Testing Systems

The significant downturn in the number of drugs approved by the US FDA has prompted newer models of drug discovery that promise to produce a larger number of possible leads. All promising leads must be put through some level of preformulation testing, creating a large burden on preformulation groups to produce results in shorter times. The technologic developments in analytical methodologies allow greater understanding of drug substances and most companies would rather quickly adopt these new techniques, particularly if they automate the testing methods. Some of the most recent introductions of new techniques include liquid chromatography (LC) or gas chromatography (GC)-MS/MS systems, use of Sirius GLpKa and lipophilicity and pION pSOL instruments, CheqSol® (chasing equilibrium solubility) measurements, nanocrystal technology, and the modulating role of solubilizers on drug efflux by P-glycoprotein (Pgp), and in silico prediction of the effect of solubilizers on Pgp are some of the newer goals of preformulation studies. There are scores of new innovations in dissolution instrumentation, drug substance stability study, and identification of degradation products. One test at the preformulation level pertains to biological transportability of the new drug substance; much of this information is gained from differential solubility analysis. Many methods like the Caco-2 cells model are now routinely used to provide initial estimates about the biological activity potential of new compounds. All of the changing regulatory requirements and technological developments place a significant burden on the preformulation team to stay alert and stay abreast of the technology.

Polymorph Screening

Some of the most significant information comes from identifying the optimal crystal form and the corresponding behavior in different humidity conditions. This information will have implications for a drug's stability and solubility and guide decisions concerning the appropriate dosage form or formulation and how it is packaged, handled, and stored. If the compound is a crystal, the next step is to identify its shape or the different shapes it can take. This information is crucial for several reasons, the most obvious of which is to ensure uniform synthesis, manufacturing, and testing of the compound within a formulation.

Drug manufacturers need to confirm that each batch of API has the same crystalline structure, and that this crystal form remains constant throughout the formulation and life of the drug product (particularly for solid oral dosage forms, powders, creams, ointments, and suspensions). The crystalline structure can also place physical constraints on the ability to manufacture a particular dosage form. For example, needle-shaped crystals tend to entangle and often do not flow well in manufacturing equipment. This can cause formulation of "hot spots," with high concentrations of the API in some areas and deficits in others. If the compound comes in different crystal shapes, then formulators will prefer the shape that is most conducive to the physical manufacture of the desired dosage form—other things being equal. For example, if drug manufacturers prefer a tablet or capsule, we may recommend that they synthesize more spherically shaped crystals rather than flat plates or needles. The crystalline structure also affects a compound's stability and solubility, which again has important implications for formulating, manufacturing, packaging, and storing pharmaceutical products and API. A trade-off may often present itself when selecting a crystal form. For example, crystalline structures that are more desirable from the standpoint of synthesis or formulation manufacture may be less advantageous when considering stability or solubility.

pKa, Partitioning, and Solubility

Critical variables that should be considered when making formulation decisions are pKa, lipophilicity, and solubility. The pKa and lipophilicity can be measured using Sirius GLpKa and a pION pSOL instrument is used to measure the intrinsic solubility of the compound. The pKa value is the pH at which acidic or basic groups attached to molecules exist as 50% ionized and 50% nonionized in aqueous solution. The pKa value provides valuable data on the interaction of an ionizable drug with charged biological membranes and receptor sites and information on where the drug may be absorbed in the digestive tract. Knowing the pKa also enables the scientist to know how much to alter the pH to drive a compound to its fully ionized or nonionized form for analytical and other purposes, such as formulation, solubility, and stability. Formulators need to know where a drug will dissolve in the digestive tract and whether that corresponds to the optimal region for absorption, especially if they are planning to create a dosage form that will be taken orally. If the drug dissolves too early, it may reprecipitate in a form that is poorly absorbed. But if a drug does not dissolve until after it travels through the stomach or small intestine, it is not likely to get absorbed. In the first case, scientists may want to create a formulation that slows the dissolution and in the second case, a formulation that speeds it up. Another option would be to formulate a dosage that could be administered by injection. Often it is preferred to use a traditional, manual test process to evaluate solubility. For example, one may place samples into three buffer solutions at different pH, shake them mechanically overnight, and then measure how much of the compound has dissolved into the solutions. The measure of the intrinsic solubility of a compound (i.e., the fundamental solubility at which the compound is completely unionized) is useful for formulators in many ways. Working over a pH range from 2 to 11, the pSOL instrument can typically determine intrinsic solubility across a range of 5-50 mg/mL. The use of the Sirius GLpKa to create lipophilicity profiles is very useful. Drugs that can be taken orally must fall into a fairly narrow window between extreme lipophilicity and extreme hydrophilicity. Many drugs cross biological lipid membranes by passive transport, and there is an optimum value of lipophilicity for each type of membrane. For example, drugs that are highly lipophilic may be easily transported or absorbed, but may get trapped inside fat storage regions, where they will be ineffective. On the other hand, a drug that is extremely hydrophilic may not penetrate the membrane and therefore, has no pharmacological effect. Hence, formulators often find lipophilicity profiles very valuable.

