Investigation on factors affecting drug delivery using polymers and phospholipids 3

CHAPTER Effect of Cyclodextrin-Drug Complexation and Drug Ionization on the Permeation of Haloperidol Across Human Skin 3.1 Introduction According to Fick’s law of diffusion, the delivery rate of molecules across the skin is dependent on their physicochemical properties, partitioning coefficients and solubilities. Higher drug solubility, increases the drug concentration in the donor phase resulting in increased permeability (Ceschel et al., 2005; Wang et al., 2005). Haloperidol (HP) is practically insoluble in water and has a basic pK of 8.3 (Lim et al., 2006). To increase the solubility, pH of 2.5-3.8 and 2.5-4.5 are used for injection and oral dosage forms respectively. However such acidic solutions can cause irritation in the site of injection (Loukas et al., 1997). Current approaches to soubilize water-insoluble drugs are complex formation with cyclodextrins, liposomes, microemulsion-based drug delivery systems and supersaturation. Cyclodextrins (CDs) are attractive candidates for increasing the aqueous solubilities of lipophilic drugs. They are cyclic oligosaccharides of D- glucopyranose units in the shape of cones, each with an outer hydrophilic surface and an inner hydrophobic cavity. The solubilization effect of CDs is due to the formation of a non-covalent water soluble inclusion complex, therefore drug-CD complexes are 47 easily dissociated and in equilibrium with free drug (Loukas et al., 1997; Liu et al., 2003). Due to the solubility, hygroscopicity and toxicity concerns of CDs, they were modified and examples are hydroxypropyl β-CDs (HP β-CDs) and randomly methylated β-CD (RM β-CDs) (Liu et al., 2003; Gibaud et al., 2005; Murthy et al., 2004). CD derivatives can influence the solubilities of drugs (Loukas et al., 1997; Liu et al., 2003; Sigurðardóttir and Loftsson 1995). They were also reported to decrease local irritation (Amdidouche et al., 1994; Hoshino et al., 1989; Ventura et al., 2006) as well as stabilize photosensitive drugs (Godwin et al., 2006). Some investigators reported that CDs increased the skin permeation rates of drugs by extracting the lipid from the skin (Bently et al., 1997; Okamoto et al., 1986; Uekama et al., 1982; Vianna et al., 1998) while others reported that CDs did not show any enhancing effect on the flux rates of drugs through the skin (Larrucea et al., 2001; Shaker et al., 2003; Williams et al., 1998). The pH of the vehicle influences the solubility and partitioning of the drug into the skin, implying that the ionized and unionized moieties of a drug influence its solubility and partitioning through stratum corneum and hence affect the skin permeation (Hadgraft and Valenta 2000; Sridevi and Diwan 2000a and b). Wagner’s group reported that pH values of donor and receptor compartments influence skin pH and change the permeability of the drug (Wagner et al., 2003). On the contrary, Sznitowska’s team reported that there were no significant differences in permeability 48 of hydrocortisone in the pH range of 1-10, and only extreme pH values affected permeability (Sznitowska et al., 2001; Thune et al., 1988). To further understand the effect of CD and pH, the aim of the present work is to investigate the solubility and permeation of a drug from CD inclusions. For this purpose the complexation of haloperidol with two derivatives of β-CD (RM β-CD and HP β-CD) at pH were studied by the phase solubility method. Molecular modeling was conducted using DM β-CD (Dimethyl--cyclodextrin) and HP β-CD. Surface tension and contact angle measurements were carried out to further elucidate the effect of CDs on the permeability of HP though human epidermis. The effect of concentrations of RM β-CD alone and then combined with limonene on the skin permeation were studied. To elucidate the influence of pH of the donor phase on skin permeability, further experiments using phosphate buffer at pH in the donor compartment alone and also in combination with RM β-CD were carried out. Then, RM β-CD was added to the receptor solution to maintain a sink condition, while the donor compartment consisted of solutions of HP in RM β-CD or propylene glycol. 