SUMMARY OF FEATURES

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T

he sixth edition of Modern Pharmacology With Clinical Applications continues our commitment to enlisting experts in pharmacology to provide a textbook that is up-to-date and comprehensive. De- signed to be used during a single semester, the book fo- cuses on the clinical application of drugs within a con- text of the major principles of pharmacology. It is meant to serve students in medicine, osteopathy, dentistry, pharmacy, and advanced nursing, as well as undergrad- uate students.

SUMMARY OF FEATURES

This edition includes a number of new or updated features that further enhance the appeal of the text.

Study Questions: Each chapter includes five to seven examination questions (following the United States Medical Licensing Examination guidelines) with detailed answers to help students test their knowledge of the covered material.

Case Studies: Appearing at the end of each chapter, case studies present students with real-life examples of

clinical scenarios and require them to apply their knowledge to solve the problem.

Refined Focus: In this edition, we chose to focus more on drug classes rather than on individual drugs, eliminate unnecessary detail such as chemical struc- tures, and maintain emphasis on structure–activity relationships in drug action and development.

Updated Information: This edition also includes new information from the clinic and the laboratory.

Emerging information has been added within chapters and when appropriate (as in the case of herbal drugs and erectile dysfunction), through the addition of new chapters.

With these revisions, we hope we have provided a book that is readable, up-to-date, comprehensive but not exhaustive, and accurate—a text that supplies both students and faculty with a clear introduction to modern pharmacotherapeutics.

Charles R. Craig Robert E. Stitzel

P r e f a c e

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I. GENERAL PRINCIPLES OF PHARMACOLOGY 1. Progress in Therapeutics 03

Robert E. Stitzel and Joseph J. McPhillips 2. Mechanisms of Drug Action 10

William W. Fleming

3. Drug Absorption and Distribution 20 Timothy S. Tracy

4. Metabolism and Excretion of Drugs 34 Timothy S. Tracy

5. Pharmacokinetics 48 Timothy S. Tracy

6. Drug Metabolism and Disposition in Pediatric and Gerontological Stages of Life 56

Jeane McCarthy

7. Principles of Toxicology 63 Mary E. Davis and Mark J. Reasor

8. Contemporary Bioethical Issues in Pharmacology and Pharmaceutical Research 73

Janet Fleetwood

II. DRUGS AFFECTING THE AUTONOMIC NERVOUS SYSTEM

9. General Organization and Functions of the Nervous

System 83

10. Adrenomimetic Drugs 96 Tony J.-F. Lee and Robert E. Stitzel 11. Adrenoceptor Antagonists 109

David P. Westfall

12. Directly and Indirectly Acting Cholinomimetics 121 William F. Wonderlin

13. Muscarinic Blocking Drugs 134 William F. Wonderlin

14. Ganglionic Blocking Drugs and Nicotine 141 Thomas C. Westfall

III. Drugs Affecting the Cardiovascular System 15. Pharmacologic Management of Chronic Heart

Failure 151

Mitchell S. Finkel and Humayun Mirza 16. Antiarrhythmic Drugs 160

Peter S. Fischbach and Benedict R. Lucchesi 17. Antianginal Drugs 196

Garrett J. Gross

18. The Renin–Angiotensin–Aldosterone System and Other Vasoactive Substances 206

Lisa A. Cassis

19. Calcium Channel Blockers 218 Vijay C. Swamy and David J. Triggle 20. Antihypertensive Drugs 225

David P. Westfall

21. Diuretic Drugs 239

Peter A. Friedman and William O. Berndt

22. Anticoagulant, Antiplatelet, and Fibrinolytic (Thrombolytic) Drugs 256

Jeffrey S. Fedan

23. Hypocholesterolemic Drugs and Coronary Heart

Disease 268

Richard J. Cenedella

IV. DRUGS AFFECTING THE CENTRAL NERVOUS SYSTEM

24. Introduction to Central Nervous System Pharmacology 281

Charles R. Craig

25. General Anesthesia: Intravenous and Inhalational

Agents 291

David J. Smith and Michael B. Howie

26. Opioid and Nonopioid Analgesics 310 Sandra P. Welch and Billy R. Martin

27. Local Anesthetics 336 J. David Haddox

28. Agents Affecting Neuromuscular Transmission 338

Michael D. Miyamoto

29. Central Nervous System Stimulants 348 David A. Taylor

30. Sedative–Hypnotic and Anxiolytic Drugs 355 John W. Dailey

31. Drugs Used in Neurodegenerative Disorders 364 Patricia K. Sonsalla

32. Antiepileptic Drugs 374 Charles R. Craig

33. Drugs Used in Mood Disorders 385 Herbert E. Ward and Albert J. Azzaro

34. Antipsychotic Drugs 397 Stephen M. Lasley

35. Contemporary Drug Abuse 406 Billy R. Martin and William L. Dewey

V. THERAPEUTIC ASPECTS OF INFLAMMATORY AND SELECTED OTHER CLINICAL DISORDERS

36. Antiinflammatory and Antirheumatic Drugs 423 Karen A. Woodfork and Knox Van Dyke

37. Drugs Used in Gout 441 Knox Van Dyke

T a b l e o f C o n t e n t s

xi

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38. Histamine and Histamine Antagonists 449 Knox Van Dyke and Karen A. Woodfork

39. Drugs Used in Asthma 458 Theodore J. Torphy and Douglas W. P. Hay

40. Drugs Used in Gastrointestinal Disorders 470 Lisa M. Gangarosa and Donald G. Seibert

41. Drugs Used in Dermatological Disorders 484 Eric L. Carter, Mary-Margaret Chren, and David R. Bickers 42. Drugs for the Control of Supragingival

Plaque 499

Angelo Mariotti and Arthur F. Hefti

VI. CHEMOTHERAPY

43. Introduction to Chemotherapy 509 Steven M. Belknap

44. Synthetic Organic Antimicrobials: Sulfonamides, Trimethoprim, Nitrofurans, Quinolones,

Methenamine 515

Marcia A. Miller-Hjelle, Vijaya Somaraju, and J. Thomas Hjelle 45. -Lactam Antibiotics 526