Poorly soluble compounds represent an estimated 60% of compounds in development and many major marketed drugs. It is important to measure and predict solubility and permeability accurately at an early stage, and interpret these data to help assess the potential for development of candidates. This requires developing an effective strategy to select the most appropriate tools to examine and improve solubility in each phase of development, and optimization of solid-state approaches to enhance solubility including the use of polymorphs, co-crystals, and amorphous solids.

Poor solubility can hinder—or even prevent—drug development. Yet the volume and level of poorly soluble compounds is dramatically increasing, leaving gaps in development pipelines. Currently only 8% of new drug candidates have both high solubility and permeability. It is important to know the solubility of drugs as it helps in the identification of potential screening and bioavailability issues. It is valuable in planning chemistry changes during biopharmaceutical evaluation, is important for the confirmation of bioavailability issues, and is also useful in early development of formulations. In drug development, solubility knowledge is needed for biopharmaceutical classification, biowaivers and bioequivalence; it is also required for formulation optimization and salt selection. In manufacture, solubility also affects the optimization of the manufacturing processes.

With this trend of increasingly insoluble drugs stretching resources, many companies are now re-evaluating their strategy. They know that there are many available technologies to measure, predict and improve solubility, and several new emerging techniques. Studies that encompass this scope would include how membrane permeation of drugs can be enhanced by means of solubilizing agents, how the solid state is characterized and modified to improve solubility and drug performance, how salt screening and selection can impact dissolution rate and oral absorption, application of nanocrystal technology to increase dissolution rate, and analysis of the use of pharmaceutical co-crystals in enhancing drug properties.

There are several new emerging methods to measure the solubility of ionizable drugs, such as using the method called CheqSol. CheqSol is a software product that processes data, and controls Sirius' existing GLpKa, PCA200, and D-PAS instrumentation. Not only does CheqSol measure equilibrium and kinetic solubility rapidly and accurately, it also provides insights into compound behavior that will be of value for the better understanding of drug bioavailability, modeling of precipitation processes, and for investigating changes of crystalline form in suspensions. Pharmaceutical scientists need to know the solubility of drug molecules during drug discovery, as well as in confirmation of bioavailability issues, human formulation design, and Biopharmaceutical Classification, which is required by the FDA. CheqSol is much faster than shake-flask methods, and, it measures both the equilibrium and the turbidimetric (or kinetic) solubilities in the same experiment. CheqSol works by monitoring the pH, as hydrochloric acid (HCl) or potassium hydroxide (KOH) solutions are carefully added to a 10-mL solution of the ionized drug until it precipitates, as detected by an abrupt decrease in the amount of light transmitted through the solution. The concentration at this point is equivalent to a kinetic solubility. Chasing equilibrium then begins—HCl and KOH are added sequentially to force the solution to become supersaturated or subsaturated, and the state of saturation is determined from subsequent small changes in the pH reading. The concentration of unionized species at the crossing points, when the pH change is zero and the sample is neither super nor sub-saturated, is equal to the intrinsic solubility. For "chasers," such as diclofenac that supersaturate and chase equilibrium, CheqSol often finds an equilibrium solubility result within 20 minutes, and confirms it several times during a 60-minute experiment. For "nonchasers," such as chlorpromazine that do not chase equilibrium, the pH after precipitation follows the Precipitation Bjerrum Curve and the software calculates the result from the shape of the curve.

Predicting aqueous solubility with in silico tools is a key drug property. It is however, difficult to measure accurately, especially for poorly soluble compounds, and thus numerous in silico models have been developed for its prediction. Some in silico models can predict aqueous solubility of simple, uncharged organic chemicals reasonably well; however, solubility prediction for charged species and drug-like chemicals is not very accurate. However, extrapolating solubility data to intestinal absorption from pharmacokinetic and physicochem-ical data, elucidating crucial parameters for absorption, and assessing the potential for improvement of bioavailability are important at the preformulation stages.

Solubilizers (e.g., organic solvents, detergents, Pluronics) are often used to solubilize drugs in aqueous solution without considering their effects on biological systems, such as (i) lipid membranes and (ii) multidrug resistance (MDR) efflux transporters (e.g., Pgp or MDR1).

The modulatory role of solubilizers on drug efflux by Pgp and in silico prediction of the effect of solubilizers on Pgp are some of the newer goals of preformulation studies.

