In order to invent a good drug for depression, researchers have been depressed for a long time.

Recently, the General Office of the National Health Commission issued the "Work Plan for Exploring Characteristic Services for Depression Prevention and Treatment" to create a social atmosphere that pays attention to the mental health of the entire population and supports and participates in depression prevention and treatment work.

Humans have been troubled by depression for a long time, and scientists have been working hard to develop drugs to relieve depression symptoms. It was not until the 1980s that a depression drug called "Prozac" came out, and depression patients got their first salvation. What bottlenecks did it have to break through in its birth? What failures did it encounter? How many bad lucks did the researchers go through and how many broken teeth did they have to bite to come up with this little pill?

Written by | Ji Zhi

In the development of psychiatry, some discoveries are crucial and have even fundamentally changed the face of the discipline. For example, Freud's psychoanalysis; Ugo Cerletti's invention of electroconvulsive therapy (ECT); John Cade's discovery of the application value of lithium salts as psychiatric drugs, etc. Among these game-changing discoveries, the famous "Prozac" undoubtedly occupies a firm place.

Prozac, whose main ingredient is Fluoxetine, is the first successfully marketed antidepressant of the Selective Serotonin Reuptake Inhibitor (SSRI) class. Its drug form is Fluoxetine hydrochloride.

Figure 1. Chemical structure of fluoxetine hydrochloride

Fluoxetine was discovered by Eli Lilly in 1972 and approved by the US FDA in 1987 for the treatment of depression, bringing hope to patients suffering from depression at the time. It was also from fluoxetine that SSRI antidepressants were gradually launched on the market, opening a new chapter in the drug treatment of depression and becoming the most widely used prescription antidepressant drugs. Thirty years have passed quietly, and more and more antidepressants based on new mechanisms and new targets have entered the market, but fluoxetine is still very commonly used in clinical practice. It is one of the essential drugs listed by the World Health Organization and an important drug that is indispensable to the basic health system [1].

The origin of the research on antidepressants can be traced back to the work of the pioneer Nathan S. Kline in the 1950s. Kline was the director of research at Rockland Hospital in Rockland County, New York. At that time, tuberculosis was rampant. While using anti-tuberculosis drugs to treat patients, clinicians accidentally discovered that such drugs had a magical psychological effect on patients - it could make patients show "unusual" happiness. The report attracted Kline's attention, and he began to conduct his own research on depression drugs. He and psychoanalyst Mortimer Ostow jointly published a report on this topic at the annual meeting of the American Psychiatric Association in 1957 [2], which was recognized by the academic community at the time. People began to believe that drug treatment could also bring hope to patients with depression. Since then, a new class of drugs - antidepressants - was born.

Early antidepressants were mostly tricyclic antidepressants (TCAs). This type of drug was first modified from anti-tuberculosis drugs. Its core structure is a seven-membered heterocyclic ring with a benzene ring on each side. It can inhibit the reuptake of norepinephrine (NA) and serotonin (5-HT) by the presynaptic membrane, increase the concentration of the two in the synaptic cleft, and prolong the time they act on the corresponding receptors, thereby exerting an antidepressant effect.

Representative tricyclic drugs include imipramine, amitriptyline, clomipramine, doxepin, desipramine, nortriptyline, and protriptyline (Figure 2). Among them, amitriptyline has a better effect on patients with depression accompanied by insomnia; imipramine is more ideal for patients with endogenous depression and menopausal depression, but has a poor effect on the depressive symptoms of psychosis; clomipramine is the first choice for the treatment of obsessive-compulsive disorder, and is also one of the drugs approved by the US FDA for the treatment of obsessive-compulsive disorder; doxepin has a better effect on various anxiety and depression states [3].

Figure 2. Common tricyclic antidepressants (TCAs)

Although tricyclic drugs have been proven to have antidepressant effects, they also have obvious toxic side effects. For example, they have a small therapeutic safety window, severe cardiac toxicity, anticholinergic effects (manifested as dry mouth, constipation, blurred vision, drowsiness, weight gain, etc.), and can cause postural hypotension. Therefore, scientists have been committed to developing drugs with fewer toxic side effects and better efficacy.

