About Author:
Dhananjay S Jadhav
*M Tech (Pharmaceutical Technology) Department of Pharmaceutical Technology,
University Institute of Chemical Technology,
North Maharashtra University,
Jalgaon -425001. Maharashtra, India.
dhananjaysjadhav@hotmail.com
Abstract
Solubility of drug candidate plays a vital role in selection of lead compound in early stage of drug development and discovery. Biopharmaceutical classification system distributes the drug candidate into different bins depending on the solubility and permeability. Two type of solubility determined at different stages of drug discovery, kinetic solubility and thermodynamic solubility. It is useful in deciding development plan and option of formulation development and to confirm result obtained from kinetic solubility data. Different problem encounter while determining the solubility, most of characteristics usually pH dependent, such as multiple and often overlapping ionization, complexation, aggregation, micelle formation, and “common ion” effect, incubation time, adsorption to micro porous filters, plastic or glass surfaces, polymorph interconversion. Above parameter should be considered while determining solubility. Different method available for measurement of the different solubility, conventional shake flask method now a day’s replaced by the high throughout solubility assay technique which considerably reduces the incubation time increased accuracy of result. Solubility determination can be done by ultraviolet absorption, nephlometry, Nuclear magnetic resonance and Potentiometric in drug discovery. The present review attempts to give a brief account of solubility and it’s importance, process of solubilisation, problems that occur while determining solubility, different types of solubility and there application, parameter to be considered while measuring solubility, different method to measure solubility, application in drug discovery in development, recent advances in solubility measurement.
REFERENCE ID: PHARMATUTOR-ART-1653
1) INTRODUCTION:
The criteria for drug molecule selection in the pharmaceutical industry are naturally focused on pharmacological activity, safety and potential clinical and commercial value. The success of a drug as a candidate for development to the market place depends on issues such as stability and bioavailability. The advent of new or improved therapies to treat intractable diseases could be delayed or compromised if the wrong choice of molecule is made at the outset. Both the stability and the bioavailability of a compound have a fundamental relationship with its solubility. Nearly all pharmaceutical drug products are required to be in a molecular dispersed form (solvated) before adsorption across biological membranes can occur. Thus, efficacy and therapeutic effect hinge on solubility. Among the five key physicochemical screens in early compound screening, Pka, solubility, permeability, stability and lipophilicity, poor solubility tops of the list of undesirable compound properties. Compounds with insufficient solubility carry a higher risk of failure during discovery and development. This happens because achieving good activity against a biological target is of paramount importance and structural features that produce good activity (e.g. lipophilic substructures) can reduce solubility. In recent years there focus to the effect of compound solubility in biological assays awareness of aqueous solubility has developed in the field of drug discovery because of the high rate of candidate attrition caused by biopharmaceutical properties [1].
Based on the Biopharmaceutics classification system developed by [2], drugs can be classified into BCS Class I (highly soluble and permeable), Class II (highly permeable but poorly soluble), Class III (highly soluble but poorly permeable), and Class IV (poorly soluble and poorly permeable). Those poorly water-soluble “brick dust compounds” (Classes II and IV), normally characterized as high molecular weights, large log P values, and poor water solubilities, generally have problems with drug bioavailability. Poor aqueous solubility is caused by two main factors: I) high lipophilicity II) strong intermolecular interactions which make the solubilization of the solid energetically costly.
What is meant by good and poorly soluble depends partly on the expected therapeutic dose and potency. “As a rule of thumb”, a compound with an average potency of 1mg/kg should have a solubility of at least 0.1g/L to be adequately soluble. If a compound with the same potency has a solubility of less than 0.01g/L it can be considered poorly soluble. [3]
BIOPHARMACEUTICS CLASSIFICATION SYSTEM: [4]
Figure: 1 Biopharmaceutical classification system
Poor aqueous solubility can often be overcome by appropriate formulation work. However, this approach is expensive and without guarantee of success. It is much better to improve solubility by chemistry means through adequate changes in the molecule itself. To this end, it is desirable to determine the aqueous solubility of candidates as early as possible in the discovery process. Even though higher throughput assays have recently become available, the generation of high quality solubility data remains a relatively expensive and time consuming activity. Therefore, the development of models to predict the aqueous solubility of drug candidates from their chemical structure has attracted considerable attention. Predictive models based on molecular descriptors also help understanding what feature(s) limits solubility and can thus provide useful information to medicinal chemists.
2) SOLUBILITY AND IT’s IMPORTANCE:
DEFINITION:
Conceptually, solubility is an easy parameter to measure but its meaning and concept of use is often different for discovery and development scientists and this can be a source of misunderstandings and controversy. In a broad sense, solubility may be defined as the amount of a substance that dissolves in a given volume of solvent at a specified temperature. More specifically, compound solubility can be defined as
a) Unbuffered solubility, usually in water, means solubility of a saturated solution of the compound at the final pH of the solution (which may be far from pH 7 due to self-buffering).
b) Buffered solubility also termed apparent solubility refers to solubility at a given pH, e.g. 2 or 7.5, measured in a defined pH-buffered system and usually neglects the influence of salt formation with counterion of the buffering system on the measured solubility value.
c) Intrinsic solubility means the solubility of the neutral form of an ionizable compound. For neutral (non-ionizable) compounds all three definitions coincide.
d) The thermodynamic solubility of a compound in a solvent is the maximum amount of the most stable crystalline form of the compound that can remain in solution in a given volume of the solvent at a given temperature and pressure under equilibrium conditions. This equilibrium balances the energy of solvent and solute interacting with them against the energy of solvent and solute interacting with each other. [5]
CLASSIFICATION OF SOLUBILITY ACCORDING TO I.P.