James F. Graumlich

46. Aminoglycoside Antibiotics 538 Steven Belknap

47. Tetracyclines, Chloramphenicol, Macrolides, and Lincosamides 544

Richard P. O’Connor

48. Bacitracin, Glycopeptide Antibiotics, and the Polymyxins 552

Mir Abid Husain

49. Drugs Used in Tuberculosis and Leprosy 557 Vijaya Somaraju

50. Antiviral Drugs 567 Knox Van Dyke and Karen Woodfork

51. Therapy of Human Immunodeficiency Virus 584 Knox Van Dyke and Karen Woodfork

52. Antifungal Drugs 596 David C. Slagle

53. Antiprotozoal Drugs 606 Leonard William Scheibel

54. Anthelmintic Drugs 621

Mir Abid Husain and Leonard William Scheibel

55. The Rational Basis for Cancer Chemotherapy 630 Branimir I. Sikic

56. Antineoplastic Agents 638 Branimir I. Sikic

57. Immunomodulating Drugs 657 Leonard J. Sauers

58. Gene Therapy 666

John S. Lazo and Jennifer Rubin Grandis

VII.DRUGS AFFECTING THE ENDOCRINE SYSTEM 59. Hypothalamic and Pituitary Gland

Hormones 677

Priscilla S. Dannies

60. Adrenocortical Hormones and Drugs Affecting the Adrenal Cortex 686

Ronald P. Rubin

61. Estrogens, Progestins, and SERMs 704 Jeannine S. Strobl

62. Uterine Stimulants and Relaxants 716 Leo R. Brancazio and Robert E. Stitzel

63. Androgens, Antiandrogens, and Anabolic Steroids 724

Frank L. Schwartz and Roman J. Miller 64. Drugs Used in the Treatment of Erectile

Dysfunction 735

John A. Thomas and Michael J. Thomas 65. Thyroid and Antithyroid Drugs 742

John Connors

66. Parathyroid Hormone, Calcitonin, Vitamin D, and Other Compounds Related to Mineral Metabolism 754 Frank L. Schwartz

67. Insulin and Oral Drugs for Diabetes Mellitus 763 Michael J. Thomas and John A. Thomas

68. Vitamins 777 Suzanne Barone

69. Herbal Medicine 785 Gregory Juckett

Index

xii TABLE OF CONTENTS

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I

1. Progress in Therapeutics 3 Robert E. Stitzel and Joseph J. McPhillips 2. Mechanisms of Drug Action 10

3. Drug Absorption and Distribution 20 Timothy S. Tracy

4. Metabolism and Excretion of Drugs ••

Timothy S. Tracy

5. Pharmacokinetics ••

Timothy S. Tracy

6. Drug Metabolism and Disposition in Pediatric and Gerontological Stages of Life ••

Jeane McCarthy

7. Principles of Toxicology ••

Mary E. Davis and Mark J. Reasor 8. Contemporary Bioethical Issues in

Pharmacology & Pharmaceutical Research ••

Janet Fleetwood

1 SECTION

I

GENERAL PRINCIPLES

OF PHARMACOLOGY

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3

Early in human history a natural bond formed between religion and the use of drugs. Those who became most proficient in the use of drugs to treat disease were the “mediators” between this world and the spirit world, namely, the priests, shamans, holy persons, witches, and soothsayers. Much of their power within the community was derived from the cures that they could effect with drugs. It was believed that the sick were possessed by demons and that health could be re- stored by identifying the demon and finding a way to cast it out.

Originally, religion dominated its partnership with therapeutics, and divine intervention was called upon for every treatment. However, the use of drugs to effect cures led to a profound change in both religious thought and structure. As more became known about the effects of drugs, the importance of divine intervention began to recede, and the treatment of patients effectively became a province of the priest rather than the gods whom the priest served. This process lead to a growing understanding of the curative powers of natural products and a decreasing reliance on supernatural intervention and forever altered the relationship between humanity and its gods. Furthermore, when the priests began to apply the information learned from treating one patient to the treatment of other patients, there was a recognition that a regularity prevailed in the natural world independent of supernatural whim or will. Therapeutics thus evolved from its roots in magic to a foundation in experience.

This was the cornerstone for the formation of a science- based practice of medicine.

CONTRIBUTIONS OF MANY CULTURES

The ancient Chinese wrote extensively on medical subjects. The Pen Tsao, for instance, was written about 2700 B.C. and contained classifications of individual medicinal plants as well as compilations of plant mixtures to be used for medical purposes. The Chinese doctrine of signatures (like used to treat like) enables us to un- derstand why medicines of animal origin were of such great importance in the Chinese pharmacopoeia.

Ancient Egyptian medical papyri contain numerous prescriptions. The largest and perhaps the most impor- tant of these, the Ebers papyrus (1550 B.C.), contains about 800 prescriptions quite similar to those written today in that they have one or more active substances as well as vehicles (animal fat for ointments; and water, milk, wine, beer, or honey for liquids) for suspending or dissolving the active drug. These prescriptions also commonly offer a brief statement of how the preparation is to be prepared (mixed, pounded, boiled, strained, left overnight in the dew) and how it is to be used (swal- lowed, inhaled, gargled, applied externally, given as an enema). Cathartics and purgatives were particularly in vogue, since both patient and physician could tell almost immediately whether a result had been achieved.

It was reasoned that in causing the contents of the gastrointestinal tract to be forcibly ejected, one simultane- ously drove out the disease-producing evil spirits that had taken hold of the unfortunate patient.

The level of drug usage achieved by the Egyptians undoubtedly had a great influence on Greek medicine and literature. Observations on the medical effects of

Progress in Therapeutics

Robert E. Stitzel and Joseph J. McPhillips

1 1

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various natural substances are found in both the Iliad and the Odyssey. Battle wounds frequently were cov- ered with powdered plant leaves or bark; their astrin- gent and pain-reducing actions were derived from the tannins they contained. It may have been mandrake root (containing atropinelike substances that induce a twilight sleep) that protected Ulysses from Circe. The oriental hellebore, which contains the cardiotoxic Veratrum alkaloids, was smeared on arrow tips to in- crease their killing power. The fascination of the Greeks with the toxic effects of various plant extracts led to an increasing body of knowledge concerned primarily with the poisonous aspects of drugs (the science of toxicol- ogy). Plato’s description of the death of Socrates is an accurate description of the toxicological properties of the juice of the hemlock fruit. His description of the paralysis of sensory and motor nerves, followed eventu- ally by central nervous system depression and respira- tory paralysis, precisely matches the known actions of the potent hemlock alkaloid, coniine.

