Clinical Trials: Study Design, Endpoints and Biomarkers, Drug Safety, and FDA and ICH Guidelines

638 Pages·2011·6.96 MB·English

by  Tom Brody PhD University of California at Berkeley

1
CHAPTER
The Origins of Drugs
I. INTRODUCTION
Drugs have a number of origins, as outlined by the bullet points:
Natural products, for example, chemicals from plants and microorganisms
l
Analogues of naturally occurring chemicals, where these chemicals reside in the
l
biosynthetic pathways of mammals
Antibodies that bind to naturally occurring targets in the body
l
Discovery that an existing drug, established as effective for a first disease, is also
l
effective for treating an unrelated second disease
Drugs identified by screening libraries of chemicals.
l
Some drugs are based on natural products, where the natural products were known
to have pharmacological effects. The term “natural products” is a term of the art that
generally refers to chemicals derived from plants, fungi, or microorganisms. Drugs that
are derived from natural products, or that actually are natural products, include war-
farin (1) penicillin (2,3) cyclosporin (4) aspirin (5,6) paclitaxel (7) fingolimod (8) and
reserpine (9). Many other drugs have structures based on chemicals that occur naturally
in the human body, that is, where the drugs are analogues of these chemicals. These
include analogues of intermediates or final products of biosynthetic pathways. Drugs
that are analogues of chemicals in biosynthetic pathways include methotrexate, cladrib-
ine, and ribavirin.
Still other drugs originated by first identifying a target cell, or target protein, and
then by preparing antibodies that bind to that target. Once a target protein is iden-
tified, this target protein (or a derivative of it) can be used as a vaccine. Moreover,
1 Wardrop D, Keeling D. The story of the discovery of heparin and warfarin. Br J Haematol. 2008;141:757–763.
2 Diggins FW. The true history of the discovery of penicillin, with refutation of the misinformation in the literature. Br
J Biomed Sci. 1999;56:83–93.
3 Fleming A. On the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation
of B. influenzae. 1929. Bull World Health Organ. 2001;79:780–790.
4 Heusler K, Pletscher A. The controversial early history of cyclosporin. Swiss Med Wkly. 2001;131:299–302.
5 Lafont O. From the willow to aspirin. Rev Hist Pharm. (Paris). 2007;55:209–216.
6 Mahdi JG, Mahdi AJ, Mahdi AJ, Bowen ID. The historical analysis of aspirin discovery, its relation to the willow tree
and antiproliferative and anticancer potential. Cell Prolif. 2006;39:147–155.
7 Socinski MA. Single-agent paclitaxel in the treatment of advanced non-small cell lung cancer. Oncologist.
1999;4:408–416.
8 Adachi K, Chiba K. FTY720 story. Its discovery and the following accelerated development of sphingosine
1-phosphate receptor agonists as immunomodulators based on reverse pharmacology. Perspect Medicin Chem.
2007;1:11–23.
9 Rao EV. Drug discovery from plants. Curr Science. 2007;93:1060.
Clinical Trials © 22001122 Elsevier Inc.
DOI: 10.1016/B978-0-12-391911-3.00001-3 All rights reserved. 1
2 Clinical Trials
drugs are also derived by using a screening assay and by testing hundreds or thou-
sands of purified candidate compounds using that assay. Where the screening method
is automated, the method is called high-throughput screening. The screening assay may
consist of tumor cells that are cultured in vitro, where a robot determines if the candi-
date drug inhibits a particular enzyme in the tumor cell, or if the candidate drug kills
the tumor cell.
II. STRUCTURES OF DRUGS
Knowledge of drug structure is important to the investigator and to clinical trial
personnel for a number of reasons. First, the issue of whether a drug is hydrophobic or
hydrophilic will dictate the excipient that needs to be used. The structure can also pro-
vide an idea of stability during long-term storage and, for example, if the drug is sensi-
tive to light. Second, the structure may dictate the route of administration, and enable
a prediction of pharmacokinetics of the drug and pathways of metabolism, transport,
and excretion. Third, the structure of the drug, and more particularly the class of
compound, can help the investigator predict adverse events that might be expected
from the drug. Fourth, FDA-submissions, such as the Investigational New Drug and
Investigator’s Brochure, typically contain a picture of the drug structure.
a. Origins of warfarin
Warfarin is a drug that is widely used to prevent blood clotting, for example in people
at risk of heart attacks or strokes (10). A natural product produced during the spoiling
of sweet clover inspired warfarin’s design. The drug was not named after any kind of
warfare, even though it is used in warfare against mice and rats. It was named after the
Wisconsin Alumni Research Foundation.
