HPLC for Pharmaceutical Scientists 2007 (Part 12)

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HPLC for Pharmaceutical Scientists 2007 (Part 12)

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Preformulation is a bridge between discovery and development where development scientists participate in selection and optimization of lead compounds. It is very critical at this stage to evaluate the developability of potential drug candidates in order to select new chemical entities and decrease the number of failures during future drug development. On average, only one out of ten new chemical entities (NCE) entering firstin-human testing reaches registration, approval, and marketing stage. The reasons for failures of development compounds include problems with biopharmaceutical properties, clinical safety, toxicology, efficacy, cost of goods, and marketing (see Figure 12-1) [1, 2]. ...

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  1. 12 ROLE OF HPLC IN PREFORMULATION Irina Kazakevich 12.1 INTRODUCTION Preformulation is a bridge between discovery and development where devel- opment scientists participate in selection and optimization of lead compounds. It is very critical at this stage to evaluate the developability of potential drug candidates in order to select new chemical entities and decrease the number of failures during future drug development. On average, only one out of ten new chemical entities (NCE) entering first- in-human testing reaches registration, approval, and marketing stage. The reasons 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 as gastrointestinal and plasma solubility, lipophilicity (LogD), permeability, first-pass metabolism, systemic metabolism, protein binding, and in vivo bioavailability are related to the solubility, chemical stability, and permeabil- ity of drug candidates and have to be considered at discovery lead selection before recommendation to the development stage. A major challenge in any drug discovery program is achieving reasonable bioavailability upon oral administration; therefore, any information that high- lights potential problems with cell permeability and absorption is valuable when reviewing structural families as leads for drug discovery. Lipinski et al. [3] have reviewed 2245 compounds selected from the United States Adopted HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc. 577
  2. 578 ROLE OF HPLC IN PREFORMULATION Figure 12-1. Reasons for attrition from 1991 to 2000. (Reprinted with permission from reference 1.) Name (USAN), International Nonproprietary Name (INN), and World Drug Index (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, the number of hydrogen bond donors, and the number of hydrogen bond acceptors. It was concluded that compounds are most likely to have poor absorption when molecular weight is >500, the calculated LogP is >5, the number of hydrogen bond donors is >5, and the number of hydrogen bond acceptors is >10. Lipinski has referred to this analysis as “rule of five” because the 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 pKa of the compound and the pH of the gastrointestinal region (Figure 12-2). Almost 63% of all drugs are ionized in aqueous solution and can 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 possible failure in future development—numerous studies are performed to fully char- acterize prospective drug candidates. The major analytical technique in each preformulation 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 con- centration and identity determination by UV and/or MS detection, respec- tively. The type of HPLC methods differ based on the specific preformulation tests that will be described below. In the early stage of preformulation, characterization of the drug molecule involves ionization constants and partition coefficient determinations, aqueous and 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
  3. INITIAL PHYSICOCHEMICAL CHARACTERIZATION (DISCOVERY SUPPORT) 579 Figure 12-2. Physical properties of the gastrointestinal tract. (Reprinted with permis- sion from reference 5.) at this stage. At the later stage of preformulation, after recommendation of NCE to development, the development support from preformulation group involves a more detailed solid-state characterization program, elaborating on moisture sorption, compressibility, melting point, particle size, shape, and surface area assessments, as well as excipient compatibility and prototype for- mulation stability evaluation. Further information on the role of preformulation in drug development process can be found in several excellent monographs [6–8] with the focus on pharmaceutical aspects of process development. 12.2 INITIAL PHYSICOCHEMICAL CHARACTERIZATION (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, pKa, and LogD for ionizable drugs and aqueous solubility. These physical characteristics can affect the absorption of drug candidate and, there- fore, drug bioaivalability. The requirements for HPLC analysis at this stage are speed and efficiency of the separation. It is critical to mention that at the early stage of discovery, very little information is available about the properties of