The Indian cultures of Central and South America, although totally isolated from the Old World, developed drug lore and usage in a fashion almost parallel with that of the older civilization. The use of drugs played an inti- mate part in the rites, religions, history, and knowledge of the South American Indians. New World medicine also was closely tied to religious thought, and Indian cultures treated their patients with a blend of religious rituals and herbal remedies. Incantations, charms, and appeals to various deities were as important as the appropriate application of poultices, decoctions, and infusions.

Early drug practitioners, both in Europe and South America, gathered herbs, plants, animals, and minerals and often blended them into a variety of foul-smelling and ill-flavored concoctions. The fact that many of these preparations were so distasteful led to an attempt to improve on the “cosmetic” properties of these mixtures to ensure that patients would actually use them.

Individuals who searched for improved product formulations were largely responsible for the founding of the disciplines of pharmacy (the science of preparing, com- pounding, and dispensing medicines) and pharmacog- nosy (the identification and preparation of crude drugs from natural sources).

There has long been a tendency of some physicians to prescribe large numbers of drugs where one or two would be sufficient. We can trace the history of this polypharmaceutical approach to Galen (A.D. 131–201), who was considered the greatest European physician after Hippocrates. Galen believed that drugs had certain essential properties, such as warmth, coldness, dry- ness, or humidity, and that by using several drugs he could combine these properties to adjust for deficien- cies in the patient. Unfortunately, he often formulated general rules and laws before sufficient factual information was available to justify their formulations.

By the first century A.D. it was clear to both physician and protopharmacologist alike that there was much variation to be found from one biological extract to another, even when these were prepared by the same individual. It was reasoned that to fashion a rational and reproducible system of therapeutics and to study pharmacological activity one had to obtain standardized and uniform medicinal agents.

At the turn of the nineteenth century, methods became available for the isolation of active principles from crude drugs. The development of chemistry made it possible to isolate and synthesize chemically pure compounds that would give reproducible biological results.

In 1806, Serturner (1783–1841) isolated the first pure active principle when he purified morphine from the opium poppy. Many other chemically pure active compounds were soon obtained from crude drug preparations, including emetine by Pelletier (1788–1844) from ipecacuanha root; quinine by Carentou (1795–1877) from cinchona bark; strychnine by Magendie (1783–

1855) from nux vomica; and, in 1856, cocaine by Wohler (1800–1882) from coca.

The isolation and use of pure substances allowed for an analysis of what was to become one of the basic con- cerns of pharmacology, that is, the quantitative study of drug action. It was soon realized that drug action is produced along a continuum of effects, with low doses producing a less but essentially similar effect on organs and tissues as high doses. It also was noted that the appear- ance of toxic effects of drugs was frequently a function of the dose–response relationship.

Until the nineteenth century, the rapid development of pharmacology as a distinct discipline was hindered by the lack of sophisticated chemical methodology and by limited knowledge of physiological mechanisms. The significant advances made through laboratory studies of animal physiology accomplished by early investigators such as Françoise Magendie and Claude Bernard provided an environment conducive to the creation of similar laboratories for the study of pharmacological phe- nomena.

One of the first laboratories devoted almost exclu- sively to drug research was established in Dorpat, Estonia, in the late 1840s by Rudolph Bucheim (1820–

1879) (Fig. 1.1). The laboratory, built in Bucheim’s home, was devoted to studying the actions of agents such as cathartics, alcohol, chloroform, anthelmintics, and heavy metals. Bucheim believed that “the investigation of drugs . . . is a task for a pharmacologist and not for a chemist or pharmacist, who until now have been expected to do this.”

Although the availability of a laboratory devoted to pharmacological investigations was important, much more was required to raise this discipline to the same prominent position occupied by other basic sciences; this included the creation of chairs in pharmacology at other

4 I GENERAL PRINCIPLES OF PHARMACOLOGY

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1 Progress in Therapeutics 5

academic institutions and the training of a sufficient number of talented investigators to occupy these positions.

The latter task was accomplished largely by Bucheim’s pupil and successor at Dorpat, Oswald Schmiedeberg (1838–1921), undoubtedly the most prominent pharmacologist of the nineteenth century (Fig. 1.1). In addition to conducting his own outstanding research on the pharmacology of diuretics, emetics, cardiac glycosides, and so forth, Schmiedeberg wrote an important medical textbook and trained approximately 120 pupils from more than 20 countries. Many of these new investigators either started or developed laboratories devoted to experimental pharmacology in their own countries.

One of Schmiedeberg’s most outstanding students was John Jacob Abel, who has been called the founder of American pharmacology (Fig 1.1). Abel occupied the chair of pharmacology first at the University of Michigan and then at Johns Hopkins University. Among his most important research accomplishments is an examination of the chemistry and isolation of the active principles from the adrenal medulla (a monobenzyl derivative of epinephrine) and the pancreas (crystallization of insulin). He also examined mushroom poisons, investigated the chemotherapeutic actions of the arsenicals and anti- monials, conducted studies on tetanus toxin, and designed a model for an artificial kidney. In addition, Abel founded the Journal of Experimental Medicine, the Journal of Biological Chemistry, and the Journal of Pharmacology and Experimental Therapeutics. His devo- tion to pharmacological research, his enthusiasm for the training of students in this new discipline, and his estab- lishment of journals and scientific societies proved criti-

cal to the rise of experimental pharmacology in the United States.

Pharmacology, as a separate and vital discipline, has interests that distinguish it from the other basic sciences and pharmacy. Its primary concern is not the cataloguing of the biological effects that result from the administration of chemical substances but rather the dual aims of (1) providing an understanding of normal and abnormal human physiology and biochemistry through the application of drugs as experimental tools and (2) applying to clinical medicine the information gained from fundamental investigation and observation.

A report in the Status of Research in Pharmacology has described some of the founding principles on which the discipline is based and that distinguish pharmacology from other fields of study. These principles include the study of the following:

• The relationship between drug concentration and biological response

• Drug action over time

• Factors affecting absorption, distribution, binding, metabolism, and elimination of chemicals

• Structure-activity relationships

• Biological changes that result from repeated drug use: tolerance, addiction, adverse reactions, altered rates of drug metabolism, and so forth

• Antagonism of the effects of one drug by another

• The process of drug interaction with cellular macromolecules (receptors) to alter physiological function (i.e., receptor theory)

F I G U R E 1 . 1

The three important figures in the early history of pharmacology are (left to right) Rudolf Bucheim, Oswald Schmiedeberg, and John Jacob Abel. They not only created new laboratories devoted to the laboratory investigation of drugs but also firmly established the new discipline through the training of future faculty, the writing of textbooks, and the founding of scientific journals and societies.