Physiological development points to new directions. The development of drugs is inseparable from the promotion of physiological research. In the early analysis of autopsy samples of patients with depression, scientists have found that the levels of 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxy-indoleacetic acid (5-HIAA) in the hindbrain of patients with depression who committed suicide were much lower than those of patients who died suddenly or had myocardial infarction [4, 5]. Does this mean that the level of 5-HT in the human body may be related to depression?

In 1969, a book titled The Structure and Function of Nervous Tissue, Volume III: Biochemistry and Disease was published. The book detailed the “latest” knowledge of central nervous physiology at the time [6] and elaborated on the synthesis, storage, release, decomposition and reuptake of 5-HT in the human body, which met the criteria for 5-HT as a neurotransmitter. At the same time, pharmacology also had evidence that, similar to NA, 5-HT was closely related to the pathology of depression. Inspired by this, scientists turned to looking for monoamine neurotransmitter reuptake inhibitors with less toxicity, hoping to improve depressive symptoms by specifically inhibiting the reuptake of 5-HT.

Now we know that 5-hydroxytryptamine (5-HT), also known as serotonin, is a monoamine neurotransmitter. Other neurotransmitters belonging to this family include dopamine (DA), norepinephrine (NE or NA), etc. These neurotransmitters can be synthesized in neurons and mediate nerve signal transmission, so they are also related to human emotional regulation.

With the development of biological disciplines, modern new drug design is often based on the identified targets, with the help of powerful computer-assisted functions, to design and synthesize effective lead compounds. Back in the 1960s, with limited physiological capabilities at the time, scientists were able to keenly observe the association between 5-HT levels and depression from the analysis of autopsy samples of patients with depression, and pioneered the concept of "selective reuptake inhibitors", which was indeed a remarkable achievement at the time.

What are reuptake inhibitors?

5-HT is synthesized in presynaptic neurons, released into the synaptic cleft, and binds to the receptor family on the postsynaptic neuron, thereby mediating the transmission of neural signals. During this process, the reuptake pump on the presynaptic neuron re-uptakes part of the free 5-HT, resulting in a decrease in 5-HT concentration.

SSRIs work by inhibiting the reuptake pump, which increases the concentration of 5-HT in the synaptic cleft. With a similar mechanism of action, tricyclic antidepressants can simultaneously inhibit the reuptake of NA and 5-HT by the presynaptic membrane, and are not selective; while fluoxetine only selectively inhibits the reuptake of 5-HT and does not affect the physiological effects of dopamine and norepinephrine, which is one of its advantages.

Figure 3. Mechanism of action of 5-HT and SSRIs [7]

Unsatisfactory structural modification In 1970, Eli Lilly's medicinal chemist Bryan Molloy and pharmacologist Robert Rathburn began to collaborate to develop a new antidepressant that would retain the therapeutic activity of tricyclic antidepressants (TCAs) while overcoming the cardiotoxicity and anticholinergic effects of TCAs. Based on early TCAs research, they used a drug-induced hypothermia model in mice to screen drugs and found that diphenhydramine and some other antihistamines also achieved the same effects as TCAs. Arvid Carlsson and colleagues at the University of Goteborg in Sweden observed that in addition to antagonizing histamine receptors, diphenhydramine could also inhibit monoamine uptake, which means that it may be effective in treating depression [8].

Based on the above findings, Molloy modified and optimized the structure of diphenhydramine and synthesized many analogs. In the first round of modification, they kept the side chain containing N-methylethylamine unchanged, and transformed the N,N-dimethyl-2-(diphenylmethoxy)-ethylamine structure of diphenhydramine into a phenoxyaniline core. They designed and synthesized a series of 3-phenoxyphenylamine compounds, and conducted pharmacological tests on these newly synthesized molecules in the Rathbun laboratory. It was found that the compound LY94939 (also known as Nisoxetine, translated into Chinese as "Nisoxetine") has the same activity as TCAs antidepressants. Subsequent in vitro target activity tests showed that Nisoxetine, like desipramine, has an inhibitory effect on the reuptake of norepinephrine, while its inhibitory effect on the reuptake of 5-hydroxytryptamine (5-HT) and dopamine (DA) is much weaker [9, 10]. Therefore, scientists continue to explore and try to find drug molecules that can inhibit 5-HT.

