05 November 2015

Biomedicine 15 years ago and today

Expectations and reality

Ilya Yasny and Peter Vlasov, XXII CENTURY 

All technologies (and biomedical ones are no exception) go through the stage of discovery, increased expectations, often a decline, and then either implementation (and a more sober attitude to them), or oblivion if expectations were not met. We decided to look about 15 years into the past (that's how much biomedical technologies need to go through the described path) in order to figure out which of the supposed "breakthrough" technologies have begun to bring real benefits today, and which have not yet reached the stage of practical implementation.

Principles of topic selectionThe articles on biology/biotechnology/medicine selected by the indexer of scientific publications Web Of Science according to the following criteria are taken:

  • publications were published in 1995-2005 .,their topic section contains the terms "perspective therapy", "breakthrough therapy", "new therapeutic modalities" and the like,
  • the publications were sorted by citation (for all the time since the release) in the sources included in the Web Of Science Core Collection – and it was the most cited works that determined the list of topics for our consideration.
  • RNA interference
Expectations

The discovery in the late 1990s of the phenomenon of the RNA interference mechanism (RNAi), which consists in blocking the synthesis of a protein with a certain RNA matrix in the case of the selection of a specific (complementary to the target matrix) RNA fragment ("antisense" RNA), was an inspiring event for biomedicine.

It seemed that the selection of the right interfering agents would allow us to disable any genes – and hence their corresponding proteins – in any cells of the body. And the specificity of the interaction of relatively long sections of nucleic acids will ensure high safety. This paradigm was projected, among other things, on the prospects for the treatment of almost any diseases by disabling certain (key) proteins – be it external pathogens (bacteria and viruses) or the cells of the person himself (for example, cancer).


Figure 1. Schematic representation of RNA interference. Interfering RNA (siRNA) penetrates into the cell, combines with proteins into the RISC complex, then it binds to the target matrix (informational) RNA (mRNA) and subsequent degradation of mRNA. This prevents the synthesis of the protein encoded by the target mRNA.Subsequently, serious problems were identified with the targeted delivery of RNA interfering molecules.

One of the problems of using RNA as a therapeutic agent was its low stability in blood plasma, where numerous enzymes lead to rapid degradation of nucleic acids. Another problem is the need to deliver RNA inside the cell, since a highly charged molecule cannot pass through the lipid bilayer of the outer cell membrane. 

The solution, which looks very promising, was chosen quite quickly – the use of viral carriers specifically "targeted" at the desired cells. 


Another way is to pack RNA into nanoparticles that would protect it from destruction by enzymes and ensure penetration into cells.

However, most of the drugs that reached phase 3 of clinical trials were ordinary RNA molecules protected from degradation by chemical modification enzymes.

RealityThen the developers were waiting for disappointment after disappointment.

It turned out that RNA drugs are too toxic. Even a length of 20-30 nucleotides is not enough for complete selectivity with respect to the target RNA, and among the three billion pairs of nucleotides in the human genome, there are bound to be other targets, binding to which causes unpleasant side effects. In addition, some degradation of RNA still occurs, and short ones are even more likely to cause side effects. As a result, almost all the few drugs that reached the market were withdrawn from it, and now there are only two drugs at the registration stage that treat Duchenne muscular dystrophy – a rare disease associated with dysfunction of the protein dystrophin, a component of muscle fibers. These RNAi agents are not classical interfering RNAs - they bind to the mRNA encoding dystrophin and cause the ribosome to skip the non–functioning site. As a result, functionally active dystrophin begins to be synthesized in the cells of sick people, and the symptoms of the disease are relieved.

Perhaps in the future, problems with non-specific RNA binding and insufficiently targeted delivery will be solved and we will see more specific RNAi-based drugs.

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Magnetic nanoparticles in cancer therapyExpectations

Soon after the technological breakthroughs that made it possible to create nanoscale objects and operate with them, it turned out that nanoparticles – especially iron oxides and some other metals – have the highest penetrating ability against the cells of living organisms.

It was assumed that cancer cells are capable of more active – in comparison with healthy cells – absorption of such particles. If the particles are made of a conductive material, then the subsequent exposure to a powerful high-frequency electromagnetic field causes a strong heating of the particles, which destroys the cells containing them. It should be noted that heating of unwanted cells/tissues – the so–called hyperthermia - has been used in clinical practice before, including in oncology, but it was with the advent of nanomaterials that this method of therapy began to look very promising.