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In the past 100 years there has been extraordinary growth in medical knowledge. This expansion of information has come about largely through the contributions of the biological sciences to medicine by a system- atic approach to the understanding and treatment of disease. The experimental method and technological advances are the foundations upon which modern medicine is built.

DRUG CONTROL AND DEVELOPMENT

Before the twentieth century, most government controls were concerned not with drugs but with impure and adulterated foods. Medicines were thought to pose problems similar to those presented by foods. Efficacy was questioned in two respects: adulteration of active medicines by addition of inert fillers and false claims made for the so-called patent (secret) medicines or nos- trums. Indeed, much of the development of the science of pharmacy in the nineteenth century was standardiz- ing and improving prescription drugs.

A landmark in the control of drugs was the 1906 Pure Food and Drug Act. Food abuses, however, were the primary target. Less than one quarter of the first thousand decisions dealt with drugs, and of these, the majority were concerned with patent medicines.

The 1906 law defined drug broadly and governed the labeling but not the advertising of any substance used to affect disease. This law gave the Pharmacopoeia and the National Formulary equal recognition as authorities for drug specifications. In the first contested criminal pros- ecution under the law, action was taken against the maker of a headache mixture bearing the beguiling name of Cuforhedake-Brane-Fude. In 1912, Congress passed an amendment to the Pure Food and Drug Act that banned false and fraudulent therapeutic claims for patent medicines.

Prescription drugs also were subject to control under the 1906 law. In fact, until 1953 there was no fixed legal boundary between prescription and nonprescrip- tion medications. Prescription medications received a lower priority, since food and patent medicine abuses were judged to be the more urgent problems.

For the next 30 years, drug control was viewed primarily as a problem of prohibiting the sale of dangerous drugs and tightening regulations against misbranding.

Until the 1930s, new drugs posed little problem because there were few of them.

MODERN DRUG LEGISLATION

The modern history of United States drug regulation began with the Food, Drug and Cosmetic Act of 1938, which superseded the 1906 Pure Food and Drug Act.

The 1938 act was viewed as a means of preventing the marketing of untested, potentially harmful drugs. An

obscure provision of the 1938 act was destined to be the starting point for some of the most potent controls the Food and Drug Administration (FDA) now exercises in the drug field. This provision allowed the prescription drug to come under special control by requiring that it carry the legend “Caution—to be used only by or on the prescription of a physician.”

A major defect of the generally strong 1938 law was its inadequate control of advertising. Regulations now require that the “labeling on or within the package from which the drug is to be dispensed” contain adequate information for the drug’s use; this requirement explains the existence of the package insert. If the pharmaceutical manufacturer makes claims for its product beyond those contained in an approved package insert, the FDA may institute legal action against the deviations in advertising.

The 1938 act required manufacturers to submit a New Drug Application (NDA) to the FDA for its approval before the company was permitted to market a new drug. Efficacy (proof of effectiveness) became a re- quirement in 1962 with the Kefauver-Harris drug amendments. These amendments established a requirement that drugs show “substantial evidence” of efficacy before receiving NDA approval. Substantial evidence was defined in the amendments as evidence consisting of adequate and well-controlled investigations, including clinical investigations, by experts qualified by scientific training and experience to evaluate the effectiveness of the drug, on the basis of which such experts could fairly and responsibly conclude that the drug would have the claimed effect under the conditions of use named on the label.

Drug regulation in the United States is continuing to evolve rapidly, both in promulgation of specific regulations and in the way regulations are implemented (Table 1.1). The abolition of patent medicines is an outstanding example, as is control over the accuracy of claims made for drugs. Since the 1962 amendments, the advertising of prescription drugs in the United States has been in- creasingly controlled—to a greater extent than in most other countries. All new drugs introduced since 1962 have some proof of efficacy. This is not to say that mis- leading drug advertisements no longer exist; manufacturers still occasionally make unsubstantiated claims.

Phase Purpose

I Establish safety

II Establish efficacy and dose

III Verify efficacy and detect adverse affects IV Obtain additional data following approval

Phases of Clinical Investigation TA B L E

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CLINICAL TESTING OF DRUGS

Experiments conducted on animals are essential to the development of new chemicals for the management of disease. The safety and efficacy of new drugs, however, can be established only by adequate and well-controlled studies on human subjects. Since findings in animals do not always accurately predict the human response to drugs, subjects who participate in clinical trials are put at some degree of risk. The risk comes not only from the potential toxicity of the new drug but also from possible lack of efficacy, with the result that the condition under treatment becomes worse. Since risk is involved, the primary consideration in any clinical trial should be the welfare of the subject. As a consequence of unethical or questionably ethical practices committed in the past, most countries have established safeguards to protect the rights and welfare of persons who participate in clinical trials. Two of the safeguards that have been es- tablished are the institutional review board (IRB) and the requirement for informed consent.

The IRB, also known as the ethics committee or human subjects committee, originally was established to protect people confined to hospitals, mental institutions, nursing homes, and prisons who may be used as subjects in clinical research. In the United States any institution conducting clinical studies supported by federal funds is required to have proposed studies reviewed and approved by an IRB.

People who volunteer to be subjects in a drug study have a right to know what can and will happen to them if they participate (informed consent). The investigator is responsible for ensuring that each subject receives a full explanation, in easily understood terms, of the purpose of the study, the procedures to be employed, the nature of the substances being tested, and the potential risks, benefits, and discomforts.

PHASES OF CLINICAL INVESTIGATION

The clinical development of new drugs usually takes place in steps or phases conventionally described as clinical pharmacology (phase I), clinical investigation (phase II), clinical trials (phase III), and postmarketing studies (phase IV). Table 1.1 summarizes the four phases of clinical evaluation.

Phase I

When a drug is administered to humans for the first time, the studies generally have been conducted in healthy men between 18 and 45 years of age; this prac- tice is coming under increasing scrutiny and criticism.

For certain types of drugs, such as antineoplastic agents, it is not appropriate to use healthy subjects because the risk of injury is too high. The purpose of phase I studies is to establish the dose level at which signs of toxicity first

appear. The initial studies consist of administering a sin- gle dose of the test drug and closely observing the subject in a hospital or clinical pharmacology unit with emergency facilities. If no adverse reactions occur, the dose is increased progressively until a predetermined dose or serum level is reached or toxicity supervenes.

