Clinical trials study design, endpoints and biomarkers, drug safety

Phase I clinical trials begin by administering a small dose of the study drug to a group of three or more patients.. Moreover, a 3-arm clinical trial can include a study drug group, plac

Drugs have a number of origins, as outlined by the bullet points:

l Natural products, for example, chemicals from plants and microorganisms

l Analogues of naturally occurring chemicals, where these chemicals reside in the biosynthetic pathways of mammals

l 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 effective for treating an unrelated second disease

l Drugs identified by screening libraries of chemicals

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.

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.

rabbits It was discovered that the best analogue was warfarin (11) Warfarin is also the active ingredient in rodent poison.

O OH

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.

OH OH O

O N N N

NH2

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

21 Socinski MA Single-agent paclitaxel in the treatment of advanced non-small cell lung cancer Oncologist

1999;4:408–416.

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.

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

O O

O

O

H

H OH

O O

O O

Paclitaxel

e Origins of cladribine

Cladribine (2-chloro-2-deoxyadenosine) is a small molecule that is a nucleotide 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)

ana-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.

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accumulation of deoxyadenosine nucleotides in the lymphocytes, that is, in lymphocytes

of people suffering from adenosine deaminase deficiency, reduces the number of cytes As a consequence, the patients suffer from severe immunodeficiency

lympho-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)

H OH

N N

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.

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 throughput screening Von Hoff et al (34) and others (35) describe a drug used for treating cancer that was identified by high-throughput screening

high-g Origins of therapeutic antibodies

Antibodies designed with the aid of animal models are used for treating 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

vari-to vascular endothelial growth facvari-tor recepvari-tor (VEGF), and is used vari-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.

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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

Light chain Heavy chain

Heavy 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 Therapeutic 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 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 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

non-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 logical issues:

pharmaco-l 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

The following concerns in vitro stability For drugs that are oligopeptides or teins, stability during manufacturing and storage is an issue because of spontaneous deamidation Aswad and co-workers have detailed the deamidation of biologicals

pro-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.

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(50,51) The following concerns in vivo stability Stability of proteins can also be promised by degradation catalyzed by contaminating proteases and by the related issue of aggregation (52,53,54) Polyethylene glycol can be connected to recombinant enzymes and cytokines to enhance stability and lifetime of the drug in the blood-stream Polypeptide drugs that are modified in this way are called pegylated polypeptides

com-Table 1.1 The 20 classical amino acids

Amino acid 1-letter abbreviation 3-letter abbreviation Amino acids with charged side group

52 Simpson RJ Stabilization of proteins for storage Cold Spring Harb Protocol 2010 May (5).

53 O’Fágáin C Storage and lyophilisation of pure proteins Methods Mol Biol 2011;681:179–202.

54 Chi EY, Krishnan S, Randolph TW, Carpenter JF Physical stability of proteins in aqueous solution: mechanism and

driving forces in nonnative protein aggregation Pharm Res 2003;20:1325–1336.

50 Paranandi MV, Guzzetta AW, Hancock WS, Aswad DW Deamidation and isoaspartate formation during in vitro

aging of recombinant tissue plasminogen activator J Biol Chem 1994;269:243–253.

51 Aswad DW, Paranandi MV, Schurter BT Isoaspartate in peptides and proteins: formation, significance, and analysis.

J Pharm Biomed Anal 2000;21:1129–1136.

(55,56,57) where the polyethylene glycol can be attached to residues of lysine or nine (58,59,60).

argi-The following concerns immunogenicity For drugs that are antibodies, enzymes, cytokines, and hormones, what is desired is that it is not immunogenic In other words, what is desired for these particular drugs is that the drug does not provoke an immune response against the drug itself But in striking contrast, for drugs that are vaccines, the purpose of the drug is to stimulate immune response against the drug itself, and (hope-fully) also to stimulate an immune response against the infecting agent or tumor that contains a similar or identical polypeptide sequence Knowledge of the amino acids is needed for understanding all of the above issues

IV ANIMAL MODELS

a Introduction

Regulatory approval for drugs requires data on safety and efficacy in animals While data on safety can be acquired from studies on mice, rats, rabbits, dogs, and primates, data on efficacy can only be acquired where there is an available animal model of the disorder in question Where the goal of the drug is to enhance wound healing, it is easy

to find a suitable animal model (all that is needed is to surgically remove a circle of skin from the animal) However, where the goal of the drug is to treat diseases such as can-cer, immune diseases, or infections, there is a need to find an appropriate animal model.While rodents spontaneously develop various cancers, it is not practical to acquire

a group of rodents with the same type of cancer, and at the same stage of the cancer, at the same time The array of tumors that occur spontaneously in rats has been exhaus-tively documented (61,62)

Separate animal models are available for various types of cancer Marks (63) identified sources of various animal cancer models Nandan and Yang (64) Kuperwasser

61 Son WC, Bell D, Taylor I, Mowat V Profile of early occurring spontaneous tumors in Han Wistar rats Toxicol Pathol

56 Hershfield MS, Roberts LJ 2nd, Ganson NJ, et al Treating gout with pegloticase, a PEGylated urate oxidase, provides

insight into the importance of uric acid as an antioxidant in vivo Proc Natl Acad Sci USA 2010;107:14351–14356.

57 Fishburn CS The pharmacology of PEGylation: balancing PD with PK to generate novel therapeutics J Pharm Sci

2008;97:4167–4183.

58 Lee H, Park TG Preparation and characterization of mono-PEGylated epidermal growth factor: evaluation of in

vitro biologic activity Pharm Res 2002;19:845–851.

59 Veronese F, Sartore L, Orsolini P, Deghenghi R U.S Pat No 5,286,637.

60 Gauthier MA, Klok HA Arginine-specific modification of proteins with polyethylene glycol Biomacromolecules

2011;12:482–493.

63 Marks C Mouse Models of Human Cancers Consortium (MMHCC) from the NCI Dis Model Mech 2009;2:111.

64 Nandan MO, Yang VW Genetic and chemical models of colorectal cancer in mice Curr Colorectal Cancer Rep

2010;6:51–59.

Mycobacterium leprae, the cause of leprosy (Hansen’s disease) A suitable small-animal

model for hepatitis C infections has not yet been found

In proposing and using animal models for disorders that have an immune nent, it is useful to be aware of the similarities (and differences) between the mouse immune system and the human immune system Mestas and Hughes (71) outline some

compo-of these similarities and differences Researchers conducting animal models on eases with an immune component, that is, cancer, autoimmune diseases, and inflamma-tory disorders, need to be aware of similarities and differences in the immune systems between their animal and humans For example, comparisons between mouse and human dendritic cells (72) T cells (73) B cells (74) NK cells (75) macrophages (76) eosinophils (77) and toll-like receptors (TLRs) (78) are available

dis-65 Kuperwasser C, Dessain S, Bierbaum BE, et al A mouse model of human breast cancer metastasis to human bone

Cancer Res 2005;65:6130–6138.

66 Soda M, Takada S, Takeuchi K, et al A mouse model for EML4-ALK-positive lung cancer Proc Natl Acad Sci USA

2008;105:19893–19897.

67 Noonan FP, Dudek J, Merlino G, De Fabo EC Animal models of melanoma: an HGF/SF transgenic mouse model

may facilitate experimental access to UV initiating events Pigment Cell Res 2003;16:16–25.

68 Menne S, Cote PJ The woodchuck as an animal model for pathogenesis and therapy of chronic hepatitis B virus

infection World J Gastroenterol 2007;13:104–124.

69 Bukh J, Thimme R, Meunier JC, et al Previously infected chimpanzees are not consistently protected against

reinfection or persistent infection after reexposure to the identical hepatitis C virus strain J Virol 2008;82:8183–8195.

70 Hamilton HK, Levis WR, Martiniuk F, Cabrera A, Wolf J The role of the armadillo and sooty mangabey monkey in

human leprosy Int J Dermatol 2008;47:545–550.

71 Mestas J, Hughes CC Of mice and not men: differences between mouse and human immunology J Immunol

2004;172:2731–2738.

72 Boonstra A, Asselin-Paturel C, Gilliet M, et al Flexibility of mouse classical and plasmacytoid-derived dendritic cells

in directing T helper type 1 and 2 cell development: dependency on antigen dose and differential toll-like receptor

ligation J Exp Med 2003;197:101–109.

73 Walzer T, Arpin C, Beloeil L, Marvel J Differential in vivo persistence of two subsets of memory phenotype CD8

T cells defined by CD44 and CD122 expression levels J Immunol 2002;168:2704–2711.

74 Frasca D, Landin AM, Riley RL, Blomberg BB Mechanisms for decreased function of B cells in aged mice and

humans J Immunol 2008;180:2741–2746.

75 Ehrlich LI, Ogasawara K, Hamerman JA, et al Engagement of NKG2D by cognate ligand or antibody alone is

insufficient to mediate costimulation of human and mouse CD8 T cells J Immunol 2005;174:1922–1931.

76 Sunderkötter C, Nikolic T, Dillon MJ, et al Subpopulations of mouse blood monocytes differ in maturation stage

and inflammatory response J Immunol 2004;172:4410–4417.