Spoiled sweet clover contains coumarin, a compound that inhibits an enzyme in
the liver, where the end-result is impaired blood clotting. Blood clotting factors are
biosynthesized in the liver, and then released into the bloodstream. Farmers in the
mid-west found that cattle bled to death during the process of de-horning, where the
cattle had eaten spoiled sweet clover. Eventually, one particular farmer in Wisconsin
brought a bucket of unclotted blood to researchers at the University of Wisconsin. The
researchers examined blood, as well as samples of spoiled sweet clover, and discovered
that the culprit was dicoumarol, a degradative product of coumarin. Researchers syn-
thesized and tested about 50 analogues of this compound. The analogues were tested in
10 Gage BF, van Walraven C, Pearce L, et al. Selecting patients with atrial fibrillation for anticoagulation: stroke risk
stratification in patients taking aspirin. Circulation. 2004;110:2287–2292.
The Origins of Drugs 3
rabbits. It was discovered that the best analogue was warfarin (11). Warfarin is also the
active ingredient in rodent poison.
O O
OH O
Warfarin
b. Origins of methotrexate and 5-fluorouracil
The natural substrate of one particular enzyme, dihydrofolate reductase, inspired the
design of methotrexate. This natural substrate is dihydrofolic acid (12). Dihydrofolic acid
is the end-product of the biosynthetic pathway of folates (13). Anti-cancer drugs that
inhibit dihydrofolate reductase were designed by synthesizing and screening chemi-
cals that resembled dihydrofolate (14,15,16). Methotrexate, which is an analogue of
dihydrofolic acid and also an analogue of folic acid, inhibits dihydrofolic acid reduc-
tase. Another anti-folate drug used in oncology is 5-fluorouracil. Fluorouracil was
invented by Charles Heidelberger (17,18). The drug was developed on the basis of
findings in the 1950s that cancer cells incorporated a larger amount of the uracil base
into the DNA than normal cells. In testing a number of halogen substituted uracils,
5-fluorouracil appeared to be the most active and promising drug. Fluorouracil is a
suicide inhibitor of thymidylate synthase. This means that the enzyme’s own catalytic
activity results in the activation of the drug, where this activation causes the drug to
react covalently with the enzyme, thereby destroying the enzyme’s catalytic activity.
11 Link KP. The discovery of dicumarol and its sequels. Circulation. 1959;19:97–107.
12 Folic acid is used as a vitamin supplement and for enzymatic studies of dihydrofolic acid reductase. But folic acid is
not a naturally occurring chemical. Folic acid is formed during the breakdown of dihydrofolic acid, upon exposure
to oxygen. Dihydrofolic acid is a natural product made by microorganisms and plants.
13 Brown GM, Williamson JM. Biosynthesis of riboflavin, folic acid, thiamine, and pantothenic acid. Adv Enzymol Relat
Areas Mol Biol. 1982;53:345–381.
14 Friedkin M. Enzymatic aspects of folic acid. Annu Rev Biochem. 1963;32:185–214.
15 Bertino JR. The mechanism of action of folate antagonists in man. Cancer Res. 1963;23:1286–1306.
16 Brody T. Folic acid, In: Machlin LJ, ed. Handbook of Vitamins Marcel Dekker, Inc. New York, 1990; pp. 453–489.
17 Muggia FM, Peters GJ, Landolph JR Jr. XIII International Charles Heidelberger Symposium and 50 Years of
Fluoropyrimidines in Cancer Therapy held on September 6 to 8, 2007 at New York University Cancer Institute,
Smilow Conference Center. Mol Cancer Ther. 2009;8:992–999.
18 Heidelberger C. On the rational development of a new drug: the example of the fluorinated pyrimidines. Cancer
Treat Rep. 1981;65 (Suppl 3):3–9.
4 Clinical Trials
H N N N
2
N N
N O
H
NH2 N
OH
O
O OH
Methotrexate
c. Origins of ribavirin
Ribavirin was discovered by synthesizing analogues of compounds participating in the
pathways of nucleotide biosynthesis. In designing, synthesizing, and testing a variety
of analogues of intermediates in nucleotide biosynthetic pathways, the result was the
discovery of ribavirin, also known as virazole (19,20). Ribavirin is the standard of care
used for treating hepatitis C virus (HCV) infections.
