Tài liệu HPLC for Pharmaceutical Scientists 2007 (Part 12) doc

The biopharmaceutical properties such first-as gfirst-astrointestinal and plfirst-asma solubility, lipophilicity LogD, permeability, first-pass metabolism, systemic metabolism, protein bindi

of failures during future drug development.

On average, only one out of ten new chemical entities (NCE) entering in-human testing reaches registration, approval, and marketing stage Thereasons for failures of development compounds include problems with bio-pharmaceutical properties, clinical safety, toxicology, efficacy, cost of goods,and marketing (see Figure 12-1) [1, 2] The biopharmaceutical properties such

first-as gfirst-astrointestinal and plfirst-asma solubility, lipophilicity (LogD), permeability,

first-pass metabolism, systemic metabolism, protein binding, and in vivo

bioavailability are related to the solubility, chemical stability, and ity of drug candidates and have to be considered at discovery lead selectionbefore recommendation to the development stage

permeabil-A major challenge in any drug discovery program is achieving reasonablebioavailability upon oral administration; therefore, any information that high-lights potential problems with cell permeability and absorption is valuablewhen reviewing structural families as leads for drug discovery Lipinski et al.[3] have reviewed 2245 compounds selected from the United States Adopted

577

HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto

Copyright © 2007 by John Wiley & Sons, Inc.

Name (USAN), International Nonproprietary Name (INN), and World DrugIndex (WDI), comparing calculated physical properties and clinical exposure.Four parameters were chosen that were associated with solubility and per-meability, namely, molecular weight, octanol/water partition coefficient, thenumber of hydrogen bond donors, and the number of hydrogen bond acceptors It was concluded that compounds are most likely to have poorabsorption when molecular weight is >500, the calculated LogP is >5, thenumber of hydrogen bond donors is >5, and the number of hydrogen bondacceptors is >10 Lipinski has referred to this analysis as “rule of five” becausethe cutoffs for each of the four parameters were all close to five or a multiple

of five The rule of five can serve as qualitative absorption/permeability predictor

The absorption of drug molecules in the gastrointestinal tract is dependent

upon the pK a of the compound and the pH of the gastrointestinal region(Figure 12-2) Almost 63% of all drugs are ionized in aqueous solution andcan exist in a neutral or a charged state, depending on the pH of the local environment [4]

Based on the major goal of preformulation—identification of possiblefailure in future development—numerous studies are performed to fully char-acterize prospective drug candidates The major analytical technique in eachpreformulation group is liquid chromatography Ninety percent of all ana-lytical equipment in preformulation groups are HPLC systems equipped with

UV and MS detection systems HPLC is a fast and reliable method for centration and identity determination by UV and/or MS detection, respec-tively The type of HPLC methods differ based on the specific preformulationtests that will be described below

con-In the early stage of preformulation, characterization of the drug moleculeinvolves ionization constants and partition coefficient determinations, aqueousand nonaqueous kinetic and equilibrium solubility determination, pH solubil-ity profile, chemical stability assessment, and salt and polymorph screening.Assessment of biopharmaceutics and toxicological screening are also essential

Figure 12-1 Reasons for attrition from 1991 to 2000 (Reprinted with permission from

reference 1.)

at this stage At the later stage of preformulation, after recommendation ofNCE to development, the development support from preformulation groupinvolves a more detailed solid-state characterization program, elaborating onmoisture sorption, compressibility, melting point, particle size, shape, andsurface area assessments, as well as excipient compatibility and prototype for-mulation stability evaluation.

Further information on the role of preformulation in drug developmentprocess can be found in several excellent monographs [6–8] with the focus onpharmaceutical aspects of process development

(DISCOVERY SUPPORT)

During the early discovery stage the medicinal chemists use in vitro activities and fast in vivo small animal studies to discover the best compound to develop.