Phase I studies are usually confined to a group of 20 to 80 subjects. If no untoward effects result from single doses, short-term multiple-dose studies are initiated.

Phase II

If the results of phase I studies show that it is reasonably safe to continue, the new drug is administered to patients for the first time. Ideally, these individuals should have no medical problems other than the condition for which the new drug is intended. Efforts are concentrated on evaluating efficacy and on establishing an optimal dose range.

Therefore, dose–response studies are a critical part of phase II studies. Monitoring subjects for adverse effects is also an integral part of phase II trials. The number of subjects in phase II studies is usually between 80 and 100.

Phase III

When an effective dose range has been established and no serious adverse reactions have occurred, large numbers of subjects can be exposed to the drug. In phase III studies the number of subjects may range from several hundred to several thousand, depending on the drug.

The purpose of phase III studies is to verify the efficacy of the drug and to detect effects that may not have sur- faced in the phase I and II trials, during which exposure to the drug was limited. A new drug application is sub- mitted at the end of phase III. However, for drugs intended to treat patients with life-threatening or severely debilitating illnesses, especially when no satisfactory therapy exists, the FDA has established procedures designed to expedite development, evaluation, and marketing of new therapies. In the majority of cases, the procedure applies to drugs being developed for the treatment of cancer and acquired immunodeficiency syndrome (AIDS). Under this procedure, drugs can be approved on the basis of phase II studies conducted in a limited number of patients.

Phase IV

Controlled and uncontrolled studies often are conducted after a drug is approved and marketed. Such studies are intended to broaden the experience with the drug and compare it with other drugs.

SPECIAL POPULATIONS

One of the goals of drug development is to provide sufficient data to permit the safe and effective use of the drug.

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A N S W E R S

1. D. There is always some degree of risk in clinical trials; the object is to minimize the risk to the patient. The primary consideration in any clinical trial is the welfare of the subject. The safety of the drug is one objective for certain clinical trials as is the efficacy of the drug in other trials.

2. A. Phase I studies are carried out in normal volun- teers. The object of phase I studies is to determine the dose level at which signs of toxicity first appear.

Phase II studies are carried out in patients in which the drug is designed to be effective in. It is conducted to determine efficacy and optimal dosage.

Phase III studies are a continuation of phase II, but many more patients are involved. The purpose of phase III studies is to verify efficacy established earlier in phase II studies and to detect adverse effects that may not have surfaced in earlier studies. Phase IV studies are conducted when the drug has been

approved and is being marketed. The purpose of these studies is to broaden the experience with the drug and to compare the new drug with other agents that are being used clinically.

3. C. John Jacob Abel occupied the first chair of a de- partment of pharmacology in the United States.

This was at the University of Michigan. Abel subsequently left Michigan to chair the first department of pharmacology at Johns Hopkins University.

Claude Bernard was an early French physiologist and pharmacologist. Rudolph Bucheim established one of the first pharmacology laboratories at the University of Dorpat (Estonia). Oswald

Schmiedeberg is considered the founder of pharmacology. He trained approximately 120 pupils from around the world, including the father of American pharmacology, John Jacob Abel.

Therefore, the patient population that participates in clinical trials should be representative of the patient population that will receive the drug when it is marketed. To a varying extent, however, women, children, and patients over 65 years of age have been underrepresented in clini- cal trials of new drugs. The reasons for exclusion vary, but the consequence is that prescribing information for these patient populations is often deficient.

ADVERSE REACTION SURVEILLANCE

Almost all drugs have adverse effects associated with their use; these range in severity from mild inconven- iences to severe morbidity and death. Some adverse ef-

fects are extensions of the drug’s pharmacological effect and are predictable, for example, orthostatic hypoten- sion with some antihypertensive agents, arrhythmias with certain cardioactive drugs, and electrolyte imbal- ance with diuretics. Other adverse effects are not predictable and may occur rarely or be delayed for months or years before the association is recognized. Examples of such reactions are aplastic anemia associated with chloramphenicol and clear cell carcinoma of the uterus in offspring of women treated with diethylstilbestrol during pregnancy. Postmarketing surveillance programs and adverse reaction reporting systems may detect such events. The best defense against devastating adverse actions is still the vigilance and suspicion of the physician.

S t u d y

Q u e s t i o n s

1. The primary consideration in all clinical trials is to (A) Determine the safety of the drug

(B) Determine the efficacy of the drug (C) Ensure that there is no risk to the subject (D) Provide for the welfare of the subject 2. To conduct reliable clinical trials with a potential

new drug, it is necessary to establish a dose level that toxicity first appears. This is commonly determined in

(A) Phase I Studies (B) Phase II Studies

(C) Phase III Studies (D) Phase IV Studies

3. The history of pharmacology includes a long list of heroes. The person considered to be the founder of American pharmacology is

(A) Claude Bernard (B) Rudolph Bucheim (C) John Jacob Abel (D) Oswald Schmeideberg

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S U P P L E M E N TA L R E A D I N G

Burks TF. Two hundred years of pharmacology: A mid- point assessment. Proc West Pharmacol Soc

2000;43:95–103.

Guarino RA. (ed). New Drug Approval Process. New York: Dekker, 1992.

Holmstead B and Liljestrand G. (eds.). Readings in Pharmacology. New York: Macmillan, 1963.

Huang KC. The Pharmacology of Chinese Herbs. Boca Raton, FL: CRC, 1993.

Lemberger L. Of mice and men: The extension of animal models to the clinical evaluation of new drugs.

Clin Pharmacol Ther 1986;40:599–603.

Muscholl E. The evolution of experimental pharmacology as a biological science: The pioneering work of Bucheim and Schmiedeberg. Brit J Pharmacol 1995;116:2155–2159.

O’Grady J and Joubert PH (eds.). Handbook of Phase I/II Clinical Drug Trials. Boca Raton, FL: CRC, 1997.

Parascandola J. John J. Abel and the emergence of U.S.

pharmacology. Pharmaceut News 1995;2:911.

Spilker, B. Guide to Clinical Trials. New York: Raven, 1991.

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10 RECEPTORS

A fundamental concept of pharmacology is that to initiate an effect in a cell, most drugs combine with some molecular structure on the surface of or within the cell.

