In the development of new drugs, biopharmaceutical studies including bioavailability considerations are vital. The analyst has crucial duties in many phases of these studies
"Over the past decade or so, pharmacy has undergone a transformation whereby both the practice and teaching of the subject have been placed on a more quantitative, rational scientific basis. As a result of this highly desirable trend, there have developed several areas of scientific endeavors which may be referred to, collectively, as the pharmaceutical sciences."
U) One of the newest and fastest growing of these areas is the rather diverse field called biopharmaceutics. This term was not even indexed in the Journal of Pharmaceutical Sciences eight years ago, but in the last two years, 57 articles were indexed under this and closely related headings. "Biopharmaceutics is the study of the factors influencing the bioavailability of a drug in man and animals and the use of this information to optimize pharmacologic or therapeutic activity of drug products in clinical applications . . . Bioavailability is a term used to indicate measurement of both the relative amount of an ad-
ministered drug that reaches the general circulation and the rate at which this occurs." (2) The pharmaceutical industry has quickly recognized the emerging field of biopharmaceutics, because it realized that not only did new, more potent therapeutic agents have to be synthesized, but drug delivery systems had to be designed that would achieve maximum effectiveness for these agents used therapeutically. This recognition has posed some challenges and responsibilities for the chemist engaged in pharmaceutical analysis. We have identified five tasks that the analyst should perform in the ideal biopharmaceutics program (the scope of these tasks may be altered by institutional structure): (1) To provide basic physical and chemical data about the drug substance that will assist the pharmacist in the design of a suitable dosage form (2) To provide suitable analytical methods that will allow the pharmacist to evaluate the content, stability, and properties of the dosage forms he has designed (3) To provide assay methods for the drug substance in blood and urine that will enable the pharmacist to evaluate the bioavailability of the drug in a particular dosage form
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(4) To assist in the review of experimental protocols for conducting studies in vivo that will establish the biovailability of the dosage form (5) To provide the analytical information that must be submitted to regulatory agencies. Task 1 : Basic Physical and Chemical Data About the Drug Substance The solubility of the drug in acidic, neutral, and alkaline solutions, the stability of the drug in solution or as a solid mixed with various pharmaceutical excipients, and the possible formation of crystalline polymorphs are examples of the types of data that are useful to the pharmacist prior to his performing preliminary formulation studies. At our company this information is gathered both by chemists and by pharmacists who belong to a preformulation group. Many drug substances have limited solubility in water at physiological pH values. It is important that the pharmacist have knowledge of a drug's solubility in order to produce a product with satisfactory bioavailability. A well-known example is the formulation of capsules of griseofulvin, an antifungal antibiotic with limited solubility in water. Marvel and coworkers (3) found that when griseofulvin capsules were made from an antibiotic that had been ground to a fine particle size, the blood concentrations of antibiotic produced after administra-
Report
Glenn A. Brewer Squibb Institute for Medical Research New Brunswick, N.J. 08903
Biopharmaceutics: The Role of the Analyst
tion were twice those found after the administration of capsules made with the unground antibiotic. The solubility of a drug may also be influenced by the crystalline form in which the compound occurs. By selecting a desirable crystalline polymorph, Aguiar et al. were able to achieve a tenfold increase in peak blood concentrations of chloramphenicol, administered as chloramphenicol palmitate suspension (4). In the foregoing examples, a knowledge of the physical properties of a drug substance was important in improving the bioavailability of these formulations. Good bioavailability is dependent on the stability of the drug substance in the formulation. Manufacturers interested in producing products with good stability to insure a long shelf life attempt to eliminate potentially unstable products early in development. Differential thermal analysis (DTA) provides a rapid method for screening of drugs and excipients for interactions that might result in an unstable formulation (5, 6). Although the DTA method cannot be considered completely accurate in predicting instability, we have enough confidence in it to avoid those combinations that it suggests will interact. In our company, data about the physical and chemical properties of a new drug substance are collected in a single reference document called an analytical profile, prepared prior to the first trial of the drug in human subjects and updated several times
during the development of a drug candidate. Recently, the Academy of Pharmaceutical Sciences published the first in a series of books that contain analytical profiles of established drug substances (7). This volume illustrates the types of data that we attempt to include in our analytical profiles of new drug substances. Task 2: Analytical Methods for the Dosage Form Assay methods to establish the quantity and stability of the drug substance are of vital importance in the evaluation of dosage forms. [A thorough discussion of this prime activity is outside the scope of this article but has been dealt with elsewhere (8, 9, 10).] In addition, tests such as those for tablet hardness, tablet disintegration time, pH of liquid formulations, dosage form uniformity, and the particle size of suspensions are needed for the proper evaluation of dosage forms. These tests, combined with specifications for raw materials and a rigidly defined procedure for manufacturing the dosage form, help to insure uniform bioavailability of successive batches of the drug. In the last few years, determination of dissolution rate has become one of the most widely discussed methods in pharmaceutical analysis. Knowledge of the dissolution rate of a drug can be useful to the pharmacist in developing a new product. The dissolution rate can also be used as one measure of the uniformity of a pharmaceutical manufacturing process, but for this purpose, it must be correlated with information about bioavailability.
