**1. Introduction**

186 Toxicity and Drug Testing

Izevbigie EB, Bryant JL, Walker A: A novel natural inhibitor of extracellular signal-regulated kinases and human breast cancer cell growth. *Exp Biol Med* 2004;229:163–169. Janssen, P.A.J., Niemegeers, C.J.E., Dony, J.G.H. The inhibitory effect of fentanyl and other

K.Saidu, J. Onah, A. Orisadipe, A.Olusola, C. Wambebe, Kgamaniel, (2000) Antiplasmodial,

Koster, R., Anderson, N., De Beer, E. J. Acetic acid for analgesic screening. Federation

O'Hara, M; Kiefer D; Farrel K and Kemper K (1998) A review of 12 commonly used

Oliver JA: Opportunities for using fewer animals in acute toxicity studies. In: *Chemical* 

Orisakwe OE, Njan AA, Afonne OJ, Akumka DD, Orish VN, Udemezue OO: Investigation

Raj PP: Pain mechanisms. In: *Pain Medicine, A Comprehensive Review*. Mosby-Year Book, St.

Sanchez-Mateo CC, Bonkanka CX, Hernandez-Perez M, Rabanal RM: Evaluation of

Sanchez-Mateo, C.C., Bonkanka, C.X., Hernandez-Perez, M., Rabanal, R.M. Evaluation of

Siegmund EA, Cadmus RA, Lu G: Screening of analgesic including aspirin-type compound

Taiwo O, Xu HX, Lee SF: Antibacterial activites of extracts from Nigerian chewing sticks.

Tona L, Cimanga RK, Mesia K, *et al*.: Antiplasmodial activity of extracts and fractions from

WHO, (2003). Assessment and monitoring of antimalarial drug efficacy for the treatment of

Wilcox ML: A clinical trial of 'AM', a Uganda herbal remedy for malaria. *J Public Health Med* 

Willcox, M.L, G. Bodeker (2000). Plant-based malaria Control: research initiative on

Zimmermann M: Ethical guidelines for investigations of experimental pain in conscious

traditional antimalaria methods. *Parasitology Today* 16: 220 – 221.

seven medicinal plants. *J Ethnopharmacol* 2004;93:27–32.

uncomplicated faciparum malaria. Geneva.

*Testing and Animal Welfare*. The National Chemicals Inspectorate, Solna, Sweden,

into the nephrotoxicity of Nigerian bonny light crude oil in albino rats. *Int J Environ* 

analgesic and topical anti-inflammatory effects of *Hypericum reflexum* L. fil. *J* 

analgesic and topical anti-inflammatory effects of *Hypericum reflexum* L. fil. *J* 

based upon the antagonism of chemically induced writhing in mice. *J Pharmacol* 

of Erythrina senegalensis. Journal of Ethnopharmacology 71,275 – 280. Kambizi L, Afolayan AJ: An ethnobotanical study of plants used for the treatment of

Lorke D: A new approach to practical acute toxicity testing. *Arch Toxicol* 1998;5:275–289. Masaba SC: The antimalarial activity of *Vernonia amygdalina* Del. (Compositae). *Trans R Soc* 

sexually transmitted disease. *J Ethnopharmacol* 2001;77:5–9.

Arzneim-Forsch *Drug Res* 1963, 6: 502-507

medicinal herbs. *Arch. Fam. Med*. 7: 523-536.

Proceedings 1959, 18: 412.

1986, pp. 119–143.

*Trop Med Hyg* 2000;94:694–695.

*Res Public Health* 2004;2:91–95.

*Ethnopharmacol* 2006;107:1–6.

*Ethnopharmacol* 2006, 107: 1-6.

*Exp Ther* 1957;119:184–186.

*Phytother Res* 1999;8:675–679.

animals. *Pain* 1983;16:109–110.

1999;21:318–324.

Louis, 1996, pp. 12–24.

morphine-like analgesic on the warm water induced tail withdrawal reflex in rats.

analgesic, and anti-inflammatory activities of the aqueous extract of the stem bark

Solubility of a drug is one of its important physico-chemical properties. More attention has been paid to the aqueous solubility since water is the unique solvent of biological systems. It is obvious that a drug should be reached to its receptors in the body through the aqueous and non-aqueous media. The chance of a low water soluble drug to be appeared in the market place is very low and nearly 40 % of the drug candidates fail to reach higher phases of the drug trials simply because of their low water solubility. The solubility in non-aqueous solvents is not too important from clinical viewpoint however these solubilities play curious roles in drug discovery and development investigations. Most of drugs are synthesized in non-aqueous media and/or extracted from natural sources using non-aqueous extracting solvents. Different polymorphs of some drugs could be produced from their crystallization using organic solvents.