  4. 580 ROLE OF HPLC IN PREFORMULATION molecule and only a few milligrams of compound is available for characteri- zation. Therefore, it is important to choose the most efficient column and the simplest mobile phase. Also, recommended is the use of more contemporary HPLC systems as UPLC from Waters employing columns with dimensions of 50 × 2.1 mm, 1.8-µm particle size and the Fast 1200 system from Agilent with column dimensions of 50 × 4.6 mm, 1.8-µm particle size, respectively, to enhance the turnaround time for sample analysis. Other platforms would include using Chromolith Speedrod® monolithic columns at high flow rates. Also, taking into consideration the short column length, gradient elution should be recommended for all HPLC methods at this stage of drug candidate characterization. The post-run equilibration time is not significant in the case where 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 of drug development, from structure activity relationships (SAR) to physiology based pharmacokinetics (PBPK) and pharmacokinetics-/pharmacodynamics (PK/PD) to help choosing some of the lead compounds. Some tests that are carried 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 bio- pharmaceutics umbrella, they will not be discussed here. The reader can find relevant information in numerous monographs [9, 10]. 12.2.1 Ionization Constant, pKa Most potential drug candidates are weak bases or acids. Solubility and many other 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 the following equilibrium: BH + ↔ B + H + (12-1) where the ionization equilibrium constant could be expressed as Ka = [B] ⋅ [H + ] (12-2) [BH + ] It is obvious from the above equilibrium that the ratio of ionic to nonionic form of the drug in the solution is controlled by the proton concentration, which is commonly represented by pH values (negative logarithm of proton concentration). Taking the negative logarithm of expression (12-2), the well- known Henderson–Hasselbalch equation could be obtained:
  5. INITIAL PHYSICOCHEMICAL CHARACTERIZATION (DISCOVERY SUPPORT) 581 pKa = pH + log [BH + ] (12-3) [B] 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 an increased affinity for lipids. Variation of the ionization state of the molecule at different pH has typical sigmoidal shape (as shown in Figure 12-3). Corresponding expression for this dependence could be derived from equation (12-2) and the mass balance of the ionic and nonionic form of the drug: q = [B] + [BH + ] (12-4) If one assumes quantity q equal to 100, then concentration of B or BH+ forms will 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 100 ⋅ 10 ( pK a − pH ) [BH + ] = (12-5) 1 + 10 ( pK a − pH ) 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 pKa values. Several different techniques are usually employed for pKa determination. They were described in detail by Comer [11]. Figure 12-3. Dependence of the relative amount (in the form of a percent) of proto- nated form on the pH of aqueous media.
  6. 582 ROLE OF HPLC IN PREFORMULATION In practice the most common technique to determine pKa value is by employing potentiometric titration based on the detection of the variations of either the conductivity or current at fixed applied potential at various pH values. The automated potentiometric titration system well known as a GLpKa or PCA200 from Sirius Analytical [12] is considered to be a good approach for pKa determination with water-soluble drugs at pH 2–8 for the new drug candidates when the amount of drug substance is limited. For poorly water- soluble compounds it is advised to use GlpKa with D-Pass or Sirius Profiler SGA 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 pKa 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 pKa could be accurately performed with very limited amount of sample. Fast HPLC method with optimum analyte retention is suitable for this purpose, but the influence of the organic mobile- phase modifier on the mobile phase pH and analyte pKa should be accounted for in order to provide the accurate calculation of the respective pKa value. Detailed discussion of the HPLC-based methods for the pKa determination is given in Chapter 4. In the case of sufficient drug supply the old-fashioned solubility method can be used for pKa determination based on the different equilibrium solubility at different pH values. This method is very precise, but time- and drug- consuming, and is described in detail in reference 6. Drug substance often contains several ionizable groups that may signifi- cantly complicate experimental measurement of the pKa. All different types of pKa determination methods are essentially based on the measurement of the titration curve. If the pKa values of several ionizable groups in the mole- cule are within 2 pH units from each other, experimental measurement become very tedious. Recent advancements in the molecular computational methods and developments of physicochemical databases for a large number of known compounds allow computer-based prediction of the pKa values on the basis of known physicochemical correlations and fast computer screening of known values for related or structurally similar compounds from the data- base. Detailed discussion of these programs is given in Chapter 10. 12.2.2 Partition and Distribution Coefficients One of the most important physicochemical parameters associated with oral absorption, central nervous system (CNS) penetration, and other pharmaco- kinetic parameters is lipophilicity of organic compounds, which determines distribution 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 as one of the major characteristics of drug-like organic molecules. It was stated that LogP should be not more than five for drug candidates to have a good