We often use determinations of dissolution rate to screen formulations of solid dosage forms. If we have several apparently equivalent formulations, the dissolution rate can be used to decide which one has the best probability of providing maximal bioavailability. Final selection must be based on tests in vivo, such as serum concentration assays. In formulating an antibiotic capsule, we found that the bioavailability varied from batch to batch. To determine which ingredients or steps in the formulation process were critical, we designed a study in which each variable could be altered slightly. Many of the capsules that were produced in this study showed differences in dissolution rate. The relationship between dissolution rate and bioavailability was determined for three lots of capsules. The dissolution rates for these capsules, as determined by the USP-NF rotating basket technique (11), are shown in Figure 1. The serum concentration curves for the same formulations are shown in Figure 2. The dissolution rate is a more sensitive detector of differences that result from changes in the manufacturing procedure than the serum concentration. When we establish specifications for the dissolution rate for this product, we may set reasonably wide limits and still be certain that we have not compromised the bioavailability. Figure 3 demonstrates that by choosing the ordinates carefully, one can obtain a correlation between serum concentrations and dissolution rates. Task 3: Analytical Methods to Determine Concentration of the Drug in Blood and Urine The determination of the concentration of a drug in the blood is a key element in the study of its bioavailability. [The Food and Drug Admin-
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istration has recently issued proposed regulations for such bioavailability studies (12).] What we really wish to determine is the effectiveness of a dosage form. The complex relationship between the serum concentration of a drug and its effectiveness was recently illustrated by Koch-Weser (13). Figure 4 shows the various factors that are not accounted for when we use a blood-level measurement as an index of the effectiveness of a drug. Although the effectiveness of some drugs can be measured by the intensity or duration of their activity on the patient, this measurement of drug activity is often subjective and usually only semiquantitative. For most drugs the site of action is not known with certainty; even when it is, it is easier and certainly more acceptable to the patient to sample his blood rather than any other tissue. The ideal blood-level assay is characterized by specificity, rapidity, and precision. We can seldom achieve all these elements in one procedure. The limiting factor is usually sensitivity, and the analyst is willing to sacrifice rapidity and precision to determine small concentrations of a drug. The rapidity of a method is of secondary importance, especially when the study involves a large number of samples. Many types of analytical methods have been used for blood-level assays, with the gas chromatographic, fluorometric, spectrophotometric, dye ex-
15
traction, polarographic, and radioisotopic methods being the most common. Chromatographic methods are usually the most specific, because the compound to be measured is separated from impurities prior to analysis. Spectrophotometric, fluorometric, and dye extraction methods are usually rapid, but related compounds often interfere with the assay. Radioisotopic, fluorometric, and po, larographic methods are usually very sensitive. Sometimes, several analytical techniques are combined in a single assay to achieve the desired results. Radioimmune and spin-labeled immune assays of drugs in body fluids are being used with increasing frequency, and we believe they deserve even greater exploitation. The radiolabeled drugs are frequently available from drug metabolism studies. Although it takes time for an animal to develop specific antibodies to a drug, one must remember that other activities can be undertaken during the waiting period. Because we usually measure microgram quantities of a drug in a blood sample, it is not necessary to develop extremely high antibody titers to have preparations that will be useful for determining the blood levels of drugs that have been given in milligram quantities. With their rapidity, specificity, and sensitivity, radioimmunoassays meet many of the criteria of a good bloodlevel assay.