The following table indicates the meanings of the terms used in statements of approximate solubilities at 200C-300C [6]
|
Descriptive term |
Approximate volume of solvent in milliliters per gram of solute |
|
very soluble |
less than 1 |
|
freely soluble |
from 1 to 10 |
|
Soluble |
from 10 to 30 |
|
sparingly soluble |
from 30 to 100 |
|
slightly soluble |
from 100 to 1000 |
|
very slightly soluble |
from 1000 to 10,000 |
|
insoluble or practically insoluble |
more than 10,000 |
The term `partly soluble' is used to describe a mixture of which only some of the components dissolve.
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3) PROCESS OF SOLUBILISATION: [7]
Figure: 2. The intermolecular forces that determine thermodynamic solubility.
a) Solvent and solute are segregated; each interacts primarily with other molecules of the same type.
b) To move a solute molecule into solution, the interactions among solute molecules in the crystal (lattice energy) and among solvent molecules in the space required to accommodate the solute (cavitation energy) must be broken. The system entropy increases slightly because the ordered network of hydrogen bonds among solvent molecules has been disrupted.
c) Once the solute molecule is surrounded by solvent, new stabilizing interactions between the solute and solvent are formed (solvation energy), as indicated by the dark blue molecules. The system entropy increases owing to the mingling of solute and solvent (entropy of mixing), but also decreases locally owing to the new short-range order introduced by the presence of the solute, as indicated by the light blue molecules.
4) PROBLEMS THAT OCCUR WHILE DETERMINING SOLUBILITY:
In HTS solubility assays solid state properties of compounds and experimental conditions can greatly affect the results of thermodynamic solubility studies
Methods should (1) allow the identification of residual solid forms (salts, complexes, hydrates, etc.) at the beginning and the end of the solubility study, (2) ensure that equilibrium between solid and solute has really been achieved, (3) be able to handle solvents with different viscosity, and (4) use analytical methods that permit the identification of compounds, degradation products, and impurities after (5) separation from the residual solid. Consequently, distribution of solids and of different types of solvents, removal of residual solids, and analytical capabilities for solids and solutes are key components of these methods.
Speciation characteristics (usually pH dependent), such as multiple and often overlapping ionization, complexation, aggregation, micelle formation, and “common-ion” effects, can hamper the interpretation of the measurements. Consideration of the Incubation time needed to reach equilibrium is being debated by practitioners and this equilibration time could greatly affect measurement of the apparent solubility. Potential experimental artifacts, such as adsorption to micro porous filters, plastic or glass surfaces, “promiscuous inhibitor” molecules forming very small (0.1-0.2 µm) particles which can pass through filters and a multitude of other confounding effects may escalate the inter-laboratory variability of the measured values. Different polymorphic forms of a compound may produce subtle differences in solubility and dissolution behavior.
5) DIFFERENT TYPES OF SOLUBILITY AND THERE APPLICATION
A) KINETIC SOLUBILITY (ANTISOLVENT PRECIPITATION):
Among the challenges facing early stage solubility testing are the sheer numbers of compounds being assessed at that stage, the scarcity of compound, and the questionable purity and crystallinity of the earliest discovery lots. All of these challenges have been partially met in a high-throughput kinetic measurement of anti-solvent precipitation commonly misnamed ‘kinetic solubility’ [8] Kinetic solubility is a misnomer, not because it is not kinetic, but because it measures a precipitation rate rather than solubility. Kinetic solubility methods are designed to facilitate high throughput (>600 compounds per week) measurements, using sub milligram quantities of compound, in a manner that closely mimics the actual solubilization process used in biological laboratories.
It is the concentration of a compound in solution at the time when an induced precipitate first appears
Solubility results obtained from kinetic measurements might not match the thermodynamic solubility results perfectly. Using this data is very risky because Kinetic solubility is determined on compounds that often have not been purified to a high degree or crystallized. The impurities and amorphous content in the material used in kinetic solubility measurements sometimes lead to a measured kinetic solubility that is higher than the true solubility by inhibiting precipitation from the aqueous medium. Because kinetic solubility experiments begin with the drug in solution, there is a significant risk of achieving super saturation of the aqueous solvent through precipitation of an amorphous or metastable crystalline form. This supersaturation can lead to a measured value that is significantly higher than the thermodynamic solubility, masking a solubility problem that will become apparent as soon as the compound is crystallized.
Owing to the nature of kinetic solubility measurements, there is no time for equilibration of the compound in the aqueous solvent of measurement. Because the compounds tested are in dimethyl sulfoxide solutions, the energy required to break the crystal lattice is not factored into the solubility measurements. In fact, it is not uncommon to divide compounds into low-, medium- and high-solubility bins, depending on their kinetic solubility values, in order to reduce the importance placed on the actual numbers obtained. Rank ordering of compounds according to solubility on the basis of kinetic data can be done only if prior a comparison of the kinetic and thermodynamic solubilities of the compounds shows that these parameters have rank agreement.
Procedure:
The compound is dissolved in dimethyl sulfoxide (because it is a strong organic solvent) to make a stock solution of known concentration. This stock is added gradually to the aqueous solvent of interest until the anti-solvent properties of the water drive the compound out of solution. The resulting precipitation is detected optically, and the kinetic solubility is defined as the point at which the aqueous component cans no longer solvate the drug.
