Gene present on Philadelphia Chromosome

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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 Chromosome. 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 the presence of large number of mature myeloid cells 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 and its causes.

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 221.

When a patient is suffering from chronic myeloid leukaemia, there are three stages and symptoms associated with them.

  • The first stage is named chronic stage. In this stage 50% of the patients do not even have symptoms. Those who do notice some irregularities 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 years.

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 capability.

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).

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.

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 kinases.

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

  • By addition of a 3' pyridyl group (Fig 1A), they found out the molecule will have enhanced cellular activity inside the cell. This will lead to the formation of the Fig 1A structure from the parent compound.
  • Introduction of Benzamide (formation of Fig 1B structure) 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 (Fig 1C).
  • Even after all these additions and substitutions, the drug had problems being orally bioactive and water soluble.
  • This problem was overcome by the addition of a highly polar molecule called N-methylpiperazine (Fig 1D).
  • This drug was initially called CGP57148B, later the name was changed to STI571. Finally, it was named as Imatinib mesylate {IUPAC name 4-[(4-methylpiperazin-1-yl) methyl]-N-[4-methyl-3-[(4-pyridin-3-ylpyrimidin-2-yl) amino] phenyl] Benzamide} or Gleevec (or Glivec) as it is more commonly known.

Pre-clinical tests

After the successful production of the drug, Imatinib was tested for its activity on enzymes and its inhibitory effects were assayed.

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. Since then numerous experiments have been done using Ph+ cell lines taken from patients and the IC50 values have been between 0.1 and 0.5µm(showing that drug penetrated cell membrane).

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.

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.

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.

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. Higher doses were also used to determine tolerated dose levels. 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. Imatinib not only has efficacy, but it also improved patients' quality of life.

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

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

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 condition. The drug is a proof that targeted rational drug design is possible. The drug has revolutionised the way cancer is seen. It has created a major paradigm shift for patients and doctors alike.

Even though Gleevec is an exceptional case because it is targeting a cancer type which is very much simpler when compared with other cancers which are 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 has approved imatinib for 10 different cancers. It is one of the major sources of income for its producer Novartis.

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