77 Dyer KD, Percopo CM, Xie Z, Yang Z, et al Mouse and human eosinophils degranulate in response to activating factor (PAF) and lysoPAF via a PAF-receptor-independent mechanism: evidence for a novel receptor

platelet-J Immunol 2010;184:6327–6334.

78 Janssens S, Beyaert R Role of Toll-like receptors in pathogen recognition Clin Microbiol Rev 2003;16:637–646.

Animal models for immune disorders include the following A rodent model for multiple sclerosis, called experimental autoimmune encephalomyelitis (EAE) (79) involves injecting animals with myelin basic protein (80) A mouse model for rheuma-toid arthritis involves injecting collagen (81) A mouse model for inflammatory bowel disease employs a strain of mice that naturally develops inflammation of the intestinal tract (82) Psoriasis (83) lupus (84) asthma (85) and other immune disorders also have well-characterized animal models.

b Estimating human dose from animal studies

The most appropriate dose of a drug for humans can be derived from animal studies The FDA provides guidance on converting effective doses from animal studies, to cor-responding doses that are likely to be effective in human subjects (86) Two approaches with animals are in common use The first is to arrive at the highest drug dose that is not toxic This approach is commonly used for small molecule drugs for cancer The second is to arrive at the dose of a drug that is optimally effective, as determined by tests sensitive to efficacy With the information of the dose in hand, investigators then scale up the dose derived from animal studies, and then calculate a dose for first use in humans Lowe et al (87) Reigner and Blesch (88) Contrera et al (89) and Sharma and McNeill (90) review methods for using animal studies to arrive at doses for humans These methods include methods based on body surface area, and methods based on

81 Cho YG, Cho ML, Min SY, Kim HY Type II collagen autoimmunity in a mouse model of human rheumatoid

arthritis Autoimmun Rev 2007;7:65–70.

82 Wilk JN, Bilsborough J, Viney JL The mdr1a/ mouse model of spontaneous colitis: a relevant and appropriate

animal model to study inflammatory bowel disease Immunol Res 2005;31:151–159.

83 Schön MP Animal models of psoriasis: a critical appraisal Exp Dermatol 2008;17:703–912.

84 Cohen PL, Maldonado MA Animal models for SLE Curr Protoc Immunol 2003;February Chapter 15:Unit 15.20.

85 Takeda K, Gelfand EW Mouse models of allergic diseases Curr Opin Immunol 2009;21:660–665.

86 U.S Dept Health and Human Services Food and Drug Administration Guidance for Industry Estimating the safe

starting dose in clinical trials for therapeutics in adult healthy volunteers U.S Dept of Health and Human Services, Food and Drug Administration 2002;24 pages.

79 Mix E, Meyer-Rienecker H, Zettl UK Animal models of multiple sclerosis for the development and validation of

novel therapies – potential and limitations J Neurol 2008;255(Suppl 6):7–14.

80 Zaller DM, Osman G, Kanagawa O, Hood L Prevention and treatment of murine experimental allergic

encephalomyelitis with T cell receptor V beta-specific antibodies J Exp Med 1990;171:1943–1955.

87 Lowe PJ, Hijazi Y, Luttringer O, Yin H, Sarangapani R, Howard D On the anticipation of the human dose in first-in-man

trials from preclinical and prior clinical information in early drug development Xenobiotica 2007;37:1331–1354.

88 Reigner BG, Blesch KS Estimating the starting dose for entry into humans: principles and practice Eur J Clin

Pharmacol 2002;57:835–845.

89 Contrera JF, Matthews EJ, Kruhlak NL, Benz RD Estimating the safe starting dose in phase I clinical trials and no

observed effect level based on QSAR modeling of the human maximum recommended daily dose Regul Toxicol

Pharmacol 2004;40:185–206.

90 Sharma V, McNeill JH To scale or not to scale: the principles of dose extrapolation Br J Pharmacol

2009;157:907–921.

per-by an appropriate safety factor, for example per-by reducing the dose per-by a factor of ten According to FDA’s Guidance for Industry, NOAEL is determined as follows For selecting a starting dose, the following is used, namely, the highest dose level that does not produce a significant increase in adverse effects in comparison to the control group.

2 MABEL approach

Another approach for arriving at a suitable dose for humans is use of the Minimal Anticipated Biological Effect Level (MABEL) approach (93) The MABEL approach provides the dose level leading to a biological effect of interest The biological effect of interest can be saturation of a drug transport mechanism, stimulation of a cell signaling pathway, or activation of a cell Calculating MABEL makes use of in vitro and in vivo information available from PK data and PD data

The concentrations of drug which need to be achieved in the bloodstream of a patient during actual treatment can be estimated by studies with cultured human and animal cells According to guidance from the EMEA (94) in vitro data with cultured

cells can be used to determine, “target binding and receptor occupancy studies in vitro

in target cells from human and the relevant animal species … concentration–response

curves in vitro in target cells from human and the relevant animal species and dose/ exposure–response in vivo in the relevant animal species.”

c Scaling up the drug dose, acquired from animal studies, for use in humans

When an appropriate dose is found from animal studies, that is, by using the NOAEL approach or MABEL approach, an appropriate first dose for use in humans can be cal-culated using body surface area measurements and by incorporating a safety factor For

91 Sawyer M, Ratain MJ Body surface area as a determinant of pharmacokinetics and drug dosing Invest New Drugs

2001;19:171–177.

92 Kouno T, Katsumata N, Mukai H, Ando M, Watanabe T Standardization of the body surface area (BSA) formula to

calculate the dose of anticancer agents in Japan Jpn J Clin Oncol 2003;33:309–313.

93 Milton MN, Horvath CJ The EMEA guideline on first-in-human clinical trials and its impact on pharmaceutical

development Toxicol Pathol 2009;37:363–371.

94 European Medicines Agency (EMEA) Guideline on strategies to identify and mitigate risks for first-in-human clinical trials with investigational medicinal products (July 2007).

any given patient, for example, in a clinical study or during ordinary every-day clinical practice, the recommended drug dose may be expressed in terms of body surface area Hence, where toxicology studies or efficacy studies with animals result in an appropri-ate dose, and where researchers have expressed this dose in terms of body surface area, the same dose may be appropriate for humans The FDA provides a conversion table, for changing a dose found appropriate for animals to a corresponding dose for humans, where this conversion is based on body surface area (95) The FDA’s conversion table also includes a factor of 0.1, where the dose arrived at by the calculation is multi-plied by 0.1, in order to ensure that the dose in humans will not be toxic (96) The resulting dose, as found with the FDA’s table, is expected to be the dose that results

in no observed adverse effect, where higher doses or concentrations would result in

an adverse effect The table provides separate conversion factors, for converting animal doses to human doses, for the mouse, rat, rabbit, dog, monkey, and pig After the inves-tigator applies the scaling factor, the resulting number is called the human equivalent dose (HED) (97) The species that generates the lowest HED is called the most sensi-tive species After arriving at the HED, the HED is further modified by applying a safety factor Thus, according to FDA’s Guidance for Industry (98) “[a] safety factor should then be applied to the HED to increase assurance that the first dose in humans will not cause adverse effects.”

95 U.S Department of Health and Human Services, Food and Drug Administration Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers (July 2005).

96 Ochoa R, Rousseaux C The role of the toxicologic pathologist in risk management Toxicol Pathol

2009;37:705–707.

97 U.S Dept Health and Human Services Food and Drug Administration Guidance for Industry Estimating the safe

starting dose in clinical trials for therapeutics in adult healthy volunteers U.S Dept of Health and Human Services, Food and Drug Administration 2002;24 pages.

98 U.S Dept Health and Human Services Food and Drug Administration Guidance for Industry Estimating the safe

starting dose in clinical trials for therapeutics in adult healthy volunteers U.S Dept of Health and Human Services, Food and Drug Administration 2002;24 pages.

Before any drug is used in humans, it must be designed (or discovered), tested in animals, and characterized Drugs are characterized by their purity, stability to storage, toxicity in animals, pharmacokinetics (PK) in animals, and efficacy in animals In the United States, an Investigational New Drug (IND) application must be submitted to the FDA in order to gain approval for initiating clinical trials (4) The IND summarizes data on the chemistry and stability of the active drug substance, studies using in vitro methods such as studies with cultured cells, data on toxicity and efficacy acquired from animal studies, and any available data on humans The term “clinical data” only refers

to data from studies on human subjects (not to data from animal studies)

The main goals of a Phase I clinical trial are to assess safety and to determine an effective dose suitable for subsequent Phase II trials In clinical trials on anti-cancer drugs, Phase I trials are often configured to determine the minimal dose that can cause signifi-cant toxicity From this particular dose, it is often assumed that the dose is that which will

be most effective against the cancer In other words, the most appropriate dose is that which is just below a dose that produces unacceptable toxicity But this method is not used to arrive at the dose used in clinical trials for diseases that are not cancers

Phase I clinical trials begin by administering a small dose of the study drug to a group of three or more patients Subsequently, cohorts of patients receive increasing doses, first by 100%, then 66%, 50%, 40%, and 33%, a progression that is loosely based

1 Sihna G Japan works to shorten “drug lag,” boost trials of new drugs J Natl Cancer Inst 2010;102:148–151.

2 European Medicines Agency (EMEA) Guideline on strategies to identify and mitigate risks for first-in-human

clinical trials with investigational medicinal products (July 2007).