However, such exploration was not favored and even obstructed by many parties. For example, an academic expert in central nervous system research, when visiting Eli Lilly every quarter, advised them to give up the idea of ​​pursuing selective inhibitors of monoamine uptake, because other scientists at the time believed that enhancing the reuptake of 5-HT would not only fail to treat depression, but might cause depression. But the research team did not give up, and thanks to the support of Slater, an advisor to Eli Lilly's CNS (Central Nervous System) Research Committee, the project was finally not interrupted.

Fluoxetine: A Difference in the Slightest Thing We must remember is that medicinal chemistry is a very delicate discipline, and even if there are only slight differences in structure, the efficacy of two molecules can be very different. In this project, a slight change in structure can also change the molecule's selection of different neurotransmitters.

In 1971, another important figure who promoted the birth of fluoxetine, David T. Wong, joined the research team. Wong was born in Hong Kong, China, and was a famous biochemist at the time. His past scientific research experience made him very familiar with the role of 5-HT in mood regulation. With his encouragement, the research team designed and synthesized a second batch of more than 50 compounds, and screened these two batches of compounds again to find molecules that may selectively inhibit 5-HT reuptake. The research team re-tested the in vitro 5-HT, NA, and DA reuptake inhibitory activity of these two batches of compounds and found that the compound LY82816 (the oxalate form of fluoxetine) showed very good selectivity for 5-HT reuptake [11].

In fact, the structure of fluoxetine is very different from that of the compounds in the same series. The figure below (Figure 4) is a summary of the structure-activity relationship of this type of compound. We can see that when the two benzene rings in the 3-phenoxyphenylpropylamino compound are unsubstituted, its reuptake inhibitory activity (Ki) for 5-HT and NA is 102nM and 200nM respectively. After the introduction of a trifluoromethyl group (i.e., fluoxetine) at the para position of the phenoxy group, the 5-HT reuptake inhibitory activity increased by 6 times (Ki=17nM). Not only that, as mentioned above, fluoxetine also has strong target selectivity, which means that while fluoxetine inhibits 5-HT reuptake, it will not interfere with the normal function of other neurotransmitters.

Figure 4. Structure-activity relationship of a series of compounds during the modification process

Reconstructing the animal model Who knew that, unlike nisoxetine, fluoxetine showed no activity in the drug-induced hypothermia model of mice! It turned out that the animal model commonly used in antidepressant research during this period was based on early tricyclic antidepressants, and was therefore particularly suitable for drugs that inhibit norepinephrine (NA) reuptake, but could not show significant in vivo activity against fluoxetine. As a matter of urgency, researchers needed to establish a 5-HT reuptake inhibition model in vivo.

After continuous exploration, Wong and other scientists established three new models to evaluate the antidepressant activity of fluoxetine. These three animal models are the forced swim test for mice, the learning helpless model, and the olfactory bulbectomy model. In these three models, fluoxetine showed good effects, which eventually led to the recognition of fluoxetine. Since then, these three models have been widely used.

After many twists and turns in the clinical trials, in 1976, after all the safety tests in animals were completed, Eli Lilly submitted a clinical research application to the FDA for the first clinical trial of fluoxetine in humans. The first phase of the clinical trial went very smoothly, and fluoxetine showed good clinical safety, and was soon ready to enter the second phase: looking for effectiveness indicators for treating patients with depression.

At the time, Eli Lilly had other more advanced candidate drugs to examine, and the lack of experienced clinical researchers in psychiatry caused the Phase II clinical trial of fluoxetine to be deadlocked. With limited resources, the Phase II clinical trial could only be conducted on a relatively small scale at the beginning. However, the unfortunate thing happened again: in this small group of people, fluoxetine was found to be ineffective!

This was a huge blow to the entire project team. Fortunately, after analysis, the research team believed that the failure of the trial might be attributed to the type of volunteer patients - because these patients had not responded to other antidepressant therapies in previous treatments. Everyone cheered up and continued to move forward, restarting the trial in another group of depression patients, and finally achieved good results.