The support from development scientist consists of providing information

about LogP, pK a, and LogD for ionizable drugs and aqueous solubility Thesephysical characteristics can affect the absorption of drug candidate and, there-fore, drug bioaivalability The requirements for HPLC analysis at this stage arespeed and efficiency of the separation It is critical to mention that at the earlystage of discovery, very little information is available about the properties of

Figure 12-2 Physical properties of the gastrointestinal tract (Reprinted with

permis-sion from reference 5.)

molecule and only a few milligrams of compound is available for zation Therefore, it is important to choose the most efficient column and thesimplest mobile phase Also, recommended is the use of more contemporaryHPLC systems as UPLC from Waters employing columns with dimensions of

characteri-50× 2.1 mm, 1.8-µm particle size and the Fast 1200 system from Agilent withcolumn dimensions of 50 × 4.6 mm, 1.8-µm particle size, respectively, toenhance the turnaround time for sample analysis Other platforms wouldinclude using Chromolith Speedrod® monolithic columns at high flow rates.Also, taking into consideration the short column length, gradient elutionshould be recommended for all HPLC methods at this stage of drug candidatecharacterization The post-run equilibration time is not significant in the casewhere short columns are used, and dwell volume is improved significantly for

a new generation of HPLC systems

Many types of modeling techniques are available in the discovery phase ofdrug development, from structure activity relationships (SAR) to physiologybased pharmacokinetics (PBPK) and pharmacokinetics-/pharmacodynamics(PK/PD) to help choosing some of the lead compounds Some tests that arecarried out by discovery include techniques related to structure determina-tion, metabolism, and permeability: NMR, MS/MS, elemental analysis,

PAMPA, CACO-2, and in vitro metabolic stability Although they are

impor-tant as a part of physicochemical molecular characterization under the pharmaceutics umbrella, they will not be discussed here The reader can findrelevant information in numerous monographs [9, 10]

bio-12.2.1 Ionization Constant, pK a

Most potential drug candidates are weak bases or acids Solubility and manyother properties of the drug molecule is dependent on its ionization state.Acids are usually considered to be proton donors and bases are proton accep-tors Any drug molecule with basic functionality in aqueous media holds thefollowing equilibrium:

(12-1)where the ionization equilibrium constant could be expressed as

(12-2)

It is obvious from the above equilibrium that the ratio of ionic to nonionicform of the drug in the solution is controlled by the proton concentration,which is commonly represented by pH values (negative logarithm of protonconcentration) Taking the negative logarithm of expression (12-2), the well-known Henderson–Hasselbalch equation could be obtained:

Ka=[ ]⋅[ ] [ +]

B HBH+

BH+↔ +B H+

This allows for the estimation of the prevailing drug form at a particular pH.Ionic form of any organic molecule is usually more soluble in aqueous media,while the neutral form is usually more hydrophobic and thus shows anincreased affinity for lipids

Variation of the ionization state of the molecule at different pH has typicalsigmoidal shape (as shown in Figure 12-3) Corresponding expression for thisdependence could be derived from equation (12-2) and the mass balance ofthe ionic and nonionic form of the drug:

(12-4)

If one assumes quantity q equal to 100, then concentration of B or BH+formswill numerically be equal to the percentage of corresponding form in the solu-tion and solving equation (12-3) with expression (12-4) one will get the expres-sion for BH+concentration expressed as a percent of ionized form

(12-5)

The inflection point of this curve corresponds to the point where pH = pKa,

and it is a common way for the determination of the drug pK avalues

Several different techniques are usually employed for pK a determination.They were described in detail by Comer [11]

a a

Figure 12-3 Dependence of the relative amount (in the form of a percent) of

proto-nated form on the pH of aqueous media

In practice the most common technique to determine pK a value is byemploying potentiometric titration based on the detection of the variations ofeither the conductivity or current at fixed applied potential at various pH

values The automated potentiometric titration system well known as a GLpK a

or PCA200 from Sirius Analytical [12] is considered to be a good approach

for pK a determination with water-soluble drugs at pH 2–8 for the new drugcandidates when the amount of drug substance is limited For poorly water-

soluble compounds it is advised to use GlpK a with D-Pass or Sirius ProfilerSGA as a pH/UV method for determination of compounds that have inher-ently lower concentration in the solution media