30 45 Time (minutes)
Figure 1. Dissolution rates of three different formulations of antibiotic capsule Formulations differ primarily in a m o u n t of lubricant ( m a g n e s i u m stéarate) used; f o r m u l a t i o n A contains the least, and f o r m u l a t i o n C the most Data presented by Wadke and Poet at the Conference on Pharmaceutical Analysis, Land O'Lakes. Wis.. 1971
Development of a blood-level assay requires information about the physical and chemical properties of the drug substance. In addition, it is desirable to have information about the concentration of the drug substance in the blood and to know whether the drug is circulating as a metabolite or as the unchanged compound. Such information is most often obtained by administering radiolabeled drugs to test animals, extracting the radioactivity from the blood, and chromatographing the extract in parallel with authentic drug substance. Since drug-metabolism studies are most frequently conducted with a radiolabeled drug substance, blood samples taken during such studies can be analyzed to give the desired information. At times it is necessary to use data from animals because the drug has not yet been approved for human use. Blood-level assays should be developed early in the study of a new drug substance; it would be useful, for example, if blood-level methods were available at the time of toxicologic studies. A blood-level assay would help to determine whether insoluble drugs that had been administered orally in such studies were actually absorbed. The development of bloodlevel assays for use at this early stage in the evaluation of a drug substance would require a much larger commitment of analytical chemists than at present, because of the large number of compounds studied.
1.0 1.5 2.0 Time (hours)
Figure 2. Serum concentrations of antibiotic in human subjects at various times after administration of single 250-mg capsule Data presented by Wadke and Poet at the Conference on Pharmaceutical Analysis, Land O'Lakes, Wis.. 1971
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3 0
φ Β
Figure 3. Relationship of mean serum concen tration of antibiotic to percentage of antibiotic dissolved after 1 hr
c 2
1.5
Data presented by Wadke and Poet at the Conference on Phar maceutical Analysis. Land O'Lakes Wis., 1971
1.0
05
20 30 40 50 60 70 % Dissolved in 1 hour
Task 4: Review of Protocols for Conducting Studies in vivo of Drug Bioavailability
Although some may argue that the analyst's role is to assay samples he receives, I contend that the analyst can do his best job only if he feels both involved in and committed to a project- For him to develop this com mitment, the analyst must be in volved in the planning of the project. The first studies in vivo for bio availability are usually carried out in animals, but the results of these studies must be confirmed in human subjects. Roth types of studies in volve the same planning process, al though they may be executed by dif ferent groups and will certainly re quire different degrees of documenta tion and approval. Any study in vivo requires an ex perimental protocol which should be
80
prepared after consultation with the various scientists involved in the study and should be reviewed by them. Analytical chemists are naturally concerned with the numbers and types of samples to be analyzed; the kind of anticoagulant to be used in the samples; the conditions and times of storage; if the samples cannot be analyzed immediately, the labeling to be used on the samples; and the con ditions under which the samples are to be shipped if the study is carried out elsewhere. In addition to these strictly analytical concerns, the fol lowing areas should also be of interest to the analyst. Selection of Subjects. Although the selection of subjects is not ex tremely important in crossover stud ies, it can influence greatly the re sults of a single-phase study, espe cially one of children. Weight, stat-
Task 5: Analytical Information Required by Regulatory Agencies
Pathologic disturbances
In the United States the filing of an Investigational New Drug application (IND) must precede any bioavailabil ity study in human subjects. For such a document, the analyst provides specifications for the drug substance, including tests that confirm the iden tity of the substance and demonstrate its purity. In addition, the analyst provides identity tests and assay methods for the drug substance in specific dosage forms. A summary of the requirements for tbe IND was re cently issued by the Food and Drug Administration (19). This report has highlighted five tasks that the analyst performs in a typical biopharmaceutics study. The analyst is responsible for: ( 1) gather ing physical and chemical informa tion about the drug substance; (2) de veloping analytical methods for the evaluation of dosage forms; (3) devel oping methods for assaying the drug substance or its metabolites in blood and urine; (4) assisting in the review of the experimental protocols used in bioavailability studies; and (5) assist ing in the preparation of the docu ments required by governmental reg ulatory agencies. Biopharmaceutical studies have become a vital phase in the development of new pharmaceuti cal products, and the analyst plays a key role in conducting these studies.
Development of tolerahce
References
Presence of other drugs
( 11 "Current Concepts in the Pharmaceu tical Sciences, Biopharmaceutics," James Swarbrick, Ed., ρ 7, Lea and Fêbiger, Philadelphia, Pa., 1970.