3 Castle G, Marshall C CHMP guideline on reducing risk in first-in-man trials: how will it affect your research

Informa UK, Ltd Oct 2007; 20–22.

4 Tamimi NA, Ellis P Drug development: from concept to marketing! Nephron Clin Pract 2009;113:c125–1231.

on the Fibonacci sequence (5) The trial ends when severe or dose-limiting toxicities (DLTs) are experienced by a large fraction of subjects at a given dosage level The dose just below that which was associated with excessive DLT is defined as the maximum tolerated dose (MTD) In the case of oncology clinical trials, the dose that is the MTD may be recommended for Phase II and Phase III clinical trials In view of the fact that

it may be impossible to predict the toxicity of a newly synthesized chemical, it essarily follows that many Phase I clinical trials include doses that are too low to be effective against cancers It has been estimated that about half of subjects treated with the lowest doses receive doses that are “subtherapeutic” (6)

nec-Where subjects are titrated with a drug, it is not the case that any give subject tially receives a lower drug dose, and then receives a higher dose, and then receives an even higher dose The ICH Guidelines (7) warn against conducting a titration scheme with any one particular subject:

ini-A critical disadvantage is that by itself, this study design cannot distinguish response to increased dose from response to increased time on drug therapy or a cumulative drug dosage effect It is therefore an unsatisfactory design when response is delayed, unless treatment at each dose

is prolonged Even where the time-until-development of effect is known to be short (from other data), this design gives poor information on adverse effects, many of which have time-dependent characteristics.

In other words, conducting the entire titration scheme with a single human subject cannot distinguish between the drug’s effects that are a consequence of only the high-est dose (the highest dose in the titration scheme), or if they are a consequence of the cumulative effects of the lowest, intermediate, and highest doses This source of con-cern applies to efficacy data and to safety data

Additional information on arriving at an optimal dose, from the ICH Guidelines, emphasizes the fact that an effective dose and an unacceptably toxic dose may be in a similar or overlapping range, or may reside in well-separated ranges, for any given drug Facts on whether these ranges are, or are not, well separated can guide the clinician in choosing the starting dose in a Phase I trial (8):

For example, a relatively high starting dose (on or near the plateau of the effectiveness dose– response curve) might be recommended for a drug with a large demonstrated separation between its useful and undesirable dose ranges or where a rapidly evolving disease process demands rapid effective intervention A high starting dose, however, might be a poor choice for

6 Koyfman SA, Agrawal M, Garrett-Mayer E, et al Risks and benefits associated with novel phase 1 oncology trial

Introduction to Regulated Clinical Trials 19

a drug with a small demonstrated separation between its useful and undesirable dose ranges

In these cases, the recommended starting dose might best be a low dose exhibiting a clinically

important effect in even a fraction of the patient population, with the intent to titrate the dose

upwards as long as the drug is well tolerated.

Phase II trials are sometimes divided into Phase IIa trials and Phase IIb trials In the Phase IIa trial, the drug is tested in a small group of (12–100) subjects In this context, the drug may be used only at a single high dose, that is, at the maximally tolerated dose In the subsequent Phase IIb trial, several dose levels may be tested (dose-ranging studies) in order to define the minimally effective dose and also to decide the optimal dose, based on efficacy and safety (9)

The final stage of drug development is the Phase III clinical trial Phase III clinical trials confirm the dose level, frequency, and timing of doses Phase III trials can involve

up to several thousands of subjects This large number of subjects is needed to ensure detection of less-frequently arising drug-related toxicities, to acquire a confident assess-ment of efficacy, and to serve as a basis for the package insert or the drug label (10)

II STUDY DESIGN

Clinical studies take various forms At its simplest, one person takes a drug, and the person’s response to the drug is measured The response can take the form of various parameters, such as blood pressure, data from an electrocardiogram (11) and titer of a given bacterium or virus, where these parameters are measured shortly before, as well

as after, taking the drug

Small clinical trials generally focus on the pharmacokinetics (PK) of a drug, the influence of food on the PK of the drug, the dose providing the greatest efficacy, and the most appropriate route of dosing Regarding the dosing route, drugs may be administered orally (per os), intramuscularly (im), intravenously (iv), subcutaneously (sc), rectally, or by inhalation

Large clinical trials include one or more study arms For example, a 2-arm clinical trial can take the form of a study drug group and a placebo group Also, a 2-arm clini-cal trial can take the form of a study drug group and an active control group The term

“active control” usually refers to an older drug that has been the standard of care for the disease of interest Moreover, a 3-arm clinical trial can include a study drug group, placebo group, and active control group The terms placebo and active control are fur-ther defined below Other features of clinical trial design include a run-in period and

a follow-up period Large clinical trials focus on safety and efficacy, but other types of

9 Tamimi NA, Ellis P Drug development: from concept to marketing! Nephron Clin Pract 2009;113:c125–1231.

10 Tamimi NA, Ellis P Drug development: from concept to marketing! Nephron Clin Pract 2009;113:c125–1231.

11 Giorgi MA, Bolaños R, Gonzalez CD, Di Girolamo G QT interval prolongation: preclinical and clinical testing

arrhythmogenesis in drugs and regulatory implications Curr Drug Saf 2010;5:54–57.

information may additionally be collected, for example data on pharmacokinetics, relations between a drug’s efficacy and the subject’s genetic makeup, and correlations between the drug’s adverse effects and the subject’s genetic makeup.

cor-The instructions for conducting a clinical trial are contained in a document called

a Clinical Study Protocol The Clinical Study Protocol may include a flow chart (or drawing or table) called a schema The schema is the best way to communicate the structure and timeline of any clinical trial Shown below are various hypothetical study schema, followed by a collection of representative schema from actual clinical trials

III THE STUDY SCHEMA

The treatment schedule or timeline used in clinical trials is represented by a diagram called a schema The schema can take the form of a flow chart, histogram, or table When included in a Clinical Study Protocol, or in a research publication, the schema aids in understanding the dosing schedule Moreover, when designing a clinical trial, the principal investigator may draw several versions of the schema, before arriving at the final version to be used in the study

Where only writing is used to describe the study design, and where the study design includes multiple branching points (or multiple segments), and where the entire study is described in a single lengthy sentence, the narrative risks ambiguity

Additionally, where only writing is used to describe study design, and where the sentences include multiple phrases, and where the exact meaning depends on the placement of each comma, ambiguity is the predictable result Moreover, the term

“biweekly,” which is often used to describe trial design, is distinguished in that it has two different meanings “Biweekly” means twice per week “Biweekly” also means once every two weeks For the above reasons, a schema should be considered for even the simplest trial designs

At its simplest, the study schema might take the form shown in Fig 2.1 This ticular study schema can be used for clinical trials in oncology, infectious diseases, immune disorders, metabolic diseases, and so on The schema shown in the figure has two arms, where one arm receives the study drug and the other arm receives a placebo, six boxes and four arrows

par-The study design may also be one that contains only one arm, that is, a single arm study, as shown in the schema in Fig 2.2 The schema contains four blocks and two arrows In a single arm study, the patient may serve as his own control In this situa-tion, the patient’s health immediately prior to enrolling in the study (baseline) is used for comparison purposes In another type of single arm study, the outcome for each patient may be compared to the outcome of a historical control The historical control group can be from the same institution or same investigators as in the present single arm study, or it can be from a totally unrelated group of investigators

Introduction to Regulated Clinical Trials 21

The schema in Fig 2.3 contains a run-in period Run-in periods are often used as

a component of the study design Run-in periods are used for a variety of reasons, as detailed in a subsequent chapter in this book The common feature of all of these run-

in periods is that they occur immediately prior to randomization of the subjects, and immediately prior to allocation of the subjects to the various study arms

The schema in Fig 2.3 contains seven boxes and six arrows The run-in period is used to test the ability of potential study subjects to adhere to a timely and orderly schedule of pill-taking Information on patient adherence is especially critical for clini-cal studies on drugs that are intended for chronic administration Drugs intended for chronic administration include atorvastatin, marketed as Lipitor®, which is used to pre-vent heart attacks, and include finastride (12,13) marketed as Proscar®, which is used to prevent prostate cancer

Arm A Study drug (dose on day 1 only) Enrollment and

randomization

for 2-arm study

Arm B Placebo (dose on day 1 only)

Evaluate disease on day 20

Evaluate disease on day 20

Interval of 7 days

or less

Figure 2.1 Schema with two study arms This schema shows the step of enrollment, the interval of

time between enrollment and treatment, and the steps of treatment and evaluation of efficacy

Enrollment in

single arm study Study drug (dose on day 1 only) Evaluate disease on day 8

Interval of 7 days

or less

Figure 2.2 Schema with one study arm The schema shows the step of enrollment, the interval of

time from enrollment to beginning treatment, and the step of evaluating efficacy of treatment

12 Moinpour CM, Lovato LC, Thompson IM Profile of men randomized to the prostate cancer prevention trial:

baseline health-related quality of life, urinary and sexual functioning, and health behaviors J Clin Oncol

2000;18:1942–1953.

