Evolution of the Homo Sapiens and His Mutant, the Clinical Investigatoriens1

This story was told to us by Dr A Sankaranarayanan, a teacher, a senior colleague and a friend. A boy was preparing for his exams and he decided to memorize an essay on a cow, expecting it in the examination. However, in his examination, he got an essay on a coconut tree. The boy, though not too great in linguistic skills, had a keen sense of manipulation. The essay went like this: ‘There was a coconut tree. There was a cow tied to the coconut tree. A cow is a very useful animal. She gives us milk…………….’2

How is this story related to the book? Actually, we were planning to write a guide for clinical investigators (the cow), when we were asked to write a book on clinical research (the coconut tree). We assure you it is not the boy trick that we will be indulging in but at places, we will be compelled to tie the cow to the coconut in order to see things in right perspective.

A more comprehensive definition of clinical research is given by the Association of American Medical Colleges Task Force on Clinical Research¹ as follows:

There is a tendency to think that to conduct research you need the latest equipment and expensive chemicals. In our opinion, nothing can be farther away from the truth. There is also a tendency to use the term ‘clinical research’ only in the context of new drug development, done at present mainly by the pharmaceutical industry. This term has broader implications than that and we feel that it should be used more widely to imply any research in humans.

Identifying novel antibiotics using microbial genomics in a laboratory of a drug company is research, as is a comparison of body weights and waist circumferences of policemen (Punjab Police, please excuse) with age-matched persons from the general population is also research. While the first requires highly qualified and experience critical mass of investigators working in sophisticated labs, the second may be conducted by a student of Biostatistics using a measuring tape and a weighing machine. These are examples of extreme levels of complexity of research but most of the research is conducted at intermediate levels.3

At this point we would like to return to the above definition and try to explain the term ‘clinical trial’. It is defined as ‘A systematic study of a pharmaceutical product on human subjects (patients or healthy volunteers) in order to discover or verify the clinical, pharmacological (including pharmacodynamics/pharmacokinetics), and/or adverse effects, with the object of determining their safety and/or efficacy’.² The term clinical trial also encompasses such systematic studies done to see the usefulness of a new surgical technique or any diagnostic or therapeutic procedure. The word ‘controlled’ is often added to signify that there is another group (the control group) with which the treatment group will be compared. Controlled clinical trials can be considered as a subset of broader term ‘clinical research’.

Although the term “clinical research” was probably used in its modern context by RM Glickman in the 1980s, the concepts of medical research are as old as man himself. According to a recent article,³ the earliest clinical research is mentioned in First Kings, Chapter 18, verses 21 to 24 when Elijah tried to show that his God was real and not his opponent Baal's. He called upon the people to dress a bullock each and instructed them to keep the dressed bullock on a heap of wood. It was agreed that the deciding test for God's realness would be God's capacity to light the fire on being evoked to do so. While Baal's heap of wood remained unlit, Elijah's Lord ignited the wood and proved his realness.

Your devilish streak may be prompted to ask, ‘Could the wood have caught fire on its own, that is, without God's intervention?’ Indeed, you are on the right track of scientific thinking. Had Elijah actually considered this possibility, he would have considered improving the study design, say, by having another group of ‘No God’ (the placebo group) along with the two groups with different Gods (the active treatment groups) and that would have answered your question.

Climbing down from the mysteries of the supernatural to the ordinary world of humans, the first systematic clinical trial was probably conducted in 1747 by James Lind, a surgeon in the British Royal Navy. Scurvy was a major health problem for British navy. Lind carried out his classic trial by dividing twelve sailors into six groups. The various groups received supplements of vinegar, sulfuric acid, cider, seawater, a mixture of nutmeg, horseradish and garlic or two oranges and a lemon. In addition, identical diets were served to all of them. Only the sailors of the last group who received two oranges and a lemon did not develop scurvy. Not only did Lind prove that a dietary supplement of oranges and lemons prevented the development of scurvy, he was also able to prove that acid (vinegar and sulfuric acid) did not have a role to play in prevention of scurvy, as was the common belief those days. The purists will find a number of drawbacks in this study by the current standards, like lack of a proper protocol, lack of randomization, blinding and above all a small sample size. Nevertheless this study certainly can be recognized as the first modern clinical trial.