3.2 Materials and Methods 3.2.1 Materials Haloperidol (HP) was purchased from Sigma, Singapore. 2-hydroxypropyl-β- cyclodextrin (HP β-CD) (degree of substitution of about 0.6) and randomly methylated-β-cyclodextrin (RM β-CD) (degree of substitution of about 1.8) were kind gifts from Roquette (Lestrem, France) and Wacker (Burghausen, Germany), 49 respectively. 3.2.2 HPLC Analysis HP concentration was quantified by HPLC from Shimadzu (Kyoto, Japan) 2010A. The analysis was carried out using a reversed-phase Waters Symmetry Shield column (3.5 m, 3.0 mm  100 mm). Mobile phase was a 55:45 volume ratio of acetonitrile and 0.05M phosphate buffer adjusted to pH using phosphoric acid, flowing at a rate of 0.4 ml/min. UV detection at wave length 254 nm, injection volume 100 μL gave a retention time of min. Standard solutions of HP (0.05 - μg/ml) were prepared in 0.03% v/v lactic acid (Lim et al., 2006). 3.2.3 Molecular Modeling Molecular modeling was carried out to elaborate the complexation modes. Dimethyl-cyclodextrin (DM β-CD) was adopted as a substitution for RM β-CD (degree of substitution = 1.8) to facilitate the determination of the stable structure of the haloperidol-RM β-CD complex, for RM β-CD is a mixture of different structures. HP β-CD, with a degree of substitution of 0.6 was used for the experimental study; four 2-hydroxypropyl groups were added on the primary hydroxyl groups of cyclodextrin (Mura et al., 1995). The structures of haloperidol, DM β-CD and HP βCD were individually minimized by MMFF94s force field using software SYBYL version 7.2 (Tripos Co., USA). The structures of haloperidol, DM β-CD and HP βCD were individually minimized by MMFF94s force field using software SYBYL version 7.2 The HP molecule, in its favorable conformation was introduced into the 50 respective DM β-CD and HP β-CD cavities and the interaction energies were computed. The most likely conformation of each complex was the one with the lowest interaction energy. 3.2.4 Phase Solubility Studies Drug-CD inclusion complexes were prepared by adding an excess concentration of HP (15 mg/ml), dissolved in water or buffer phosphate (pH 5), using RM β-CD and HP β-CD solutions of different concentrations (0, 0.01, 0.05, 0.1, 0.2, 0.3 M). The suspensions were shaken on a horizontal rotary shaker in the absence of light for days and finally filtered through a membrane filter (Millipore filters®, 0.45 μm pore size, 25 mm diameter) to obtain clear solutions. All samples were prepared in triplicates. The concentrations of HP in the inclusion complexes were determined by the HPLC assay. 3.2.5 Surface Tension and Contact Angle Measurements Surface tensions of RM β-CD and HP β-CD solutions and each formulation used in the permeation study were measured using the method described previously in section 2.2.3. The wettability of the excised human skin sample was determined by sessile drop contact angle using a Rame-Hart 100 goniometer (USA). Drops were placed on the surface using a micrometer with a flat tip needle. For contact angle measurements excised human skin similar to that used in permeation studies was employed. This was done because of its availability and its potential for elucidation of the mechanism of drug permeation studies. Skin samples were prepared as those for the permeation 51 studies. 