Liposomal solubilization is an effective approach for the delivery of potent, insoluble drug candidates. However, careful consideration of the various lipid and drug properties along with an emphasis on manufacturing conditions is needed for the successful development of a marketable formulation.

Increasing dissolution rates using nanocrystal technologies is becoming common. The NanoCrystal Technology was developed by Elan Corporation (Dublin 2, Ireland). For poorly water-soluble compounds, Elan's proprietary NanoCrystal technology can enable formulation and improve compound activity and final product characteristics. The NanoCrystal technology can be incorporated into all dosage forms, both parenteral and oral, including solid, liquid, fast-melt, pulsed release, and controlled release dosage forms. Poor water solubility correlates with slow dissolution rate, and decreasing particle size increases the surface area, which leads to an increase in dissolution rate. This can be accomplished predictably and efficiently using NanoCrystal technology (4). NanoCrystal particles are small particles of the drug substance, typically less than 1000 nm in diameter, which are produced by milling the drug substance using a proprietary wet milling technique (Fig. 1). The NanoCrystal particles of the drug are stabilized against agglomeration by surface adsorption of selected GRAS (generally regarded as safe) stabilizers. The result is an aqueous dispersion of the drug substance that behaves like a solution—a NanoCrystal colloidal dispersion, which can be processed into finished dosage forms for all routes of administration.

Nanonization is a formulation technology that can universally be applied to all drugs—each drug can be transferred to drug nanocrystals. The main production technologies available to produce drug nanocrystals have their advantages and limitations. The reduction of solid particles to nanoparticles is achieved by high-pressure homogenization.

Figure 1 The NanoCrystal® technology. Source: Courtesy of Elan Corporation, Dublin 2, Ireland.

Salt Screening

Recent trends in combinatorial chemistry have resulted in the synthesis of large molecular weight (MW) lipophilic drugs. Converting the free acid/base form to a salt is an important option to explore when trying to improve solubility and oral bioavailability. Of the 21 new molecular entities approved by the FDA in 2003, 10 were salt forms. Selection of the right counter ion with optimum physiochemical characteristics is crucial to drug development. Consideration of the new compound's physical-chemical properties, processability under various manufacturing conditions, and bioavailability must be made. A complete range of characterization tools for a complete salt screen would include:

• X-ray powder diffraction analysis (XRPD)

• thermal analysis [DSC, thermogravimetric analyzer (TGA), thermo-mechanical analyzer (TMA)]

• microscopy (light and polarized)

• dynamic vapor sorption—moisture absorption and desorption

• density (intrinsic and bulk)

• solubility analysis in various media

• dissolution (including intrinsic dissolution testing)

• particle size analysis (optical, laser light, and light obscuration)

Scheme 1 describes a salt screening decision-making tree.

SOLID-STATE CHARACTERIZATION Powder Properties

Powders are masses of solid particles or granules surrounded by air (or other fluid) and it is the solid plus fluid combination that significantly affects the bulk properties of the powder. It is perhaps the most complicating characteristic because the amount of fluid can be highly variable.

Find lontzable Functional Group - Preparation of Various Salt Forms -pKa Determination - Selection of Appropriate Counterions

High

Chemical Stability

Good

Crystallinity [XRPD and DSC Analysis) Physical Stability

Option -Amorphous Solid May Have Higher Solubility

Scheme 1 Salt selection tree. Source: Courtesy of Cardinal Health, Dublin, Ohio, U.S.A.

Powders are probably the least predictable of all materials in relation to flow ability because of the large number of factors that can change their rheological properties. Physical characteristics of the particles, such as size, shape, angularity, size variability, and hardness will all affect flow properties. External factors, such as humidity, conveying environment, vibration, and perhaps most importantly, aeration, will compound the problem. The more common variables would include:

• Powder or particle variables: o particle size o size distribution o shape o surface texture o cohesivity o surface coating o particle interaction o wear or attrition characteristic o propensity to electrostatic charge o hardness o stiffness o strength o fracture toughness

• External factors influencing powder behavior: o flow rate o compaction condition o vibration o temperature o humidity o electrostatic charge

Find lontzable Functional Group - Preparation of Various Salt Forms -pKa Determination - Selection of Appropriate Counterions

High

Chemical Stability

Good

Crystallinity [XRPD and DSC Analysis) Physical Stability

Option -Amorphous Solid May Have Higher Solubility

Scheme 1 Salt selection tree. Source: Courtesy of Cardinal Health, Dublin, Ohio, U.S.A.

o aeration o transportation experience o container surface effects o storage time

Another characteristic of powders is that they are often inherently unstable in relation to their flow performance. This instability is most obvious when a free flowing material ceases to flow. This transition may be initiated by the formation of a bridge in a bin, by adhesion to surfaces, or by any event that may promote compaction of the powder. The tendency to switch in this way varies greatly from one powder to another, but can even be pronounced between batches of the same material.