There are various methods for solubility determination of drugs which is discussed in this chapter. The experimental determination is tedious and time-consuming process and sometimes there is restrictions in the availability of enough amount of a drug candidate to be used in the solubility measurements, especially in the early stages of drug discovery investigations in which only small amount of a drug is synthesized/extracted and large number of preliminary biological tests should be carried out. To cover this limitation, and in order to provide a faster and easier tool, mathematical models have been developed to correlate/predict the solubility of drugs. These models are discussed in this chapter to provide an overall view for a pharmaceutical scientist who is working in the research and development department of a company and/or a research laboratory within academia. In addition to the accurate calculations which are expected from these models, the simplicity of the required computations is another parameter which should be taken into account, since more complex computations did not attract more attention in the pharmaceutical industry.

#### **1.1 Solubility and dissolution**

When talking about solubility, there are two concepts which might be confused with each other: solubility and dissolution. The term solution (i.e. thermodynamic solution) is used to define the state which is thermodynamically stable and shows the neat result of an

Experimental and Computational Methods Pertaining to Drug Solubility 189

only form of the solubilized drug in the solution. *SM* is also called intrinsic solubility or *S*<sup>0</sup> . In early stages of drug discovery, only small amount of the new drug is available and its purity is not assured. In this stage, the solubility determination in acidic and/or basic solutions could be used in practice. Increased apparent solubility in acidic or basic medium reveals that the new drug is a basic or an acidic solute. No increase in the solubility means that the drug is a nonelectrolyte. Increased solubility in both acidic and basic media indicates either zwitterionic or amphoteric behaviour. The intrinsic solubility of a drug could be determined from apparent solubility data at various pH values. When the purity of a drug candidate is not assured, a phase-solubility diagram, i.e. the solubility at different solute:solvent ratios, is recommended. In this diagram, the co-solute effect (self association, complexation, solubilization) increases the solubility and the common ion effect decreases the solubility and no change in the solubility

Salt formation of weak acidic or basic drugs is one of their solubility increasing methods since the ionized species have greater solubility in water and other polar solvents and a number of drugs are marketed as their salt forms. The most common salts used for salt formation of acidic drugs are sodium, potassium, calcium and zinc and those for basic drugs are hydrochloride, sulphate, mesylate, maleate, phosphate, tartrate, citrate and besylate (Wells, 1988). Different slats of a given drug possess various solubilities. As an example, the solubility of lamotrigine with the counterions of tartrate, saccharinate, succinate and fumarate are 2.63, 1.37, 0.61 and 0.43 millimole per liter (Galcera and Molins, 2009). The selection of the salt of a drug is mainly carried out by trial and error basis considering practical issues such as cost of raw materials, ease of crystallization, percent yield, thermal stability and hygroscopicity of the resulting salt. Black et al. (2007) investigated the salt formation of 17 salt forms of ephedrine and reported their physicochemical properties and tried to develop a relationship between these properties which was not successful. Any model representing the properties of salt forms of drugs is a highly in demand subject in the pharmaceutical industry. As an example, the relation between the dielectric constant of the solvent and the solubility of drugs in their salt form, can be mentioned (Fakhree et al., 2010).

Polymers and macromolecules are important parts of drug design and development. The emerging technology of proteins, peptides, DNA and RNA sequences as pharmaceutical active ingredients makes it necessary for consideration of their physicochemical properties in pharmaceutical sciences, including solubility. For the beginning, in terms of macromolecules, it is better to use dispersion versus solubility in a medium and this makes a difference between their solubility in comparison with small organic molecules. The dispersion of the macromolecules in the solution results in formation of new properties for the solution such as increase in viscosity, light scattering, molecular network formation (e.g. gel) etc (Sinko and Martin, 2006). Another important note about macromolecules, is the fact that they have been produced in an aqueous medium and have philia to watery media (not always, but in most of the cases). Hence, they are sensitive to presence of organic solvents and might be precipitated by addition of the organic solvents (unlike small organic nonelectrolyte molecules which dissolve in organic media more than aqueous solutions).

might mean that drug is pure and no interaction exists.

**1.4 Solubility of pharmaceutical macromolecules** 

**1.3 Solubility of salt form of drugs** 

equilibrium between a solute (the compound which is going to be dispersed molecularly in another medium which is called solvent) and its dissolved form in the medium. The dissolving process is the migration of the molecules of the solute to the solvent medium and makes the solution which after reaching a steady state is called homogenous solution and can be represented by the following equilibrium:

How much a solute is molecularly dispersed in the solvent is called solubility and the rate of dissolving is called dissolution. Hence, the solubility value is a thermodynamic property while the dissolution rate is a kinetic one. In other words, time has no effect on solubility value and is not important in its related subjects, but it is important in dissolution related subjects.