In 1981, the Phase III clinical trial of fluoxetine began and was successfully completed in 1983. The results showed that fluoxetine was effective in treating depression with mild side effects. The clinical results of these trials were compiled in more than 100 2-inch binders and submitted to the US FDA for marketing application. It took more than seven years from the first human administration to the completion of all trials and submission of materials [12].

But the test for fluoxetine and the research team was not over yet.

Almost at the same time as the drug rush, in 1982, Astra Pharmaceuticals was the first to launch Zimelidine, a drug for the treatment of depression, in Europe. Coincidentally, Zimelidine is also a selective 5-HT reuptake inhibitor (SSRI)[13]. Eli Lilly lost the first opportunity to launch SSRI to the market.

However, soon after Zimelidine was launched on the market, it was revealed that it could cause some rare side effects, especially flu-like syndrome. In order to avoid greater losses, AstraZeneca immediately terminated all research on SSRI. This undoubtedly gave Eli Lilly a ray of hope.

In October 1983, representatives of Eli Lilly's R&D team met with the FDA Advisory Committee, hoping that they could comprehensively evaluate the clinical research results of fluoxetine and the possibility of approval for marketing. Two years later, in October 1985, the FDA Advisory Committee held a meeting to discuss the application for fluoxetine's marketing. Although some people questioned that fluoxetine might produce a flu-like reaction similar to that caused by zimelidine, the members of the Advisory Committee believed that there was a clear difference in the structures of the two, and that this special side effect might be unique to zimelidine, and they unanimously recommended the approval of fluoxetine for the treatment of depression.

While the research team was anxiously waiting, on December 29, 1987, the FDA finally approved fluoxetine hydrochloride for marketing. In January 1988, Eli Lilly successfully launched the 5-HT reuptake inhibitor fluoxetine under the trade name "Prozac" for the treatment of depression (Figure 4). The pressure and doubts that the research team had endured for many years were finally replaced by joy at this moment.

Figure 5. Eli Lilly successfully launched the 5-HT reuptake inhibitor fluoxetine, with the trade name Prozac. Image source: https://images.app.goo.gl/8Lmz3h34kAts7iYr9

Fluoxetine has a definite efficacy and mild side effects. It has been widely popular since its launch, with sales increasing year by year. In 1988, global sales exceeded US$100 million, and in 1992, it exceeded the US$1 billion mark, becoming the first central nervous system drug to exceed the US$1 billion mark in annual sales. In 1999, sales reached US$2.613 billion, making it the sixth best-selling drug in the world. Fortune magazine listed fluoxetine as one of the "drugs of the century". After the patent expired in 2001, sales in the patented drug market declined significantly, but the total number of prescriptions for fluoxetine was not affected [14].

Fluoxetine's success has given new directions to major pharmaceutical companies. A large number of companies have joined the development of 5-HT reuptake inhibitors. Funding and the efforts of researchers have led to the entry of a series of SSRIs antidepressants such as paroxetine, sertraline, venlafaxine, and citalopram into the market in just a few years, bringing new hope to patients with depression.

Only faith Back to the beginning, why is the advent of Prozac one of the events that changed the development of psychiatry? In addition to its good antidepressant effect, more importantly, Prozac has reshaped the public's understanding of mental disorders to a certain extent, redefined the concept of clinical depression, and changed the direction of psychiatry from psychotherapy to biological treatment.

Because Prozac is very safe and has minimal risk of interaction with other drugs, doctors who were reluctant to prescribe tricyclic drugs before began to use drugs to treat patients with milder symptoms, which to a certain extent lifted the stigma of depression at the time and made more people dare to reveal their illness. Therefore, Prozac has undoubtedly become an iconic drug in the history of psychiatry, and its status is unshakable.

The successful launch of fluoxetine also left valuable experience for scientific researchers. The researchers went through various tests in 16 years, and each step was extremely difficult. Their successful experience provided a very good reference for the subsequent new drug research and development: initially, the lack of a suitable evaluation model led to the invalidity of fluoxetine in vivo animal testing (human in vitro testing); then, the company gave priority to the development of other drugs, resulting in the suspension of fluoxetine clinical research; at the same time, the wrong patient selection led to the failure of clinical research; finally, the side effects of similar drugs led to the company's decision to suspend research and development. If it were not for the researchers' tenacious perseverance and unremitting pursuit of truth, the research on fluoxetine might have died long ago. And this spirit is particularly valuable today when people are eager for quick success and instant benefits.