[4]: Latorre M, Rinaldi C, Applications of magnetic nanoparticles in medicine: magnetic fluid hyperthermia // P R Health Sci J. 2009.

RealityTo date, it can be stated that this approach has not yet caused a revolution in cancer treatment.

As is often the case, the results of research on cells and on animals are poorly transferred to humans, although some successes in early clinical studies have been achieved: 

[6]: Johannsen et al., Magnetic nanoparticle hyperthermia for prostate cancer // Int J Hyperthermia 2010

The main problem of clinical application was the indiscriminate penetration of nanoparticles into the tumor, uneven or incomplete penetration of particles into the tumor and the complexity of standardization of the procedure itself. It turned out that for the selective delivery of particles to the tumor, it is not enough to simply introduce them into the bloodstream, you either need to inject them directly into the tumor, or supply them with antibodies that will direct them to the tumor. Both improvements have their drawbacks and add complexity to the procedure.

[9]: Dutz S, Hergt R. Magnetic particle hyperthermia--a promising tumour therapy? // Nanotechnology 2014

As a result, at the moment, other methods of cancer treatment, some of which will be discussed below, have turned out to be more promising and are developing faster.

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Humanized antibodiesSeveral therapeutic solutions are presented below, united by the unity of the approach: after choosing a specific protein target, whose function is important in regulating the biochemistry of a certain disease, antibodies are selected for this protein, that is, protein molecules that specifically bind to the target, block its function, change the course of biochemical reactions and – ideally – stop the development of the disease.

 

Attempts to treat various pathologies with antibodies have been made since the 1960s, however, the use of this approach in therapy was crowned with success only after the appearance of methods that "substitute" a noticeable part of them for human antibodies in grown antibodies - otherwise, antibodies obtained not in the human body themselves cause an immune response in a patient whose immune system recognizes antibodies as a foreign (and therefore probably harmful) protein object. That is why antibodies modified by "substituting" their "building blocks" for human ones were called "humanized" – "humanized". Currently, approaches have been developed and applied to obtain completely human antibodies "in vitro", bypassing the stage of animal immunization.
  • Antibodies to VEGFExpectations
Bevacizumab, a drug that later received the medical name Avastin, was the fruit of attempts to block the activity of a protein – vascular endothelial growth factor (VEGF), long chosen by scientists as a promising target in cancer therapy.

The fact is that the mentioned protein is a key regulator of vascular growth, angiogenesis – and this process is actively underway during the development and metastasis of cancerous tumors.


RealityAfter entering the market, the drug became a "blockbuster" (that is, its annual sales exceeded $ 1 billion) and entered the standards of therapy for a number of late-stage oncological diseases: colon and rectal cancer, lung, kidney, ovary, breast and some others.

Studies of Avastin in various combinations are actively continuing. However, it is now clear that, despite the fact that in some patients Avastin causes a stop in tumor growth or even its regression, then some of them have a powerful relapse, which is no longer treated. Apparently, under the action of Avastin in a tumor deprived of nutrients, the selection of the most malignant populations of cancer cells takes place. In addition, the use of Avastin is associated with quite severe side effects (since VEGF plays a role in the normal functioning of blood vessels and wound healing) – thrombosis, hypertension and bleeding, especially in the gastrointestinal tract. Due to the fact that the risk of side effects outweighed the potential benefit in women with breast cancer, the FDA does not recommend using Avastin for this indication anymore.
  • Antibodies to EGFRExpectations
The epidermal growth factor receptor (EGFR) became a target for cancer therapy when it turned out that overexpression or simply increased activity of this protein (for example, due to mutations) sends a signal to cells about unlimited division, and that it is this signaling cascade that is activated in many types of cancer.

The first widely used drug-blocker of the discussed target was Cetuximab (Cetuximab), also known by the trade name Erbitux.


RealityErbitux and its later analogue Panitumumab (Vectibix, Vectibix) have occupied a fairly large niche in the therapy of cancers expressing EGFR, primarily in head and neck cancers, as well as in colon and rectal cancers.

This approach proved ineffective in the treatment of lung and pancreatic cancer. The use of anti-EGFR antibodies is associated with its side effects – primarily dermatological, such as scabies, conjunctivitis, dermatitis, skin infections, since EGFR is necessary for the normal functioning and renewal of skin and epithelial integuments.
  • Antibodies to HER2Expectations
The Her2 protein – tyrosine protein kinase of the epidermal growth factor receptor – plays a key role in a variety of cellular signaling cascades, and an imbalance of these cascades can lead to the development of certain types of cancer.