The solubility is important in stable forms including liquid formulations and dissolution is important in transient states including the release of the drug from its formulation to biological fluids and permeability (Sinko and Martin, 2006). In pharmaceutical sciences, especially in formulation, designing a stable liquid formulation requires the knowledge on the solubility value and an effective drug delivery to the body mostly depends on the dissolution rate which is affected by the solubility (Allen et al., 2006). However, they both affect each other based on Noyes-Whitney equation (Sinko and Martin, 2006):

$$\frac{d\mathcal{W}}{dt} = \frac{DA\left(\mathcal{C}\_S - \mathcal{C}\right)}{L} \tag{1}$$

where *dW/dt* is the rate of dissolution, *A* is the surface area of the solid which is in direct contact with the molecules of the solvents, *C* is the concentration of the solute in the medium (dissolved amount), *CS* is the concentration of the solute in the diffusion layer, *D* is the diffusion coefficient, and *L* is the thickness of the diffusion layer.

Based on the discussed topics, solubility and dissolution are in relation with each other, but not the same. So, they must not be used in place of each other as the consequences can be awful! For example, a drug substance might be highly soluble, but dissolves slowly (or vice versa). So, in the formulation of such compounds, the difference between solubility and dissolution must be considered.

#### **1.2 Solubility of base form of drugs**

The apparent solubility ( *SApp* ) of a weak electrolyte is expressed by:

$$S\_{App} = S\_M + S\_I \tag{2}$$

in which *SM* is the molecular form of the drug and *SI* is the ionized form of the drug in the solution. For strong electrolytes, *SI* is predominant whereas for nonelectrolytes *SM* is the only form of the solubilized drug in the solution. *SM* is also called intrinsic solubility or *S*<sup>0</sup> . In early stages of drug discovery, only small amount of the new drug is available and its purity is not assured. In this stage, the solubility determination in acidic and/or basic solutions could be used in practice. Increased apparent solubility in acidic or basic medium reveals that the new drug is a basic or an acidic solute. No increase in the solubility means that the drug is a nonelectrolyte. Increased solubility in both acidic and basic media indicates either zwitterionic or amphoteric behaviour. The intrinsic solubility of a drug could be determined from apparent solubility data at various pH values. When the purity of a drug candidate is not assured, a phase-solubility diagram, i.e. the solubility at different solute:solvent ratios, is recommended. In this diagram, the co-solute effect (self association, complexation, solubilization) increases the solubility and the common ion effect decreases the solubility and no change in the solubility might mean that drug is pure and no interaction exists.

#### **1.3 Solubility of salt form of drugs**

188 Toxicity and Drug Testing

equilibrium between a solute (the compound which is going to be dispersed molecularly in another medium which is called solvent) and its dissolved form in the medium. The dissolving process is the migration of the molecules of the solute to the solvent medium and makes the solution which after reaching a steady state is called homogenous solution and

*X Y Solid Liquid Concenteration*

How much a solute is molecularly dispersed in the solvent is called solubility and the rate of dissolving is called dissolution. Hence, the solubility value is a thermodynamic property while the dissolution rate is a kinetic one. In other words, time has no effect on solubility value and is

The solubility is important in stable forms including liquid formulations and dissolution is important in transient states including the release of the drug from its formulation to biological fluids and permeability (Sinko and Martin, 2006). In pharmaceutical sciences, especially in formulation, designing a stable liquid formulation requires the knowledge on the solubility value and an effective drug delivery to the body mostly depends on the dissolution rate which is affected by the solubility (Allen et al., 2006). However, they both

> *dW DA C C <sup>S</sup> dt L*

where *dW/dt* is the rate of dissolution, *A* is the surface area of the solid which is in direct contact with the molecules of the solvents, *C* is the concentration of the solute in the medium (dissolved amount), *CS* is the concentration of the solute in the diffusion layer, *D* is the

Based on the discussed topics, solubility and dissolution are in relation with each other, but not the same. So, they must not be used in place of each other as the consequences can be awful! For example, a drug substance might be highly soluble, but dissolves slowly (or vice versa). So, in the formulation of such compounds, the difference between solubility and

in which *SM* is the molecular form of the drug and *SI* is the ionized form of the drug in the solution. For strong electrolytes, *SI* is predominant whereas for nonelectrolytes *SM* is the

(1)

**Solution** 

*S SS App M I* (2)

not important in its related subjects, but it is important in dissolution related subjects.