The nature of powders therefore is such that an adverse combination of environmental factors can cause an otherwise free flowing powder to block or flow with difficulty. Conversely, a very cohesive powder may be processed satisfactorily if the handling conditions are optimized.

Given the complex nature of powders, it is not surprising that processing difficulties are very common. Being able to predict flow performance would bring about many operational advantages, such as reducing stoppages and improving product quality. To achieve this, we need to know how a given powder is affected by the variables mentioned earlier, and also to have a reliable indicator of the potential instability of the powder. Predicting flow ability performance in a particular plant therefore requires knowledge of the handling and processing conditions as well as the flow ability characteristics of the material under these conditions. It means that the process conditions relevant to flow ability need to be determined. These might include the level of static and dynamic head produced in a storage bin or hopper, the amount of aeration that occurs, the opportunity to adsorb moisture, become electrostatically charged or be consolidated due to vibration. Another factor that can affect powder flow includes an increase in the amount of finer particles as a result of attrition. All or at least the most important of these factors then needs to be quantified regarding how they affect flow ability.

Slight compaction, a small vibration, or the smallest amount of aeration can significantly affect flow ability. This is the main reason why traditional methods of flow ability measurement have not been suitable as a basis for repetitive testing. In all traditional techniques, the packing condition and the air content are largely unknown quantities, and so the results will vary accordingly. When making an assessment, it is essential to know what was tested and the condition of the powder when tested. In addition to the packing problem, traditional flow ability measurements are prone to operator error, have poor repeatability and, for the most part, are very time-consuming. An automated test and analysis system is needed that takes only minutes, is very repeatable, and is independent of the operator.

The most important innovation required in relation to traditional techniques is a way of classifying powders so that flow ability performance of each powder can be measured and recorded along with its processing experience. Eventually, such a database of information could remove much of the uncertainty from processing and provide a reference base for the development of new powders. It would allow each production machine to be classified in terms of the powders that could be efficiently processed.

Ideally, the classification of powders would provide more than just flow ability data, such as flow rate and compaction indices. It would also include data describing the robustness and stability of the powder, for example, vulnerability to segregation, attrition, and vibration. Given this, then the two key issues of powder processing could be addressed. First, will the powder flow satisfactory— does it have flowability properties that suit the process? And second, is the powder robust—will it be adversely affected by being processed?

Freeman Technology and the FT4 Powder Rheometer offer real benefits to all users of powders (5). These include:

• the more efficient use of powder handling systems by reducing stoppages and optimizing throughput;

• improved product quality by introducing quality conformance checks at all stages of production; and

• overall—improved competitiveness.

Microscopy

Significant advances have been made in the field of microscopy over the past decade, allowing study of nanocrystals and elemental analysis using small samples. Some of the spectroscropic and microscopic methods available include:

• Energy dispersive X-ray spectrometry (EDS) for quick and easy elemental analysis of samples in the SEM. Minimum detection limit of 0.1% by weight.

• Wavelength dispersive X-ray spectrometry (WDS) for a more detailed elemental analysis of samples in the SEM. JEOL Four-Crystal Spectrometer attached to the JSM-35C SEM can be used for 1-mm spot analysis, digital and analog line scans, and X-ray image mapping, elements detection from Be to U, minimum detection limit of 0.01% by weight, fully quantitative results by extended (p-p-z.

• Inductively coupled plasma-atomic emission spectroscopy (ICP) provides trace level and bulk elemental analyses of solid and liquid samples. Using Varian Analytical Instruments Liberty 100 air pass inductively coupled plasma atomic emission sequential spectrometer, minimum detection limits better than 1 ppb by weight (element/line dependent) bulk solid acid digestion (for powders, residue, ingots, and so on) and liquid analyses can be performed. Analysis of all elements from Li to U (excluding N, O, F, S, and noble gases), 0.75-m Czerny Turner monochromator with holographic grating allows high intensity spectra up to four peak orders with 0.006-nm resolution through a wavelength range of 189-900 nm.

• Surface analysis (AES/XPS): Electron spectroscopy for elemental analysis of surfaces, sensitive to as low as two atomic layers. Physical electronics model PHI-570 Auger Electron Spectroscopy/X-ray Photoelectron Spectroscopy System is a double pass cylindrical mirror energy analyzer with dual anode (Mg/Al) X-ray source and has a rapid sample introduction probe. It can detect elements at the first five to ten atomic layers of sample and detect all elements except H and He.

