**1.6.2 Solubility decreasing**

190 Toxicity and Drug Testing

The solubility of proteins is influenced by the ratio of the hydrophobic and hydrophilic residues of amino acids and their arrangement in the final structure of the protein (Bolen, 2004). For example, globular proteins have hydrophobic residues in their core and hydrophilic residues in their surface. It is also affected by the pH and ionic strength of the water, presence of organic solvents and other polymers (Burgess, 2009). When talking about

1. in-vitro low solubility due to structural properties of the protein (hydrophobic

For increasing a protein's aqueous solubility, one of the strategies is addition of additives such as L-arginine and L-glutamic acids. Fusion of peptides and proteins is another method which is addition of a solubilizing sequence of amino acids or protein to the structure of the low soluble protein. Mutation in the hydrophobic amino acids sequences to hydrophilic ones is another strategy. However, this might not work in all of the cases (Trevino et al., 2008). Another approach is screening to find a more soluble homologue of that protein in

For understanding the dissolution of a drug in the human body fluids, it is crucial to focus on the solubility of drugs in more realistic environment and to acquire larger amount of experimental data for simulating the solubility at different pHs, in the presence of bile salts etc which exists in the real solubilization media within human body. Solubility data of drugs in biorelevant media are increasingly required in early phases of drug discovery to predict

Solubility modification of drugs is required in separation, purification, analysis and formulation investigations and different methods are used to achieve the

Several methods have been used to enhance the aqueous solubility of drugs including cosolvency, hydrotropism, complexation, ionisation, use of the surface active agents, crystal structure modifications and addition of ionic liquids. These methods have been discussed in details in the literature (Myrdal and Yalkowsky, 1998). Mixing a permissible non-toxic organic solvent with water, i.e. cosolvency, is the most common and feasible technique to enhance the aqueous solubility of drugs. The common cosolvents which, are used in the pharmaceutical industry are ethanol, propylene glycol, glycerine, glycofural, polyethylene glycols (mainly 200, 300 and 400), N,N-dimethyl acetamide, dimethyl sulfoxide, 2-propanol, dimethyl isosorbide, N-methyl 2-pyrrolidone (NMP) and room temperature ionic liquids (Rubino, 1990; Mizucci et al., 2008; Jouyban et al., 2010a). Their applications and possible side effects have been discussed in the literature (Spiegel and Noseworthy, 1963; Tsai et al., 1986; Patel et al., 1986; Golightly et al., 1988; Rubino, 1990). Hydrotropes are a class of

the solubility of proteins, there are different kinds of low solubility for the proteins:

hydrophobic, residue charge, and β-sheets in the structure, and 4. low solubility due to conformational changes (Trevino et al., 2008).

2. in-vivo low solubility due to over expression of the protein in an organism (*E. coli*), 3. amyloid formation which results in aggregation of the proteins because of their

residues),

other organisms (Waldo, 2003).

**1.6 Solubility modifications** 

**1.6.1 Solubility increasing** 

increased/decreased solubility values.

**1.5 Solubility of drugs in biological fluids** 

the bioavailability of a drug after oral administration.

In precipitation and crystallization processes as a part of extraction and purification of the pharmaceutically related compounds, lowering the solubility is desirable. Lowering the solubility for pharmaceutical compounds might include using of temperature alteration, addition of antisolvent, using of a low soluble salt or ester of the drug, and producing low soluble polymorphs (Blagden et al., 2007; Widenski et al., 2009).

Precipitation or crystallization both can be used in this regard depending on the rate of solubility decreasing. If it is happened quickly, then the solid state might be in amorphous form and the process called precipitation. If the lowering of solubility takes place in a controlled way that crystal growth can happen, then the process called crystallization. Precipitation of proteins and macromolecules such as DNA and RNA are other examples for this kind of solubility modification. In protein biosynthesis and extraction, different methods of desolubilization are used which include: salting out, isoelectric point precipitation, precipitation with organic solvents, addition of non-ionic hydrophilic polymers, flocculation by polyelectrolytes, and addition of polyvalent metallic ions (Burgess, 2009). Another reason making it desirable to precipitate macromolecules such as proteins, DNA, and RNA is pre-treatment of biological analytes before starting analyses.