Application:
a) kinetic solubility data are intended only to assess feasibility for biological assays
b) discovery teams use kinetic solubility data to assess structure–solubility relationships and pre-clinical formulation activities,
B) EQUILIBRIUM SOLUBILITY (THERMODYNAMIC SOLUBILITY):
It is the concentration of compound in a saturated solution when excess solid is present, and solution and solid are at equilibrium. Thermodynamic (equilibrium) solubility represents the saturation solubility of a compound in equilibrium with an excess of undissolved substance at the end of the dissolution process. Thermodynamic solubility is often regarded as being the ‘true’ solubility of a compound and as the ‘gold standard’ for development needs. However, these values are not absolute numbers and depend, like kinetic values, on a multitude of compound properties and experimental factors.
Although thermodynamic solubility is the most theoretically and experimentally rigorous parameter, it is neither practical nor useful to measure it in the earliest discovery stages when purity, physical form and compound supply are all in question. At this stage, kinetic solubility measurements facilitate the rapid binning of large numbers of compounds for which little material is available.
It is based on the simple principle (add excess solid to the solvent of choice, stir the system for an infinite amount of time, and then measure the concentration of the resulting solution), the practical aspects of this process can be daunting. Because few researches have the patience to stir their solutions for an infinite amount of time to ensure equilibrium has been reached between the solution and the most stable crystalline form.
Application:
1. It is useful in deciding development plan & option of formulation development
2. To confirm earliest kinetic solubility assay result
3. To develop efficient cleaning validation protocol for cleaning of pilot plant &equipment
6) PARAMETER TO BE CONSIDERED WHILE MEASURING SOLUBILITY:
A) pH of solution
Most pharmaceutical compounds are weakly ionizable acids or bases, or combinations of these two ionization types. The solubility of non-ionizable compounds is a single value that reflects a simple balance between the molar free energy of the solid drug and that of the drug interacting with a polar aqueous solvent; for an ionizable drug, however, the ionizability of both the drug and the solvent must be considered. Because the extent of drug ionization changes with the extent of solvent ionization (i.e. the pH), the solid-state to solution-state equilibrium of the drug will also change with pH; thus, the measured solubility has to be viewed in the context of the pH of the solution at equilibrium and the pKa values of the compound [9].
The pH–solubility relationship of ionizable compounds is based on the Henderson–Hasselbach relationship, which relates the solubility of the completely ‘unionized’ compound (So, intrinsic solubility) to both the solubility measured at a given pH (S) and the pKa of the compound. The Henderson–Hasselbach equation takes slightly different forms for acidic and basic compounds, which can be written as,[10]
Thus, it can be seen that the solubility of an acid increases with pH at pH values greater than the pKa. For bases, the solubility value increases with decreasing pH at pH values less than pKa of the compound.
pH-dependent regions of solubility:
It is clear from the above equations that pH has an enormous effect on the solubility of ionizable compounds. In general, the pH– solubility profile can be divided into four different regions according to the physical interactions that dominate (Figure 3). [11]
Figure: 3 PH–solubility profile for a compound with a single, basic pKa value of 5. The four regions of pH- dependent solubility – salt plateau, pH max, ionized compound and unionized compound
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a) The intrinsic solubility region (pH > 7 in Figure 3). This region is defined as the pH range in which the compound is completely unionized in solution and has the lowest solubility. In this pH range, any compound that precipitates from solution will precipitate as the unionized free form, regardless of the initial salt form.
b) The ionizing portion of the curve and the region of the steepest slope. This region begins around the pKa value (_pH 4–5.5 in Figure 3). At the pKa, there are equal concentrations of ionized and unionized forms of the compound in solution. Every pH unit change on either side of the pKa will give a tenfold change in the amount of ionized drug in solution. Precipitate formed in this pH range can be in either the free form or the salt form, depending on the strength of the solid state interactions. Figure 1 shows the pH–solubility profile for a base with a single pKa. The ionized portion of the curve is more complex for compounds with multiple ionization sites.
c) pHmax. This region corresponds to the pH that yields maximum solubility of the compound (_pH 4 in Figure 1), where the ionizing portion of the curve meets the salt plateau on the pH–solubility profile. At this point, the equilibrium solid state will be a salt: that is, completely ionized drug associated with an oppositely charged counterion through columbic interactions.
d) The salt plateau(pH>4 in Figure1). In this pH range, the salt solubility of the compound prevails. The solubility of the compound is almost constant: its value is dependent on the strength of solid-state interactions with the counterions forming the salt and is given by the solubility product, Ksp, which is defined as the product of the concentrations of ion and counterion in solution:
Dissolution
Drug. salt <---------------> drug ion + salt counterion
Ksp = [drug ion] [salt counterion]
S = (Ksp)1/2
The Ksp value for a given salt of a compound is a constant value. Therefore, the drug concentration in saturated solutions is a function of the counterion concentration. As the counterion concentration in solution increases, the dissolved drug concentration decreases to maintain the Ksp. This is an important concept, especially for hydrochloride salts of poorly soluble compounds, because the active drug concentration that can be achieved is a function of the chloride concentration in the solvent or the gastrointestinal tract on oral dosing [12, 13]. Equations describing the concentrations of ionized and unionized species and salt in solution as a function of pH are described.
Case study
The solubility of an ionizable molecule is measured in water. The measured concentration at equilibrium is 1.5 mg/ml. Is this solubility sufficient to deliver an intravenous solution of 2.0 mg/ml at pH 7.4? Without knowing the pH of the final solution and the pKa of the molecule, this question is impossible to answer. Care must be taken to consider solubility always in the context of pH and pKa.
Similarly, if the measured solubility falls on the steep portion of the pH–solubility profile, small changes to the pH can have a marked effect on the solubility. To maintain a drug concentration of 2.0 mg/ml in an intravenous solution at pH 7.4, the formulator has to ensure that the pH–solubility profile has sufficient margins with respect to the pH and the desired concentration in solution to prevent the compound from precipitating.