3.2.6 In vitro Skin Permeation Studies Permeation studies of drug alone or as complexes with RM β-CD were performed using a flow-through diffusion cell apparatus described earlier in section 2.2.8. The donor compartment was filled with ml of formulations containing mg/ml drug. The first receptor phase was isotonic phosphate buffer saline pH 7.4 (PBS). Samples from the receptor phase were collected every hour over a 30-h period, and the amount of HP permeated was analyzed by HPLC. The steady state flux (J) was estimated from the slope of the straight line portion of the cumulative HP absorbed against time profile. Experiments were carried out in triplicates. The effect of RM β-CD on the skin permeation of HP was studied using two sets of experiments. First a concentration dependent effect of RM β-CD (0, 0.01, 0.05, 0.1 M) was studied. Further, synergistic effect of RM β-CD in combination with limonene 0.1% v/v in propylene glycol (PG) solution was investigated. The effect of pH on the permeability of the drug was studied at pH 5. The combined effects of ionization and 0.01 M RM β-CD were also observed. In another set of experiments, PBS in the receiver solution was replaced by 0.01% w/v RM β-CD, while the donor compartment consisted of HP in 0.01 M RM β-CD or PG solutions. 52 3.3 Results and Discussion 3.3.1 Molecular Modeling The hypothetical structures of the complexes formed by haloperidol and cyclodextrins are presented in Fig. 3.1. For the haloperidol-DM β-CD complex, the computed total energy is -40.7 kcals/mol and the steric energy is -30.587 kcals/mol. For the haloperidol-HP β-CD complex, total energy is -39.6 kcals/mol and the steric energy is -29.848 kcals/mol. The differences in energy values indicate that the interaction between haloperidol and DM β-CD might be stronger than that of haloperidol and HP β-CD. 53 a (1) a (2) a (3) a (4) b (1) b (2) b (3) b (4) Fig. 3.1 (a) Hypothetical structure of the haloperidol-DM β-CD complex, and (b) haloperidol-HP β-CD complex. (1) Side view; (2) Side view with electron surface; (3) Top view; and (4) Top view with electron surface. 3.3.2 Solubility Studies The solubilities of HP in phosphate buffer of pH solutions with and without RM βCD or HP β-CD are presented in Fig. 3.2. This pH was selected as it is the same pH of the skin and may therefore minimize skin irritation. The highest increase in drug solubility occurred for RM β-CD, indicating that this oligosaccharide complexed 54 more of the drug than HP β-CD. Molecular modeling supports the results obtained for solubility profile such that the increase in solubility was due to greater interaction of HP with DM β-CD. The solubilization profile in Fig. 3.2 is linear for all formulations indicating the formation of a 1:1 complex irrespective of the ionization of the drug (Loukas et al., 1997). More solubilization was achieved when the drug was in its degree of ionized form in RM β-CD, resulting in a 128-fold increase of the intrinsic solubility of the drug. When phase solubility experiments were performed with CD in the presence of buffer, the change in solubility was higher than in the presence of CD alone, indicating a synergistic effect. Methylated CDs have been observed to have larger cavity volumes than HP β-CD. Consequently, RM β-CD can easily accommodate the hydrophobic drugs such as HP (Torque et al., 2004). Concentration of HP (mg/ ml) 12 RM β-CD buffer pH R = 0.9909 HP β-CD buffer pH RM β-CD R = 0.9895 HP β-CD R = 0.9624 R = 0.9698 0 0.1 0.2 0.3 RM β-CD and HP β-CD concentration (M) Fig. 3.2 Phase solubility of haloperidol in CD solutions (n=3). 