  7. INITIAL PHYSICOCHEMICAL CHARACTERIZATION (DISCOVERY SUPPORT) 583 TABLE 12-1. Preferable Dosing Form for Different LogP Regions LogP Dosing Form Low 2 pH units above pKa and for acids >2 pH units below pKa. 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 dis- tribution coefficient at a pH where analyte is in its neutral state. This is not a constant and will vary according to the protogenic nature of the molecule. The choice of partition solvent has been a subject of debate. Different type of solvents have been used for the determination 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, and propylene 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, K 0 for phenol in w hexane is only 0.11 while in octanol it is equal to 29.5. There were several attempts 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 drug absorption in gastrointestinal tract. The classical measurement of LogP is the shake flask method [17]. A known amount of drug is dissolved in a flask containing both octanol phase and aqueous buffer at controlled pH to ensure the existence of only nonionic form (at least two units from the drug pKa). The flask is shaken to equilibrate the sample between two phases. There must be no undissolved substance present in both phases. After the system reaches its equilibrium, which is time- and temperature-dependent, the concentration of drug is analyzed by HPLC in both phases. Partitioning coefficient is calculated as c0 Kw = 0 (12-6) cw
  8. 584 ROLE OF HPLC IN PREFORMULATION This method allows for the accurate determination of K 0 only within the − w 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 the water–organic interface, and shaking of this two-phase system will create a stable emulsion difficult for analytical sampling. The ultracentrifugation at speed of 14,000 rpm for 15–20 min can be enough in most cases to separate two 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 K 0 is used, and it is denoted as LogP: w LogP = log( Kw ) 0 (12-7) The biggest challenge for the use of HPLC in the LogP measurement is the determination of the drug concentration in the octanol phase. If the octanol solution is being injected onto the reversed-phase column, it can modify the stationary phase, shift the analyte retention, and lead to an incorrect mea- surement due to the retention shift. To avoid this problem the dilution in the corresponding mobile phase is recommended. Also, when LogP is more than four, the concentration of drug in water phase is very small, causing a detec- tion problem with UV detection. This becomes even more troublesome if the compound of interest has a weak UV chromophore. The use of MS detection and 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 in their 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. The estimation of LogP for unknown compounds can be made on the basis of this calibration curve. This method is straightforward, but requires the previous knowledge of pKa values for ionizable compounds to avoid the possible ion- ization that will lead to incorrect determination of values of LogP. Recently, 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 of compound in correlation with its distribution properties in RP HPLC [19, 20]. The retention factor was used to calculate a distribution coefficient between stationary phase and mobile phase. In case of Sirius Profiler LDA automated system, a set of molecules with known LogP values was used to calibrate the system and convert the chromatographic retention time into octanol/water partition coefficients. The system could cover the LogP range from −1 to 5.5 by choosing between three different methods and different column lengths
  9. INITIAL PHYSICOCHEMICAL CHARACTERIZATION (DISCOVERY SUPPORT) 585 ranging from 1 to 25 cm, but was recently removed from the market. The well- known automated pH titrator from Sirius, GlpKa, can be used as well to deter- mine the octanol/water partition coefficient. The measurement is based on a two-phase acid/base titration in a mixture of water/octanol [21]. Partition coefficient discussed above represents oil/water equilibrium dis- tribution of only neutral forms of a substance. The distribution at different pH is described by LogD, which is the logarithm of the ratio of the concentrations of all forms of analyte in oil and water phases at particular pH. Logarithm of distribution 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 on the pH. It is usually assumed that in the oil-phase drug molecule could exist in only nonionic form; thus the distribution coefficient, D0 , for basic drug B w could be written as [ B]oil Dw = 0 (12-8) [B]water + [BH + ]water If LogP and pKa for a studied drug is known, then it is possible to express D0 w as a function of pH of aqueous phase through these values using equations (12-3) and (12-6)–(12-8). Resulting expression is Log(D0 ( pH)) = LogP − Log[1 + 10 pK a − pH ] w (12-9) Figure 12-4 represents the comparison of the pH dependencies of ionic form of a basic drug with LogD. LogD Figure 12-4. Normalized dependence of the protonated form of the base (solid) and its LogD dependence on the aqueous pH (dashed).