B) Polymorph:
Polymorph is a solid material with at least two different molecular arrangements that give distinct crystal species. The achievement of system equilibrium means that sufficient time must be allowed for all change in the composition to stop. These two conditions of solubility bring with them the problem that the equilibrium attained. If the system is truly at equilibrium will be between the most stable polymorphic form of the drug substance and the drug in solution, irrespective of the starting polymorphic form of the drug. There is still a risk that this foreshortened incubation will not be sufficient for metastable crystal forms to convert to the most stable form, and that the measured concentration will represent the apparent (intrinsic) solubility of a different crystal form. This risk must be taken into consideration when running a solubility experiment with material that is not known to be the most stable crystalline form. [14]
Figure: 4 A schematic of the equilibriums that are possible for a metastable drug substance (Form I) in solution. Forms II, III and IV are polymorphic forms of the initial drug and will be present in concentrations dependent on the equilibrium between the form and the drug in solution.
Above Figure illustrates why the expectation of an accurate solubility measurement can be difficult to achieve by the shake and-assay method. In this example, the least stable polymorphic form of a drug substance is introduced to a solution. In time, a second equilibrium is established between the drug in solution and a second polymorph. The quantity of solid is governed by the equilibrium constant for the dissolution process. Ultimately, the majority of solid will be the least soluble (most stable polymorphic form) in equilibrium with the drug in solution. The other polymorphic forms will be present in quantities dependent on their equilibrium constants. Quantification of the solubility for a metastable form can, therefore, result in considerable error depending on the time point selected for the assay of the solution phase.
High melting point =strong lattice=hard to remove a molecule=low dissolution rate (and vice versa)
The stable polymorph will have the slowest dissolution rate, and so there may be occasion when it would be desirable to speed the dissolution by using a metastable form is then, It will convert back to the stable form during the product’s shelf, so manufactures check for the existence of polymorphism and ensure that they use the same appropriate polymorphic form every time they make a product [15]
Example,
a) Abbott’s antiviral drug Ritonavir: the slow precipitation of a new stable polymorph of Ritonavir from dosing solutions demanded an emergency reformulation to ensure consistent drug release characteristics
b) Chlormphenicol palmitate suspension the stable α-polymorph produces low serum levels, whereas the metastable β-polymorph yields much higher serum levels when the same dose is administered.
C) Pseudo polymorphism (solvates &hydrates)
It is possible for materials to crystallize and in so doing to trap molecules of the solvent within the lattice. If the solvent used is water, the material will be described as a hydrate. If the solvents other than water are present in a crystal lattice the material is called a solvate. Hydrates often have very different properties from the anhydrous form, as like polymorphism the most usual situation is for anhydrous form to have a faster dissolution rate than the hydrous form. [15]
Example,
a) Hydrous theophylline have slower dissolution due to the water could hydrogen bond between two drug molecules and tie the lattice together; this would give a much stronger, more stable lattice. Anhydrous theophylline rise to high concentration n solution and then falls again until the amount dissolved is the same as that recorded for the hydrate. Because anhydrous form prepares supersaturated solution first and hydrous forms have less dissolution rate and form saturated solution.
b) Hydrous form having the high dissolution rate e.g. erythromycin.
D) Thermodynamic equilibrium:
Thermodynamic equilibrium will always seek the overall lowest energy state (most stable) of the system; thus, only the ‘real’ equilibrium solubility reflects the balance of forces between the solution and the most stable, lowest energy crystalline form (most stable) of the solid. The less solid-state energy stabilization that has to be overcome, the more molecules that can be accommodated in the solution state before the energy required to break a molecule out of its crystal lattice overwhelms the energy returned from solute–solvent interactions and the increase in system entropy. [16] Thus, the most stable crystal (lowest energy crystal) form will also have the lowest solubility. Although solubility experiments that begin with a metastable solid form might measure a higher apparent solubility, given enough time the limiting solubility of the most stable form will eventually dominate (Figure 5). [17]
Figure: 5. Relationship between energy and solubility. Solid forms are found in energy minima, representing the favorable intermolecular interactions that hold molecules together in a crystal form. The deepest trough represents the lowest energy crystal form, giving rise the lowest (thermodynamic) solubility, St. Other minima represent metastable solid forms, which have differing degrees of intermolecular energy stabilization that yield different apparent solubilities, Sa1 and Sa2. These metastable forms, if provided with activation energy, will eventually convert to the lowest energy form and yield thermodynamic solubility. In the absence of intermolecular interactions (gas phase), the solubility reflects only the interaction between solute and solvent, shown as Sg. The inset provides another view of the relationship between energy and solubility: whereas the isolated molecule and solvated molecule have fixed energies, different solid states provide different energy barriers that must be overcome to achieve dissolution.
E) Temperature: [18]
Generally in many cases solubility increases with the rise in temperature and decreases with the fall of temperature but it is not necessary in all cases. However we must follow two behaviors In endothermic process solubility increases with the increase in temperature and vice versa For example: solubility of potassium nitrate increases with the increase in temperature In exothermic process solubility decrease with the increase in temperature For example: solubility of calcium oxide decreases with the increase in temperature Gases are more soluble in cold solvent than in hot solvent
F) Pressure:
The effect of pressure is observed only in the case of gases. An increase in pressure increases of solubility of a gas in a liquid. E.g. carbon dioxide is filled in cold drink bottles (such as coca cola, Pepsi 7up etc.) under pressure
G) Complexation & Coprecipitation:
Coprecipitation with PVP can markedly influence the dissolution of drugs in most instance coprecipitation as well as complexation is employed for enhancing the dissolution characteristics of the drug substance. Due to the formation of energetic amorphous form, other has attributed the effect to the drug being molecularly dispersed in the polyvinylpyrollidie. [19]
Example, Hydroflumethiazide-PVP coprecipitates.