This was however, an isolated example of a classical clinical trial that included concurrent control groups (sea water, acid) and most of the practice 4of medicine in those years was based on personal experiences of reputed senior professors, which was passed from one edition of a textbook to another and from one book to another. The distinction between research and treatment was not very clear. Much of the treatment was experimental in nature.

While telling the story of Elijah, we had seeded you with the possibility of having an additional group of ‘No God’ or no ‘intervention’. It was to answer the very legitimate question which many of you would have thought- could the wood have caught fire without assistance from God? Use of placebo or an inert substance matching in appearance to the substance being investigated, was meant to answer such a question only. The first documented use of allocation of placebo to a control and a treatment group (by alternating patients) is credited to Fibiger, who, in 1898, used it for the treatment of diphtheria.⁴ What Fibiger did was to give placebo or the investigational agent to alternate patients. However, his design lacked the two techniques now recognized as the most significant advancements in the design of clinical trials: randomization and blinding.

In 1933, Evans and Hoyle published the first placebo-controlled trial in patients with angina in the Quarterly Journal of Medicine. They subjected 66 patients of angina to various treatment periods, that of placebo or the active treatment. A good 37.5 percent of the patients reported improvement while on placebo. The investigators were thus able to demonstrate what can be called the placebo effect and were able to validate their statement: “The value of remedies in relieving anginal pain cannot be judged unless the observations are properly controlled.”

Randomization as a method of allocation of treatments to different patients was not recognized until the late 1930s, although the technique was used by Fisher in 1926 for agricultural experiments. It was Professor, later Sir Austin Bradford Hill, who, in a series of articles for the Lancet in 1937, argued for the use of this technique. He used this technique later in a trial of streptomycin in tuberculosis patients.

Fold and coworkers, in 1937, showed the importance of observer bias and introduced the concept of blinding, when they published their study on the effect of xanthines in the treatment of angina. In their study, they had to question the patients and in order to eliminate the bias arising out of leading questions, the investigators refrained themselves from informing which patient got which agent.

The three disciplines that contributed immensely to the modernization of clinical research in those years were chemistry, pharmacology and statistics. It is interesting to note how developments in each of these specialties occurred either concurrently or just one after another. For instance, isolation of morphine (the active ingredient) from opium by Seturner in 1815 provided the investigators with an opportunity to use a particular dose of a compound and observe its effect thereby leading to formulation of one of the most fundamental principles of pharmacology: the ‘dose-response relationship’. The development of analytical chemistry enabled the scientists to elucidate another important issue: the ‘structure activity relationship’.5

However, despite tremendous achievements in other areas like physiology and therapeutics along with the ones mentioned above, clinical research lacked something. Most workers were not willing to accept that the difference between two drugs could be due to chance and not due to the different treatments. Protopharmacology was the term used to describe events that occurred after medication was taken and it assumed that medication caused the event. The conversion of such thinking into “statistical” thinking took several decades.

Now we know that science is statistical. In other words, science tells us how things happen within certain limits and what is the likelihood of their happening within limits again. Statistics tells us what these limits are and the probability and frequency of a similar thing happening again. Professor Bradford Hill presented basic principles of experimental design in a series of articles in 1937, which were later reprinted in the form of a textbook on medical statistics. Smith and coworkers can be credited with the first clinical trial to use a formal statistical analysis. It was a study of antibody production after yellow fever vaccination by two different methods. It was in 1966 that Schor and Karton legitimized the now well-known criterion of p<0.05 for a difference between two groups to be considered not due to chance. Statistics will be discussed in detail in Part B.

The discoveries of diphtheria and tetanus antitoxins in France and Germany in the 1890s provided the companies with biological sources of treatment that were useful for the patients and were therefore marketable. Despite these successes, the pharmaceutical industry of this time still did not invest majorly in research. The initial attempts of pharmaceutical companies for carrying out research were limited to the fields of quality, purity and quantity. Drug-related research was mostly carried out in academic institutes.

World War II changed the scene with pharmaceutical companies joining hands with the governments for drug-related research and production. Production of dried plasma and penicillin was the result of such efforts. Initially, the efforts of the industry were mainly directed towards production of antibiotics. The period between 1960 to 1980 is considered as a period of industry maturation. The pharmaceutical industry started concentrating on research for conditions other than infective conditions. Oral contraceptives, antihypertensives, nonsteroidal antiinflammatory agents (NSAIDs), anxiolytics, and several others were borne out of this change in attitude.