  10. 586 ROLE OF HPLC IN PREFORMULATION At high pH, the neutral form of a drug (basic compound) has a distribu- tion coefficient equal to its partitioning coefficient. With the decrease of the pH of the aqueous phase, the degree of drug ionization increases, thus increas- 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 its content in oil phase. There is no plateau region in the LogD curve at low pH for basic compounds (Figure 12-4). On the other hand, for acidic compounds, there is a plateau region in the LogD curve at low pH (pHs below the pKa); and then as the pH increases, the more ionic equilibrium is shifted toward the ionized form of the acid, which continually increases its concentration in the aqueous 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 acidic compounds. These are only the theoretical dependencies; real behavior of actual mole- cule usually is significantly altered due to different types of intermolecular interactions. Molecular solvation, association, hydrogen bonding, and counte- rions all have a significant effect on drug ionization constant and partitioning and distribution coefficients. Detailed and comprehensive discussion of these effects could be found in the book by Avdeef [22]. 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 dosage form development. Only a drug in solution can be absorbed by the gastroin- testinal track. The rate of dissolution and the intestinal permeability of the drug molecules are dependent on the aqueous solubility—that is, the higher the solubility, the faster the rate of dissolution. An excellent monograph describing the theory of solubility and solubility behavior of organic com- pounds was written by Grant and Higuchi [23]. For additional information on solubility, the reader can be referred to references 24–27. Solubility is expressed as the concentration of a substance in a saturated solution 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 of drug development because it affects drug bioavailability evaluation; in many cases, solubility-limited absorption has been reported. Only a compound that is in solution is available to cross the gastrointestinal membrane. The solubil- ity measurements in aqueous buffered systems at different pHs are used to mimic gastrointestinal human or animal fluids. Solubility determination in DMSO is very important at the early stages of lead candidate selection because of the increasing use of 10 mM DMSO solution as a stock solution for biological testing for very slightly soluble lead candidates [29]. In general,
  11. INITIAL PHYSICOCHEMICAL CHARACTERIZATION (DISCOVERY SUPPORT) 587 TABLE 12-2. Solubility Definitions by US Pharmacopeia [28] Parts of Solvent Required Descriptive Term for One Part 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 10,000 and over aqueous solubility is measured in simple buffered 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 intestinal fluid, 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 experimental methods based on high-throughput solubility measurements are used to deter- mine the apparent solubility of potential lead candidates as well as in silico predictions [34] to quickly assess aqueous solubility. These methods are described 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 necessary for 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 buffered solution in excess (saturated solution), and the suspension is shaken on a mechanical shaker until the system reaches the equilibrium between two phases, solid and liquid. Sometimes the equilibration time is very long and can vary 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 when possible if they may be prone to photodegradation. To check the equilibrium condition, several HPLC measurements should be determined at several time points. 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,
  12. 588 ROLE OF HPLC IN PREFORMULATION TABLE 12-3. Variation of Aqueous Solubility in the Literature [37] Compound Solubility Range (g/mL) Estradiol 0.16–5.00 Indomethacin 4.00–14.0 Griseofulvin 8.00–13.0 Progesterone 7.90–200 Digoxin 28.0–97.9 Riboflavine 66.0–99.9 Dexamethasone 89.1–121 Hydrocortisone 280–359 TABLE 12-4. Biopharmaceutical Classification of Drug Substances Class Solubility Permeability Class 1 High solubility High permeability Class 2 Low solubility High permeability Class 3 High solubility Low permeability Class 4 Low solubility Low permeability and even the amount of excess solids [37]. Table 12-3 shows some examples of reported aqueous solubility range for commercial drugs. Aqueous solubility of ionizable molecules at different pH values is an important 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 to define the class of a drug substance in the Biopharmaceutics Classification System (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 important parameters 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 con- ditions are strictly defined by the FDA. The pH solubility profile of the test drug substance should be determined at 37°C in aqueous media with a pH in the range of 1–7.5. Standard buffer solutions described in the USP are considered to be appropriate for use in these studies. A number of pH condi- tions are used bracketing the pKa value for the respective test substance. For example, for a drug with a pKa of 5, solubility should be determined at