This molecular structure is called a receptor. The combi- nation of the drug and the receptor results in a molecular change in the receptor, such as an altered configura- tion or charge distribution, and thereby triggers a chain of events leading to a response. This concept applies not only to the action of drugs but also to the action of nat- urally occurring substances, such as hormones and neurotransmitters. Indeed, many drugs mimic the effects of hormones or transmitters because they combine with the same receptors as do these endogenous substances.

It is generally assumed that all receptors with which drugs combine are receptors for neurotransmitters, hormones, or other physiological substances. Thus, the dis- covery of a specific receptor for a group of drugs can lead to a search for previously unknown endogenous substances that combine with those same receptors. For example, evidence was found for the existence of endogenous peptides with morphinelike activity. A series of these peptides have since been identified and are col- lectively termed endorphins and enkephalins (see Chapter 26). It is now clear that drugs such as morphine merely mimic endorphins or enkephalins by combining with the same receptors.

DRUG RECEPTORS AND BIOLOGICAL RESPONSES

Although the term receptor is convenient, one should never lose sight of the fact that receptors are in actuality

molecular substances or macromolecules in tissues that combine chemically with the drug. Since most drugs have a considerable degree of selectivity in their actions, it follows that the receptors with which they interact must be equally unique. Thus, receptors will interact with only a limited number of structurally related or comple- mentary compounds.

The drug–receptor interaction can be better appreci- ated through a specific example. The end-plate region of a skeletal muscle fiber contains large numbers of receptors having a high affinity for the transmitter acetylcholine. Each of these receptors, known as nicotinic receptors, is an integral part of a channel in the postsynaptic membrane that controls the inward movement of sodium ions (see Chapter 28). At rest, the postsynaptic membrane is relatively impermeable to sodium.

Stimulation of the nerve leading to the muscle results in the release of acetylcholine from the nerve fiber in the region of the end plate. The acetylcholine combines with the receptors and changes them so that channels are opened and sodium flows inward. The more acetylcholine the end-plate region contains, the more receptors are occupied and the more channels are open. When the number of open channels reaches a critical value, sodium enters rapidly enough to disturb the ionic bal- ance of the membrane, resulting in local depolarization.

The local depolarization (end-plate potential) triggers the activation of large numbers of voltage-dependent sodium channels, causing the conducted depolarization known as an action potential. The action potential leads to the release of calcium from intracellular binding sites.

The calcium then interacts with the contractile proteins, resulting in shortening of the muscle cell. The sequence of events can be shown diagrammatically as follows:

Mechanisms of Drug Action

William W. Fleming

2 2

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2 Mechanisms of Drug Action 11

Ach receptor → Nainflux → action potential

→ increased free Ca → contraction

where Ach acetylcholine. The precise chain of events following drug–receptor interaction depends on the particular receptor and the particular type of cell. The important concept at this stage of the discussion is that specific receptive substances serve as triggers of cellular reactions.

If we consider the sequence of events by which acetylcholine brings about muscle contraction through receptors, we can easily appreciate that foreign chemicals (drugs) can be designed to interact with the same process. Thus, such a drug would mimic the actions of acetylcholine at the motor end plate; nicotine and car- bamylcholine are two drugs that have such an effect.

Chemicals that interact with a receptor and thereby initi- ate a cellular reaction are termed agonists. Thus, acetyl- choline itself, as well as the drugs nicotine and car- bamylcholine, are agonists for the receptors in the skeletal muscle end plate.

On the other hand, if a chemical is somewhat less similar to acetylcholine, it may interact with the receptor but be unable to induce the exact molecular change necessary to allow the inward movement of sodium. In this instance the chemical does not cause contraction, but because it occupies the receptor site, it prevents the interaction of acetylcholine with its receptor. Such a drug is termed an antagonist. An example of such a compound is d-tubocurarine, an antagonist of acetyl- choline at the end-plate receptors. Since it competes with acetylcholine for its receptor and prevents acetylcholine from producing its characteristic effects, admin- istration of d-tubocurarine results in muscle relaxation by interfering with acetylcholine’s ability to induce and maintain the contractile state of the muscle cells.

Historically, receptors have been identified through recognition of the relative selectivity by which certain exogenously administered drugs, neurotransmitters, or hormones exert their pharmacological effects. By apply- ing mathematical principles to dose–response relation- ships, it became possible to estimate dissociation con- stants for the interaction between specific receptors and individual agonists or antagonists. Subsequently, methods were developed to measure the specific binding of radioactively labeled drugs to receptor sites in tissues and thereby determine not only the affinity of a drug for its receptor, but also the density of receptors per cell.

In recent years much has been learned about the chemical structure of certain receptors. The nicotinic receptor on skeletal muscle, for example, is known to be composed of five subunits, each a glycoprotein weighing 40,000 to 65,000 daltons. These subunits are arranged as interacting helices that penetrate the cell membrane completely and surround a central pit that is a sodium

ion channel. The binding sites for acetylcholine (see Chapter 12) and other agonists that mimic it are on one of the subunits that project extracellularly from the cell membrane. The binding of an agonist to these sites changes the conformation of the glycoprotein so that the side chains move away from the center of the channel, allowing sodium ions to enter the cell through the channel. The glycoproteins that make up the nicotinic receptor for acetylcholine serve as both the walls and the gate of the ion channel. This arrangement represents one of the simpler mechanisms by which a receptor may be coupled to a biological response.

SECOND-MESSENGER SYSTEMS

Many receptors are capable of initiating a chain of events involving second messengers. Key factors in many of these second-messenger systems are proteins termed G proteins, short for guanine nucleotide–

binding proteins. G proteins have the capacity to bind guanosine triphosphate (GTP) and hydrolyze it to guanosine diphosphate (GDP).

G proteins couple the activation of several different receptors to the next step in a chain of events. In a number of instances, the next step involves the enzyme adenylyl cyclase. Many neurotransmitters, hormones, and drugs can either stimulate or inhibit adenylyl cyclase through their interaction with different receptors;

these receptors are coupled to adenylate cyclase through either a stimulatory (G_S) or an inhibitory (G₁) G protein. During the coupling process, the binding and subsequent hydrolysis of GTP to GDP provides the energy needed to terminate the coupling process.

The activation of adenylyl cyclase enables it to cat- alyze the conversion of adenosine triphosphate (ATP) to 35-cyclic adenosine monophosphate (cAMP), which in turn can activate a number of enzymes known as ki- nases. Each kinase phosphorylates a specific protein or proteins. Such phosphorylation reactions are known to be involved in the opening of some calcium channels as well as in the activation of other enzymes. In this system, the receptor is in the membrane with its binding site on the outer surface. The G protein is totally within the membrane while the adenylyl cyclase is within the membrane but projects into the interior of the cell. The cAMP is generated within the cell (see Figure 10.4).