HPLC is another convenient method for measurement of the NCE pK a

values As was shown by Melander and Horvath [13], the retention of any ionizable analyte closely resembles the curve shown in Figure 12-3 Chro-

matographic determination of the pK a could be accurately performed withvery limited amount of sample Fast HPLC method with optimum analyteretention is suitable for this purpose, but the influence of the organic mobile-

phase modifier on the mobile phase pH and analyte pK ashould be accounted

for in order to provide the accurate calculation of the respective pK a value

Detailed discussion of the HPLC-based methods for the pK adetermination isgiven in Chapter 4

In the case of sufficient drug supply the old-fashioned solubility method can

be used for pK a determination based on the different equilibrium solubility

at different pH values This method is very precise, but time- and consuming, and is described in detail in reference 6

drug-Drug substance often contains several ionizable groups that may

signifi-cantly complicate experimental measurement of the pK a All different types

of pK a determination methods are essentially based on the measurement of

the titration curve If the pK avalues of several ionizable groups in the cule are within 2 pH units from each other, experimental measurementbecome very tedious Recent advancements in the molecular computationalmethods and developments of physicochemical databases for a large number

mole-of known compounds allow computer-based prediction mole-of the pK avalues onthe basis of known physicochemical correlations and fast computer screening

of known values for related or structurally similar compounds from the base Detailed discussion of these programs is given in Chapter 10

data-12.2.2 Partition and Distribution Coefficients

One of the most important physicochemical parameters associated with oralabsorption, central nervous system (CNS) penetration, and other pharmaco-kinetic parameters is lipophilicity of organic compounds, which determinesdistribution of a molecule between the aqueous and the lipid environments.The lipophilicity in the form of LogP was included in Lipinski’s rule of five asone of the major characteristics of drug-like organic molecules It was statedthat LogP should be not more than five for drug candidates to have a good

oral absorption property In Table 12-1, some LogP values for various types ofdosage forms are given.

The partition coefficient itself is a constant and is defined as the ratio ofconcentration of compound in aqueous phase to the concentration in an

immiscible solvent, as the neutral molecule In practical terms the neutral

mol-ecule exists for bases >2 pH units above pKaand for acids >2 pH units below

pK a In practice, log P will vary according to the conditions under which it is

measured and the choice of partitioning solvent LogP is the logarithm of tribution coefficient at a pH where analyte is in its neutral state This is not aconstant and will vary according to the protogenic nature of the molecule.The choice of partition solvent has been a subject of debate Different type

dis-of solvents have been used for the determination dis-of partitioning coefficient

[14], but the majority of the data are generated using water–n-octanol

parti-tioning Octanol was chosen as a simple model of a phospholipid membrane.However, it has shown serious shortcomings in predicting blood–brain barrier

or skin penetration Other solvents such as chloroform, cyclohexane, andpropylene glycol dipelargonate (PGDP) have been used for modeling biolog-ical membranes

Octanol is a hydrogen-bonding solvent, and thus it shows certain specificity

in its ability to dissolve some components For example, K0

w for phenol inhexane is only 0.11 while in octanol it is equal to 29.5 There were severalattempts to rationalize solvent effects using solubility parameters [15], dielec-

tric constant [16], and others, but none appear to be consistent n-Octanol gives

the most consistent results with other physicochemical properties and drugabsorption in gastrointestinal tract

The classical measurement of LogP is the shake flask method [17] A knownamount of drug is dissolved in a flask containing both octanol phase andaqueous buffer at controlled pH to ensure the existence of only nonionic form