  13. INITIAL PHYSICOCHEMICAL CHARACTERIZATION (DISCOVERY SUPPORT) 589 pH = pKa, pH = pKa + 1, pH = pKa − 1, pH = 1, and pH = 7.4 Concentration of the drug substance should be determined using a stability-indicating assay that can distinguish drug substance from its degradation products if observed. In order to be classified as highly soluble, the FDA BCS requires that the highest human dose be soluble in 250 mL of aqueous medium over a pH range 1–7.5 [38]. The identification of specific class for the drug candidate is critical for future development of dosage forms. Different platforms are used for solubility measurements: UV; HPLC with UV detection; or HPLC with MS detection. UV spectrophotometry is the sim- plest and fastest method, unfortunately with limited applicability. In most cases the drug substance available for the study in the preformulation stage is not pure enough to provide an adequate absorbance–concentration relationship of drug substance itself. In this case, HPLC with UV detection is the most applicable technique to use. Fast gradient methods on short columns could be successfully 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 the appropriate dilution of the different solutions prepared at the different pH values. In some cases, drug substance does not have chromophores with a molar absorbtivity sufficient for accurate quantitation using UV detection. If HPLC with 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 MS detector allows the use of fast gradient HPLC separation methods, which does not require significant development time. Practically in all assays used in preformulation, the quantitation of only drug substance is required and MS detection provides an accurate quantitation. Identification of pharmaceutically acceptable vehicles that afford sufficient solubilization while maximizing physiological compatibility for preclinical pharmacokinetic evaluation is critical. The most frequently used solubilization techniques 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 with cylodextrins [40]. By using the solubilization techniques, the enhancement in solubility of poor water-soluble compounds can be significant compared to aqueous solubility and can facilitate the absorption of drug molecules in the gastrointestinal tract when delivered in solution form. The requirements for HPLC methods include careful selection of the mobile phases to avoid sample precipitation or emulsification. At the same time, chromatographic conditions should provide positive retention of the drug substance so it won’t elute with the void volume. The solubility measurement at several time points can be used for prelim- inary solution stability evaluation of new drug candidates. If degradation is observed during the solubility evaluation, further HPLC method development
  14. 590 ROLE OF HPLC IN PREFORMULATION should be oriented not only to determine drug substance concentration, but also on the separation of degradation products from the active. 12.3 CHEMICAL STABILITY HPLC is a major tool in preformulation stability testing of potential drug can- didate. The design of stability testing in the early stage of drug development is not strictly defined by FDA guidance, and different approaches are taken by different pharmaceutical companies. However, there are several major components to a comprehensive stability testing with a goal to achieve maximum information within the shortest period of time: • Development of a sensitive and reliable HPLC method of separation • Solution-state stability as a function of pH, temperature, and light • Chemical solid-state stability evaluation as a function of temperature and humidity • Identification of degradation products followed by structure elucidation and possible description of degradation mechanism To achieve this goal, the best approach is to perform forced degradation studies at the preformulation stage of drug development with most viable can- didates, which may include the free base or acid and several corresponding salt forms. The FDA and ICH guidance provides very little information about strategies and principles for conducting forced degradation studies, including problems with poorly soluble drugs and exceptionally stable compounds. The stressing condition should be regulated based on the requirements to produce enough degradation products to evaluate the possible routes of degradation, but not to unduly overstress the drug and obtain aberrant results. Sufficient exposure is achieved when a drug substance has degraded >10% from its original amount or after an exposure in excess of the energy provided by an accelerated storage condition. The goal is to mimic what would be observed in formal stability studies under ICH conditions [41]. Another major concern is related to the use of a co-solvent to dissolve the sufficient amount of drug for determination and detection of degradation products. In general, acetoni- trile or methanol is used as common co-solvent for forced degradation studies. It was shown that when acetonitrile was used as a co-solvent compared to no co-solvent system, the number of degradation products increased and led to a consequent change in the degradation pathway [42]. The recommendation in this case is to prepare the samples in several co-solvents and compare the behavior of methanol versus acetonitrile for a specific drug candidate. Forced degradation studies based on FDA guidelines are carried out in solution. This involves conditions that are more severe than in accelerated solid-state sta- bility testing. For example, these include temperatures in excess of 40°C,