7) DIFFERENT METHOD TO MEASURE SOLUBILITY
A) Conventional shake flask method: [20]
This method for determination of thermodynamic solubility of the solute depends on the equilibrium between the solute in solution & solid state. After equilibrium point there is no change in the concentration further.
Procedure:
Take 1 gm of solute dissolve in the solvent of choice in a beaker .stir the solution for specific period of time & allow the solution to reach equilibrium carry out the analysis of the final solution after equilibrium is achieved & find out the concentration of solution . Find out the concentration from calibration curve. It gives you the apparent solubility of solute.
B) High speed DSC (hyper-DSC) tool to measure the solubility of a drug within a solid or semi-solid matrix
Principle:
This method is used for the determination of solubility of in drug in polymer matrix. The method was based on the simple principle that the fraction of drug solubilised within the matrix does not contribute to the melting endotherm associated with the dispersed drug fraction. [21] Thus, by plotting the measured ?Hf values versus the drug concentrations for a range of loadings and extrapolating to zero ?Hf, the solubility of the drug in the polymer could be estimated. Here Changes in storage modulus ?G associated with melting of the drug within the silicone matrix were linearly related to drug loading, and extrapolation to zero (?G) provided an estimate of solubility, once again at the drug’s melting point. The accuracy of the equipment was ascertained running an indium standard Hyper DSC (HDSC) may overcome the disadvantage associated with conventional thermal methods such as DSC for determining solubility in polymer systems whereby solubility can only be measured at the drug melting temperature. The fast heating rates (In HDSC) do not inhibit the sample from responding to the energy imputed (as seen by no changes in heats of fusion for melt transitions between heating rates) but can affect and inhibit kinetically controlled transitions such as recrystallisation, polymorph interconversion, therefore, in this case the high heating rates may inhibit further solubilisation due to the increase in solubility profile caused by increasing the temp that may otherwise occur during slower scans.
The calculation of the area under the transition peak permitted the evaluation of the energy associated with the phase transition, ?Hf and so the calculation of solubility. The area under the melting endotherm was calculated from the onset to the end of the peak using the Pyris series 5 data analysis package, (Perkin-Elmer). A blank (non-drug containing) silicone film was also measured to ensure that there were no background thermal events.
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Example, solubility of metroindazole in silicon matrix is determined by the HDSC is .0216%w/w at 4000c/min
Figure.6. DSC Data at 400 ?C/min heating rate of the 1 (– - - –), 2.5 (– - –), 5(– – –), 7.5 (- -) and 10% (—) (w/w) metronidazole: silicone samples.
C) Determination of solubility by heating & equilibrium method:
Materials and methods: [22]
It is based on simple principle that the heating of solution takes less time to attain equilibrium. In the assay condition
Procedure:
a) An excess amount of the drug to be tested is put in to pure water (1 mL) in a 2-mL disposable crimp-top glass vial.
b) The suspension formed is then heated in a sealed glass vial in an autoclave (121°C for 20 minutes) or sonicated in an ultrasonic bath (e.g. at 70°C for 1 hour). After cooling to ambient temperature, the vial was opened, a small amount of the solid drug was added to the vial to promote drug precipitation, and the vial resealed.
c) After equilibration at ambient temperature (22°C–23°C) in a sealed vial under constant agitation for 3 to 7 days, the suspension was filtered through a 0.45-μm membrane filter (discarding approximately the first third of the filtrate), and the solution analyzed by HPLC. The time needed to reach equilibrium solubility was determined by analyzing samples of the equilibrating solution at different time points to establish constant drug solubility
D) Potentiometric approaches:
Potentiometric based approaches were first introduced into solubility profiling by Avedeef. [23] Determinations are based on the shift in pH values due to loss of compound, due to precipitation.
a) pSOL Instrument:
A simplified “pSOL procedure”, termed “chasing equilibrium” that allows reduction in measurement times was first described by. The equilibrium solubility in this method is actively sought by “changing the concentration of the neutral form by adding HCl or KOH titrants and monitoring the rate of change of pH due to precipitation or dissolution” pH/solubility profiles of compounds are not directly measured with this method and are accessible only via application of Henderson–Hasselbach relationships; potential aggregation phenomena are not considered. pSOL was judged to be very economical for ionizable compounds in terms of compound consumption (∼100 microgram)[24]
Use:
a) Recently, we used the pSol method for a detailed analysis of solubility supersaturation effects to identify a more rational approach for getting crystalline material in aqueous solutions in cases where standard crystallizations procedures did not work the generation of pH/solubility profiles is sometimes necessary to support interpretation of permeability and distribution results in discovery and early development.
b) The method is also accepted by the FDA for assessing the solubility for BCS classification PH/solubility profiles measured by the pSol method have been referred to as the industry ‘gold standard’ and to have major advantages compared to pH solubility profiles determined in a series of buffer system.
b) Cheqsol solubility instrument:
Introduction:
The new method (patent applied for) provides rapid results (20 – 80 minutes) for kinetic and equilibrium solubility of organic acids, bases and ampholytes. Although many other methods have been described for the measurement of solubility. [25]
Figure.7. The instrument setup used for CheqSol experiments:
Working:
The solubility measurement is based on the rate of pH change in the solution due to precipitation or dissolution.