Two factors were mainly responsible for these changes: (i) development of several governmental/institutional regulations for research in humans and 6(ii) advancement in and integration of fields like analytical chemistry, medicinal chemistry, immunocytology, genetics and above all, statistics. Research in medicinal chemistry has made it possible for generation of libraries of thousands of compounds. Assay techniques like immunocytology, immunofluorescence, high performance liquid chromatography and now development of gene and protein arrays have made it possible for researchers to carry out targeted drug development. Mechanization and incorporation of software technology has made it possible to screen several thousand compounds in a relatively short period of time. These developments have been the basis of high throughput screening techniques in drug development process.

Added to this is the refinement in clinical trial designs, the increasing trend of conducting multicentric trials, backed with efficient data handling systems and the ongoing universalization (harmonization) of regulatory requirements have all ensured that the new drug development process is highly streamlined and efforts to reduce this time are gaining momentum.

These advances, accompanied with recognition of ‘Clinical Pharmacology’ as a separate specialty, catapulted the growth in the field of clinical research with the result that the pharmaceutical industry today is amongst the richest, well-connected, influential and most profit-making industries. Most of the drugs are now introduced by pharmaceutical industry with hardly any new drug development occurring in the academia. The cost of new drug development is one important factor that has contributed to this change. As research gets more and more sophisticated, as guidelines for the conduct of clinical research become more and more stringent, the cost of new drug development keeps on increasing.

The cost of new drug development is estimated to be between 0.8 to 1.3 billion dollars which is nearly five times the cost in 1970's. This cost includes the costs of failures; out of 5000 compounds tested only 5 reach the stage of human testing. Of these only one may become a successful marketed drug. The process of new drug development is also time-consuming: it takes about 10–15 years for a chemical to reach the market as a drug. The number of new drugs which got FDA approval in 2006 has decreased dramatically to approximately 13 from a value nearing 200 nearly a couple of decades back.⁵ Once a new drug gets introduced in the parent country, the company tries to market it in other countries; there is generally some delay in this process and this is termed as “drug-lag”. In India, the drug-lag has been declining over the past few years and is currently less than one year.⁶

Therefore, it is now realized that clinical research is not merely about new drug development, it is also about assessment of safety of drugs especially in the postmarketing phase. There are several examples of how unsafe drugs have been marketed and billions of dollars earned. What follows below is the story of a novel type of drugs known as cyclooxygenase (COX)-2 inhibitors which were launched with a lot of promise of gastrointestinal safety as compared to the older NSAIDs. This story is being told to emphasize upon the following things: (i) Pharmaceutical companies can use their influence quite 7effectively; (ii) The drug regulatory bodies, including the top of the mill kinds like the US FDA can and do make mistakes; (iii) It is important to pay attention to the conflicting viewpoints that keep emerging; (iv) Clinical research is an ongoing process, and (v) Dissemination of information is as important as research itself.

The COX-2 Inhibitor Story (Based on an article “COX-2 inhibitors - a CLASS act or just VIGORously promoted” by the authors published in Medscape Gen Med 2004;6:e37;Reprinted with permission from Medscape.Com).

[In 1990, Fu et al⁷ detected a novel COX protein in monocytes stimulated by interleukin and a year later Kujubu et al⁸ identified a gene with considerable homology to COX-1. Further research demonstrated that this novel protein (termed COX-2) was an inducible enzyme with increased expression in inflammation. On the other hand, COX-1 was named a “housekeeping” enzyme as it was expressed constitutively, with relatively ubiquitous presence. It was also recognized as the main source of cytoprotective prostaglandins in the gastric mucosa. Since the conventional NSAIDs inhibited both COX-1 and COX-2, it was postulated that the efficacy of NSAIDs (attributable to COX-2 inhibition) could be achieved without gastrointestinal toxicity (due to COX-1 inhibition).