DOSAGE Completeness of absorption Apparent volume of distribution Rate of elimination CONCENTRATION IN SERUM WATER Diffusion Active transport CONCENTRATION AT SITE OF ACTION Functional state
INTENSITY OF EFFECT
ure, and age of experimental and con trol groups should be matched as closely as possible. Κ alow has written about the significance of pharmaco genetics in drug evaluations (14). Diet of Subjects. In most studies the diet of the subjects should be rig idly controlled. The amount of fat in the diet affects the absorption of the water-insoluble antibiotic griseofulvin ( 75). The absorption of tetracycline is reduced when the diet includes milk or other calcium-containing products (76). The rate of absorption of many drugs is altered if the stomach is full rather than empty. Effects of Other Medication. The use of other drugs during a bloodlevel study is generally forbidden, but even prior use of drugs by an experi mental subject can influence greatly the blood levels of the drug under in vestigation. Mannering (17) and Conney (78) list more than 200 com pounds that may increase the metab olism of a drug substance and thus alter blood levels of that drug in a particular subject.
Figure 4. "Dose-effect relation of drugs in man and factors that influ ence it" Simplification of figure published by J. Koch-Weser in N. Engl. J. Med.. 287, 227 (1972)
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(2) "Guidelines for B i o p h a r m a c e u t i c a l Studies in M a n , " ρ 17, A P h A Academy of P h a r m a c e u t i c a l Sciences, February 1972.. (3) J . R. Marvel, D. A. Schlichting, C. Denton, E . J . Levy, a n d M . M . C a h n , J. Invest. Dermatol, 42, 197 (1964). (4) A. J . Aguiar, J. Kic, J r . , A. W . Kinkel, a n d J . C. S a m y n , J. Pharm. Sci., 56, 847(1967). (5) H . J a c o b s o n a n d G. Reier, ibid., 58, 631(1969). (6) H . J a c o b s o n a n d I. Gibbs, p a p e r in p r e p a r a t i o n for J. Pharm. Sci. (7) "Analytical Profiles of Drug S u b s t a n c e s , " Vol I, K. Florey, E d . , Academ ic Press, N e w York, Ν . Υ., 1972. (8) T . Higuchi a n d E . B r o c k m a n n - H a n s sen, Pharm. Anal., Interscience (1961). (9) Anon., " T h e Stability a n d Stability T e s t i n g of P h a r m a c e u t i c a l s , a n Annotat ed B i b l i o g r a p h y , " 1939-1963 a n d 19641970, P h a r m a c e u t i c a l M a n u f a c t u r e r s (10) J. W . S u t h e r l a n d , D. E . Williamson, a n d J. E . Thervogt, Anal. Chem., 43, 206R(1971). (11) " N a t i o n a l F o r m u l a r y , " XIII, ρ 802, M e t h o d I, American P h a r m a c e u t i c a l Association, Washington, D.C., 1970. (12) Anon., Fed. Regist., 38, 885 (1973). (13) J . Koch-Weser, N. Engl. J. Med., 287, 227(1972). (14) W. Kalow, Rep. Ross Conf. Pediat. Res., 5 8 , 4 8 ( 1 9 6 8 ) . (15) R. G. Crounse, J. Invest. Dermatol., 37,529(1961). (16) K. E . Price, Z. Zolli, J r . , J . C. Atkin son, a n d H . G. L u t h e r , Antibiot. Che mother., 7 , 6 7 2 ( 1 9 5 7 ) . (17) G. J . M a n n e r i n g , "Selected Pharmacological T e s t i n g M e t h o d s , " Vol 3, M a r c e l Dekker, N e w York, N.Y., 1968. (18) A. H . Conney, Pharmacol. Rev., 19, 317(1967). (19) FDA Pap., ρ 5 ( J u n e 1971). Based on a talk given at the Eastern Analytical Symposium, Atlantic City, N.J., October 31November 2, 1972.
G l e n n A . B r e w e r is t h e a s s i s t a n t d i r e c t o r of a n a l y t i c a l r e s e a r c h of t h e S q u i b b I n s t i t u t e for M e d i c a l R e search. H e received his B S degree from H o b a r t College a n d M S a n d P h D d e g r e e s f r o m t h e U n i v e r s i t y of Wisconsin (biochemistry). As a senior research biochemist at Commercial Solvents Corp., he isolated a n d char acterized new antibiotic substances. D r . B r e w e r j o i n e d S q u i b b i n 1956 a n d s p e n t s e v e r a l y e a r s d e v e l o p i n g fer m e n t a t i o n p r o c e s s e s . I n 1960 h e joined the Analytical Research De p a r t m e n t as a research supervisor.