**Solvent molecules Solute molecules** 

affect each other based on Noyes-Whitney equation (Sinko and Martin, 2006):

diffusion coefficient, and *L* is the thickness of the diffusion layer.

The apparent solubility ( *SApp* ) of a weak electrolyte is expressed by:

dissolution must be considered.

**1.2 Solubility of base form of drugs** 

can be represented by the following equilibrium:

Salt formation of weak acidic or basic drugs is one of their solubility increasing methods since the ionized species have greater solubility in water and other polar solvents and a number of drugs are marketed as their salt forms. The most common salts used for salt formation of acidic drugs are sodium, potassium, calcium and zinc and those for basic drugs are hydrochloride, sulphate, mesylate, maleate, phosphate, tartrate, citrate and besylate (Wells, 1988). Different slats of a given drug possess various solubilities. As an example, the solubility of lamotrigine with the counterions of tartrate, saccharinate, succinate and fumarate are 2.63, 1.37, 0.61 and 0.43 millimole per liter (Galcera and Molins, 2009). The selection of the salt of a drug is mainly carried out by trial and error basis considering practical issues such as cost of raw materials, ease of crystallization, percent yield, thermal stability and hygroscopicity of the resulting salt. Black et al. (2007) investigated the salt formation of 17 salt forms of ephedrine and reported their physicochemical properties and tried to develop a relationship between these properties which was not successful. Any model representing the properties of salt forms of drugs is a highly in demand subject in the pharmaceutical industry. As an example, the relation between the dielectric constant of the solvent and the solubility of drugs in their salt form, can be mentioned (Fakhree et al., 2010).

#### **1.4 Solubility of pharmaceutical macromolecules**

Polymers and macromolecules are important parts of drug design and development. The emerging technology of proteins, peptides, DNA and RNA sequences as pharmaceutical active ingredients makes it necessary for consideration of their physicochemical properties in pharmaceutical sciences, including solubility. For the beginning, in terms of macromolecules, it is better to use dispersion versus solubility in a medium and this makes a difference between their solubility in comparison with small organic molecules. The dispersion of the macromolecules in the solution results in formation of new properties for the solution such as increase in viscosity, light scattering, molecular network formation (e.g. gel) etc (Sinko and Martin, 2006). Another important note about macromolecules, is the fact that they have been produced in an aqueous medium and have philia to watery media (not always, but in most of the cases). Hence, they are sensitive to presence of organic solvents and might be precipitated by addition of the organic solvents (unlike small organic nonelectrolyte molecules which dissolve in organic media more than aqueous solutions).

Experimental and Computational Methods Pertaining to Drug Solubility 191

amphiphilic molecules that cannot form organized structures, such as micelles, in water but they increase the aqueous solubility of drugs. Often strong synergistic effects are observed when hydrotropes are added to aqueous surfactant or polymer solutions. Caffeine and nicotinamide are well known hydrotropic agents and their ability to solubilize a wide variety of therapeutic drugs including riboflavin (Lim and Go, 2000) has been demonstrated. Complexation of drugs is another solubilization technique and there are a number of reports on complexation of drugs by cyclodextrins. Ionization is applicable for weak electrolytes

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

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

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;

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.,

and the solubility of some drugs could be increased by changing pH of the solution.

soluble polymorphs (Blagden et al., 2007; Widenski et al., 2009).

**1.6.2 Solubility decreasing** 

before starting analyses.

Gibaldi et al., 2007).

2006; Gibaldi et al., 2007).

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


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 other organisms (Waldo, 2003).

### **1.5 Solubility of drugs in biological fluids**

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 the bioavailability of a drug after oral administration.

#### **1.6 Solubility modifications**

Solubility modification of drugs is required in separation, purification, analysis and formulation investigations and different methods are used to achieve the increased/decreased solubility values.

#### **1.6.1 Solubility increasing**

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 amphiphilic molecules that cannot form organized structures, such as micelles, in water but they increase the aqueous solubility of drugs. Often strong synergistic effects are observed when hydrotropes are added to aqueous surfactant or polymer solutions. Caffeine and nicotinamide are well known hydrotropic agents and their ability to solubilize a wide variety of therapeutic drugs including riboflavin (Lim and Go, 2000) has been demonstrated. Complexation of drugs is another solubilization technique and there are a number of reports on complexation of drugs by cyclodextrins. Ionization is applicable for weak electrolytes and the solubility of some drugs could be increased by changing pH of the solution.