(at least two units from the drug pK a) The flask is shaken to equilibrate thesample between two phases There must be no undissolved substance present

in both phases After the system reaches its equilibrium, which is time- andtemperature-dependent, the concentration of drug is analyzed by HPLC inboth phases Partitioning coefficient is calculated as

This method allows for the accurate determination of K0

w only within the −

1000 to +1000 region or approximately within six orders of magnitude span.These experiments could be complicated by solubility and equilibration kinet-ics and the properties of a substance For example, if a studied compound has a property of nonionic surfactant, it will be mainly accumulated at thewater–organic interface, and shaking of this two-phase system will create astable emulsion difficult for analytical sampling The ultracentrifugation atspeed of 14,000 rpm for 15–20 min can be enough in most cases to separatetwo phases Actual equilibration of the system is tested by several measure-ments of the equilibrium concentration at different time intervals

Because of the wide range of partitioning coefficient values, in most cases

the decimal logarithm of K0

wis used, and it is denoted as LogP:

(12-7)

The biggest challenge for the use of HPLC in the LogP measurement is thedetermination of the drug concentration in the octanol phase If the octanolsolution is being injected onto the reversed-phase column, it can modify thestationary phase, shift the analyte retention, and lead to an incorrect mea-surement due to the retention shift To avoid this problem the dilution in thecorresponding mobile phase is recommended Also, when LogP is more thanfour, the concentration of drug in water phase is very small, causing a detec-tion problem with UV detection This becomes even more troublesome if thecompound of interest has a weak UV chromophore The use of MS detectionand proper ionization mode is recommended to increase the sensitivity.Direct HPLC experiment can be used for estimation of LogP, but this tech-nique is valid only for neutral molecules or for ionized molecules analyzed intheir neutral state [18] The following is a brief description of this method.Compounds with known LogP is injected onto C18 hydrophobic column,and the respective retention factors are used to create a calibration curve Theestimation of LogP for unknown compounds can be made on the basis of thiscalibration curve This method is straightforward, but requires the previous

knowledge of pK a values for ionizable compounds to avoid the possible ization that will lead to incorrect determination of values of LogP Recently,

ion-an automated isocratic liquid chromatography system, dedicated to the measurement of LogP, Profiler LDA, was introduced into the market by Sirius-Analytical, Ltd There were numerous attempts to use the retention time ofcompound in correlation with its distribution properties in RP HPLC [19, 20].The retention factor was used to calculate a distribution coefficient betweenstationary phase and mobile phase In case of Sirius Profiler LDA automatedsystem, a set of molecules with known LogP values was used to calibrate thesystem and convert the chromatographic retention time into octanol/waterpartition coefficients The system could cover the LogP range from −1 to 5.5

by choosing between three different methods and different column lengths

LogP=log K( )w0

ranging from 1 to 25 cm, but was recently removed from the market The

well-known automated pH titrator from Sirius, GlpK a, can be used as well to mine the octanol/water partition coefficient The measurement is based on atwo-phase acid/base titration in a mixture of water/octanol [21]

deter-Partition coefficient discussed above represents oil/water equilibrium tribution of only neutral forms of a substance The distribution at different pH

dis-is described by LogD, which dis-is the logarithm of the ratio of the concentrations

of all forms of analyte in oil and water phases at particular pH Logarithm ofdistribution coefficient at pH 7.4 is often used to estimate the lipophilicity of

a drug at the pH of blood plasma

As follows from the definition, the distribution coefficient is dependent onthe pH It is usually assumed that in the oil-phase drug molecule could exist

in only nonionic form; thus the distribution coefficient, D0

w, for basic drug Bcould be written as

of a basic drug with LogD

Log D pH LogP Log p pH

+ water

LogD

Figure 12-4 Normalized dependence of the protonated form of the base (solid) and

its LogD dependence on the aqueous pH (dashed)

