Success Story of Gleevec (Imatinib): a product of rational drug design

I know Ankur recently posted a short essay on Gleevec.

I am writing something on the same drug. I did an essay on Gleevec during my Masters programme. This post has some parts of my old essay. I hope this gives you all a good idea on rational design, in particular the “magic bullet” drug.

Introduction

During the early 1990s, George Daley, R Van Etten and David Baltimore successfully induced Chronic Myeloid Leukaemia in mice using the p210bcr/abl gene present on Philadelphia Chromosome1. This experiment confirmed that the genes Bcr/Abl gene expression is the major reason for the pathophysiology of chronic myeloid leukaemia (CML) in most cases. CML is a haematological stem cell disorder characterized by myeloid cells hyperplasia in the bone marrow. After studies on the gene, they found out tyrosine kinase was highly active in the cells and this lead to CML. The next logical step was to find a drug which has inhibiting capacity for the Bcr and Abl genes. After a series of screening processes, a highly effective drug was found out called Imatinib. The drug was highly successful in eliminating the disease symptoms and helped the patients live a better life even after diagnosis with CML.

To begin understanding the discovery process of Imatinib, it is essential to understand the disease.

Chronic Myeloid Leukaemia (CML)

CML is characterized by the reciprocal translocation between chromosome 9 and 22, resulting in Bcr-Abl fusion. This can be cytogenetically visible if we look at the shortened state of chromosome 222.

When a patient is suffering from CML, there are three stages:

  • The first stage is named chronic stage. In this stage 50% of the patients do not even have symptoms. Few complain about fatigue, pain and feeling full (loss of appetite). The blood workup of the patient will show 10% of abnormal white blood cells in the plasma and more than usual WBCs inside the bone marrow.
  • The second phase is called accelerated phase, where up to 20% abnormal white cells are found in the blood. During this phase, patients generally complain about fatigue, bruising, fever, night sweats, infection, bone and abdominal pain.
  • Third phase is blast crisis phase where the symptoms become much more noticeable. They include fatigue, bleeding, fever, weight loss, complications from infection and gout due to rapid cell turnover.

For Patients who were diagnosed with CML, the median survival was estimated to be about 5 to 6 years (before using Imatinib). Some patients, however, have an aggressive course from the outset and die within a year of diagnosis; other patients survive for 20 or more years3.

By the numerous experiments and studies done on this disease it is now clear that the Bcr-Abl gene fusion protein product is responsible for CML (90%).

Biology of Abl gene and Bcr gene

The Abl gene is responsible for production of a protein which works as a non-receptor for tyrosine kinase. The Abl gene is ubiquitously present in hematopoietic cells, but usually decreases with myeloid maturation. Tyrosine kinase is an enzyme which phosphorylates a substrate using phosphate group from ATPs. The Abl phosphorylation is tightly regulated process. If this controlling region is lost (as in case of fusion with Bcr), then it will lead to uncontrolled kinase activity.

Bcr gene is much more complex because they have many functional motifs. It is also involved in phosphorylation and GTP binding. The first exon present on this gene has oncogenic property because they have the codons responsible for production of proteins involved in Bcr-Abl fusion. It also has serine and threonine kinase enzymatic activity and it has autophosphorylation capability4.

Biology of Bcr-Abl gene

Studies on p210bcr/abl shows that they are pleiotropic molecules critical for the development of CML and they have effect on DNA repair leading to instability (might lead to disease progression) (Razelle Kurzrock, 2005).

Inhibition of tyrosine kinase activity

After the detailed study of Abl tyrosine kinase activity, medicinal chemists started working on inhibitors for this enzyme. They worked on several possible compounds which showed inhibitory effect. Some of the molecules that showed inhibition included benzopyranones and benzothiopyranones and the tyrphostin classes of compounds. However, they showed limited selectivity (affected normal tyrosine kinase present in the rest of the body) or showed less potency at cellular level (Michael Deininger et al, 2005).

Discovery of Imatinib

After working on many compounds the chemists landed on a 2-phenylaminopyrimidine derivative. This compound had low potency and poor specificity, inhibiting both serine/threonine and tyrosine kinases5.

Using this as the parent molecule, they started designing a specific tyrosine kinase inhibiting molecule.

  • By addition of a 3’ pyridyl group, they found out the molecule will have enhanced cellular activity inside the cell.
  • Introduction of Benzamide  will increase the “anti-tyrosine kinase” activity.
  • If “flag methyl” group is added to the 6 –position instead of anilino phenyl ring, it will lead to enhanced activity against tyrosine kinase.
  • To overcome problems of oral bioavailability and water solubility a highly polar molecule called N-methylpiperazine  was added.
  • This drug was initially called CGP57148B (changed to STI571) later it was called Imatinib {IUPAC name 4-[(4-methylpiperazin-1-yl) methyl]-N-[4-methyl-3-[(4-pyridin-3-ylpyrimidin-2-yl) amino] phenyl] Benzamide} or Gleevec as it is more commonly known.