13 Moinpour CM , Darke AK , Donaldson GW, et al Longitudinal analysis of sexual function reported by men in the

prostate cancer prevention trial J Natl Cancer Inst 2007;99:1025–1035.

Figure 2.4 provides one more hypothetical schema This schema highlights facets

of clinical trials that are ancillary to the study drug These additional facets include the step of enrollment, the steps of allocation and randomization, the taking of blood sam-ples, and an amendment to the Clinical Study Protocol The goal of the amendment was to get permission to increase the drug dose

a Examples of schema from clinical trials

The following provides a selection of representative schema from real clinical trials Oncology clinical trials are usually more complex than clinical trials for other dis-eases, because of the use of multiple drugs, often with staggered dosing schedules, and

Study drug Twice a day for 5 weeks

Placebo Twice a day for 5 weeks

Allocation

&

randomi-zation

Study drug Once a day for 5 weeks

Placebo Once a day for 5 weeks Enrollment

Amendment to Clinical Study Protocol

Blood samples taken at baseline (on day 1, before administering drug) and the first day of each 1 week cycle

Blood samples taken at baseline (on day 1, before administering drug) and the first day of each 1 week cycle

Figure 2.4 Schema of a 2-arm study where the study design was changed during the study The

schema shows the date when the study design was changed Also shown are dates when blood ples were withdrawn

sam-Exclude subjects who were not able

to follow the schedule

Include subjects who had followed the schedule

Randomize

Daily placebo for

5 years

Daily study drug for

5 years

Figure 2.3 Schema including a run-in period The study design includes a run-in period that screens

for the ability of potential subjects to follow the treatment schedule

Introduction to Regulated Clinical Trials 23

sometimes used in combination with radiation therapy For this reason, the following schema are from oncology clinical trials

Study designs sometimes include a decision tree Typically, decision trees in clinical trials require assessment of the response at a particular date, and deciding whether to increase or decrease the dose given to each individual subject, or deciding to remove the subject from the trial

As is evident, various schema emphasize different aspects of the trial, for example details on the run-in period, schedules of drug dosing, decision trees that take into account drug toxicity, and schedules for blood samples collection

The concepts illustrated by the following study schema are as follows The term

“staging” refers to identifying the stage (severity) of a particular cancer

l Staging (14)

l Staging followed by re-staging (15)

l Schemas showing a decision tree (16,17)

l Schema for a dose-escalation clinical trial (18,19,20)

l Run-in period that determines expression of a biomarker (21)

l Schema indicating days for evaluating pharmacokinetics (22)

l Schema with a run-in period where risk for an adverse event is assessed (23)

l What to do about placebo dosing, where two arms of the clinical trial receive two different volumes of a study drug (24)

14 Blumenschein Jr GR, Khuri FR, von Pawel J, et al Phase III trial comparing carboplatin, paclitaxel, and bexarotene with carboplatin and paclitaxel in chemotherapy-naive patients with advanced or metastatic non-small-cell lung

cancer: SPIRIT II J Clin Oncol 2008;26:1879–1885.

15 Czito BG, Willett CG, Bendell JC, et al Increased toxicity with gefitinib, capecitabine, and radiation therapy in

pancreatic and rectal cancer: phase I trial results J Clin Oncol 2006;24:656–662.

16 Baselga J, Zambetti M, Llombart-Cussac A, et al Phase II genomics study of ixabepilone as neoadjuvant treatment

for breast cancer J Clin Oncol 2009;27:526–534.

17 Katsumata N, Watanabe T, Minami H, et al Phase III trial of doxorubicin plus cyclophosphamide (AC), docetaxel,

and alternating AC and docetaxel as front-line chemotherapy for metastatic breast cancer: Japan Clinical Oncology

Group trial (JCOG9802) Ann Oncol 2009;20:1210–1215.

18 Marshall J, Chen H, Yang D, et al A phase I trial of a Bcl-2 antisense (G3139) and weekly docetaxel in patients with

advanced breast cancer and other solid tumors Ann Oncol 2004;15:1274–1283.

19 Moore M, Hirte HW, Siu L, et al Phase I study to determine the safety and pharmacokinetics of the novel Raf

kinase and VEGFR inhibitor BAY 43-9006, administered for 28 days on/7 days off in patients with advanced,

refractory solid tumors Ann Oncol 2005;16:1688–1694.

20 Van Cutsem E, Verslype C, Beale P, et al A phase Ib dose-escalation study of erlotinib, capecitabine and oxaliplatin in

metastatic colorectal cancer patients Ann Oncol 2008;19:332–339.

21 Dy GK, Miller AA, Mandrekar SJ, et al A phase II trial of imatinib (ST1571) in patients with c-kit expressing

relapsed small-cell lung cancer: a CALGB and NCCTG study Ann Oncol 2005;16:1811–1816.

22 Marshall J, Chen H, Yang D, et al A phase I trial of a Bcl-2 antisense (G3139) and weekly docetaxel in patients with

advanced breast cancer and other solid tumors Ann Oncol 2004;15:1274–1283.

23 Ganz PA, Hussey MA, Moinpour CM, et al Late cardiac effects of adjuvant chemotherapy in breast cancer survivors

treated on Southwest Oncology Group protocol s8897 J Clin Oncol 2008;26:1223–1230.

24 Reck M, von Pawel J, Zatloukal P, et al Phase III trial of cisplatin plus gemcitabine with either placebo or

bevacizumab as first-line therapy for nonsquamous non-small-cell lung cancer: AVAil J Clin Oncol

2009;27:1227–1234.

l Run-in period that determines response to chemotherapy, where chemotherapy is provided before initiation of the clinical trial (25)

l Two study arms, where one arm uses sequential dosing with two drugs, while the other arm uses concurrent administration of the same two drugs (26)

l Comparing neoadjuvant therapy with adjuvant therapy (27)

l Schema where each study arm uses both neoadjuvant therapy, followed by surgery, followed by adjuvant therapy (28)

l Two study arms, where the first arm uses a specific order of administration of two different drugs, while the second arm uses the reverse order of administration of the two drugs (29)

l Patients receiving three different drugs, each drug having its own different schedule (30)

Burstein et al (31) provide an interesting thumbnail diagram depicting a dozen ferent schema for various oncology clinical trials Chittick et al (32) provide a particu-larly interesting schema from a clinical trial on infections

dif-b Sequential treatment versus concurrent treatment – the Perez schema

In a clinical trial with three arms, Perez et al (33) disclosed the first arm as the control arm, the second arm as a sequential arm, and the third arm as a concurrent arm (Fig 2.5)

In the control arm, patients received an active control (AC) followed by paclitaxel The abbreviation “AC” refers to doxorubicin, which is an anthracycline (AC) drug

25 Hanna NH, Sandier AB, Loehrer PJ Sr, et al Maintenance daily oral etoposide versus no further therapy following induction chemotherapy with etoposide plus ifosfamide plus cisplatin in extensive small-cell lung cancer: a Hoosier

Oncology Group randomized study Ann Oncol 2002;13:95–102.

26 Perez EA, Suman VJ, Davidson NE, et al Cardiac safety analysis of doxorubicin and cyclophosphamide followed

by paclitaxel with or without trastuzumab in the North Central Cancer Treatment Group N9831 adjuvant breast

cancer trial J Clin Oncol 2008;26:1231–1238.

27 Gianni L, Baselga J, Eiermann W, et al Phase III trial evaluating the addition of paclitaxel to doxorubicin followed by cyclophosphamide, methotrexate, and fluorouracil, as adjuvant or primary systemic therapy: European Cooperative

Trial in Operable Breast Cancer J Clin Oncol 2009;27:2474–2481.

28 Untch M, Möbus V, Kuhn W, et al Intensive dose-dense compared with conventionally scheduled preoperative

chemotherapy for high-risk primary breast cancer J Clin Oncol 2009;27:2938–2945.

29 Puhalla S, Mrozek E, Young D, et al Randomized phase II adjuvant trial of dose-dense docetaxel before or after

doxorubicin plus cyclophosphamide in axillary node-positive breast cancer J Clin Oncol 2008;26:1691–1697.

30 Sekine I, Nishiwaki Y, Noda K, et al Randomized phase II study of cisplatin, irinotecan and etoposide combinations

administered weekly or every 4 weeks for extensive small-cell lung cancer (JCOG9902-DI) Ann Oncol

2003;14:709–714.

31 Burstein HJ, Prestrud AA, Seidenfeld J, et al American Society of Clinical Oncology clinical practice guideline:

update on adjuvant endocrine therapy for women with hormone receptor-positive breast cancer J Clin Oncol

2010;28:3784–3796.