• Scanning electron microscopy for high resolution and high magnification photographs. Can also perform elemental analysis with EDS and WDS attachments. JEOL JSM6320F & JSM-35C research-grade SEM can provide imaging from 10 x to 400,000 x. Using analytical electron microscopy very high magnification images with excellent depth of focus can be obtained. This is especially important when rough surface structures are being examined. In addition, information about the chemical composition at the microlevel and the phase composition of the sample under study can be directly obtained. Using SEM, such as the Fraunhofer Institut Für Fertigungstechnik und Angewandte Material for Schung it is possible to magnify structures up to 500,000 times with high depth of focus. A finely focused electron beam allows structures down to 0.001 mm to be resolved. The acceleration voltage of the electron beam directed at the sample surface can be varied between 300 V and 30 kV. The emitted secondary and back-scattered electrons give information about the topology of the sample. Back-scattering electrons can also be used to produce material contrast images.

• X-ray diffraction (XRD) for phase analysis, crystallographic information, residual stress, texture analysis, and reflectometry on powders, bulk, or thin films. Philips X'Pert PRO, and a second Philips dual diffractometer system with automated PC control, independent theta/20, sample spinner, and 21 sample changer can be used for crystallography and Rietveld analysis of samples; flat, irregular, thin films, or in glass capillaries.

• Fourier transform infrared spectroscopy (FT-IR) is useful for identifying organic and inorganic compounds by comparison with library references. Perkin Elmer System 2000 offers near IR, mid IR, far IR: 15,000-15,030 cm, transmittance (T), specular reflectance (SR; Ref. 6) and diffuse reflectance (DR), horizontal and vertical attenuated total reflectance (ATR) microscope (>10-mm spot, 10,00010,580 cm)21.

Thermal Analysis

Materials characterization requires the measurement of molecular and macroscopic properties. Thermal analysis techniques determine calorimetric and mechanical properties, such as heat capacity, mechanical modulus, sample mass, and dimensional changes in temperature ranges between — 150°C and 1600°C. Thermal analysis utilizes DSC, TGA, TMA, and dynamic mechanical analysis instrumentation supplemented by software products, accessories, consumables, and documentation. Applications are frequently found in research and QC environments. They cover the characterization of materials, process development, and evaluation as well as safety investigations. All METTLER TOLEDO thermal analysis products belong to the latest generation STARe family. The associated METTLER TOLEDO FP900 series includes instruments for the rapid determination of physical properties, such as melting, boiling, dropping, or softening points.

Microthermal analysis is a recently introduced thermoanalytical technique that combines the principles of scanning probe microscopy with thermal analysis via replacement of the probe tip with a thermistor. This allows samples to be spatially scanned in terms of both topography and thermal conductivity, whereby placing the probe on a specific region of a sample and heating, it is possible to perform localized thermal analysis experiments on those regions.

Molecular Spectroscopy

The foundations for fluorescence correlation spectroscopy (FCS) were already laid in the early 1970s, but this technique did not become widely used until single-molecule detection was established almost 20 years later with the use of diffraction-limited confocal volume element. The analysis of molecular noise from the GHz- to the Hz-region facilitates measurements over a large dynamic range covering photophysics, conformational transitions and interactions as well as transport properties of fluorescent biomolecules. From the Poissonian nature of the noise spectrum the absolute number of molecules is obtainable. Originally used for the analysis of molecular interactions in solutions, the strength of FCS lies also in its applicability to molecular processes at either the surface or interior of single cells. Examples of the analysis of surface kinetics including on and off rates of ligand-receptor interactions will be given. The possibility of obtaining this type of information by FCS will be of particular interest for cell-based drug screening.

Recrystallization, grinding, compaction, and freeze-drying are frequently used in the pharmaceutical industry to obtain a desirable crystalline form of bulk powder and excipients. These processes affect not only the surface area, but also the crystalline disorder of the powder materials. Because both these parameters may affect the bioavailability of a drug through the rate of dissolution, it is necessary to control the conditions under which the pharmaceutical drug powders are produced. The extent of disorder in a crystalline solid may induce the hygroscopicity of the drug in addition to the flow, mechanical properties, and chemical stability. Because the qualities of a pharmaceutical preparation depend on the characteristics of the bulk powders and excipients, controlling the production process is important. An amorphous solid-state powder may determine the bioavailability of a slightly water-soluble drug because the property affects solubility and hence, absorption of the drug in the gastrointestinal tract. However, the amorphous form has problems regarding stability and hygroscopicity, resulting in transformation to a more stable crystalline form during preservation.

Therefore, in order to control the quality of pharmaceutical solid dosage products, techniques for the evaluation of crystallinity of the bulk powders and/or excipients are needed. XRD, DSC, FT-Raman spectroscopy, and microcalorimetry are currently the most widely used methods to evaluate crystallinity.