This realization rekindled the efforts of the pharmaceutical industry to produce safer NSAIDs via selective inhibition of COX-2, and this class of agents (celecoxib and rofecoxib) was introduced in 1999. By October 2000, celecoxib and rofecoxib had sales exceeding 3 billion in the US alone and a prescription volume in excess of 100 million for the 12-month period ending in July 2000.⁹ Moreover, the sales of celecoxib alone increased from US$ 2623 million in 2000 to US$ 3114 million in 2001.¹⁰ Most of the credit for this more than 80 percent increase in sales could be attributed to an extensively publicized and widely distributed study – the Celecoxib Long-term Arthritis Safety Study (CLASS), published in JAMA in 2000.¹¹ The impact of the study can be gauged from the fact that about 30,000 reprints of CLASS were bought from the publisher and it was cited more than 10 times as commonly than any other article published in the same issue.¹² No less influential was another trial – VIGOR (VIOXX Gastrointestinal Outcomes Research), a double-blind trial conducted at 301 centers in 22 countries.¹³ Both these trials concluded that COX-2 inhibitors were associated with significantly fewer adverse effects than the conventional NSAIDs.

However, the progress that we have made in science is because scientists tend to question everything, and not surprisingly, this hypothesis began to show cracks when, in the late 1990s, it was shown that within 40 minutes of oral challenge with acid, there was a marked upregulation of COX-2 in the rat stomach.¹⁴ Several subsequent studies demonstrated a crucial protective role for COX-2 and provided evidence that COX-2 contributes to mucosal defense.^15,16 This realization led some workers to postulate that inhibition of COX-2 may delay ulcer healing and cause exacerbation of inflammation.^17,18 Several workers, including ourselves, showed worsening of gastric ulcers or necrosis in the small intestine in the presence of altered gastric mucosa.^19-21

As the debate on the toxicity of COX-2 inhibitors intensified, in November 2001, JAMA published two letters^22,23 drawing attention to the fact that the conclusions given in the CLASS trial differ from the complete information that was available to the US Food and Drug Administration (FDA).²⁴8

The CLASS study, funded by the manufacturers of celecoxib, compared celecoxib, 800 mg/day with ibuprofen, 2400 mg/day and diclofenac, 150 mg/day in patients with osteoarthritis or rheumatoid arthritis. The two primary endpoints were the incidence rates for upper GI complications (bleeding, perforation or obstruction) and symptomatic ulcers during the first six months of treatment. It was concluded that celecoxib was associated with a lower incidence of symptomatic ulcers and ulcer complications combined as compared to the traditional NSAIDs. CLASS was reported as a single trial whereas actually it was the combined analysis of two separate trials with protocols that differed markedly from each other in design, outcomes, duration of follow up and analysis; moreover, both the trials were of longer duration. Two comparisons planned were celecoxib with ibuprofen and celecoxib with diclofenac with the primary endpoint being ulcer related complications (and not symptomatic ulcers as reported) and a follow up of 15 months (for complications) and 12 months (for ulcers) in both the trials. Analyzed accordingly, the number of ulcer-related complications were similar in both the groups and almost all the ulcer complications that had occurred during the second half of the trials were in the celecoxib group.²⁵ Moreover, using a preplanned (by the FDA) definition of ulcer-related complications, a non-significant trend in favor of diclofenac was observed.²⁶ Even more disturbing was the fact that, though these results were available at the time of submission of the manuscript, no mention was made in the publication. In addition, when the above-mentioned contradictions were pointed out, the authors’ replies²⁷ failed to refer to these results.

The other “landmark” trial, VIGOR, recruited rheumatoid arthritis patients to either 50 mg/day rofecoxib or 500 mg/day naproxen twice daily. The primary endpoint was confirmed clinical upper GI events (gastroduodenal perforation or obstruction), upper GI bleeding and symptomatic gastroduodenal ulcers. After a median follow-up of 9 months, it was shown that rofecoxib was associated with a significantly fewer clinically important upper GI events than naproxen. Like in the case of the CLASS trial, the US FDA reviewed the VIGOR study data and presented it on its website.²⁸ It was shown that when all serious adverse events are included (and not just GI events), naproxen treated patients had fewer serious events as compared to rofecoxib (7.8% versus 9.3%, RR 0.81, 95% CI 0.62-0.97). The cumulative risk of developing serious cardiovascular thrombotic events (mainly myocardial infarction) was 1.7 percent in the rofecoxib group compared to 0.7 percent in the naproxen group. Moreover, there were significantly more withdrawals in the rofecoxib group as compared to naproxen group, and they were due to hypertension, edema, hepatotoxicity, heart failure or pathological laboratory findings. These safety data as well as their analyses were subject of the design and protocol of the VIGOR trial, but were not provided in the publication, only the GI data favoring rofecoxib was mentioned. The Editor's note to the publication²⁹ mentioned that “……… 11 of the 13 principal authors have had financial associations with the company, which sponsored the study……………….. The other two principal authors are………………. employees of …….. (the company)”.