32 Chittick GE, Zong J, Blum MR, et al Pharmacokinetics of tenofovir disoproxil fumarate and ritonavir-boosted

saquinavir mesylate administered alone or in combination at steady state Antimicrob Agents Chemother

2006;50:1304–1310.

33 Perez EA, Suman VJ, Davidson NE, et al Cardiac safety analysis of doxorubicin and cyclophosphamide followed

by paclitaxel with or without trastuzumab in the North Central Cancer Treatment Group N9831 adjuvant breast

cancer trial J Clin Oncol 2008;26:1231–1238.

Introduction to Regulated Clinical Trials 25

In the sequential arm, patients received the same drugs as in the active control arm, but followed by an antibody (trastuzumab)

In the concurrent arm, patients received the same drugs as in the active control arm, but with the antibody given at the same time (concurrently) as paclitaxel Trastuzumab, which has the trade name Herceptin®, is an antibody that binds to HER2

The goal of the study was to determine which treatment was least toxic to the heart Arm A, which did not include the antibody, was the least cardiotoxic (cardiac damage in 0.3% of subjects) Arm B was more toxic (2.8% cardiotoxic) Arm C was the most toxic (3.3% cardiac damage), but only slightly more toxic than Arm B

As a general proposition, sequential chemotherapy is preferred over combination chemotherapy, where reducing toxicity is especially needed, for example, with patients who are elderly (34) In other words, toxicity is expected to be a bigger problem where two drugs are administered on the same day, and a lesser problem where two different drugs are administered on separate days Raetz et al (35) provide a dramatic exam-ple of sequential chemotherapy, but for a different reason In this study, three differ-ent blocks of chemotherapy were administered Each of these three blocks delivered

a different collection of drugs The goal of using these three blocks of time (non- overlapping blocks) of chemotherapy was to ensure a lengthy period of remission

34 Miles D, von Minckwitz G, Seidman AD Combination versus sequential single-agent therapy in metastatic breast

cancer Oncologist 2002;7(Suppl 6):13–19.

35 Raetz EA, Borowitz MJ, Devidas M, et al Reinduction platform for children with first marrow relapse of acute

lymphoblastic leukemia: a Children’s Oncology Group Study J Clin Oncol 2008; 26:3971–3978.

Sequential arm AC three times per week for 4 cycles, where each cycle was 3 weeks

Concurrent arm AC three times per week for 4 cycles, where each cycle was 3 weeks

Paclitaxel once per week for

12 weeks

Paclitaxel once per week for 12 weeks Paclitaxel once per week for 12 weeks and antibody, once per week for 12 weeks

Antibody once a week for 52 weeks

Antibody once a week for 40 weeks

Figure 2.5 Schema of a 3-arm study The first arm received an active control treatment The second

arm received small molecule drug plus antibody on different, non-overlapping weeks (sequential treatment), while the third arm received small molecule drug and antibody at the same time, that is, during the same week (concurrent treatment)

c Neoadjuvant chemotherapy versus adjuvant chemotherapy – the Gianni schema

Gianni et al (36) provide a schema for a 3-arm clinical trial where two arms use vant chemotherapy and where one arm uses neoadjuvant chemotherapy (Fig 2.6).The term “adjuvant chemotherapy” refers to chemotherapy that follows surgery In contrast, the term “neoadjuvant chemotherapy,” sometimes called “induction chemo-therapy,” refers to chemotherapy that precedes surgery

adju-In one adjuvant arm, patients received surgery followed by A, which was followed by CMF In another adjuvant arm, patients received surgery followed by AT, which was followed

by CMF But in the neoadjuvant arm, patients received AT, and then CMF, and finally surgery

“A” refers to doxorubicin, a drug that is an anthracycline (A) “T” refers to taxel, a drug that is a taxane (T) “CMF” refers to the combination of cyclophospha-mide, methotrexate, and 5-fluorouracil The take-home lesson from this schema is as follows In designing the clinical trial, the investigator is free to test surgery followed by drugs (adjuvant), as well as drugs followed by surgery (neoadjuvant)

pacli-d Neoadjuvant chemotherapy plus adjuvant chemotherapy – the Untch schema

The clinical study of Untch et al (37) uses a schema where, for every patient, therapy was given before surgery and also after surgery (Fig 2.7) Hence, the schema involved neoadjuvant therapy as well as adjuvant therapy, for every single patient

chemo-It is interesting to point out that one study arm involved concurrent apy (both drugs at once) while the other study arm involved sequential chemotherapy (drugs administered, not on the same day, but on different days)

chemother-Randomize and allocate subjects

to the three arms.

Gianni study

4 cycles

of AT Surgery 4 cycles of CMF

4 cycles

of A Surgery 4 cycles of CMF

4 cycles

of CMF

4 cycles

Figure 2.6 Schema showing 3 arms where subjects received adjuvant therapy or neoadjuvant

therapy The first two arms received adjuvant therapy, that is, surgery prior to drugs The third arm received neoadjuvant therapy, that is, drugs for downsizing the tumors prior to surgery

36 Gianni L, Baselga J, Eiermann W, et al Phase III trial evaluating the addition of paclitaxel to doxorubicin followed by cyclophosphamide, methotrexate, and fluorouracil, as adjuvant or primary systemic therapy: European Cooperative

Trial in Operable Breast Cancer J Clin Oncol 2009;27:2474–2481.

37 Untch M, Möbus V, Kuhn W, et al Intensive dose-dense compared with conventionally scheduled preoperative

chemotherapy for high-risk primary breast cancer J Clin Oncol 2009;27:2938–2945.

Introduction to Regulated Clinical Trials 27

CMF refers to the combination of three drugs, cyclophosphamide, methotrexate, and 5-fluorouracil

e Forwards sequence and reverse sequence – the Puhalla schema

The clinical trial of Puhalla et al (38) contains two arms (Fig 2.8) Subjects in the first arm received drugs in this order: D followed by AC In detail, subjects received docetaxel on the first day of each cycle, for 4 cycles, where each cycle was 14 days long This was followed by doxorubicin (an anthracycline drug) plus cyclophospha-mide, on the first day of each cycle, for 4 cycles Each cycle was 2 weeks long

Now, this is about the second arm Subjects in the second arm received drugs in the reverse order, that is, AC followed by D The investigators chose a 2-arm study design, where each arm was the reverse of the other, because it was not possible to pre-dict which order would provide the better outcome

The type of trial design, where one arm uses a “forward sequence” of two drugs, and where another arm uses the “reverse sequence” of the two drugs, sometimes finds

a basis in the mechanism of action of certain drugs The following specifically cerns chemotherapy involving paclitaxel and gemcitabine As explained by Paccagnella

con-et al (39) for a clinical trial on lung cancer, “[s]everal reports have shown that the sequence of administration of gemcitabine and paclitaxel may also affect the efficacy of this combination chemotherapy, since paclitaxel increases the concentration of active metabolite of gemcitabine (dFdCTP) when administered first.”

39 Paccagnella A, Oniga F, Bearz A, et al Adding gemcitabine to paclitaxel/carboplatin combination increases survival

in advanced non-small-cell lung cancer: results of a phase II–III study J Clin Oncol 2006;24:681–687.

38 Puhalla S, Mrozek E, Young D, et al Randomized phase II adjuvant trial of dose-dense docetaxel before or after

doxorubicin plus cyclophosphamide in axillary node-positive breast cancer J Clin Oncol 2008;26:1691–1697.

epirubicin + paclitaxel

epirubicin + paclitaxel

epirubicin

+ paclitaxel

epirubicin + paclitaxel

Arm A (4 cycles over 12 weeks) Untch study Surgery

Rando-mize

CMF (3 drugs)

epirubicin epirubicin epirubicin paclitaxel paclitaxel paclitaxel

Figure 2.7 Schema showing 2-arm study Subjects in the first arm received chemotherapy before

as well as after surgery Subjects in the second arm also received chemotherapy prior to and after surgery The neoadjuvant chemotherapy in arm A was concurrent; that in arm B was sequential

f Both arms received three drugs, each arm at a different schedule – the Sekine schema

In a clinical trial of lung cancer, Sekine et al (40) administered three drugs to all jects in arm A and the same three drugs to all subjects in arm B (Fig 2.9) Different schedules were used for each of the three drugs The two arms were different from each other in that different schedules were used for administering the drugs

sub-The three drugs shown on the schema, cisplatin, irenotecan, and etoposide, are small molecule drugs Subjects also received a fourth drug, granulocyte colony stimu-lating factor (G-CSF) G-CSF is a polypeptide, not a small molecule For clarity in pre-sentation G-CSF is not shown on the schema Blood samples and chest X-rays were taken at various intervals (not shown) Blood counts were run twice a week, and chest X-rays were conducted once a week Chest X-rays are used during the diagnosis and treatment of lung cancer (41)

g Staging – the Blumenschein schema

The schema of Blumenschein et al (42) reveals three different steps in the trial design (Fig 2.10) These are the step of stratifying patients according to disease stage, the step

of randomization, and the step of drug administration Although most clinical trials in

Arm A 4 cycles of docetaxel (once at start of cycle), each cycle being 2 weeks long, followed by 4 cycles of AC (once at start of cycle), each cycle being 2 weeks long

Arm B 4 cycles of AC (once at start of cycle), each cycle being 2 weeks, followed by

4 cycles of docetaxel (once at start of cycle), each cycle being 2 weeks long

Figure 2.8 Schema showing a 2-arm study In this 2-arm study drugs were administered in a

“forward” sequence for arm A, and in a “reverse” sequence for arm B

40 Sekine I, Nishiwaki Y, Noda K, et al Randomized phase II study of cisplatin, irinotecan and etoposide combinations

administered weekly or every 4 weeks for extensive small-cell lung cancer (JCOG9902-DI) Ann Oncol

2003;14:709–714.