At high pH, the neutral form of a drug (basic compound) has a tion coefficient equal to its partitioning coefficient With the decrease of the

distribu-pH of the aqueous phase, the degree of drug ionization increases, thus ing its total concentration in the aqueous phase As the pH decreases, the ionic equilibrium is shifted toward the protonated form of a drug, which con-tinually increases its concentration in the aqueous phase and decreases itscontent in oil phase There is no plateau region in the LogD curve at low pHfor basic compounds (Figure 12-4) On the other hand, for acidic compounds,

increas-there is a plateau region in the LogD curve at low pH (pHs below the pK a);and then as the pH increases, the more ionic equilibrium is shifted toward theionized form of the acid, which continually increases its concentration in theaqueous phase and decreases its content in the oil phase This results in the absence of plateau in the LogD curve at high pH (pH > pKa) for acidiccompounds

These are only the theoretical dependencies; real behavior of actual cule usually is significantly altered due to different types of intermolecularinteractions Molecular solvation, association, hydrogen bonding, and counte-rions all have a significant effect on drug ionization constant and partitioningand distribution coefficients Detailed and comprehensive discussion of theseeffects could be found in the book by Avdeef [22]

mole-12.2.3 Solubility and Solubilization

Aqueous solubility is one of the most important physicochemical properties

of a new drug candidate because it affects both drug absorption and dosageform development Only a drug in solution can be absorbed by the gastroin-testinal track The rate of dissolution and the intestinal permeability of thedrug molecules are dependent on the aqueous solubility—that is, the higherthe solubility, the faster the rate of dissolution An excellent monographdescribing the theory of solubility and solubility behavior of organic com-pounds was written by Grant and Higuchi [23] For additional information onsolubility, the reader can be referred to references 24–27

Solubility is expressed as the concentration of a substance in a saturatedsolution at a defined temperature The US Pharmacopeia (USP) gives the solubility definitions shown in Table 12-2

Solubility measurements are generally carried out in the early stages ofdrug development because it affects drug bioavailability evaluation; in manycases, solubility-limited absorption has been reported Only a compound that

is in solution is available to cross the gastrointestinal membrane The ity measurements in aqueous buffered systems at different pHs are used tomimic gastrointestinal human or animal fluids Solubility determination inDMSO is very important at the early stages of lead candidate selectionbecause of the increasing use of 10 mM DMSO solution as a stock solution forbiological testing for very slightly soluble lead candidates [29] In general,

solubil-aqueous solubility is measured in simple buffered solubil-aqueous media In practice,the aqueous medium of the gastrointestinal track is a mixture of salts and sur-factants, and the recipes to mimic the fasted (fasted state simulated intestinalfluid, FaSSIF) [30] and fed state (fed state simulated intestinal fluid, FeSSIF)[31] may be used when the influence of gastrointestinal fluid on oral absorp-

tion of NCE is studied especially for in vivo/in vitro correlation experiments

[32] It was reported that for some compounds the solubility in FaSSIF and FeSSIF will be higher than the solubility in aqueous buffers at the same

pH [33]

At the early stage of candidate selection the different experimentalmethods based on high-throughput solubility measurements are used to deter-mine the apparent solubility of potential lead candidates as well as in silicopredictions [34] to quickly assess aqueous solubility These methods aredescribed in details in references 5 and 35 In the later stages of preformula-tion when the drug candidate is in a well-characterized crystalline solid state,more precise determination of the equilibrium aqueous solubility is necessaryfor designing appropriate formulations The old-fashioned shake flask method

is recommended to measure equilibrium aqueous solubility [36] at this stage.The procedure is very simple The compound in solid state is added to bufferedsolution in excess (saturated solution), and the suspension is shaken on amechanical shaker until the system reaches the equilibrium between twophases, solid and liquid Sometimes the equilibration time is very long and canvary from 2 hours to a few days or weeks, which is dependent upon the numer-ous factors that affect solubility Solution stability may also be a concern, as

an additional precaution the solutions should be protected from light whenpossible if they may be prone to photodegradation To check the equilibriumcondition, several HPLC measurements should be determined at several timepoints The system is considered to be in equilibrium when the solubility mea-surements between several time points remain constant