  15. CHEMICAL STABILITY 591 extreme high and low pH values, oxidation by 3% hydrogen peroxide, and light conditions exceeding ICH guideline [43]. As a part of discovery support, these forced degradation studies are performed on discovery batch material to identify future problems with drug candidates and to eliminate the recommendation of unstable molecules to develop or to help define proper storage conditions for early-phase material— that is, store at low temperature, protect from light, and ensure tight packag- ing. As a part of preformulation studies, this forced degradation testing is not a part of formal stability program for clinical batches, but sheds light in regard to possible thermolitic, hydrolitic, oxidative, and photolitic degradation mech- anisms for the prospective drug candidate. At this stage it is critical to develop a suitable HPLC separation method, not only based on UV detection and peak purity check, but also one that is compatible with MS detection. Preferably, columns with 3-µm particles and not more than 15 cm in length (i.d. could be 3.0 or 4.6 mm) should be used, and mobile phases compatible with MS detec- tion are recommended. As a starting point, a C8 column that is stable from 2 to 11 or a phenyl hexyl column that is stable from 2 to 10 could be selected and a gradient could be employed from 5% acetonitrile to 95% acetonitrile. 0.05 v/v% TFA could be used in both acetonitrile and water mobile phases. Development of stability-indicating methods are discussed in the method development chapter (Chapter 8). Despite the usual situation in the prefor- mulation research environment when all tests should have been done yester- day, an analyst should carefully develop a stability-indicating HPLC method because in most cases the conditions of this method will be used as a starting point for most, if not all, further HPLC methods during the development process of a particular drug in the downstream formulation development. Unfortunately, the isolated drug substance and drug-product-related degra- dation products are not available at this early stage, and the peak purity analy- sis using UV diode array detection along with mass spectrometric detection should be performed. Once the initial stability-indicating method is developed, the forced degra- dation studies are carried out and the pathways for degradation may be elucidated. Four major degradation processes are usually distinguished: oxidation, hydrolysis (H+ or OH−), photolysis (light), and catalysis (effect of trace metal ions, Fe2+, Fe3+, Cu2+, Co2+, etc.). Temperature is an integral part of all these processes. According to the Arrhenius equation, the reaction constant is related to the temperature as follows: K = A exp − a  E (12-10)  RT  where Ea is activation energy, R is the gas constant, and T is temperature in degrees kelvin. The higher the temperature the higher the reaction constant, and this leads to the increase of the degradation rate.
  16. 592 ROLE OF HPLC IN PREFORMULATION Degradation is a chemical transformation of the drug substance and can be expressed as a chemical reaction with the specific kinetics. These reactions can have different orders, which are characterized by the different rate of parent compound decomposition. The most common are zero, first and second order reactions. It is not a subject of this chapter to discuss reaction kinetics in details; however, specific preformulation-related discussions can be found in reference 6, and a general approach with examples is very well described by Martin [44]. Zero-order reactions are usually of self-disintegration type, where decom- position is independent of the concentration of reactants (including drug substance). For this reaction the decrease of the drug substance amount has a linear dependence versus time. In the first-order reaction, the decomposition is dependent on the concen- tration of one reactant (drug substance) and the decrease of the substance concentration is exponential. In the second-order reaction, the decomposition is dependent on the concentration of two reactants (e.g., drug substance and water in a hydrolytic degradation). The rate of the decrease of the substance amount is reciprocal to the drug concentration. Usually the determination of the amount of drug substance at four or more different time points of the degradation experiment is necessary for the deter- mination of the reaction order and construction of the degradation curve, which can then be used to determine the rate constant at a particular temperature. If the reaction order is known, then rate constant could be calculated from just two points. For example, for the first-order reaction the rate constant is expressed as ln  [C ]  = − Kt (12-11)  [C0 ]  where [C] is a drug concentration at time t, [C0] is the original drug concen- tration (at time t0 = 0), and K is the reaction constant. Subtracting the same equations for time moments t1 and t2 from each other, it is possible to calcu- late the rate constant: [C ] ln  1   [C 2 ]  K= (12-12) t 2 − t1 Note. Only the ratio between initial concentration C0 of parent compound to the concentration Ct at defined time point should be used in any kinetic calculations as described in detail by Martin [44]. It is not mathematically accurate to select the starting concentration as a base value and calculate all concentrational variations relative to the starting concentration.