O
NH
N 2
N
HO N
O
OH OH
Ribavirin
d. Origins of paclitaxel
Paclitaxel (Taxol®), an anti-cancer drug, was discovered in extracts of the Pacific yew tree,
Taxus brevifolia. In 1963, a crude extract from Pacific yew bark was found to have activity
against tumors in experimental animals (21). In 1991, the active component, paclitaxel,
was approved by the FDA as an anti-cancer drug. Paclitaxel, which is in a class of drugs
19 Witkowski JT, Robins RK, Sidwell RW, Simon LN. Design, synthesis, and broad spectrum antiviral activity of
1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide and related nucleosides. J Med Chem. 1972;15:1150–1154.
20 Te HS, Randall G, Jensen DM. Mechanism of action of ribavirin in the treatment of chronic hepatitis C. Gastroenterol
Hepatol. 2007;3:218–225.
21 Socinski MA. Single-agent paclitaxel in the treatment of advanced non-small cell lung cancer. Oncologist.
1999;4:408–416.
The Origins of Drugs 5
called taxanes, acts on the cytoskeleton of the cell. Specifically, the drug acts on tubulin,
disrupts the normal behavior of the cytoskeleton in mediating cell division, and causes cell
death (22). Docetaxel (Taxotere®) is a semi-synthetic analogue of paclitaxel (23) having a
mechanism and anti-cancer properties similar to those of paclitaxel. Docetaxel can be syn-
thesized using a precursor extracted from needles of the European yew, Taxus baccata (24).
O
O OH
H C O
2
O CH2
H H C
2
N CH
O 2
O
H C
2
O OH O H O CH
2
OH O
O
Paclitaxel
e. Origins of cladribine
Cladribine (2-chloro-2-deoxyadenosine) is a small molecule that is a nucleotide ana-
logue. Cladribine is an analogue of deoxyadenosine. After administration, cladribine
enters various cells and once inside the cell, an enzyme catalyzes the attachment of
three phosphate groups. The result is the conversion of cladribine to cladribine tri-
phosphate. Cladribine triphosphate, in turn, inhibits DNA synthesis, inhibits DNA
repair, and results in apoptosis (death of the cell). The drug is most active in cells with
high levels of the deoxycytidine kinase, such as lymphocytes (25). Cladribine is used
for treating multiple sclerosis and a type of leukemia (hairy cell leukemia).
The connection between deoxynucleotides and killing lymphocytes, as it applies to
cladribine, is as follows. Inherited deficiencies of the enzyme adenosine deaminase inter-
fere with lymphocyte development while sparing most other organ systems (26). The
22 Pusztai L. Markers predicting clinical benefit in breast cancer from microtubule-targeting agents. Ann Oncol. 2007;18
(Suppl 12):xii,15–20.
23 Bissery MC, Guénard D, Guéritte-Voegelein F, Lavelle F. Experimental antitumor activity of taxotere (RP 56976,
NSC 628503), a taxol analogue. Cancer Res. 1991;51:4845–4852.
24 Verweij J. Docetaxel (Taxotere): a new anti-cancer drug with promising potential? Br J Cancer. 1994;70:183–184.
25 Piro LD, Carrera CJ, Beutler E, Carson DA. 2-Chlorodeoxyadenosine: an effective new agent for the treatment of
chronic lymphocytic leukemia. Blood. 1988;72:1069–1073.
26 Carson DA, Kaye J, Seegmiller JE. Lymphospecific toxicity in adenosine deaminase deficiency and purine nucleoside
phosphorylase deficiency: possible role of nucleoside kinase(s). Proc Natl Acad Sci USA. 1977;74:5677–5681.
6 Clinical Trials
accumulation of deoxyadenosine nucleotides in the lymphocytes, that is, in lymphocytes
of people suffering from adenosine deaminase deficiency, reduces the number of lympho-
cytes. As a consequence, the patients suffer from severe immunodeficiency.
Carson et al. (27) realized that the elimination of adenosine deamidase activity
can halt lymphocytes that are pathological, such as the lymphocytes in leukemia (leu-
kemia is a cancer of lymphocytes). This elimination was accomplished by cladribine.
Cladribine, in effect, mimicks the inherited disease (adenosine deaminase deficiency)
because cladribine resists the effects of adenosine deaminase. Cladribine naturally
resists deamination catalyzed by adenosine deaminase. (For cladribine to be effective
in destroying lymphocytes, it is not necessary that patients be suffering from adenosine
deaminase deficiency.) Just as the normally occurring deoxyadenosine kills lympho-
cytes in people with the genetic disease of adenosine deaminase deficiency, cladribine
kills lymphocytes when administered to normal humans (28). It was about ten years
after the use of cladribine to treat leukemia that cladribine was first used to treat mul-
tiple sclerosis (29,30).