Near-infrared (NIR) spectroscopy is becoming an important technique for pharmaceutical analysis. This spectroscopy is simple and easy because no sample preparation is required and samples are not destroyed. In the pharmaceutical industry, NIR spectroscopy has been used to determine several pharmaceutical properties, and a growing literature exists in this area. A variety of chemoinfometric and statistical techniques have been used to extract pharmaceutical information from raw spectroscopic data. Calibration models generated by multiple linear regression (MLR) analysis, principal component analysis, and partial least squares regression analysis have been used to evaluate various parameters.

X-Ray Diffraction

The determination of the average morphology is often a "bottle neck" in elucidating other important behaviors of large quantities of crystalline powders used in pharmaceutical development and processing.

X-rays are electromagnetic radiation of wavelength about 1 A (10210 m), which is about the same size as an atom. They occur in that portion of the electromagnetic spectrum between gamma rays and the ultraviolet. The discovery of X-rays in 1895 enabled scientists to probe crystalline structure at the atomic level. X-ray diffraction has been in use in two main areas: for the fingerprint characterization of crystalline materials and the determination of their structure. Each crystalline solid has its unique characteristic X-ray powder pattern, which may be used as a "fingerprint" for its identification. Once the material has been identified, X-ray crystallography may be used to determine its structure, that is, how the atoms pack together in the crystalline state and what the interatomic distance and angle are. X-ray diffraction is one of the most important characterization tools used in solid-state chemistry and materials science. The size and the shape of the unit cell for any compound can be most easily determined using the diffraction of X-rays.

It is possible to use XRD techniques to estimate the average shape and "habit" of organic crystalline material using a single crystal. The relative intensities of the peaks in an XRPD pattern from a sample exhibiting a "standard" preferred orientation correlates with the shape of the crystallites present. Models have been developed to yield a quantitative "enhancement" factor for each face. The combined simple forms morphology (CSM) of the material can be produced by indexing the observed faces and modifying the simulated Bravais-Friedel-Donnay-Harker (BFDH) morphology (7). The average shape of crystallites can be estimated from the CSM by multiplying each face by its enhancement factor.

Stability Testing

The regulatory authorities clearly define the protocols for the testing of drug products for stability during the shelf life. However, testing of drug substances at the preformulation level for stability evaluation offers several advantages and opportunities once the drug substances enter the formulation stage. First, it provides a clear idea about which types of dosage forms can be used. A highly unstable protein drug cannot be placed in anything but a highly preserved and protected parenteral form, as an example. The development of stability testing protocols start with the development of stability indicating methods, the details of which can be readily found in any pharmaceutical analysis text or through the website of the US FDA. The Q1A R2 (Stability Testing of New Drug Substances and Products) is a good starting place (8). Similar guidelines are provided for biotechnology and botanical products.

Moisture Isotherm

The crystalline structure can significantly affect a compound's tendency to absorb moisture, which can impact sample handling for analytical testing, formulation, stability, and product shelf life in areas of varying humidity. For example, if a compound attracts water as it is exposed to rising humidity levels, each kilogram of material will contain more water and less of the compound as the humidity level increases. This could carry over to the formulation, where, as a consequence of the higher moisture content in the API, a subpotent formulation could be manufactured. Hence, drug manufacturers need this information in order to adequately control humidity and ensure uniform moisture to compound ratios across batches. They also need the information to design specific formulations and packaging that will maintain the stability of a finished product when it is shipped and stored in different environments. A compound may remain stable in the controlled humidity and temperature of a lab or manufacturing facility, but degrade if it acquires water when trucked across country or stored on a pharmacy shelf. For example, compound A may exist in two salt forms—a nonhygroscopic monohydrochloride form and a hygroscopic dihydrochloride form. The client needs to use the dihydrochloride form for a solid oral dosage.

Increasing moisture levels in the formulation leads to localized areas of high concentrations of hydrochloric acid in the vicinity of the API molecules. The decision must then be made whether to develop a capsule formulation or a tablet formulation, as each have their own unique set of manufacturing and packaging challenges. While the basic generation of a capsule formulation may be the quickest route, initially, the need for extensive coating and or packaging may slow the entire process. These measures would be required to protect the product from moisture, as the generation of hydrochloric acid in the formulation could result in partial digestion of the capsule shell. For a tablet formulation, the application of a moisture impermeable coating may be a simpler process and more options may be available to package the material in a protective environment. Knowing this information at the beginning of the process may save the client considerable time while providing some early direction to the development of an appropriate and successful formulation. Experimental data can generate a lot of information regarding the characteristic behavior of the molecule. In addition to understanding the hygroscopicity of the compound, drug manufacturers should know whether their compound's vapor sorption is "reproducible" as humidity levels go up and down. One of our most useful instruments is the vapor sorption analyzer, which, among other things, subjects samples to increases and decreases in relative humidity (RH).