41 Gohagan JK, Marcus PM, Fagerstrom RM, et al Final results of the Lung Screening Study, a randomized feasibility

study of spiral CT versus chest X-ray screening for lung cancer Lung Cancer 2005;47:9–15.

42 Blumenschein Jr GR, Khuri FR, von Pawel J, et al Phase III trial comparing carboplatin, paclitaxel, and bexarotene with carboplatin and paclitaxel in chemotherapy-naive patients with advanced or metastatic non-small-cell lung

cancer: SPIRIT II J Clin Oncol 2008;26:1879–1885.

Introduction to Regulated Clinical Trials 29

CP administered for 4 cycles, each cycle is 3 weeks long Stratify subjects

CP is administered for 4 cycles, each cycle is 3 weeks long

Bexarotene administered once per day

Figure 2.10 Schema showing a 2-arm study The schema shows the steps of stratification and

ran-domization The daily administration of bexarotene is unambiguous, as the schema shows a separate arrow for each of the 28 days of administration of this drug

Arm B 1 2 3 4 5 6 7 8 9 10 11 12 13 weeks

Irinotecan (days 1, 8, 15 of these weeks) Etoposide (days 1–3 of these weeks) Cisplatin (day 1 of these weeks)

Arm A 1 2 3 4 5 6 7 8 9 10 11 12 13 weeks

Irinotecan (day 1 of these weeks)

Etoposide (days 1–3 of these weeks)

Cisplatin (day 1 of these weeks) Rando-

mize

Figure 2.9 Schema showing a 2-arm study Each arm received the same three drugs, but with

differ-ent timing for each of the three drugs

oncology stratify the patients before randomization, this particular schema is unique

in that the published flow chart actually includes a box showing the step of tion Subjects in the two arms received the same drugs, except that arm B received an additional drug, bexarotene CP refers to carboplatin plus paclitaxel Carboplatin is in a class of drugs called platinum drugs Paclitaxel is in a class of drugs called taxanes

stratifica-h Staging and restaging – the Czito schema

In a clinical study involving chemotherapy and radiation, Czito et al (43) provided a schema that indicates steps where staging occurred (Fig 2.11) Although staging is used

in clinical trials in oncology, infections, immune diseases, and other disorders, it is only

on occasion that the step of staging is actually depicted in the schema Staging was specifically included in this schema to emphasize the fact that staging was done at two different points during the course of the clinical trial

The trial was a single-arm trial Because the trial had only one arm, the subjects were not randomized In other words, subjects were not randomized to be treated with arm A drugs or arm B drugs, because there was only one arm As this was a Phase I trial, the main purpose was to determine the dose-limiting toxicity (DLT), that is, to determine a dose suitable for using in a subsequent Phase II trial

Chemotherapy was given every single day for 38 days Radiation was given on the same days as the drugs, except not on Saturdays or Sundays The days for radiation treatment are made unambiguous by the schema, as it shows arrows only on week days (not on weekends)

i Methodology tip – staging

The term staging refers to measuring the parameters of a cancer, with respect to a set

of criteria accepted by the medical profession For most cancers, there are established,

4-week interval

Restaging Capecitabine plus gefitinib daily, for 38 consecutive days

Radiation only on weekdays

2-week interval

Staging

Czito study

Figure 2.11 Schema of a single-arm clinical trial Staging of the tumors occurred at two time points,

as indicated

43 Czito BG, Willett CG, Bendell JC, et al Increased toxicity with gefitinib, capecitabine, and radiation therapy in

pancreatic and rectal cancer: phase I trial results J Clin Oncol 2006;24:656–662.

Introduction to Regulated Clinical Trials 31

published criteria for determining if the cancer is at, for example, stage I, stage II, stage III, or stage IV By staging, what is meant is the determination of the size, number, and location of tumors, and the comparison of these values with established standards for

different stages for the cancer By restaging, what is meant is repeating the

determi-nation of the size, number, and location of the tumors, and comparing these values with the standard for staging of the cancer in question Staging encompasses the acts

of determining if the tumors are resectable, locally advanced, or metastatic Staging can

be accomplished using computed tomography, endoscopic ultrasound, and surgical evaluation

j Decision tree – the Baselga schema

The schema of Baselga et al (44) depicts two points where a decision needed to be made (Fig 2.12) These two points are indicated by the forked arrows The decision involved assessing the tumor’s response to therapy (at that time point) for every indi-vidual patient Based on the tumor’s response, the patient was assigned to one of two different therapies

The Baselga clinical trial could be characterized as a single arm trial, because there was no control group and no placebo group The goal of the trial was to detect cor-relations between gene expression, as determined by measuring gene expression in tumor biopsies, and positive response to drug treatment The expression of 200 genes was measured, where these genes were chosen before the study This clinical trial involved neoadjuvant chemotherapy Neoadjuvant refers to the fact that chemotherapy was administered before surgery (not after surgery) Where the schema reads “tumors shrink or remain stable” or “tumors progress,” this indicates that the physician had examined the patient, determined the status of the tumors, and made the decision to use the treatment that is disclosed in the subsequent (lower) arrows on the flow chart

Where trial design includes a decision point or decision tree, the medical writer should use forked arrows in the schema to indicate the decision

k Decision tree – the Katsumata schema

Decision trees occur in the schema of Katsumata et al (45) where the decision is gered by an increase (if any) in tumor size (Fig 2.13) At each decision point, the deci-sion took the form of crossing over to the alternate therapy, or of continuing with the

trig-44 Baselga J, Zambetti M, Llombart-Cussac A, et al Phase II genomics study of ixabepilone as neoadjuvant treatment

for breast cancer J Clin Oncol 2009;27:526–534.

45 Katsumata N, Watanabe T, Minami H, et al Phase III trial of doxorubicin plus cyclophosphamide (AC), docetaxel,

and alternating AC and docetaxel as front-line chemotherapy for metastatic breast cancer: Japan Clinical Oncology

Group trial (JCOG9802) Ann Oncol 2009;20:1210–1215.

same type of drug The letter “A” represents doxorubicin, an anthracycline compound (46) C represents cyclophosphamide Thus, AC means the combination of doxorubicin plus cyclophosphamide D represents docetaxel.

Tumors progress

If tumor progress, discontinue treatment

Tumors shrink or remain

stable

If tumors shrink or remain stable, continue to next cycle of anthracycline and complete 6 cycles of anthracycline

Repeat mammogram

Surgery

Biopsy, mammogram, and gene expression Baselga study

Tumors progress

If tumor progress discontinue treatment with ixabepilone

Begin 6 cycles of anthracycline

Figure 2.12 Schema with boxes showing decision points Forked arrows indicate time points when

tumors were assessed, and when a decision was made to proceed with the indicated course of action

46 O’Shaughnessy J, Twelves C, Aapro M Treatment for anthracycline-pretreated metastatic breast cancer Oncologist

2002;7(Suppl 6):4–12.

Introduction to Regulated Clinical Trials 33

The decision to switch drugs was triggered by detection of tumor progression either during the first 18 weeks of treatment or after completion of the first 18 weeks

of treatment In the words of the investigators, “[o]n treatment failure or disease gression during or after treatment, patients were crossed over from AC to D, or from D

The following defines the over design According to Donner (48) the over design involved a single group of subjects, where each subject served as his own control for comparing two treatments Subjects were randomized to one of two treat-ment sequences, where half the subjects received the treatments in the order EC, the other half in the order CE The advantage of this design is that it allows the effects of the treatments to be compared within the same subjects

cross-The cross-over design may be used for various different goals One goal is to gain regulatory approval for only one of the drugs, that is, for a treatment scheme that uses only one of the two drugs Another goal is to gain regulatory approval for the

Randomization.

Katsumata study

CA once a week for 18 weeks

D once a week for 18 weeks CA once a weekfor 18 weeks

D once a week for 18 weeks

Decision: If, during treatment, or at the 18 week time point, tumors have progressed, then immediately switch from CA to D

Decision: If, during treatment, or at the 18 week time point, tumors have progressed, then immediately switch from D to CA

Figure 2.13 Schema showing a 2-arm study Each of the two arms incorporated a cross-over design

48 Donner A Approaches to sample size estimation in the design of clinical trials – a review Stat Med 1984;3:199–214.

47 Katsumata N, Watanabe T, Minami H, et al Phase III trial of doxorubicin plus cyclophosphamide (AC), docetaxel,

and alternating AC and docetaxel as front-line chemotherapy for metastatic breast cancer: Japan Clinical Oncology

Group trial (JCOG9802) Ann Oncol 2009;20:1210–1215.

combination of the two drugs, for a treatment scheme where drug A is given for a period of time followed by drug B for a separate period of time.