Pre-clinical tests

During the pre clinical tests, imatinib was assayed for inhibition of the enzyme.

In-vitro analysis

During the experiments it became clear that this specifically inhibited only tyrosine kinase and it did not affect threonine kinase or serine kinase or any growth receptors (Ex: Epidermal growth factor receptors or VEGF – R1 and R2).

The experiments were repeated on cell lines (containing Abl active forms). It showed inhibition with 50% inhibitory concentration being 0.1 – 0.35 µm (Buchdunger et al, 1996). Since then numerous experiments have been done using Ph+ cell lines taken from patients and the IC50 values have been between 0.1 and 10 µm (showing that drug penetrated cell membrane).

No. of colonies % of inhibition for 1µm conc. No. of colonies % of inhibition for 10µm conc.
AMuLV cells 97 ± 3 71 20 94
v-sis cells 152 ± 16 83 20 94

In vivo analysis

To test the anti tumour activity of imatinib, syngeneic mice were transformed by transferring Bcr-Abl gene. Experiments done at Ciba Geigy (now Novartis) showed that imatinib is orally absorbed effectively in mice and relevant concentration in plasma is seen with half life of 1.3 hrs. Use of 160mg/kg of imatinib on mice consecutively for 11 days showed assured continuous blocking of p210bcr/abl but it did not affect any other cancer type which is Bcr-Abl negative (Buchdunger et al, 1996).

Using protein structure predictions, they found out that imatinib intimately reacts (engaging at least 21 amino acids) with Abl Kinase. This binding will create conformational changes in the enzyme leading to obstruction of ATP binding site (competitive inhibitor). Therefore, phosphorylation cannot take place and the tumour cells cannot proliferate.

Clinical Side effects that might create problems.

There were some clinical side effects observed in rats, dogs and monkeys when the imatinib was being tested. Dogs and monkeys showed reduced sperm count but there is limited data on fertility of men who have undergone treatment with imatinib.

Imatinib is also teratogenic in rats and hence women are advised to use conception and avoid pregnancies.

Clinical trials – Imatinib Monotherapy

Phase I

The phase I studies on the drug were done in June 1998, to determine the tolerated dosage level. Patients (diagnosed with CML in chronic stage and who had failed IFN treatment) were treated with 300mg of the drug and the results were very promising. Complete haematological response was shown by the patients. There were minimal side effects like Nausea, periorbital oedema and rashes.

Phase II

Phase II trials started in late 1999. Tremendous improvement was shown by patients who were treated with imatinib on a daily basis. The disease progression free survival rate was as high as 89.2%. The drug was eliminated predominantly by hepatic metabolism and had a plasma half life period of 18 hours (hence daily dosage is recommended).

Phase III

In the third phase studies, imatinib was administered along with IFN and cytarabine (anti-metabolic agent). The results were very positive showing 87% complete cytogenetic response (table 2). Imatinib not only has efficacy, but it also improved patients’ quality of life (Michael Deininger et al 2005).

Table 2: Imatinib versus IFN + cytarabine in patients with chronic CML (newly diagnosed)
n = 553 Complete hematologic response complete cytogenic response Progression-free survival, 14 months
Imatinib 95.3 87 92.1
IFN + cytarabine 55.5 8.8 73.5

After passing all the three phases of clinical trials, imatinib was approved by FDA in the year 2001.

It also was a feature article in TIME magazine as “magical bullet” curing cancer.

New problems emerge everyday regarding this drug. One such is the problem of resistance, but by using low doses and combining the imatinib dose with other forms of therapy will help in overcoming the resistance problem.

Conclusion

In 2009, Lasker-DeBakey Clinical Medical Research Award, often called the “American Nobel Prize” was awarded to Dr Brian J. Druker who shared the honour with Nicholas B. Lydon and Charles L. Sawyers for developing targeted treatments for CML and converting a fatal cancer into a manageable chronic condition6. The drug is a proof that targeted rational drug design is possible. It has created a major paradigm shift for patients and doctors alike.

Gleevec is an exceptional case because it is targeting a cancer type which simpler when compared with other cancers (usually influenced by complex interaction of genetic and environmental factors).  The drug has changed the way how drug designing is looked upon. By 2009, FDA had approved imatinib for many other different cancers.

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One response to “Success Story of Gleevec (Imatinib): a product of rational drug design

  1. To the timid and hesitating everything is impossible because it seems so.

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