Incidentally, the cardiovascular adverse effects of COX-2 inhibitors were also not unexpected. It was postulated, even before their introduction in 1999, that COX-2 inhibitors may decrease vascular prostacyclin (PGI₂) production and may adversely 9affect the balance between prothrombotic and antithrombotic eicosanoids.³⁰ Unlike the platelet inhibition afforded by COX-1 inhibitors, COX-2 inhibitors do not have this property. In contrast, by decreasing vasodilatory and antiaggregatory PGI₂ production, COX-2 antagonists may tip the balance in favor of prothrombotic eicosanoids (thromboxane A₂) and may lead to increased cardiovascular thrombotic events.³¹

It was thus not surprising that within less than one year of their marketing, several cases of ischemic complications in patients receiving COX-2 inhibitors were reported. Moreover, as predicted, urinary levels of a metabolite of thromboxane A₂ were markedly elevated. However, since most of the patients receiving them had connective tissue diseases, which themselves predispose to thrombosis, the generalizability of these results was questionable. Later, a meta-analysis showed that serious thrombotic cardiovascular effects were found to be twice as high with rofecoxib as with naproxen (RR 2.38; 95% CI 0.39 to 4.00).³² Moreover, the FDA analysis of the VIGOR data showed that the increased risk in serious thrombotic cardiovascular events was seen in patients in whom aspirin was indicated (RR 4.89; 95% CI 1.41 – 16.88) as well as in those in whom aspirin was not indicated (RR 1.89; 95% CI 1.03 – 3.45).

Moreover, COX-2 inhibitors were also shown to increase BP,³³ and more patients in the VIGOR trial developed hypertension in the rofecoxib group compared with the naproxen group. There was a 4.6 mmHg increase in the mean systolic BP and 1.7 mmHg increase in the diastolic BP in the rofecoxib group as compared to a 1.0 and 0.1 mmHg increase in systolic and diastolic BP, respectively with naproxen. It is well-established that a 2-mmHg reduction in diastolic BP results in about a 40 percent reduction the rate of stroke and a 25 percent reduction in the rate of MI, which makes the cardiovascular adverse effects of selective COX-2 inhibitors even more important.

The striking parallels - misleading presentation of the VIGOR data and the data manipulation in the CLASS trial provided insight into the growing role of commercialization of the whole process of drug development. Quick, favorable results and rapid entry into the market seem to be the main driving force for the drug companies. Not surprisingly though, for each day's delay in gaining approval, the manufacturer may lose, on an average, US$ 1.3 million.³⁴ Unfortunately, such examples are not uncommon; Stelfox et al, found that authors whose work showed drugs to be safe/effective had a higher frequency of financial relationships with the drugs’ manufacturers than authors whose work did not.³⁵ Finally, with increasing reports on cardiac adverse effects, the company voluntarily withdrew rofecoxib in 2004. The story interestingly does not end here. In early 2005, an FDA advisory panel recommended that the company be allowed to resume sales of rofecoxib (Vioxx). This surprising, to say the least, decision came on the same day that the panel also voted to recommend allowing two other COX-2 inhibitors celecoxib (Celebrex) and valdecoxib (Bextra) to remain on the market, but with stronger warning labels. This 10good news led these companies’ shares to rise sharply, by more than 11 percent and 5 percent, respectively.

As a result, in May 2005, a new center was established to look at adverse drug reaction monitoring data and this center would be independent of the FDA committee that looks after the approval process.

The whole of the history of the drug industry is characterized by a three-step sequence, use of a drug → mishap → a new law. The US Pharmaceutical industry provides a classical example of these sequences (Table 1.2.1).

During the passage of one of these Acts, President William Howard Taft had noted: “There are none so credulous as sufferers from disease. The need is urgent for legislation that will prevent the raising of false hopes of speedy cures of serious ailments by misstatements of facts as to worthless mixtures on which the sick will rely while their disease progresses unchecked.” This statement, said many years ago, remains valid even today.