The acid and base titrants were 0.5 M HCl and KOH, and were delivered to the titration vessel through capillaries, by precision dispensers capable of delivering reproducible aliquots of known liquid volume. Deionized water of resistivity >1014O cm is used throughout the experiment. The sample quantity is selected to ensure that when fully neutral, the concentration would be above its intrinsic solubility and would most probably precipitate if the pH has the right value. Occasionally the method has to be adjusted by increasing the sample quantity. Before each experiment, accurate pKa values were measured at 25°C and at 37°C on the Sirius GLpKa titrator. The correct pKa value is required as an error of 1 unit in the pKa will induce an error of 1 logarithmic unit on the solubility scale, log (1/S). Usually the drug solubility has different values at room temperature, 25°C, or at the body temperature, 37°C. The instrument setup allows measurements at different temperatures in a thermostated environment. The apparatus used to perform the solubility determinations was a GLpKa titrator and a D-PAS spectrometer, manufactured by Sirius Analytical Instruments Ltd (Forest Row, East Sussex, and UK). The UV absorption of the solution was continuously monitored in the titration vial by a fibre optic dip-probe. The software was RefinementPro 2 and CheqSol. All titrations were performed in 0.15 M KCl solution, to mimic the ionic strength of the blood, under argon atmosphere and with degassed reagents. The solubility assays are sensitive to carbon dioxide; therefore the measurements have to be performed under argon atmosphere, in air-tight vessels. A schematic titration head is shown in Figure 7. The D-PAS spectrometer provides higher sensitivity, allowing the use of a reduced sample quantity. It also allows the measurement of extreme pKa values, e.g. below 2 and/or above 12.Future extensions of both methods regarding determination of solid state properties seem to be possible.
E) Use of 1H NMR to facilitate solubility measurement for drug discovery compounds
NMR is a powerful technique for solution sample analysis. It provides detailed information of each component in the solution and can also be used for quantitative analysis. Quantitation can be carried out using internal standard using isolated aromatic resonances, without a calibration curve, which makes using NMR for quantitation convenient and provides a time saving over the HPLC-UV method. With NMR, there is also lesser of an issue with dynamic range found in HPLC-UV with higher concentration samples. With more concentrated samples, an HPLC UV system will yield peaks that are non-quantifiable due to topping out of the detector. This leads to the further step of making sample dilutions prior to analysis. This is not the case with NMR. The added expense of an NMR system versus an HPLC-UV could be prohibitive. The NMR method is fast, and sample preparation is simple if the formulation materials are made ahead of time in larger batches. In addition, the process can be easily automated. Therefore, solubility determination by NMR provides an easy and practical approach to screening Discovery formulations. [26]
TSP was used as the internal standard. TSP has a single proton signal that is isolated from the signals of the formulation excipients and the compounds of interest, and therefore, eliminates the potential for interference with peaks resulting from the compound of interest. The TSP can be prepared in bulk D2O facilitating the process. Also, Since the NMR method is nondestructive, although some recovery process would be required, samples can be reanalyzed over time to yield information on the stability of a compound in any formulation.
Procedure
Approximately 15mg of each compound weigh into 4mL scintillation vials. Three milliliters of the appropriate formulation vehicle is then added and vortex the sample. Samples were placed on an end-over-end rotator and equilibrated for 2 days. The pH is measured from the suspensions at the end of the equilibration period. For analysis, the suspension is filter using 0.22_m Millipore PVDF syringe filters. After discarding the first 5 drops, the remaining portion is subject to NMR analysis.
One potential draw back
NMR solubility analysis is the relatively lower sensitivity compared with UV detection and mass spectrometry techniques, which limits analysis of samples having low solute concentrations.
F) A fully automated kinetic solubility screen in 384-well plate format using Nephelometry:
Principle
Laser nephelometry is the measurement of forward scattered light when a laser beam is directed through a solution. The more particulate there is in the solution, the greater the amount of forward scattered light (measured as counts). [27]
Limitations
Method includes any factors that result in turbidity or light scattering could interfere with data.
a) Colored compounds may be misassigned as insoluble in determining the precipitation point when turbidimetric method is used, leading to lower solubility values.
b) Imperfections including scratches on the wells in the plates or any foreign materials in the wells could scatter light and produce a false positive reading.
Commercial solutions are available, including the BD Gentest Solubility Scanner (Flow cytometry) (www. bdbioscience.com), Millipore MultiScreen® and MultiScreen®, and BMG LabTech NEPHELOstar (nephelometry)
G) Solubility measurement by UV absorption:
Kinetic solubility in high-throughput assays can be also quantities by UV plate readers or HPLC- UV. In these methods, a drug solution in DMSO is diluted with aqueous media. After precipitation, the saturated solution is passed through a 96-well filter plate. The drug concentration in the filtrate is quantities by UV absorption, and solubility is calculated using a calibration curve.
UV absorption methods can achieve a limit of detection down to 1 μM 6. They provide a more accurate determination of solubility because the value is determined based on absorption of drug molecules against a calibration curve. However, the methods cannot distinguish multiple chromophores that overlap in their absorption. False positive results can be generated if any impurities in the compounds or excipients absorb light in the wavelength region of interest.
Commercial equipment
Solvinert filter plates (direct UV) (www.Millipore. com), uSOL Evolution™ (direct UV)(www.pio-inc.com)The µDISS Profiler™ instrument (Pion)( [28]
H) Noyes–Whitney template titration method:
A potentially more reliable method for the determination of solubility is the. Potentiometric analysis is achieved by titrating acid or base with the solid drug. Bjerrum Difference Plots can then be constructed from the titrations; these shows the average number of protons bound in relation to pH, and provide approximate solubilities, which are then refined via iterative, least squares fit analysis1. This method has been applied successfully to the calculation of solubility for a series of polymorphs (Yet not published)
8) INCREASING THE SOLUBILITY OF IONIZABLE COMPOUNDS:
Having examined the shape of the pH–solubility profile for ionizable compounds, it is worth discussing the ways that this profile can be used to engineer solubility improvements for ionizable compounds.