Whether or not a particular agonist has any effect on a particular cell depends initially on the presence or absence of the appropriate receptor. However, the na- ture of the response depends on these factors:

• Which G protein couples with the receptor

• Which kinase is activated

• Which proteins are accessible for the kinase to phosphorylate

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The variety of possible responses is further increased by the fact that receptor-coupled G proteins can either activate enzymes other than adenylate cyclase or can directly influence ion channel functions.

Many different receptor types are coupled to G proteins, including receptors for norepinephrine and epinephrine (- and -adrenoceptors), 5-hydroxytrypta- mine (serotonin or 5-HT receptors), and muscarinic acetylcholine receptors. Figure 2.1 presents the structure of one of these, the 2-adrenoceptor from the human kidney. All members of this family of G protein–coupled receptors are characterized by having seven membrane-enclosed domains plus extracellular and intracellular loops. The specific binding sites for ag- onists occur at the extracellular surface, while the inter- action with G proteins occurs with the intracellular por- tions of the receptor. The general term for any chain of events initiated by receptor activation is signal trans- duction.

THE CHEMISTRY OF DRUG–RECEPTOR BINDING

Biological receptors are capable of combining with drugs in a number of ways, and the forces that attract the drug to its receptor must be sufficiently strong and long-lasting to permit the initiation of the sequence of events that ends with the biological response. Those forces are chemical bonds, and a number of types of bonds participate in the formation of the initial drug–receptor complex.

The bond formed when two atoms share a pair of electrons is called a covalent bond. It possesses a bond energy of approximately 100 kcal/mole and therefore is strong and stable; that is, it is essentially irreversible at body temperature. Covalent bonds are responsible for the stability of most organic molecules and can be broken only if sufficient energy is added or if a catalytic agent that can facilitate bond disruption, such as an enzyme, is present. Since bonds of this type are so stable at physiological temperatures, the binding of a drug to a receptor through covalent bond formation would result in the formation of a long-lasting complex.

Although most drug–receptor interactions are read- ily reversible, some compounds, such as the anticancer nitrogen mustards (see Chapter 56) and other alkylat- ing agents form relatively irreversible complexes.

Covalent bond formation is a desirable feature of an antineoplastic or antibiotic drug, since long-lasting inhibi- tion of cell replication is needed. However, covalent bond formation between environmental pollutants and cellular constituents may result in mutagenesis or car- cinogenesis in normal, healthy cells.

The formation of an ionic bond results from the electrostatic attraction that occurs between oppositely charged ions. The strength of this bond is considerably less (5 kcal/mole) than that of the covalent bond and di- minishes in proportion to the square of the distance between the ionic species. Most macromolecular receptors have a number of ionizable groups at physiological pH (e.g., carboxyl, hydroxyl, phosphoryl, amino) that are available for interaction with an ionizable drug.

The hydrogen atom, with its strongly electroposi- tive nucleus and single electron, can be bound to one strongly electronegative atom and still accept an electron from another electronegative donor atom, such as nitrogen or oxygen, and thereby form a bridge (hy- drogen bond) between these two donor atoms. The formation of several such bonds between two molecules (e.g., drug and receptor) can result in a relatively stable but reversible interaction. Such bonds serve to maintain the tertiary structure of proteins and nu- cleic acids and are thought to play a significant role in establishing the selectivity and specificity of drug–

receptor interactions.

Van der Waals bonds are quite weak (0.5 kcal/mole) and become biologically important only when two atoms are brought into sufficiently close contact. Van der Waals forces play a significant part in determining drug–receptor specificity. Like the hydrogen bonds, several van der Waals bonds may be established between two molecules, especially if the drug molecule and a receptor have complementary three-dimensional confor- mations and thus fit closely together. The closer the drug comes to the receptor, the stronger the possible binding forces that can be established. Slight differences in three-dimensional shape among a group of agonists

NH₂

A P A L A A A L A Y A

S G G E R S G G Y A N A S G S GW P P R Q G Y S A G A

A A

A

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K RR YC T S L S KR

S R A HH H

S L PP YE S R S AS R

L G P PS SA TL A G

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I L A Y G

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TW S R T R A A G R P R G G A P L R R G G R R R A G A E G G A G G A D G G A G P G A A G G A A A G L G M TPS A G D P G Y R

F I Y Y G Y

A A G P

E X T R A C E L L U L A R

I N T R A C E L L U L A R

F I G U R E 2 . 1

Primary structure of the human kidney ²-adrenoceptor. The amino acid sequence is represented by the one-letter code.

(Reprinted with permission from Regan JW et al. Cloning and expression of a human kidney cDNA. Proc Natl Acad Sci USA 85:6301, 1988.)

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2 Mechanisms of Drug Action 13

and therefore slight differences in fit or strength of bonding forces that can be established between agonists and receptor form the basis for the structure–activity re- lationships among related agonists.

DYNAMICS OF DRUG–RECEPTOR BINDING

The drug molecule, following its administration and passage to the area immediately adjacent to the recep- tor surface (sometimes called the biophase), must bond with the receptor before it can initiate a response.

Resisting this bond formation is a random thermal agitation that is inherent in every molecule and tends to keep the molecule in constant motion. Under normal circumstances, the electrostatic attraction of the ionic bond, which can be exerted over longer distances than can the attraction of either the hydrogen or van der Waals bond, is the first force that draws the ionized molecule toward the oppositely charged receptor surface.

This is a reasonably strong bond and will lend some stability to the drug–receptor complex.

Generally, the ionic bond must be reinforced by a hydrogen or van der Waals bond or both before significant receptor activation can occur. This is true because unreinforced bonds are too easily and quickly broken by the energy of thermal agitation to permit sufficient time for adequate drug–receptor interaction to take place. The better the structural congruity (i.e., fit) between drug and its receptor, the more secondary (i.e., hydrogen and van der Waals) bonds can form.

Even if extensive binding has taken place, unless covalent bond formation has occurred, the drug–receptor complex can still dissociate. Once dissociation has occurred, drug action is terminated. For most drug–receptor interactions, there is a continual random association and dissociation. The frequencies of association and dissociation are a function of the affinity between the drug and the receptor, the density of receptors, and the con- centration of drug in the biophase. The magnitude of the response is generally considered to be a function of the concentration of the drug–receptor complexes formed at any moment in time.