In comments about the desirability of a cross-over scheme used in clinical practice, Katsumata et al (49) state that, “[a]lternating chemotherapy is an approach designed to produce maximal antitumor activity by alternating non-crossresistant regimens of che-motherapy Alternating chemotherapy has been suggested to be effective in Hodgkin’s disease and small-cell lung cancer.” In this type of cross-over design, it is desirable that there are “carry-over effects.” In other words, it is hoped that the carry-over effects produced by the cross-over design improve the efficacy of the treatment

The ICH Guidelines (50) provide a general comment and warning on the over study design These comments refer to a cross-over design where the goal is to evaluate the efficacy of just one of the two drugs used in the study (not where it is the goal to evaluate the efficacy of the combination of the two drugs, when the two drugs are administered sequentially):

cross-A randomized multiple cross-over study of different doses can be successful if drug effect develops rapidly and patients return to baseline conditions quickly after cessation of therapy, if responses are not irreversible (cure, death), and if patients have reasonably stable disease This design suf- fers, however, from the potential problems of all cross-over studies: it can have analytic prob- lems if there are many treatment withdrawals; it can be quite long in duration for an individual patient; and there is often uncertainty about carry-over effects (longer treatment periods may minimize this problem), baseline comparability after the first period, and period-by-treatment interactions.

The cross-over design is sometimes used in clinical trials where there is a placebo arm (51,52) Where one group of subjects is initially assigned to the placebo arm, those subjects are assigned to receive the placebo until the endpoint of interest arrives Typically, the endpoint of interest, at least in oncology clinical trials, is growth of the tumors beyond a predetermined point The event where a tumor grows beyond a pre-determined point is known as “progression.” For any given subject in the placebo arm,

at the time that this endpoint is reached, that subject is then crossed over to the drug (the active treatment) that was used in the study drug arm Where a cross-over design

is used in clinical trials involving a placebo, this design can improve recruitment of

49 Katsumata N, Watanabe T, Minami H, et al Phase III trial of doxorubicin plus cyclophosphamide (AC), docetaxel, and alternating AC and docetaxel as front-line chemotherapy for metastatic breast cancer: Japan Clinical Oncology

Group trial (JCOG9802) Ann Oncol 2009;20:1210–1215.

50 ICH Harmonised Tripartite Guideline Dose–response information to support drug registration E4 March 1994,

Introduction to Regulated Clinical Trials 35

subjects in the trial who are not particularly eager to be assigned to the placebo ment (53,54)

treat-l Methodology tip – what is “tumor progression”?

In the clinical trial of Katsumata et al (55) increases in tumor size were compared to

an accepted standard The accepted standard was as follows It was an increase in tumor dimensions of 20% or greater, in the interval between starting chemotherapy and a subsequent tumor assessment

The term progression, in the context of oncology, refers to an increase in tumor size

and number, where the increase progresses beyond a certain minimal limit set by these criteria Progression can be with reference to the RECIST criteria (56,57,58) Some investigators prefer to use an older set of criteria, the WHO response criteria (59,60) The Katsumata study assessed tumors with respect to the WHO criteria

m Methodology tip – unit of drug dose expressed in terms of body

surface area

The following explains the unit used in the drug dose, “doxorubicin 40 mg/m2,” used above in the study of Katsumata et al (61) In the words of the investigators, Arm A

of the clinical trial received, “doxorubicin 40 mg/m2 plus cyclophosphamide 500 mg/

m2 (AC) every 3 weeks for six cycles.” The investigators also wrote that patients in Arm B of the trial received “docetaxel 60 mg/m2 (D), administered by i.v infusion over the course of 1 h every 3 weeks for six cycles.”

53 Ma BB, Britten CD, Siu LL Clinical trial designs for targeted agents Hematol Oncol Clin North Am

2002;16:1287–1305.

54 Gray R, Manola J, Saxman S, et al Phase II clinical trial design: methods in translational research from the

Genitourinary Committee at the Eastern Cooperative Oncology Group Clin Cancer Res 2006;12:1966–1969.

55 Katsumata N, Watanabe T, Minami H, et al Phase III trial of doxorubicin plus cyclophosphamide (AC), docetaxel,

and alternating AC and docetaxel as front-line chemotherapy for metastatic breast cancer: Japan Clinical Oncology

Group trial (JCOG9802) Ann Oncol 2009;20:1210–1215.

56 Eisenhauer EA, Therasse P, Bogaerts J, et al New response evaluation criteria in solid tumours: revised RECIST

guideline (version 1.1) Eur J Cancer 2009;45:228–247.

57 Therasse P, Arbuck SG, Eisenhauer EA, et al New guidelines to evaluate the response to treatment in solid tumors

European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States,

National Cancer Institute of Canada J Natl Cancer Inst 2000;92:205–216.

58 Schwartz LH, Bogaerts J, Ford R, et al Evaluation of lymph nodes with RECIST 1.1 Eur J Cancer

2009;45:261–267.

59 World Health Organization: Handbook for Reporting Results of Cancer Treatment Geneva, Switzerland: World Health

Organization; 1979, publication 48.

60 Park JO, Lee SI, Song SY, et al Measuring response in solid tumors: comparison of RECIST and WHO response

criteria Jpn J Clin Oncol 2003;33:533–537.

61 Katsumata N, Watanabe T, Minami H, et al Phase III trial of doxorubicin plus cyclophosphamide (AC), docetaxel,

and alternating AC and docetaxel as front-line chemotherapy for metastatic breast cancer: Japan Clinical Oncology

Group trial (JCOG9802) Ann Oncol 2009;20:1210–1215.

The question is, therefore, what is the meaning of the unit: mg/m2?

Drug doses are sometimes expressed in terms of body surface area (meters squared) According to Felici et al (62) and others (63,64) many anti-cancer drugs have a nar-row therapeutic window This means that a small change in dose can lead to poor anti-tumor effects or an unacceptable toxicity The rationale for using body surface area is

to normalize the drug dose among patients Using body surface area in the unit of drug dosing seems to work best for drugs where there is a relationship between body surface area and a pharmacokinetic parameter, such as the parameter of half-life in the bloodstream

n Run-in period – the schema of Dy

The schema of Dy et al (65) discloses a run-in period (Fig 2.14) Where a clinical trial includes a run-in period, it occurs before randomization of subjects and before allocat-ing subjects to the various arms of the trial

Run-in periods are used for a variety of purposes, for example for determining if patients are willing or capable of taking medications on time, or if patients find the study drug to be intolerably toxic

As shown in the schema in Fig 2.14, the run-in period was used to screen patients for expression of a biomarker, c-kit Where tumors were negative for c-kit, the sub-ject was not included in the trial Where tumors were positive for c-kit, patients were included in the trial, and were then treated with imatinib

Pre-registration

Test if tumor is positive for the biomarker, c-kit

Dy study

Exclude patient from trial if tumor is c-kit negative

Include patient from trial if tumor is c-kit positive

Imatinib, continual infusion for 28 days

Figure 2.14 Study schema of a 1-arm trial The schema includes a run-in period that is used to

determine eligibility of each potential subject

62 Felici A, Verweij J, Sparreboom A Dosing strategies for anticancer drugs: the good, the bad and body-surface area

Eur J Cancer 2002;38:1677–1684.

63 Sawyer M, Ratain MJ Body surface area as a determinant of pharmacokinetics and drug dosing Invest New Drugs

2001;19:171–177.

64 Kouno T, Katsumata N, Mukai H, Ando M, Watanabe T Standardization of the body surface area (BSA) formula to

calculate the dose of anticancer agents in Japan Jpn J Clin Oncol 2003;33:309–313.

65 Dy GK, Miller AA, Mandrekar SJ, et al A phase II trial of imatinib (ST1571) in patients with c-kit expressing

relapsed small-cell lung cancer: a CALGB and NCCTG study Ann Oncol 2005;16:1811–1816.