Special case of apparent solubility: salts
Pharmaceutical salts pose a special case of apparent solubility that has important implications for drug discovery and development Salts are formed when a compound that is ionized in solution forms a strong ionic interaction with an oppositely charged counterion and maintains that interaction through crystallization. The resulting solid comprises charged drug molecules and their associated oppositely charged counterions. The essential characteristic of salts that makes them so attractive in pharmaceutical applications is that the columbic attraction between the drug molecule and counter ion changes the potential energy landscape of the solid state and leads to stronger interactions between the charged active pharmaceutical ingredient and polar aqueous solvents.
This can result in enhanced dissolution rates and higher apparent solubility on physiologically relevant timescales, resulting in more effective drug delivery in vivo. Caution must be applied when discussing salt solubilities: the ionizable drug molecule that facilitates salt formation complicates the discussion of solubility, as mentioned in the previous section. Because salts have such a crucial role in pharmaceutical drug delivery, it is important to understand how the choice of counterion for a salt affects solubility.
Optimizing factors that affect solubility
Following the example of Bogardus and Blackwood , a simple relationship can be developed among pHmax, pKa, intrinsic solubility and salt solubility that can be applied to a specific salt of any monoprotic compound:
Ksp, it must be recalled, is related to the constant solubility achieved on the salt plateau and depends on the identity of the salt. Because so(equilibrium solubility) and pKa depend on the properties of the drug, not on those of the counterions, this equation can be used to describe the pH at which maximum solubility is achieved. It can also rationalize the improvements in solubility that can be achieved through physical and chemical manipulation of the drug molecule.
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a) First, simply by increasing the strength of the pKa, the whole pH–solubility curve can be shifted (Figure 4a). This is useful for pharmaceutical compounds because it can be used to ensure that the region of maximum solubility corresponds to the physiologically relevant pH range, or that the solubility of a solution formulation is governed by the desired solid form (free form or salt).
b) Increases in the intrinsic solubility of the unionized molecule might lead to increases in solubility across the whole pH–solubility profile (Figure 4b). Although this is an attractive possibility, it can be difficult to achieve because the factors governing intrinsic solubility (e.g. stability of the solid state or strength of intermolecular interactions) can be difficult to predict and to alter systematically.
c) A higher Ksp value signifies increased solubility along the salt plateau and at pHmax, but it will not change the shape or position of the rest of the pH–solubility curve (Figure 4c). Nonetheless, raising Ksp is perhaps the most valuable scheme for enhancing the solubility of pharmaceutical molecules because, unlike changes in So or pKa, it does not require alterations to molecular structure that could adversely affect pharmaceutical activity. Although it can be both simple and fruitful to enhance solubility by exploiting Ksp, unfortunately the opposite effect can also be achieved if ionized molecules in solution encounter counterions with which they form a less soluble salt. Similarly, excess counterions can drive the drug out of solution through the common ion effect
Figure: 8 Strategies to increase solubility in the physiological pH range by altering physical chemical properties.
a) By strengthening the pKa of a weak base by 1 or 2 pH units, the whole pH–solubility curve can be shifted so that the region of maximum solubility (salt plateau and pHmax) overlaps with the physiologically relevant pH range.
b) Increased intrinsic solubility (So) can lead to increased solubility at every pH, although the solubility product (Ksp) or common ion effects can effectively limit solubility at the salt plateau.
c) Effect of enhanced salt solubility (increased Ksp) on the pH–solubility profile.
9) APPLICATION IN DRUG DISCOVERY IN DEVELOPMENT:
Throughout the various phases of discovery and development, solubility information serves a wide range of needs. In the early stages, solubility is used to characterize compounds belonging to a chemical series and to determine whether these compounds are soluble enough for structure–activity relationship screens. As compounds advance past structure–activity relationship screens, solubility data are used to assess absorption, distribution, metabolism and elimination parameters and to develop formulations for safety screens, pre-clinical and early clinical use. In drug development, solubility knowledge is needed for biopharmaceutical classification, biowaivers and bioequivalence. It is also required for formulation optimization and salt selection. In manufacturing, solubility affects the optimization of manufacturing processes
a)Compound characterization in during drug discovery and development.
b) For bioassay optimization: [29]
In recent years there has been a growing awareness of solubility related limitations on many aspects of drug discovery. Initially, the focus was on the role of compound solubility in drug absorption and pharmacokinetics. Recent concerns have extended this focus to the effect of compound solubility in biological assays. The work of Lipinski raised awareness that low solubility will limit compound absorption after oral dosing. As a result, minimum solubility levels were recommended for candidates, based on their permeability and therapeutic dose, and fundamental properties, such as solubility, were regularly measured.
Figure: Discovery bioassay process with regard to solubility.
c)To avoid a bottleneck between lead optimization and the entry into human (EIH):
Due to patent protection to pharmaceutical compound the pharma industries try to reduce delay in entry of drug into the market. The initial selection of the lead compound is depends on the solubility characteristics to reduce the failure in subsequent stage of drug development.
d)Focus of solubility in different stages of the drug discovery and development:
Figure: 9 Focus of solubility assays along the drug discovery–development process: discovery and development areas which need solubility information.
e)Used in quality control and bioequivalence studies and preparation of biowaiver. The concept underlying the Biopharmaceutics Classification System (BCS) finally lead to introducing the possibility of waiving in vivo bioequivalence studies in favour of specific comparative in vitro testing in order to conclude bioequivalence of oral immediate release products with systemic actions. This approach is meant to reduce unnecessary in vivo bioequivalence studies however, is restricted to non-critical drug substances in terms of solubility, permeability, and therapeutic range, and to non-critical pharmaceutical forms. Although frequently discussed, BCS-based biowaivers are still rarely used probably attributed to uncertainties on both, pharmaceutical companies and regulatory authorities. Substantial differences of biowaiver dossiers and respective assessments contribute to the impression that a common understanding is lacking on a successful use of the BCS concept to support biowaivers. It is intended to reach an optimal and harmonised application of biowaiver principles within the European community by means of preparing an annex to the guideline on Bioavailability and Bioequivalence.