In the 90s, when it became clear that the increased expression of Her2 is characteristic of a large proportion of breast cancer cases in women, work began on obtaining targeted antibodies to this protein. In 2000, active clinical trials of these antibodies in combination with chemotherapy began. The first antibody of this type, which has been widely used, was the drug Trastuzumab (Herceptin).

[12]: Slamon et al., Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2 http://www.ncbi.nlm.nih.gov/pubmed/11248153 // N Engl J Med 2001

RealityHerceptin has become a blockbuster due to its effectiveness in the treatment of breast cancer expressing HER2, but it also has drawbacks: not all patients respond to treatment, its use can cause heart failure, and over time, many develop resistance to the drug and the tumor begins to grow again – there is a selection for cells that do not express HER2.

Nevertheless, Herceptin has become a big step forward compared to conventional chemotherapy, allowing to prolong the life of many patients and improving its quality.
  • TNF-alpha and inflammatory diseasesExpectations
As it turned out, a protein called Tumor necrosis Factor alpha (TNF–alpha), initially identified - as it is easy to guess from the name – as an important regulator of oncogenesis processes, is responsible for the work of immune T cells in general, and is a key regulator of their activation in acute and chronic inflammation.

The selection of antibodies capable of specifically suppressing the activity of this protein promised to become a promising therapeutic approach. 


Since the 2000s, three TNF-alpha binding antibodies have been registered for the treatment of rheumatoid arthritis - Infliximab (Remicade), Adalimumab (Humira) and Etanercept (Enbrel).

RealityAll of them also received registration for psoriasis, and Remicade and Humira also received registration for inflammatory bowel diseases.

The mentioned diseases are autoimmune – that is, the body's immune system attacks its own proteins, which leads to chronic inflammation and damage to tissues and organs. In the case of rheumatoid arthritis, mainly the joints are damaged, in the case of psoriasis, the skin is damaged, and inflammatory bowel diseases affect the large and small intestines. All of them are united by the presence of increased amounts of TNF-alpha at the site of the lesion and blood flow.

All anti-TNF agents have severe side effects, therefore they are indicated only on the 3rd - 4th line of therapy in patients with moderate or severe course of the disease (usually after nonsteroidal anti-inflammatory, corticosteroids, immunosuppressants). Nevertheless, the drugs slow down the development of the disease, and the benefits of them exceed the risk so much that for several years in a row they occupy the top three lines in the list of medicines with maximum annual sales (total sales of these three drugs exceed $ 20 billion).

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ImatinibExpectations

The drug, also known as "Glivek", has become the embodiment of the paradigm of the so-called "rational drug design".

This approach has become possible relatively recently, with a new round of development of molecular biology, physics and chemistry. Its essence is as follows: knowing the details of the biochemical cascades in cells and the protein agents involved in them, it is possible to choose the structure of the substance so that it binds to specific target proteins and regulates their activity in particular and the corresponding cascades in general. The study of protein activity in some types of cancer has given grounds to consider proteins of the protein kinase family as potential targets. The initial experimental screening – large-scale testing of a large number of candidate molecules – allowed scientists to select substances that block the activity of these proteins. Subsequent modification and improvement of inhibitory properties led to the appearance of the drug under discussion in the late 90s. 

[14]: Capdeville et al., Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug // Nat Rev Drug Discov 2002

RealityThe creation of Glivek allowed to reverse the therapy of severe oncological diseases of the blood, for example, chronic myelogenous leukemia and acute lymphoblastic leukemia.

These diseases occur due to chromosomal aberration, as a result of which the Bcr-Abl protein appears in bone marrow cells, which is a chimera of two proteins, and leads to oncological cell degeneration. Glivec was created specifically to block the activity of this protein, which made it possible to fight not just rapidly dividing cells, as chemotherapy does (killing normal cells, among other things), but – specifically – with malignant cells, because only they carry mutant Bcr-Abl. Of course, as the first experiment of this kind, Glivec turned out to be imperfect, since it suppressed not only Bcr-Abl, but also a number of other proteins, causing its side effects. However, he opened the way to a number of drugs created on the same principle.

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RapamycinExpectations

Appearing under the name Sirolimus, this drug of natural (bacterial) origin was originally used as an antifungal.

