A. Patel1,2, C.P. Page1, M.B. Brown3,4, S.A. Jones2

Prof. Robert Hider and Dr. Kong Xiole

MedPharm Ltd. and the EPSRC for providing the funding for this research project

References

Acknowledgements

1.  Serajuddin AT (2007). Salt formation to improve drug solubility. Advanced Drug Delivery Reviews 59(7): 603-616.

2.  Dai J, Mendonsa SD, Bowser MT, Lucy CA, Carr PW (2005). Effect of anionic additive type on ion pair formation constants of basic pharmaceuticals. Journal of Chromatography A 1069(2): 225-234.

Introduction and Aims Methods

1The Sackler Institute of Pulmonary Pharmacology, Institute of Pharmaceutical Science, King’s College London, 150 Stamford Street, London, SE1 9NH, UK 2Drug Delivery Research Group, Institute of Pharmaceutical Science, King’s College London, 150 Stamford Street, London, SE1 9NH, UK.

3MedPharm Ltd, 50 Occam Road, Surrey Research Park, Guildford, Surrey, GU2 7AB, UK 4School of Pharmacy, University of Hertfordshire, College Lane, Hatfield, Herts, AL10 9AB, UK

For an ion-pair to form however an excess amount of counter ion is required. This can be supplied in the formulation. The aim of this work was to form a salbutamol sulphate ion-pair and to characterise its transport properties. The objectives of this work were:

To determine the affinity constant between salbutamol and sulphate in solution using Fourier transform infra red (FTIR) spectroscopy.

To assess how salbutamol transported across a model membrane when presented as an ion- pair.

-1 0 1 2 3 40

25

50

75

100 pK = 1.505R2 = 0.9877

- Log free sulphate (M)

SAL

unbo

und

(%)

Figure 2. FTIR binding affinity plot for salbutamol (SAL) sulphate (2:1) in solution. Data expressed as percentage (%) unbound against the negative log of free sulphate concentration.

0 50 100 150 200 2500

50100150200250300350400450500550

SB:SS 1 : 0

SB:SS 1 : 0.7

SB:SS1 : 0.006

Time (min)

Cum

ulat

ive

Mas

s pe

r Ar

ea (µ

g/cm

2 )

Figure 4. Cumulative mass of salbutamol permeation per unit area for salbutamol base (SB) and salbutamol in the presence of sulphate (SS) into de - ionised water (pH 7.4) across RCM (n = 5). Each point represents the mean ± SD.

Salbutamol formed an ion-pair in solution. The strength of the interaction was considered to be in-line with values reported in the literature (Dai et al., 2005). Molecular modelling of the bound complex showed multiple hydrogen bonds formed between the salbutamol - sulphate ion-pair. The transport data showed that the physicochemical properties of salbutamol had been modified by the presence of sulphate.

Results and Discussion

An investigation into salbutamol sulphate ion pair interactions and their influence on transport across a hydrophilic membrane

The affinity constant (pK) was used to calculate the percentage of salbutamol bound to sulphate in solution using speciation software. At 0.0673 sulphate concentration 68% of salbutamol (0.00178 M) was bound to sulphate (table 2).

The steady state flux of salbutamol (0.00178 M) in the presence of 0.0673 M sulphate concentration was 1.54 ± 0.17 µg/cm2/min between 30-120 minutes (P ≤ 0.0001, two-tailed, unpaired, Student’s t-test) (table 2).

Atoms Atom number Bond distance (Å)

H ammonium – O alkoxide H(28)-O(42) 1.8609

H ammonium – O alkoxide H(72)-O(43) 1.7691

H ammonium – O alkoxide H(44)-O(42) 1.7168

H alcohol – O oxo H(24)-O(41) 1.7925

H alcohol – O oxo H(74)-O(41) 1.8284

Figure 3. (a) 3D conformational model of salbutamol sulphate ion-pair (2:1), (carbon atoms in grey, oxygen atoms in red, nitrogen atoms in blue, sulphate atom in yellow & hydrogen atoms in white); (b) structural formula of salbutamol sulphate with the atom numbers and (c) the intermolecular hydrogen bonds formed between salbutamol sulphate and the bond distance calculated using ChemBio 3D version 12.0.2..

Conclusion

Figure 1. Schematic representation of a charged ion (A+) & a neutral ion-pair [A+B-] absorption across the membrane.

Pharmaceutical salts are important as converting an acidic or basic drug into a salt has the ability to modify the physicochemical properties of the parent molecule (Serajuddin, 2007). If the salt associates in solution it can reduce the overall charge of the parent molecule, hence modifying the solution state properties.

Fourier transform infra red (FT-IR) spectroscopy:

All spectras were obtained with a universal Omni-Cell system (SpecAc, UK) for the analysis of liquids equipped with CaF2 windows (Mylar spacer at 0.1 mm).

A resolution of 4 cm-1 was employed for the scans captured over the 550 – 4000 cm-1 range.

Test solutions of salbutamol base & salbutamol in the presence of sulphate were prepared in deuterated water & deuterated methanol (80:20) at pH 6.5.

Salbutamol transport:

Transport of salbutamol across regenerated cellulose membrane (RCM, Medicell International, London, U.K) was assessed using individually calibrated Franz cells (MedPharm Ltd, UK) at 37°C over a period of four hours.

The donor solution consisted of salbutamol (0.00178 M) & salbutamol in the presence of increasing sulphate concentration (0.00000673 - 0.0673 M) made up to volume in de - ionised water (pH 7.4).

Salbutamol content was determined using HPLC (LOD = 1.25 µg/ml & LOQ = 4.15 µg/ml, between 1 & 100 µg/ml with a CV ≤ 2%).

A reduction in salbutamol transport across RCM was observed with an increase in sulphate concentration (fig. 4).

SAL concentration (M)

SAL bound to sulphate (%)

Steady state flux of salbutamol in the presence sulphate (µg/cm2/min)

0 0 3.09 ± 0.29 0.00000673 0.02 2.22 ± 0.22 0.0000213 0.06 2.23 ± 0.13 0.0000673 0.20 1.75 ± 0.47 0.000213 0.64 2.09 ± 0.22 0.000673 2.00 1.39 ± 0.14

0.0673 67.87 1.54 ± 0.17

Using FT-IR the N - H bend of salbutamol (1606 cm-1) was shown to be sensitive to the presence of sulphate (1617 cm-1). A peak height ratio (1606 cm-1 and 1617 cm-1) change was detected when in the presence of increasing sulphate concentration. A log plot of salbutamol to sulphate ion-pair binding was constructed from the peak height ratio using GraphPad Prism (Fig. 2).

Table 2. The flux (µg/cm2/min) of salbutamol base (SAL, 0.00178 M) in the presence of increasing sulphate concentration (pH 7.4) through RCM over 4 hours. Fux represents mean ± SD (n = 5).

3D modelling determined the number of hydrogen bonds formed between salbutamol and sulphate (figure 3).

At a 2:1 salbutamol to sulphate ion-pair complex 5 hydrogen bonds are formed between the parent drug and counter ion.

(a) (b)

(C)

42

43

41 28;  44

             72

74

24