Recrystallization is another process which is used in pharmaceutical sciences and means to dissolve a compound in a medium, and by modifying the physicochemical conditions made the dissolved compound to crystallize again. This technique is widely used in crystal engineering technology which can produce amorphous, different polymorphs, and psudopolymorphs of a drug (Blagden et al., 2007). This is important in modification of pharmaceutically interested physicochemical properties such as compressibility in formulation process, size of particles, dissolution rate, as well as solubility (Allen et al., 2006; Gibaldi et al., 2007).

The above mentioned processes are related to preformulation processes. In formulation of pharmaceutical active ingredients the desire for lowering solubility can be seen in designing of sustained release and depot dosage forms or drug delivery systems (Allen et al., 2006; Gibaldi et al., 2007). For making a sustained release dosage form of a drug, different formulation techniques such as use of polymeric matrix, osmotic pumps, and crystallization of a poorly water soluble compound are used. For designing a depot drug delivery system, possible solutions include: use of low soluble salts or esters of a drug (e.g. methylprednisolone acetate), addition of additives (e.g. zinc and insulin), very concentrated non-aqueous solutions of drug (e.g. Leuprolide and NMP), and depot dosage forms (e.g. implants of low soluble compounds such as sex hormones) (Strickley, 2004; Allen et al., 2006; Gibaldi et al., 2007).

Experimental and Computational Methods Pertaining to Drug Solubility 193

the higher viscosity of the saturated solutions, e.g. in mixed solvents, should be kept in mind as a limitation. A combination of filtration and centrifugation is also could be used. The UV spectrophotometric analysis is the most common and the easiest analytical method. The next is the HPLC methods both in isocratic and gradient elution modes. The HPLC analysis could also detect the possible impurities or degradation products if a highly selective method was used. X-ray diffraction (XRD) and differential scanning calorimetry (DSC) of the residual solid separated from the saturated solution confirm the possible solid

The synthetic method (Hankinson and Thompson, 1965; Ren et al., 2005; Yang et al., 2008; Yu et al., 2009) which is so called laser monitoring technique (Li et al., 2006), last crystal disappearance method (Hao et al., 2005) and dynamic method (Peisheng and Qing, 2001; Weiwei et al., 2007; Wang et al., 2008) is based on disappearance of the solid drug (from the mixture of solvent and drug) monitored by a laser beam. The history of this method backs to 1886 and first introduced by Alexejew and then modified by other research groups (Ward, 1926). The disappearance of drugs could be achieved either by changing the temperature or by addition of a known amount of the solvent. It is claimed that the synthetic method is much faster and more reliable than analytical method (Yang et al., 2008). Figure 1 illustrates a schematic representation of the most completed set up used in

Fig. 1. Schematic representation of the synthetic method for determination of solubility of drugs; 1, magnetic stirrer; 2, laser generator; 3, jacketed glass vessel; 4, condenser pipe; 5, thermometer; 6, thermocouple; 7, rotor; 8, photoelectric transducer; 9, controller; 10, laser strength display; 11, constant temperature bath; 12, workstation. (Figure is reproduced from

The solubility apparatus consisted of a jacketed glass vessel (varying from 60 to 250 mL) maintained at the desired temperature by circulating water that was provided by a constanttemperature bath. The water temperature was controlled by a workstation with a temperature accuracy of (0.1 K) achieved continuous stirring, and a condenser (or a

phase transformations during equilibration.

**2.1.2 Synthetic method** 

the synthetic method.

Ren et al., 2005).

Also low solubility is useful when stability of a pharmaceutical compound is low in its solubilized form (Sinko and Martin, 2006). Hence, suspension formulations (i.e. ready to use and lyophilized powder for suspension preparation) might be a useful strategy.

In the recent decade, emerging technologies such as micro-formulation, microencapsulation, nano-formulation, and nano-encapsulation are using solubility decreasing principals as a part of their processes. This is usually done by addition of antisolvent and fine particle stabilizers to gain a suspension with micro/nano-sized particles.