To summarize, the pathway of discovery of cladribine for multiple sclerosis was as
follows. First, it was known that an inherited genetic disease involved the accumula-
tion of deoxyadenosine nucleotides in the cell, and resulted in death of lymphocytes.
Second, researchers developed a drug that, when administered to a human subject,
mimicked the effects of this disease (due to the inability of adenosine deaminase to act
on the drug). Third, the drug was used to treat leukemia. Fourth, the drug was used to
treat multiple sclerosis (31).
NH
2
N
N
HO N N CI
O
OH H
27 Carson DA, Wasson DB, Taetle R, Yu A. Specific toxicity of 2-chlorodeoxyadenosine toward resting and proliferating
human lymphocytes. Blood. 1983;62:737–743.
28 Piro LD, Carrera CJ, Beutler E, Carson DA. 2-Chlorodeoxyadenosine: an effective new agent for the treatment of
chronic lymphocytic leukemia. Blood. 1988;72:1069–1073.
29 Sipe JC, Romine JS, Koziol JA, McMillan R, Zyroff J, Beutler E. Cladribine in treatment of chronic progressive
multiple sclerosis. Lancet. 1994;344:9–13.
30 Beutler E, Koziol JA, McMillan R, Sipe JC, Romine JS, Carrera CJ. Marrow suppression produced by repeated doses
of cladribine. Acta Haematol. 1994;91:10–15.
31 Giovannoni G, Comi G, Cook S, et al. A placebo-controlled trial of oral cladribine for relapsing multiple sclerosis.
New Engl J Med. 2010;362:416–426.
The Origins of Drugs 7
f. Origins of drugs in high-throughput screening
A number of drugs and drug candidates were discovered by high-throughput screening.
Wigle et al. (32) describe antibiotics that were found by high-throughput screening. White
et al. (33) describe drugs for treating inflammatory diseases that were discovered by high-
throughput screening. Von Hoff et al. (34) and others (35) describe a drug used for treating
cancer that was identified by high-throughput screening.
g. Origins of therapeutic antibodies
Antibodies designed with the aid of animal models are used for treating vari-
ous cancers and immune diseases. For example, antibody drugs include trastuzumab
(Herceptin®) (36) which binds to epidermal growth factor, and which is used to treat
breast cancer. Antibody drugs also include bevacizumab (Avastin®) (37) which binds
to vascular endothelial growth factor receptor (VEGF), and is used to treat a variety of
cancers. Moreover, an antibody drug used to treat various immune diseases is natali-
zumab (Tysabri®) (38). This antibody binds to a protein called integrin, which occurs
on the surface of white blood cells. Natalizumab is used to treat two immune diseases,
namely, multiple sclerosis and Crohn’s disease.
Developing antibody drugs includes the step of refining the polypeptide sequence
of the antibody into a drug suitable for administering to humans (39,40,41). This
refinement step is called humanization (42). Humanization refers to the process of
using genetic engineering to convert any protein of animal origin, to a protein that
can be injected into people, where the injected protein fails to elicit an immune reac-
tion against itself.
32 Wigle TJ, Sexton JZ, Gromova AV, et al. Inhibitors of RecA activity discovered by high-throughput screening: cell-
permeable small molecules attenuate the SOS response in Escherichia coli. J Biomol Screen. 2009;14:1092–1101.
33 White JR, Lee JM, Young PR, et al. Identification of a potent, selective non-peptide CXCR2 antagonist that inhibits
interleukin-8-induced neutrophil migration. J Biol Chem. 1998;273:10095–10098.
34 Von Hoff DD, LoRusso PM, Rudin CM, et al. Inhibition of the hedgehog pathway in advanced basal-cell carcinoma.
New Engl J Med. 2009;361:1164–1172.
35 Zhou BB, Zhang H, Damelin M, Geles KG, Grindley JC, Dirks PB. Tumour-initiating cells: challenges and
opportunities for anticancer drug discovery. Nat Rev Drug Discov. 2009;8:806–823.
36 Verma S, Lavasani S, Mackey J, et al. Optimizing the management of her2-positive early breast cancer: the clinical
reality. Curr Oncol. 2010;17:20–33.
37 Eskens FA, Sleijfer S. The use of bevacizumab in colorectal, lung, breast, renal and ovarian cancer: where does it fit?
Eur J Cancer. 2008;44:2350–2356.
38 Coyle PK. The role of natalizumab in the treatment of multiple sclerosis. Am J Manag Care. 2010;
16(Suppl 6):S164–S170.
39 Kent SJ, Karlik SJ, Cannon C, et al. A monoclonal antibody to alpha 4 integrin suppresses and reverses active
experimental allergic encephalomyelitis. J Neuroimmunol. 1995;58:1–10.