3.3.3 Surface Tension and Contact Angle Measurments The surface tensions of aqueous solutions of different concentrations of RM β-CD and HP β-CD are shown in Fig. 3.3. A remarkable change in the surface tension of 55 pure water occurred when RM β-CD or HP β-CD was added, indicating that these systems have effect on the surface tensions of pure water. The methylated compounds showed the most reduction (Evrard et al., 2004; Thompson 1997). From Fig. 4.3 it is evident that surface tension reached a constant value after certain concentration suggesting the formation of super molecular aggregates of RM β-CD and HP β-CD (Leclercq et al., 2007; Binkowski-Machut et al., 2006). Critical micelle concentration values were determined from the sharp changes in the slope of the surface tension versus log [CD] plot. CMC values for RM β-CD and HP β-CD were 4.4 mM and 3.2 mM respectively. The aqueous solution of naturally occurring β-CD does not have any surface activity (Leclercq et al., 2007; Lu et al., 1997). 65 55 RM β-CD HP β-CD Surface Tension (mN/m) 75 45 -8 -7 -6 -5 -4 -3 log [β-CD] (M) -2 -1 Fig. 3.3 Surface tension of RM β-CD and HP β-CD (n=3). Based on above results, a possible mechanism for the formation of large micelle assemblies was deduced as shown in Fig. 3.4. Particle size analysis, as observed by the light scattering, supported this hypothesis (Binkowski-Machut et al., 2006). However as compared with conventional surfactants these aggregates not behave 56 a) Water RM β-CD 0.1 M Cumulative HP μg\cm RM β-CD 0.05 M RM β-CD 0.01 M 0 10 15 20 25 30 25 30 Time (h) b) 20 PG Cumulative HP μg\cm PG-Limonene-RM β-CD 0.01M 15 PG-Limonene 10 0 10 15 20 Time (h) Fig. 3.5 Permeation profile of haloperidol across human epidermis. Influence of (a) different RM β-CD concentrations, (b) limonene and RM β-CD, (c) ionization and RM β-CD, (d) RM β-CD and sink condition in receptor compartment, R and D denote receptor and donor compartments of the flow-through diffusion cells, respectively (n=3). 63 c) 14 Buffer pH 12 Water Cumulative HP μg\cm RM β-CD 0.01M-Buffer pH 10 0 10 15 20 25 30 20 25 30 Time (h) d) 10 D: PG, R: PBS D: PG, R: RM β-CD Cumulative HP μg\cm D: RM β-CD 0.01M, R: PBS D: RM β-CD 0.01M, R: RM β-CD 0 10 15 Time (h) Fig. 3.5 Permeation profile of haloperidol across human epidermis. Influence of (a) different RM β-CD concentrations, (b) limonene and RM β-CD, (c) ionization and RM β-CD, (d) RM β-CD and sink condition in receptor compartment, R and D denote receptor and donor compartments of the flow-through diffusion cells, respectively (n=3). 64 3.4 Conclusion The effects of surface tension, contact angle and solubility on the skin permeation of haloperidol were investigated using randomly methylated β-cyclodextrin (RM β-CD) and hydroxypropyl β-cyclodextrin (HP β-CD). Molecular modeling was carried out to explain the complexation modes and the solubility data. Highest increase in drug solubility was observed when the drug was in its degree of ionized form in RM β-CD, resulting in a 128-fold increase in the intrinsic solubility of the drug. Surface tension measurements indicate a surface active effect for RM β-CD and HP β-CD. Contact angle measurements showed that vehicles with higher skin wettability increased the contact of the drug with skin surface and therefore resulted in significantly higher drug permeation across human epidermis (p[...]... a and b; Singh et al., 2005) Both ionized and unionized moieties of drug molecules contributed to the total flux, (Jtot) which can be calculated using the following equation: J tot  K punion * C union  K pion * C ion (3- 1) Where Kpunion and Kpion are the dependent drug permeabilities and Cunion and Cion are the dependent concentrations of ionized and unionized moieties, respectively (Hadgraft and. .. PG-Limonene-RM β-CD 0.01M 15 PG-Limonene 10 5 0 0 5 10 15 20 Time (h) Fig 3. 5 Permeation profile of haloperidol across human epidermis Influence of (a) different RM β-CD concentrations, (b) limonene and RM β-CD, (c) ionization and RM β-CD, (d) RM β-CD and sink condition in receptor compartment, R and D denote receptor and donor compartments of the flow-through diffusion cells, respectively (n =3) 63 c)... tension and contact angle values of the solutions (n =3) Formulations Surface Tension (mN/m) ± SD Contact angle ± SD water 70 .3 ± 0.25 91.6 ± 3. 