While most of these developments followed the sequence of use of a drug → mishap → a new law, there have been other notable developments in regulations which were not related to any major damage. One of these needs particular mention, the FDA Modernization Act (FDAMA) of 1997. This Act permits FDA to approve a marketing application for a new drug (or biological) product on the basis of adequate and well-controlled trials establishing that the drug (or biological) product has an effect on a surrogate endpoint that is reasonably likely, based on epidemiologic, therapeutic, pathophysiologic, or other evidence, to predict clinical morbidity. Details of this Act may be obtained from the following URL: www.fda.gov/opacom/7modact.html.

The FDA also launched a fast track program designed to facilitate the development and expedite the review of new drugs that are intended to treat serious or life threatening conditions and that demonstrate the potential to address unmet medical needs (Section 506).11

Another important landmark was the birthing of ICH, the International Conference on Harmonization. The need for harmonization arose from rapid increase in laws, regulations and guidelines for testing safety, quality and efficacy of new products; regulatory agencies differing in their technical requirements for similar guiding principles; duplication of time consuming and expensive testing, globalization of pharmaceutical industry and medical research and delays in new drugs reaching beneficiaries around the world. Since 1991, the European Union, the United States, and Japan have been collaborating in the government- and industry-sponsored International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use. European Commission, European Federation of Pharmaceutical Industries’ Associations, Ministry of Health and Welfare of Japan, Japan Pharmaceutical Manufacturers Association, FDA and Pharmaceutical Research and Manufacturers of America were the key founder members of ICH. WHO, Canada and European Federation of Pharmaceutical Industries’ Associations acted as observers.

ICH working groups have issued guidelines addressing various aspects of clinical research, including data management, reporting of research findings, safety of subjects, and research on special populations. The ICH process is a five-step process of consensus building, start of regulatory action, regulatory consultation, adoption of a tripartite harmonized text and finally its implementation. These guidelines are available as Common Technical Documents (CTD) and can be obtained the Internet at www.ich.org. The topics are broadly categorized into the following—Quality (Q), Safety (S), Efficacy (E) and Multidisciplinary (M). Within each of the above, the topics are further subdivided. The Quality section of the Common Technical Document (M4Q) provides a format for presenting data pertinent to Chemistry, Manufacturing and Controls (CMC) information in a registration dossier. The table of contents includes sections on Drug Substance and Drug Product. Provision has been made for including region of specific information. A new section on Pharmaceutical Development has been included to replace the Development Pharmaceutics Report (currently a part of the EU submission requirements). Addition of Quality Overall Summary, a new document, has been a recent improvization in this section.

The CTD Safety (M4S) Guideline delineates the structure and format of the nonclinical summaries and nonclinical study reports in Modules 2 and 4, respectively. A nonclinical overview, not exceeding 30 pages covers the integrated and critical assessment of the pharmacologic, pharmacokinetic, and toxicologic evaluation of the new chemical entity (NCE).

Within nonclinical summaries, thirty-four templates are provided for the preparation of the Nonclinical Tabulated Summaries, and 31 example tables are provided. 13

CTD-Efficacy (M4E) describes the structure and format of the clinical data in an application, including summaries and detailed study reports. There are two high level clinical summaries in Module 2 of the CTD: the Clinical Overview, a short document that provides a critical assessment of the clinical data; and the Clinical Summary, a longer document that focuses on data summarization and integration. Clinical Study Reports and raw data (where applicable) are included in Module 5 of the CTD.

In conclusion, clinical research, like other sciences of modern era, is an evolving science. There was a time when a blow on the head was used as a method of producing general anesthesia. The modern clinical research has evolved from this kind of primitive thinking to become a most powerful weapon of mass preservation. This has become possible because the investigators have been prepared to learn form their past mistakes and to change. Clinical research is now conducted based on well-formulated scientific principles, conducted according to certain well-tested guidelines and is now more of teamwork in which besides clinicians (specialists or super specialists), clinical pharmacologists and biostatisticians play equally important roles. It is extremely unlikely that we would see disasters of the magnitude of the thalidomide or the sulfanilamide tragedy today although the COX-2 inhibitor story tells us that we should remain vigilant.