  17. CHEMICAL STABILITY 593 TABLE 12-5. Rate Constant and Half-Life Equations Order Integrated Rate Equation Half-Life Equation 0 x = kt t1 = a 2 2k 1 a kt 0.693 log = t1 = a − x 2.303 2 k 2 x 1 = kt t1 = a( a − x ) 2 ak Source: Reprinted with permission from reference 45, p. 289. Based on the known rate constant, the half-life (the period of time required for a drug to decompose to one half the original concentration), can be deter- mined as shown in Table 12-5. Measurements of the rate constants for at least three different tempera- tures allows for the calculation of the activation energy and prediction of the temperature dependencies of the drug degradation based on the Arrhenius equation. The relationship between the rate constant and the temperature is given by the Arrhenius equation: k = Ae − E0 RT (12-13) or Ea 1 log k = log A − (12-14) 2.303 RT where k is the rate constant, R is the gas constant, A is an Arrhenius factor (constant), T is the temperature (in Kelvin), and Ea is activation energy. A plot of the logarithm of rate constant versus the reciprocal of the absolute tem- perature defines a straight line of slope −Ea /R and intercept log A [45]. The activation energy can be determined at the different forced degradation con- ditions (heat, light, peroxide). In all types of degradation assays the use of LC-MS detection is desirable since it allows for selective detection and quantitation and sometimes allows for structural elucidation of the degradation products. In some cases, tauto- merization or intramolecular rearrangements could lead to the formation of degradation products with the same molecular weight. These molecules are usually indistinguishable from the parent compound using MS with molecu- lar ion detection. The employment of LC-NMR technique may be needed to further elucidate the structures.
  18. 594 ROLE OF HPLC IN PREFORMULATION TABLE 12-6. Ionization Constants and Relative Usage Rate for the Most Common Counterions Basic Drugs Acidic Drugs Anion pKa % Cation pKa % Hydrochloride −6.1 43 Potassium 16.0 10.8 Sulphate −3.0 7.5 Sodium 14.8 62 Mesylate −1.2 2.0 Calcium 12.9 10.5 Maleate 1.9 3.0 Magnesium 11.42 1.3 Phosphate 2.2 3.2 Diethanolamine 9.7 1.0 Salycilate 3.0 0.9 Zinc 9.0 3.0 Tartrate 3.0 3.5 Choline 8.9 0.3 Lactate 3.1 0.8 Aluminium 5.0 0.7 Citrate 3.1 3.0 Alternatives 8.8 Benzoate 4.2 0.5 Succinate 4.2 0.4 Acetate 4.8 1.4 Alternatives 30.2 12.4 SALT SELECTION For ionic drugs the salt form can be considered as an alternative to increase the solubility. Drug substance usually is more soluble in aqueous media in its ionic form. Low solubility of the neutral form of the drug substance suggests the necessity to formulate it in the form of salt. The reader is referred to ref- erence 46 for more information about the properties, selection, and use of salt forms for future drug development. Examples of commonly used salt counte- rions are shown in Table 12-6. Salt form selection is mainly covered by solid-state charactezation methods, and HPLC is only used to determine the solubility and solid/solution stability of different salt forms. The requirements for HPLC method development is the same as for solubility/stability determination described previously, and the same HPLC method may be applied. 12.5 POLYMORPHISM Polymorphism is an ability of the drug substance to form crystals with differ- ent molecular arrangements giving distinct crystal species with different phys- ical properties such as solubility, hygroscopicity, compressibility, and others. This phenomenon is well known within pharmaceutical companies. The reader can find additional information in references 47 and 48. The determination of possible polymorphic transition and existence of thermodynamically unstable forms during preformulation stage of drug development is important. Typical methods used for solid-state characterization of polymorphism are DSC,
  19. POLYMORPHISM 595 FT/IR, microscopy, and X-ray powder diffraction [49, 50]. HPLC is used to evaluate chemical stability of different polymorphic forms as well as for solu- bility determination, and this parameter is very critical for drug development, because the difference in solubility can lead to different bioavailability of solid dosage form, especially if the bioavailability is dissolution-limited. An example of how polymorphism can affect final product solubility can be shown on Abbott Laboratories products and on Norvir oral liquid and Norvir semisolid capsules, with Ritonavir as an active ingredient. Ritonavir was not bio- available in the solid state, and both formulations contained ritonavir in ethanol/water solutions. At the time there was no crystal form control required from