DOSE–RESPONSE RELATIONSHIP

To understand drug–receptor interactions, it is necessary to quantify the relationship between the drug and the biological effect it produces. Since the degree of effect produced by a drug is generally a function of the amount administered, we can express this relationship in terms of a dose–response curve. Because we cannot always quantify the concentration of drug in the biophase in the intact individual, it is customary to corre- late effect with dose administered.

In general, biological responses to drugs are graded;

that is, the response continuously increases (up to the maximal responding capacity of the given responding system) as the administered dose continuously increases. Expressed in receptor theory terminology, this means that when a graded dose–response relationship exists, the response to the drug is directly related to the number of receptors with which the drug effectively in- teracts. This is one of the tenets of pharmacology.

The principles derived from dose–response curves are the same in animals and humans. However, obtain- ing the data for complete dose–response curves in humans is generally difficult or dangerous. We shall therefore use animal data to illustrate these principles.

Quantal Relationships

In addition to the responsiveness of a given patient, one may be interested in the relationship between dose and some specified quantum of response among all individ- uals taking that drug. Such information is obtained by evaluating data obtained from a quantal dose–response curve.

Anticonvulsants can be suitably studied by use of quantal dose–response curves. For example, to assess the potential of new anticonvulsants to control epileptic seizures in humans, these drugs are initially tested for their ability to protect animals against experimentally induced seizures. In the presence of a given dose of the drug, the animal either has the seizure or does not; that is, it either is or is not protected. Thus, in the design of this experiment, the effect of the drug (protection) is all or none. This type of response, in contrast to a graded re- sponse, must be described in a noncontinuous manner.

The construction of a quantal dose–response curve requires that data be obtained from many individuals.

Although any given patient (or animal) either will or will not respond to a given dose, a comparison of individuals within a population shows that members of that population are not identical in their ability to respond to a particular dose. This variability can be expressed as a type of dose–response curve, sometimes termed a quantal dose–response curve, in which the dose (plotted on the horizontal axis) is evaluated against the percentage of animals in the experimental population that is protected by each dose (vertical axis). Such a dose–

response curve for the anticonvulsant phenobarbital is illustrated in Figure 2.2A. Five groups of 10 rats per group were used. The animals in any one group received a particular dose of phenobarbital of 2, 3, 5, 7, or 10 mg/kg body weight. The percentage of animals in each group protected against convulsions was plotted against the dose of phenobarbital. As Figure 2.2A shows, the lowest dose protected none of the 10 rats to which it was given, whereas 10mg/kg protected 10 of 10. With the intermediate doses, some rats were protected and some

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were not; this indicates that the rats differ in their sensitivity to phenobarbital.

The quantal dose–response curve is actually a cu- mulative plot of the normal frequency distribution curve. The frequency distribution curve, in this case re- lating the minimum protective dose to the frequency with which it occurs in the population, generally is bell shaped. If one graphs the cumulative frequency versus dose, one obtains the sigmoid-shaped curve of Figure 2.2A. The sigmoid shape is a characteristic of most dose–response curves when the dose is plotted on a geo- metric, or log, scale.

Therapeutic Index Effective Dose

The quantal dose–response curve represents esti- mates of the frequency with which each dose elicits the desired response in the population. In addition to this information, it also would be useful to have some way to express the average sensitivity of the entire population to phenobarbital. This is done through the calculation of an ED₅₀(effective dose, 50%; i.e., the dose that would protect 50% of the animals). This value can be obtained from the dose–response curve in Figure 2.2A, as shown by the broken lines. The ED₅₀for phenobarbital in this population is approximately 4mg/kg.

Lethal Dose

Another important characteristic of a drug’s activity is its toxic effect. Obviously, the ultimate toxic effect is death. A curve similar to that already discussed can be constructed by plotting percent of animals killed by phenobarbital against dose (Fig. 2.2B). From this curve, one can calculate the LD₅₀(lethal dose, 50%). Since the degree of safety associated with drug administration de- pends on an adequate separation between doses produc- ing a therapeutic effect (e.g., ED₅₀) and doses producing toxic effects (e.g., LD₅₀), one can use a comparison of

these two doses to estimate drug safety. Thus, one estimate of a drug’s margin of safety is the ratio LD₅₀/ED₅₀; this is the therapeutic index. The therapeutic index for phenobarbital used as an anticonvulsant is approximately 40/4, or 10.

As a general rule, a drug should have a high therapeutic index; however, some important therapeutic agents have low indices. For example, although the therapeutic index of the cardiac glycosides is only about 2 for the treatment and control of cardiac failure, these drugs are important for many cases of cardiac failure.

Therefore, in spite of a low margin of safety, they are often used for this condition. The identification of a low margin of safety, however, dictates particular caution in its use; the appropriate dose for each individual must be determined separately.

It has been suggested that a more realistic estimate of drug safety would include a comparison of the lowest dose that produces toxicity (e.g., LD₁) and the highest dose that produces a maximal therapeutic response (e.g., ED₉₉). A ratio less than unity would indicate that a dose effective in 99% of the population will be lethal in more than 1% of the individuals taking that dose.

Figure 2.2 indicates that Phenobarbital’s ratio LD₁/ED₉₉ is approximately 2.

Protective Index

The margin of safety is only one of several criteria to be used in determining a drug’s clinical merit. Clearly, the therapeutic index is a very rough measure of safety and generally represents only the starting point in determin- ing whether a drug is safe enough for human use.

Usually, undesirable side effects occur in doses lower than the lethal doses. For example, phenobarbital in- duces drowsiness and an associated temporary neuro- logical impairment. Since anticonvulsant drugs are intended to allow people with epilepsy to live normal

ED₅₀

1 2

20 40 60

Animals Protected (%)

80 100

3

Phenobarbital (mg/kg)

LD₅₀

10 20 30 50 100

20 40 60

Animals Killed (%)

80 100

Phenobarbital (mg/kg)

A B

5 7 10

F I G U R E 2 . 2

Quantal dose–response curves based on all-or-none responses. A. Relationship between the dose of phenobarbital and the protection of groups of rats against convulsions. B. Relationship between the dose of phenobarbital and the drug’s lethal effects in groups of rats. ED50, effective dose, 50%; LD50, lethal dose, 50%.