13 RM β-CD 0.01M 57.5 ± 0.68 − RM β-CD 0.05M 54.8 ± 0 .31 − RM β-CD 0.1M 54.1 ± 0.22 52.0 ± 3. 82 PG 36 .2 ± 0.21 40.0 ± 4.54 PG-Limonene 36 .2 ± 0.06 − PG-Limonene-RM β-CD 36 .2 ± 0.05 − buffer pH 5 59.2 ± 0.42 54.11 ± 6.58 buffer pH 5- RM β-CD 56.5 ± 0. 03 − From... solution and RM β-CD 0.01 M in donor compartment (Fig 3. 5d) The flux was enhanced by a 1.6fold increase compared to that of the control (p < 0.05) It was found that the critical contact angle value, which distinguishes between penetration and non-penetration, helped to optimize the drug permeation rate By comparing Fig 3. 5a and b, PG with lower contact angle, resulted in higher drug permeation rate,... 0 .35 2.74 ± 0.14 0.09 ± 0.01 0.77 ± 0. 13 * In a and b, receiver solution is PBS * In c, receiver solution is RM β-CD 0.01% w/v Fig 3. 5c shows the effect of pH alone and in combination with RM β-CD on permeation rate of HP Ionization of the donor solution with pH 5 increased the flux rate but did not result in any significant difference compared with the control (p > 0.05) However the flux of the ionized... denote receptor and donor compartments of the flow-through diffusion cells, respectively (n =3) 64 3. 4 Conclusion The effects of surface tension, contact angle and solubility on the skin permeation of haloperidol were investigated using randomly methylated β-cyclodextrin (RM β-CD) and hydroxypropyl β-cyclodextrin (HP β-CD) Molecular modeling was carried out to explain the complexation modes and the solubility... shown in Fig 3. 5b and Table 3. 2, the combination of 0.01 M RM β-CD with 0.1% v/v limonene in PG solution improved percutaneous absorption by 1.4 fold compared to the control, but not significantly (p > 0.05) However, the drug permeation profiles were completely different and the lack of significant difference between the flux in limonene and limonene RM β-CD solutions suggests high permeation enhancing... solutions had lower surface tension of 59.2 ± 0.42 mN /m, addition of RM βCD further decreased the surface tension to 56.5 ± 0. 03 mN/m Addition of limonene did not demonstrate any decrease in interfacial tension of the PG (36 .2 ± 0.06 mN/m) when compared to pure PG solutions (36 .2 ± 0.21 mN/m), indicating that limonene does not possess any surface active effect (see Table 3. 1) 57 Table 3. 1 Surface tension... 2004) Fig 3. 4 Schematic aggregation of CD Surface tension and contact angle values of the solutions used are stated in Table 3. 1 It was observed that surface tension of water (70 .3 ± 0.25 mN/m) decreases with increase in the concentration of RM β-CD The interfacial tension of RM β-CD 0.05 and 0.1 M were of similar values, 54.8 ± 0 .31 and 54.1 ± 0.22 (mN/m), respectively, however surface tension of RM... 7.4) at body temperature (37 oC), n =3 Haloperidol release from PVA and RM β-CD electrospun fibers showed an immediate release as the fibers were rapidly dissolved within a few seconds upon contact with the aqueous medium In contrast, the PLA and PLGA fiber mat yielded a pronounced prolongation of drug release Continuous controlled release of the drug was observed from fibers containing PLA or PLGA As . CD solutions (n =3) . 3. 3 .3 Surface Tension and Contact Angle Measurments The surface tensions of aqueous solutions of different concentrations of RM β-CD and HP β-CD are shown in Fig. 3. 3. A remarkable. equation: ionpionunionpuniontot CKCKJ **  (3- 1) Where K punion and K pion are the dependent drug permeabilities and C union and C ion are the dependent concentrations of ionized and unionized moieties,. 47 CHAPTER 3 Effect of Cyclodextrin -Drug Complexation and Drug Ionization on the Permeation of Haloperidol Across Human Skin 3. 1 Introduction According to Fick’s law of diffusion, the delivery