Figure 6. Two important scientists in the development of fluoxetine: Bryan B. Molloy and David T. Wong. Image source: Left: https://www.invent.org/inductees/bryan-b-molloy; Right: blogs.oregonstate.edu/)

The development of science is a process of continuous accumulation. The pioneers of neuroscience provided the physiological basis and pioneering design ideas, and the research team verified the ideas through in-depth thinking and unremitting efforts. They did not give up despite doubts and ridicule, and finally achieved breakthrough results. As David T. Wong said when he reviewed the development of fluoxetine thirty years later:

It is essential that ideas are steadily championed by passionate believers to achieve the final goal.

References

[1] https://apps.who.int/iris/bitstream/handle/10665/93142/EML_18_eng.pdf;jsessionid=9633BBC4D3838AC972A59899C442C306?sequence=1

[2] Loomer, HP, Saunders, JC, & Kline, NS (1957). A clinical and pharmacodynamic evaluation of iproniazid as a psychic energizer. Psychiatric Research Reports, 8, 129–141.

[3] Feighner JP (1999). Mechanism of action of antidepressant medications. The Journal of clinical psychiatry, 60 Suppl 4, 4–13.

[4] Shaw, DM, Camps, FE, & Eccleston, EG (1967). 5-Hydroxytryptamine in the hind-brain of depressive suicides. The British journal of psychiatry : the journal of mental science, 113(505), 1407–1411.

[5] Bourne HR, Bunney WE Jr, Colburn RW, et al. Noradrenaline, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid in hindbrains of suicidal patients. Lancet. 1968;2(7572):805-808.

[6] Bailey OT. The Structure and Function of Nervous Tissue, vol 3, Biochemistry and Disease. JAMA. 1970;212(9):1528–1529. doi:10.1001/jama.1970.03170220082030

[7] Wong DT, Perry KW, Bymaster FP. Case history: the discovery of fluoxetine hydrochloride (Prozac). Nat Rev Drug Discov. 2005;4(9):764-774. doi:10.1038/nrd1821

[8] Molloy, BB, Wong, D. T. & Fuller, R.W. The discovery of fluoxetine. Pharmaceutical News 1, 6–10 (1994).

[9] Wong, DT, Horng, JS, & Bymaster, FP (1975). dl-N-methyl-3-(o-methoxyphenoxy)-3-phenylpropylamine hydrochloride, Lilly 94939, a potent inhibitor for uptake of norepinephrine into rat brain synaptosomes and heart. Life sciences, 17(5), 755–760. https://doi.org/10.1016/0024-3205(75)90531-7

[10] Wong DT, Bymaster FP. Effect of nisoxetine on uptake of catecholamines in synaptosomes isolated from discrete regions of rat brain. Biochem Pharmacol. 1976;25(17):1979-1983. doi:10.1016/0006-2952(76)90053-8

[11] Wong DT, Bymaster FP, Horng JS, Molloy BB. A new selective inhibitor for uptake of serotonin into synaptosomes of rat brain: 3-(p-trifluoromethylphenoxy). N-methyl-3-phenylpropylamine. J Pharmacol Exp Ther 1975;193(3):804-11

[12] Wong DT, Bymaster FP, Reid LR, Threlkeld PG. Fluoxetine and two other serotonin uptake inhibitors without affinity for neuronal receptors. Biochem Pharmacol. 1983;32(7):1287-1293. doi:10.1016/0006-2952(83)90284-8

[13] Bengtsson BO, Wiholm BE, Myrhed M, Wålinder J. Adverse experiences during treatment with zimeldine on special licence in Sweden. Int Clin Psychopharmacol. 1994;9(1):55-61. doi:10.1097/00004850-199400910-00009

[14] https://www.psychologytoday.com/us/blog/freud-fluoxetine/202003/the-story-prozac-landmark-drug-in-psychiatry