FDA for semisolid formulation, and only one form was identified at the development stage. After many successful lots of semisolid capsules, suddenly one lot did not pass the dissolution testing and when the content of the capsule was analyzed by microscopy and X-ray, the different polymorphic form of Ritonavir was identified with significantly low solubility compared to original crystal form [51].The product was recalled from market and was reformulated. It was a rare example of a dramatic effect of the existence of multiple crystal forms of a commercial pharmaceutical and showed the importance of poly- morphic screening for all type of pharmaceutical dosage forms. When the exis- tence of polymorphism for new chemical entity is identified, the property of practical interest is the relative thermodynamic stability of the identified poly- morphs; that is, are they monotrops (one is more stable than the other at any temperature) or enantiotrops (a transition temperature Tt exists below and above which the stability order is reversed)? Temperature dependence of the solubility for different polymorphic forms allows easy analysis of the existence of monotrops and enantiotrops and determination of transition temperature from the solubility ratio of the polymorphs [52]. As can be seen from Figure 12-5, intersecting solubility curves (dependence of the logarithm of the Figure 12-5. Intersecting solubility curves (dependence of the logarithm of the satu- ration concentration on the inverse temperature) indicate an enantiotropic nature of the polymorps, while parallel curves are indicative for monotropic polymorphs. The intercept for enantiotrops corresponds to the transition temperature.
  20. 596 ROLE OF HPLC IN PREFORMULATION saturation concentration on the inverse temperature) indicate an enantiotropic nature of the polymorps, while parallel curves are indicative for monotropic polymorphs. The intercept for enantiotrops corresponds to the transition tem- perature, Tt, which can be easily determined from the graph. In general, the most thermodynamically stable form that has a lower solubility and better stability is accepted for development. It was reported previously that the more thermodynamically stable polymorph is more chemically stable than a metastable polymorph due to different factors such as higher density, opti- mized orientation of molecules, and hydrogen bonding in the crystal lattice [48, 53]. The HPLC method development requirements using short columns and fast HPLC to determine the assay concentration for each polymorph at the dif- ferent temperatures are the same as for solubility determination. However, for stability evaluation of the different polymorphs a stability-indicating HPLC method should be used. 12.6 PREFORMULATION LATE STAGE (DEVELOPMENT SUPPORT) After a new chemical entity has been selected to move forward to develop- ment, the preformulation scientist supports the studies related to formulation, toxicology, and pharmacology. Based on the previous knowledge about the properties of novel chemical entity obtained during the late discovery and nomination, the stability studies of API are performed based on ICH guidance [43]. Typically, by using a GMP batch with selected solid-state form, the solution-state stability and solid-state stability studies are performed at various conditions. In general, the three conditions used for solid-state stability eval- uation—25/60, 40°C/75% RH, and 50°C dry conditions—at several time points up to 3 months (initial, 2 weeks, 6 weeks, 3 months) are reasonable to evalu- ate storage conditions of API and the impact of heat and humidity. For solution-state stability, it is important to evaluate (a) the stability at pH 1, 2, 4, 7, and 10, at ambient and elevated temperature, (b) the influence of ICH light, and (c) oxidation by peroxide. To support toxicology studies, the stabil- ity of API suspension at different strengths in aqueous solutions with corre- sponding excipients are evaluated after 1 day, 2 days, 3 days, and 8 days on potency and stability. HPLC techniques are used for all these types of stabil- ity testing, and GLP requirements are applied to HPLC methods. The system suitability for the method needs to be defined and the figures of merit such as linearity, LOD, LOQ, and solution stability in the diluent need to be performed to qualify this as a stability-indicating HPLC method at this stage. Since the degradation products are not yet identified in this stage, it is advisable to use detection systems, which have universal response and also provide high sensitivity. MS is probably the most sensitive detector that also
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