10) RECENT ADVANCES IN SOLUBILITY MEASUREMENT:
For any type of HT solubility assay it is important to organize an appropriate workflow to achieve (1) short turnaround times, (2) a seamless integration into existing instruments and processes, (3) a reduction of human errors, (4) an improvement of data consistency, and (5) an effective integration of data management. The introduction of new assays must weigh the benefits of setting up robotic systems to augment productivity through increased automation against the confines of budgetary constraints on capital investments and selecting equipment that fits company needs.
In early development, the challenge will be in the future to develop profiling capabilities that use limited quantities of material for screening programs in which several formulation technologies are tested in a rapid and expeditious manner to determine which strategy offers the highest improvement in solubility and to identify the best suited technology for further development.
The workflow for the Handling of these solvents in development is usually not yet established and the required cooperation with compound management, informatics specialists, and assay automation specialists is often not in place .New technologies using microfluidic systems could here be beneficial for the distribution of low volumes such as micro droplet devices or the physical contact-free fluid transfer by the acoustic droplet ejection technology.[30] In contrast, the systems available for automated solid compound distribution are already more advanced, except for situations with very low compound availability or with inappropriate solid state compound properties.
Enabling strategies need to be established to test only those solvents with impact on decision making in drug discovery and development. In discovery, this may result in an extension of solvents to be tested, particularly in the lead optimization phase.
Another approach to reduce the number of HT screenings could be the systematic application of multivariate techniques such as principal component analysis (PCA), finger-print-based methods or design of experiment (DOE). With the increasing number of solubility values, data interpretation activities will become as important as the underlying data for knowledge generation.
User-friendly, efficient information processing systems need to be assembled and tools for the visualization of compound solubility as a function of various physicochemical properties (solid state properties, pKa, log D, etc.) and solvent parameters (solvent composition, pH, stability, temperature, etc.) have to be established. Rapid data visualization will help to uncover intimately linked compound and solvent properties that positively or negatively affect each other and may thus facilitate and speed up decision making in drug discovery and development; examples are the recently reported “self-organized” solubility-and PAMPA excipient plots, the plotting of solubility profiles as a function of substituent’s , the bioavailability score, solubility-pH plots for forced degradation studies or the solvent miscibility plots .
However, newer approaches that substitute balances and the time consuming weighing process, such as powder distribution to chips, may soon replace these techniques and speed up assays further. Well-less, high density techniques that spot and dry compound solutions onto polystyrene sheets (ChemCard) might be another option. This technique has already been used for solubility testing and observed correlations with equilibrium solubility may be improved further if analytical techniques for solid state characterization of the dried material could be applied. Current X-ray and Raman spectroscopy will probably not be applicable for such small samples, however the recently Introduced Fourier transform infrared (FTIR) in combination with attenuated total reflection (ATR)could be an option. This technique has been successfully used to study formulation stability, drug polymorphism and dissolution processes.
Finally, drug dissolution, will also be a topic for solubility assays in the future. Current HT assays determine solubility after a defined time but do not take the compounds' dissolution rate into account which is an important parameter of drug bioavailability, in particular for class II and IV compounds of the BCS system. Consequently, in addition to equilibrium solubility assays, miniaturized assays for intrinsic or powder dissolution would be highly desirable in the future.
11) CONCLUSION:
Among the five physicochemical screens in early compound screening, pKa, solubility, permeability, stability and lipophilicity, poor solubility tops of the list of undesirable compound properties. Solubility of drug candidate plays a vital role in selection of lead compound in early stage of drug development and discovery. Biopharmaceutical classification system distributes the drug candidate into different bins depending on the solubility and permeability. Two type of solubility determined at different stages of drug discovery kinetic solubility and thermodynamic solubility. Solubility screening in drug discovery and development focuses on the solubility screening in discovery and solubility screening in development. Kinetic solubility determined at early stage of drug development when the compounds have not been purified to a high degree or crystallized and compound available in sub milligram quantities. Kinetic solubility designed to facilitate high throughput (>600 compounds per week). Another equilibrium solubility (Thermodynamic solubility), It is true solubility of compound. It is neither practical nor useful to measure it in the earliest drug discovery stage. It is useful in deciding development plan and option of formulation development and to confirm result obtained from kinetic solubility data.
Different problem encounter while determining the solubility, most of characteristics usually pH dependent, such as multiple and often overlapping ionization, complexation, aggregation, micelle formation, and “common ion” effect, incubation time, adsorption to micro porous filters, plastic or glass surfaces, polymorph interconversion. Above parameter should be considered while determining solubility. Different method available for measurement of the different solubility, conventional shake flask method now a day’s replaced by the high throughout solubility assay technique which considerably reduces the incubation time increased accuracy of result. Solubility determination can be done by ultraviolet absorption, nephlometry, Nuclear magnetic resonance and Potentiometric in drug discovery
Acknowledgement:
The authors would like to acknowledge, to the Prof. Vinod Mokale Head of Division of Pharmaceutical Technology ,School of chemical Technology, North Maharashtra University Jalgaon for providing necessary facilities and encouragement.
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