But subsequently, the range of its potential activities has greatly expanded. In 2000, researchers drew attention to the ability of rapamycin to suppress angiogenesis – vascular growth. This process is especially intense in cancerous tumors. As it turned out, rapamycin, by inhibiting the intracellular protein mTOR, blocks the activity of the key protein of angiogenesis, the VEGF protein we mentioned earlier, and also inhibits the activity of the immune system. This has opened up prospects for the use of rapamycin and its derivatives as anticancer and immunosuppressive drugs, primarily in transplantation, breast cancer, kidney cancer, lymphomas. 

RealityIn recent years, there have been loud words about the reliable effect of prolonging the life of laboratory animals with this drug – but there are no results of similar tests on humans, which, coupled with the known side effects of the drug (suppression of the immune system, cancer risks), gives researchers reason to warn against its use as an "elixir of life".



Amphotericin B and feverExpectations

Amphotericin B, an antibiotic of natural origin, was isolated and investigated back in the 1950s.

Over time, it was found out that, being a very large molecule – a macrocycle – it is able to integrate into cell membranes and form ion channels with very high conductivity. This causes the release of intracellular components – in particular, in this way it is possible to cause lysis of fungal parasites. In 2000, this drug was considered as the most promising in the treatment of fevers caused by fungal infections.


Figure 2. The complex of amphotericin B and ergosterol forms an annular structure, which, when embedded in the cell membrane, creates an ion channel in it.[16]: Walsh et al., Liposomal amphotericin B for empirical therapy in patients with persistent fever and neutropenia.

National Institute of Allergy and Infectious Diseases Mycoses Study Group // N Engl J Med 1999

Despite the wide list of life-threatening side effects (especially renal toxicity and multiple organ failure occur), amphotericin B remains the only treatment for a number of systemic and most dangerous fungal infections.

Later, the liposomal form of amphotericin B was developed – a common strategy for improving drugs, consisting in the fact that the molecule is "packed" into liposomes – balls of lipids inside which the drug is located. This change allowed to reduce side effects somewhat and increase the effectiveness in the treatment of a number of infections.

Figure 3. Liposome in section. Liposomes are quite often used for packaging drugs, as this allows them to be protected from destruction in the blood and improve delivery to cells. Liposomes from the outside are sometimes modified with molecules for more targeted delivery only to the right cells.[19]: Moen et al., Liposomal amphotericin B: a review of its use as empirical therapy in febrile neutropenia and in the treatment of invasive fungal infections




InterferonsExpectations

Interferons were discovered in the middle of the 20th century, and for a long time great hopes were pinned on them as a means of treating cancer, viral infections and inflammatory diseases.

With the progress of biology and medicine, it became clear that proteins of different types are combined under the name "interferons", but they all perform a signaling function when protecting the body from foreign objects.

The property of interferons to activate the immune response and lead to a decrease in tumors in animals, to reduce the number of virus particles in artificially infected animals gave rise to the claim that a universal cure for cancer and viral diseases has been found. Of course, as often happens, such categorical statements turned out to be premature.


As the effect of interferons on humans was studied, it became clear that as single agents they do not help everyone.

In addition, the constant use of interferons is impossible, since they cause flu-like effects and other undesirable phenomena. If you can put up with such effects with short-term therapy, then for long-term treatment, for example, inflammatory diseases, such a drug is not suitable. Now interferons are still used in the treatment of viral hepatitis B and C, multiple sclerosis, some types of cancer, but as safer and more effective drugs enter the market, interferons give way to them as too indiscriminate. 

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ConclusionThe examples considered are far from exhaustive – such interesting and broad topics as thrombolytics and antioxidants, on which great and unfulfilled hopes were pinned in the treatment of stroke; cancer immunotherapy, which finally gave really working drugs that made a breakthrough in the treatment of melanoma and lung cancer (and promising even more); stem cells cells that have not yet allowed us to achieve real success.

We will try to devote the following similar reviews to these topics.

However, the examples given are enough to make sure that drug development achieves the greatest success in those areas where a solid scientific foundation has been laid, where understanding the mechanisms of pathology development allows us to assume what can affect them, create model animals, understand how to build a research program on humans. Conversely, the current lack of understanding of how the brain works, the immune system, and how cancer progresses leads to blind and fruitless attempts to "grope" for a drug that, by a lucky chance, will work with Alzheimer's disease or stomach cancer. At the same time, as mentioned above, the statements of some scientists and specialists in biomedicine and pharmacology in general sound especially speculative, who, having barely discovered some mechanism that seems important to them, or having received a drug that works on mice, hurry to report that they have found a cure for aging or cancer. 

We hope that this article will help readers to be more sober about such bold predictions, but also give reason for optimism about the progress of biomedicine in the future.

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05.11.2015
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