Introduction to Regulated Clinical Trials 37

While all clinical trials have inclusion criteria and exclusion criteria, whether they

be in oncology, infectious diseases, immune disorders, the clinical trial of Dy et al (66)

is unique in that the schema clearly identifies one of the inclusion criteria The rion shown in the flow chart is positive expression of c-kit

crite-Once enrolled in the trial, patients received repeated cycles of imatinib, each cycle lasting 28 days, for a total time of 16 weeks Imaging was obtained after every other 28-day cycle, where imaging provided data on tumor size and number

o Methodology tip – c-kit and imatinib

The following concerns c-kit and imatinib C-kit is a membrane-bound protein that has tyrosine kinase activity (67) The ligand of c-kit is stem cell factor In other words, c-kit is the receptor of stem cell factor Binding of stem cell factor to c-kit activates various cell-signaling pathways that are needed for proliferation, differentiation, and survival Mutations in c-kit that increase the signaling activity of c-kit occur in various cancers, for example acute myeloid leukemia, gastrointestinal tumors, testicular cancer, and melanoma Where the tumor contains a mutation in c-kit, the result is reduced survival, compared to patients bearing tumors having normal c-kit Imatinib, an inhibi-tor of tyrosine kinase, has proven to be dramatically successful in treating a variety of cancers

p Run-in period – the Hanna schema

The run-in period of Hanna et al (68) took the form of a miniature clinical trial where the goal was to determine if patients would respond favorably to a combination

of three drugs (Fig 2.15)

The three drugs were etoposide, ifosfamide, and cisplatin After treatment with these three drugs during the run-in period, the patient’s tumors were measured to determine tumor size and number Where tumor size and number remained constant,

or where tumors shrank, patients were then enrolled in the clinical trial, randomized, and assigned to arm A and arm B But where the combination of the three drugs failed

to control tumors, the patient was excluded from further study

As shown in the schema (Fig 2.15), patients with tumors controlled by the three drugs were randomized and assigned to arm A (control arm) where patients received

no other drug, or to arm B, where patients received etoposide (study drug arm)

66 Dy GK, Miller AA, Mandrekar SJ, et al A phase II trial of imatinib (ST1571) in patients with c-kit expressing

relapsed small-cell lung cancer: a CALGB and NCCTG study Ann Oncol 2005;16:1811–1816.

67 Roberts KG, Smith AM, McDougall F, et al Essential requirement for PP2A inhibition by the oncogenic receptor

c-KIT suggests PP2A reactivation as a strategy to treat c-KIT cancers Cancer Res 2010;70:5438–5447.

68 Hanna NH, Sandier AB, Loehrer Sr PJ, et al Maintenance daily oral etoposide versus no further therapy following induction chemotherapy with etoposide plus ifosfamide plus cisplatin in extensive small-cell lung cancer: a Hoosier

Oncology Group randomized study Ann Oncol 2002;13:95–102.

Belani et al (69) also provide a schema with a run-in period, where the run-in period takes the form of a miniature clinical trial occurring before randomization

In the words of these authors, “[a]fter the completion of two cycles of chemotherapy, patients were reassessed with chest CT to ensure the absence of metastatic progres-sion In the absence of metastatic progression, patients were randomly assigned to one

of two different…regimens.” The particular type of trial design used by Hanna et al (70) Belani et al (71) is called, “randomized discontinuation” (72) A run-in period in

a clinical trial that has a randomized discontinuation feature serves to enrich the study population for patients likely to respond positively to the study drug

q How to maintain blinding of the treatment, when the study

drug and the control treatment are provided by different-sized

pills (or by different volumes of solutions) – the Reck schema

The following concerns studies requiring a “double dummy” design, such as the study

of Reck et al (73) detailed below (Fig 2.16) This concerns trials with two different

Enroll subjects,

Hanna study

Increase in tumor size or number

Subject excluded from trial

Control arm (no drug) Etoposide arm (study drug)

Run-in period:

etoposide, ifosfamide, and cisplatin (4 cycles, each cycle lasting

3 weeks)

Tumors stable or reduced

Randomize subjects

Figure 2.15 Study schema of a 2-arm study that included a run-in period The run-in period was used

to determine eligibility of the potential study subjects

69 Belani CP, Wang W, Johnson DH, et al Phase III study of the Eastern Cooperative Oncology Group (ECOG 2597): induction chemotherapy followed by either standard thoracic radiotherapy or hyperfractionated

accelerated radiotherapy for patients with unresectable stage IIIA and B non-small-cell lung cancer J Clin Oncol

2005;23:3760–3767.

70 Hanna NH, Sandier AB, Loehrer Sr, PJ et al Maintenance daily oral etoposide versus no further therapy following induction chemotherapy with etoposide plus ifosfamide plus cisplatin in extensive small-cell lung cancer: a Hoosier

Oncology Group randomized study Ann Oncol 2002;13:95–102.

71 Belani CP, Wang W, Johnson DH, et al Phase III study of the Eastern Cooperative Oncology Group (ECOG 2597): induction chemotherapy followed by either standard thoracic radiotherapy or hyperfractionated

accelerated radiotherapy for patients with unresectable stage IIIA and B non-small-cell lung cancer J Clin Oncol

2005;23:3760–3767.

72 Fu P, Dowlati A, Schluchter M Comparison of power between randomized discontinuation design and upfront

randomization design on progression-free survival J Clin Oncol 2009;27:4135–4141.

73 Reck M, von Pawel J, Zatloukal P, et al Phase III trial of cisplatin plus gemcitabine with either placebo or bevacizumab

as first-line therapy for nonsquamous non-small-cell lung cancer: AVAil J Clin Oncol 2009;27:1227–1234.

Introduction to Regulated Clinical Trials 39

study arms Subjects in each study arm are assigned to receive a different number

of pills (or a different injected volume) For example, subjects in arm A may receive

1 pill/day and subjects in arm B receive 4 pills/day To provide another example, jects in arm A receive an injection of 5 mL/day, and subjects in arm B receive an injec-tion of 20 mL/day This poses the vexing problem of designing the placebo arm

sub-The problem can be articulated as follows What approach should be used for the arm C (placebo arm) of the study?

The FDA has specifically recognized this situation, writing, “[s]ome trials may study more than one dose of the test treatment…[i]n these cases, it may be easier for the investigator to use more than one placebo (double-dummy) than to try to make all treatments look the same.” Thus, the best solution might be for arm C (placebo) to receive an injection of 5 mL/day of placebo and arm D (also placebo) to receive an injection of 10 mL/day of placebo (74) Thus, for maintaining blinding in this situation,

a common approach is to use a double dummy

In the clinical trial of Reck et al (75) the formulation of the study drug, zumab, was in liquid form, and was injected in patients in two different study arms, one arm receiving a smaller dose and the other arm receiving a larger dose The two different doses were 7.5 mg/kg body weight and 15 mg/kg body weight

bevaci-The problem facing the investigators was how to configure the placebo bevaci-The study drug was available in a vial of only one size, and was available at only one concentra-tion Thus, some of the study drug patients needed to receive the contents of one vial,

74 Dept of Health and Human Services Food and Drug Administration Guidance for Industry E10 Choice of control

group and related issues in clinical trials (May 2001).

75 Reck M, von Pawel J, Zatloukal P, et al Phase III trial of cisplatin plus gemcitabine with either placebo

or bevacizumab as first-line therapy for nonsquamous non-small-cell lung cancer: AVAil J Clin Oncol

2009;27:1227–1234.

Randomize.

Reck study

Bevacizumab (5.0 mg/kg) + CG

Bevacizumab (7.5 mg/kg) + CG

Placebo (high dose) + CG

Placebo (low dose) + CG

Pool all placebo data for analysis

Figure 2.16 Study schema with four arms

and other study drug patients needed to receive the contents of two vials To avoid bias

in the trial, two different placebos were used, one with a small volume and the other with a larger volume, where the volumes corresponded to those of the study drug.The end-result was that any physician administering a large volume would not likely be capable of guessing if the large volume dose contained placebo or study drug Bevacizumab or placebo was administered intravenously and concurrently with che-motherapy every 3 weeks on day 1

Patients in all four arms of the trial received cisplatin and gemcitabine (CG) Cisplatin was administered on day 1, and gemcitabine was administered on days 1 and

8, of each 21-day cycle Drug administration was continued for 6 cycles Regarding the two placebo groups, Reck et al (76) stated that, “[p]atients assigned to high- and low-dose placebo were pooled into one placebo group for all analyses.”

Another example of using two different placebos, each corresponding to a lower dose and higher dose of study drug, comes from the example of cladribine for treating multiple sclerosis (77)

The following provides two situations where a double dummy design can be used

In all cases, the double dummy design is a technique for retaining the blind when administering supplies in a clinical trial, when the two treatments cannot be made identical (78)

The first design, which is from an example from the ICH Guidelines (79) involves

a 2-arm study where the first arm receives a study drug and the second arm receives another drug, that is, an active control drug (this study design does not involve any pla-cebo group):

l Arm A All subjects in arm A receive a round pill that is the active control drug

plus a flat pill that is the placebo

l Arm B All subjects in arm B receive a flat pill that is the study drug plus a round

pill that is the placebo

The second design, also shown below, is a 4-arm study, where two arms receive only drugs, and where the remaining two arms receive only placebo:

l Arm A Round pill study drug.

l Arm B Round pill placebo.

l Arm C Flat pill active control drug.

l Arm D Flat pill placebo.

76 Reck M, von Pawel J, Zatloukal P, et al Phase III trial of cisplatin plus gemcitabine with either placebo

or bevacizumab as first-line therapy for nonsquamous non-small-cell lung cancer: AVAil J Clin Oncol

2009;27:1227–1234.

77 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.

78 ICH Harmonised Tripartite Guideline Statistical Principles for Clinical Trials E9 Feb 1998 (46 pages).

79 ICH Harmonised Tripartite Guideline Statistical Principles for Clinical Trials E9 Feb 1998 (46 pages).