However, the equilibrium solubility values are very difficult to obtain,because they are affected by many factors such as crystalline form of a sub-stance, particle size distribution, temperature, composition of aqueous phase,

TABLE 12-2 Solubility Definitions by US

Pharmacopeia [28]

Parts of Solvent Required

and even the amount of excess solids [37] Table 12-3 shows some examples ofreported aqueous solubility range for commercial drugs.

Aqueous solubility of ionizable molecules at different pH values is animportant characteristic because it indicates the potential substance behavior

in the stomach and intestinal tract and its potential impact on bioavailability.Moreover, it also provides important information for formulation scientists todefine the class of a drug substance in the Biopharmaceutics ClassificationSystem (BCS), a regulatory guidance for bioequivalence studies The BCS is

a scientific framework proposed by the FDA to classify drug substances based

on their aqueous solubility and intestinal permeability and defines importantparameters in the selection of drug candidates into development According

to the BCS, drug substances are classified as shown in Table 12-4

An objective of preformulation scientist is to determine the equilibrium solubility of a drug substance under physiological pH to identify the BCS class

of drug candidate for further development For BCS classification the test ditions are strictly defined by the FDA The pH solubility profile of the testdrug substance should be determined at 37°C in aqueous media with a pH

con-in the range of 1–7.5 Standard buffer solutions described con-in the USP are considered to be appropriate for use in these studies A number of pH condi-

tions are used bracketing the pK a value for the respective test substance

For example, for a drug with a pK of 5, solubility should be determined at

TABLE 12-3 Variation of Aqueous Solubility in the

Different platforms are used for solubility measurements: UV; HPLC with

UV detection; or HPLC with MS detection UV spectrophotometry is the plest and fastest method, unfortunately with limited applicability In most casesthe drug substance available for the study in the preformulation stage is notpure enough to provide an adequate absorbance–concentration relationship

sim-of drug substance itself In this case, HPLC with UV detection is the mostapplicable technique to use Fast gradient methods on short columns could besuccessfully used in most cases as described in Chapter 17 Some software programs such as ACD/LogD Sol Suite [39] can be used to estimate the solu-bility as a function of pH and can be used as a starting point to estimate theappropriate dilution of the different solutions prepared at the different pHvalues

In some cases, drug substance does not have chromophores with a molarabsorbtivity sufficient for accurate quantitation using UV detection If HPLCwith UV detection is used as a basic quantitation technique, then MS detec-tion as a complementary technique is desirable in most cases LC-MS is essen-tially preferable in most preformulation assays High selectivity of the MSdetector allows the use of fast gradient HPLC separation methods, which doesnot require significant development time Practically in all assays used in preformulation, the quantitation of only drug substance is required and MSdetection provides an accurate quantitation

Identification of pharmaceutically acceptable vehicles that afford sufficientsolubilization while maximizing physiological compatibility for preclinicalpharmacokinetic evaluation is critical The most frequently used solubilizationtechniques include pH manipulation for ionizable compounds; use of co-solvents such as PEG 400, ethanol, DMSO, and propylene glycol; micellar solubilization with surfactants such as Tween 80 or SLS; complexation withcylodextrins [40] By using the solubilization techniques, the enhancement insolubility of poor water-soluble compounds can be significant compared toaqueous solubility and can facilitate the absorption of drug molecules in thegastrointestinal tract when delivered in solution form

The requirements for HPLC methods include careful selection of themobile phases to avoid sample precipitation or emulsification At the sametime, chromatographic conditions should provide positive retention of thedrug substance so it won’t elute with the void volume

The solubility measurement at several time points can be used for inary solution stability evaluation of new drug candidates If degradation isobserved during the solubility evaluation, further HPLC method development