These studies involve weighing out a small sample and exposing it to a very dry atmosphere at low or high temperatures. Once the sample dries to a prespeci-fied level, the humidity is raised in increments, for example, from 5% to 95%, while keeping the temperature constant. By weighing the sample at each increment, one can determine how much moisture the compound acquires at a given percentage of RH. This is very important information for predicting stability and shelf life. We can then determine whether vapor desorption is reproducible by weighing the sample as we decrease the humidity in the same increments. The kinetics of absorption can also be studied by subjecting samples to the same incremental humidity changes, but by varying the times spent by different samples at each percentage of RH. In addition, the percentage of RH at which a transition occurs between an amorphous and crystalline structure can be determined. An API may be more stable, more processable, or more soluble (giving the impression of different pharmacological activity) in one form or another and hence, must be formulated, packaged, and handled to maintain that form. In general, four possible basic vapor sorption profiles are observed—the compound can be found to be non-hygroscopic, vapor sorption is reproducible, vapor sorption curves demonstrate a degree of hysteresis, or the vapor sorption is nonreproducible either because of deliquescence, development of nonreversible hydrates, or other reasons.

Vapor sorption is reproducible when the rate at which a compound acquires moisture during humidity increases is matched by the rate at which it loses moisture during humidity decreases. If the rates are the same, then scientists can control the moisture/compound ratio simply by controlling the humidity levels, without having to consider the specific history of the material. Hence, if a batch of material has a certain water/compound ratio in facility A and acquires more water as it is moved across a humid environment to facility B, one can restore the original ratio by insuring that B's humidity level is the same as A's (and waiting for an adequate amount of time). If vapor sorption is not reproducible, then one will need to know how the absorption rate differs from the desorption rate, and the precise humidity conditions that the material undergoes as it is transported from A to B, as the history of the material will affect the amount of moisture present in the material. Of course, whether vapor sorption is reproducible or not, it takes time to raise or lower the water content under certain humidity and temperature conditions. What is considered to be an adequate time can be ascertained experimentally by preformulation studies of the kinetics of vapor sorption for a particular compound, which focuses not only on the amount of water absorbed or released, but also on the time it takes for the processes to occur.

While the moisture content of the sample at any given RH is dependent on the history of the sample, all the moisture gained by the sample in the adsorption phase is eventually lost in the desorption phase.

While the majority of experiments conducted using the vapor sorption analyzer involve monitoring weight changes at constant temperatures and varying humidity levels, the instrument can also be used to measure changes in weight when incrementally altering temperature, while maintaining a constant humidity.

Other methods and instruments to test the effects of temperature include the DSC and the TGA. DSC measures the amount of energy (as heat) absorbed or released as a sample is heated, cooled, or held at constant temperature. These measurements provide information on effects such as glass transition, crystallization, and melting point, and provide quantitative insight into the composition of a sample. When the sample is heated, inorganic salts first split off their water of crystallization, and then other volatile components evaporate. The weight loss indicates the amount of water or volatile components in the sample. TGA also helps formulators to understand decomposition behavior. Although some similar information can be obtained with the vapor sorption analyzer, TGA is a specialized instrument that allows users to measure effects at much higher temperatures. They can even set the starting and ending temperatures and control the speed at which the temperature rises or falls.

Excipient Compatibility

Whereas the choice of excipients starts with the stages of formulation, some excipients are historically used in specific drug formulations; for example, if the newly discovered drug is a cephalosporin for use as an intravenous product, compatibility with arginine or sodium carbonate would be advised as these are the most commonly used active excipients used for solubilization. Similarly, for drugs that are likely to be compressed, compatibility with common ingredients of compression and disintegration are plausible choices at this stage. The relative emphasis on excipient interaction would depend on how the company research is planned; in many situations, the preformulation group is more closely aligned with the drug discovery group and many of these studies are left to the formulation group.

TRANSPORT ACROSS BIOLOGICAL MEMBRANES Drug Efflux and Multidrug Resistance Studies

The problem of MDR has gained increasing importance in recent years, particularly in the fields of tumor therapy and treatment of bacterial and fungal infections. One of the major mechanisms responsible for development of MDR is overexpression of drug efflux pumps. These membrane-bound, ATP-driven transport proteins efflux a wide variety of natural product toxins and chemotherapeutic drugs out of the cells and, give rise to decreased intracellular accumulation of these compounds. Thus, inhibition of efflux pumps is a versatile approach for overcoming MDR, and several compounds are in clinical Phase III studies. The main target is Pgp, which is responsible for MDR in tumor cells, and transport systems in Staphylococcus aureus, Pseudomonas aerugiosa and Escherichia coli. Due to the fact that 3D structures of the proteins at atomic resolution were not available, drug development was performed solely on the basis of ligand design. However, electron microscopy studies as well as X-ray structures of three bacterial efflux pumps may open the door to target-based drug design in the near future.