40 Yednock TA, Cannon C, Fritz LC, et al. Prevention of experimental autoimmune encephalomyelitis by antibodies
against alpha 4 beta 1 integrin. Nature. 1992;356:63–66.
41 Brody T. Multistep denaturation and hierarchy of disulfide bond cleavage of a monoclonal antibody. Analyt Biochem.
1997;247:247–256.
42 Presta LG. Molecular engineering and design of therapeutic antibodies. Curr Opin Immunol. 2008;20:460–470.
8 Clinical Trials
Antibodies take the form of four polypeptides, two light chains and two heavy
chains, as indicated in the diagram below. The first light chain and first heavy chain
are covalently attached to each other by disulfide bonds, to form a first complex. The
second light chain and second heavy chain are covalently attached to each by disul-
fide bonds to form a second complex. The first complex and second complex are also
covalently attached to each other by way of disulfide bonds.
Light chain
Heavy chain
Heavy chain
Light chain
As an example of an antibody drug, the amino acid sequence of the light chain and
the amino acid sequence of the heavy chain of trastuzumab are shown below (43).
The amino acid sequence of the light chain of trastuzumab, as found at the cited
accession numbers (44,45) is shown below. The light chain, shown below, has 214
amino acids.
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFT
LTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
The amino acid sequence of the heavy chain of this antibody, which has 451 amino acids
and can be found at the cited accession number (46) is shown below.
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISA
DTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD
ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK
43 Fong S, Hu Z. T herapeutic anti-HER2 antibody fusion polypeptides. U.S Pat Appl Publ. 2009/0226466. 2009;Sept. 10.
44 Cho HS, Mason K, Ramyar KX, et al. GenBank Accession No. PDB:1N8Z_A (submitted November 21, 2002).
45 http://www.drugbank.ca/drugs/DB00072
46 Trastuzumab (DB00072) DrugBank Accession No. DB0072. Creation date June 13, 2005, updated June 2, 2009.
The Origins of Drugs 9
The three-dimensional structure of this antibody drug can be found at www.drugbank.
ca/drugs/DB00072
Let us dwell on the structure of the light chain and heavy chain for a moment. In
testing and marketing any polypeptide drug, pharmaceutical companies are concerned
with the following drug stability issues. First, it is the case that long-term storage of
polypeptides results in the spontaneous deamination of residues of glutamine (Q) and
asparagine (N). Deamination can occur at various steps in the manufacturing process,
during shipment, and during storage. Also, oxidation of cysteine (C) residues can occur
during manufacturing, shipping, and storage. These types of damage may lower the
potency of polypeptide drugs. The reader will be able to find the locations of Q, N,
and C in the polypeptide chains of trastuzumab.
III. THE 20 CLASSICAL AMINO ACIDS
The following reviews the 20 classical amino acids. Twenty classical amino acids exist,
and these are listed, along with their abbreviations, in Table 1.1. The non-classical amino
acids include homocysteine, selenocysteine (47) methionine sulfoxide, ornithine,
gamma-carboxyglutamate (GLA) (48) phosphotyrosine, hydroxyproline (49) sarcosine,
and betaine. A protein is a long polypeptide that is a linear polymer of amino acids,
typically about 100 to 500 amino acids in length. The term oligopeptide refers to shorter
polymers of amino acids in the range of about ten to 50 amino acids. Some non-
classical amino acids, such as homocysteine, exist only in the free state, and do not
become incorporated into any polypeptide. But other non-classical amino acids, such
as gamma-carboxyglutamate and phosphotyrosine, occur in naturally occurring pro-
teins because of a process called post-translational modification.
Knowledge of the amino acids is needed to understand the following pharmaco-
logical issues:
Stability during manufacturing and storage
l
Point of attachment of polyethylene glycol (PEG)
l
Unwanted immunogenicity
l
Immunogenicity that is desired and essential for drug efficacy.
l
The following concerns in vitro stability. For drugs that are oligopeptides or pro-
teins, stability during manufacturing and storage is an issue because of spontaneous
deamidation. Aswad and co-workers have detailed the deamidation of biologicals
47 Brody T. Nutritional Biochemistry, 2nd ed. San Diego, CA: Academic Press. 1999;21, 825–827.
48 Brody T, Suttie JW. Evidence for the glycoprotein nature of vitamin K-dependent carboxylase from rat liver. Biochim
Biophys Acta. 1987;923:1–7.
49 Brody,T. Nutritional Biochemistry, 2nd ed. San Diego, CA: Academic Press. 1999;21, 619–623.