-1160 cm −1 can be associated with the stretching frequency of primary and secondary C-OH groups [20], those bands disappearance in the complex.
The intensity and shape of bands between 800-1500 cm −1 changed dramatically for the inclusion compound as compared to those for pure JMP, γ-CD, and physical mixture. Thus indicating that the vibrating and bending of the guest molecule (JMP) was restricted due to the formation of inclusion complex [17,20,21].

X-Ray Diffraction Analysis.
The XRD pattern of pure drug presented several diffraction peaks indicating the crystalline nature of the drug (Figure 5), the γ-CD also exhibited a typical crystalline diffraction pattern. Among the physical mixture showed several peaks attributable both to the crystalline drug and γ-CD. The coevaporated product showed a single very broad band in witch the diffraction peaks of drug and γ-CD disappeared. This phenomenon confirmed that an inclusion complex between drug and γ-CD was formed and which indicates the formation of a new crystalline phase [21][22][23][24][25][26].  cavity and are very important for the study of the interaction of guest molecules with cyclodextrins. The NMR spectra ( Figure 6) and the partial 1 H NMR spectra in DMSO-d 6 of pure γ-CD and co-evaporated product (Figure 7) show that the inclusion of JMP into the γ-CD induces upfield changes in the 1 H NMR chemical shift values for both the protons H3 and H5 with (Δδ = 0.0306 ppm and Δδ = 0.0106 ppm), respectively. It is noteworthy that NMR allows clear distinction between inclusion and any other possible external interaction processes, with large effects observed on the proton located in the hydrophobic cavity (H3 and H5), clearly proving inclusion in aqueous medium. On the other hand, protons located on the outside experience no or very small shift [27,28].
As shown in (Figure 7) and in the partial 1 H NMR spectra in DMSO-d 6 of pure JMP and coevaporated product (Figure 8), the singlet peak at the value of the chemical shift of 4.16 ppm which can be attributed to the methyl group belonging to the portion of the conjugated carbon chain of pure JMP disappears in the association complex.
However, it is probably that (-CH=CH-CH=CH-CH 2 -C | H-CH3) part of JMP structure, which is highly hydrophobic, was completely embedded inside the lipophilic core of γ-cyclodextrin.

Conclusion
The results of this study clearly evidence that the Josamycin propionate can be efficiently complexed with γ-cyclodextrin in a relatively high proportion forming an inclusion complex.
The properties of complex were characterized by phasesolubility techniques, X-ray diffraction, FTIR spectroscopy, and 1 H NMR spectroscopy. Thus, the pharmaceutical property of aqueous solubility of Josamycin propionate can be improved.