The lipophilicity of drug molecules (represented as the logarithm of the n-octanol/water partition coefficient) often strongly correlates with their pharmacological and toxic activities. It is therefore, not surprising that there is considerable interest in developing mathematical models capable of accurately predicting their value for new drug candidates. The key importance of lipophilicity in bio-studies is discussed for ^-blockers. Examples of their lipophilicity-dependent pharmacological properties including pharmacokinetic, pharmacodynamic, and clinical aspects are reviewed. Comprehensive lipophilicity compilations of ^-blockers are not available so far. Log P calculations with 10 programs for 30 clinically relevant ^-blockers are presented for the first time in this review.

Modulators and inhibitors of multidrug efflux transporters, such as Pgp, are used to reduce or inhibit MDR, which leads to a failure of the chemotherapy of, for example, cancers, epilepsy, bacterial, parasitic, and fungal diseases. Binding and transport of first-, second-, and third-generation modulators and inhibitors of Pgp take into account the properties of the drug (H-bonding potential, dimensions, and pKa values) as well as the properties of the membrane.

Gram-positive lactic acid bacteria possess several MDRs that excrete out of the cell a wide variety of mainly cationic lipophilic cytotoxic compounds as well as many clinically relevant antibiotics. These MDRs are either proton/drug antiporters belonging to the major facilitator superfamily of secondary transporters or ATP-dependent primary transporters belonging to the ATP-binding cassette superfamily of transport proteins.

It is increasingly recognized that efflux transporters play an important role, not only in chemo protection, for example, MDR, but also in the absorption, distribution, and elimination of drugs. The modulation of drug transporters through inhibition or induction can lead to significant drug-drug interactions by affecting intestinal absorption, renal secretion, and biliary excretion, thereby changing the systemic or target tissue exposure of the drug. Few clinically significant drug interactions that affect efficacy and safety are due to a single mechanism and there is considerable overlap of substrates, inhibitors, and inducers of efflux transporters and drug metabolizing enzymes, such as CYP3A. As well, genetic polymorphisms of efflux transporters have been correlated with human disease and variability of drug exposure.

In Vitro-In Vivo Correlation

The in vitro-in vivo correlation (IVIVC) is an extremely useful exercise at the pre-formulation level that determines how scale up and post approval changes or Biowaiver principles would be exploited. Conceptually, IVIVC describes a relationship between the in vitro dissolution/release versus the in vivo absorption. This relationship is an important item of research in the development of drug delivery systems. In vitro dissolution testing serves as a guidance tool to the formu-lator regarding product design and in quality control. Especially, it is of specific importance for modified release dosage forms, which are intended for the purpose of prolonging, sustaining, or extending the release of drugs. By applying mathematical principles, such as linear system analysis or moment analysis, data describing in vitro and in vivo processes can be obtained. Developing a predictable IVIVC depends upon the complexity of the delivery system, its formulation composition, method of manufacture, physicochemical properties of the drug, and the dissolution method. Several sophisticated commercial dissolution methods are available along with the software to develop IVIVC models; these will bediscussed elsewhere in the book.

Caco-2 Cell Studies

Caco-2 monolayer, a model for human drug intestinal permeability, is of great interest. Kinetics of intestinal drug absorption, permeation enhancement, chemical moiety, structure-permeability relationships, dissolution testing, in vitro/in vivo correlation, and bioequivalence are studied using Caco-2. The Caco-2 cell line is heterogeneous and is derived from a human colorectal adenocarcinoma. Caco-2 cells are used as in vitro permeability models to predict human intestinal absorption because they exhibit many features of absorptive intestinal cells. This includes their ability to spontaneously differentiate into polarized enterocytes that express high levels of brush border hydrolases, and form well-developed junc-tional complexes. Consequently, it becomes possible to determine whether passage is transcellular or paracellular based on a compound's transport rate. Caco-2 cells also express a variety of transport systems including di-peptide transporters and Pgp. Due to these features, drug permeability in Caco-2 cells correlates well with human oral absorption, making Caco-2 an ideal in vitro permeability model. Additional information can be gained on metabolism and potential drug-drug interactions as the drug undergoes transcellular diffusion through the Caco-2 transport model. The Millipore MultiScreen Caco-2 assay system is a reliable 96-well platform for predicting human oral absorption of drug compounds (using Caco-2 cells or other cell lines whose drug transport properties have been well characterized).

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