What are “HDAC” inhibitors?

HDAC inhibitors are a group of drugs, which have a wide range of actions, including the ability to modify the activity of genes. HDAC stands for “Histone De-ACetylase” and the HDAC inhibitors are drugs which inhibit this enzyme. They may turn out to be every important in sickle cell disease.

Histones are large protein molecules which are closely associated with DNA in all the cells of the body. Normally, DNA is tightly wound around the histone proteins in a coil, a bit like a spring. In this state the DNA is said to be condensed and the genes encoded, by that section of DNA, are inactive. In order for the genes to become active the DNA has to dissociate from the histone proteins, when it does so it relaxes, unwinds and becomes much less tightly coiled. In this state the genes, in this part of the DNA, are accessible to transcription factors and other chemicals, which bind to the DNA and initiate gene activity. The degree of association between histones and DNA is therefore one of the ways in which we control the activity of our genes. If the DNA is tightly wound around the histone proteins the genes are inactive,  but if the DNA and histones dissociate, and the DNA unwinds, then the genes are activated.

How closely histone proteins and DNA are associated depends upon the degree to which lysine residues, in the histone proteins, are acetylated; the more acetyl groups attached to the histone proteins the less they bind to the DNA, the more the DNA unwinds and therefore the more active the genes become. The degree of acetylation is dependent on a balance between two groups of enzyme, the histone acetylases and the histone de-acetylases, which brings us back to the HDAC inhibitors.

In the upper picture the DNA (purple) is tightly wound around the histone proteins (Pale blue) and the  genes are inaccessible and inactive. In the lower picture acetyl groups have been attached to the lysine molecules (red) on the histones, allowing the DNA to uncoil or relax and for the exposed genes to become activated. The balance between the activity of histone acetylases (HAT) and de-acetylases (HDAC) determines the state of the DNA and the activity of the genes.

In the upper picture the DNA (purple) is tightly wound around the histone proteins (pale blue) and the genes are inaccessible and inactive. In the lower picture acetyl groups (red) have been attached to the lysine residues on the histones, allowing the DNA to uncoil or relax and for the exposed genes to become activated. The balance between the activity of histone acetylases (HAT) and histone de-acetylases (HDAC) determines the state of the DNA and the activity of the genes.

Because the group of drugs we are interested in, the HDAC inhibitors, inhibit the removal of acetyl groups from the histone proteins they tend to preserve the DNA in the open, unwound, relaxed state, and so they promote gene activity.

Why is this important in sickle cell disease? The key gene we are interested in is the gamma globin gene which controls the production of foetal haemoglobin (Hb F). After birth the gamma globin gene is silenced, during the gamma-beta switch, and in adults the level of Hb F is normally very low. However, a high level of Hb F protects against the effects of sickling by preventing the “crystallisation” of sickle haemoglobin molecules. Anything which re-activates the gamma globin genes in adults, and maintains a high level of Hb F, would therefore have a beneficial effect in sickle cell disease. This is the way that hydroxycarbamide works, but unfortunately this drug does not work in everyone, and there are side effects which can sometimes be difficult to manage. The HDAC inhibitors offer another way of re-activating the gamma globin genes.

The more red cells that contain Hb F and the higher the concentration of Hb F in each red cell the less severe will be the sickle cell disease.

The more red cells that contain Hb F and the higher the concentration of Hb F in each red cell the less severe will be the sickle cell disease. Phenotype is a word used to describe the severity of a disease.

A long time ago now, in 1988, doctors working in California noticed that in babies born to mothers, who had diabetes, the switch from making Hb F to making adult haemoglobin (Hb A) was delayed. They concluded that this was due to a substance in the baby’s blood called butyric acid. This substance is produced in the mother, as a result of her diabetes, and is transferred, in the womb, across the placenta to the baby. The scientists confirmed this by showing that if you infused butyric acid into new born lambs the same effect was produced, namely the switch from making Hb F to Hb A was delayed.

This was a new and fascinating observation and the same doctors went on to see whether butyric acid could be used in patients. In 1993 they published their observations in 3 patients with sickle cell disease and 3 patients with severe beta-thalassaemia. The patients were given intravenous (IV) infusions of arginine butyrate for 2-3 weeks at a time. The Hb F level increased in all six patients by 6-45%. In 1999 another group confirmed these findings, and demonstrated that if you gave IV arginine butyrate as “pulsed therapy” (treatment for 4 days every 1-3 weeks) then the patients had a sustained increase in Hb F levels, which rose from an average pre-treatment value of 7.2% to an average post-treatment value of 21.0%. A very significant response. The key is thought to be 30%; if all of a patient’s red cells contain 30% Hb F, then the effects of any sickle haemoglobin would be completely neutralised.

Unfortunately, very little happened over the next 10 years. This was partly because doctors were starting to use hydroxycarbamide, which also increases Hb F, but is much easier to administer, because it can be taken by mouth. In contrast, arginine butyrate is destroyed very quickly in the body and has to be given by a continuous IV infusion through an indwelling IV line, such as a porta-cath.

Despite this relative lack of interest, there were some important observations on the use of arginine butyrate to treat chronic leg ulcers in patients with sickle cell disease. Leg ulcers are notoriously difficult to manage and heal, but in 2002 a group in Philadelphia noticed that in some patients with leg ulcers, who were treated with arginine butyrate, the ulcers healed much more quickly. This observation was confirmed in 2010 when it was shown that after 3 months IV treatment, 78% of ulcers had healed, compared with only 24% in the patients who received standard management. It was unclear to the investigators whether it was the butyrate or arginine component of the infusion which was helpful.

The next important advances were presented at the American Society of Haematology Conference in Georgia in 2012 by a group from Harvard. They had used an in vitro, or test tube, culture method, in which they grew red cell precursors in a test tube and exposed them to various chemicals to see which was the best at promoting Hb F production. They screened hundreds of molecules and identified a small number, which produced a very significant increase in Hb F synthesis, without causing widespread acetylation of DNA and generalised gene activation. These compounds only inhibited a small number of histone de-acetylases, those in class I and class II, and seemed to be specific to the genes producing haemoglobin. Very importantly they can to be given orally and they are currently under investigation in clinical trials on patients with sickle cell disease.

Two drugs, vorinostat and panobinostat (LBH589) are being tested in phase I/II clinical trials for safety and effectiveness in Massachusetts and Georgia respectively; the trials are both due to be completed late in 2014. A third drug, givinostat (ITF2357), was reported by an Italian group, from Milan, in the British Journal of Haematology this month, to significantly increase the synthesis of Hb F, again in test tube cultures of red cell precursors. It is likely that givinostat will shortly enter clinical trial in sickle cell disease patients.

All of these drugs have also been used as anti-cancer agents and are currently being investigated for use in cutaneous T-cell lymphoma, multiple myeloma, acute myeloid leukaemia and Hodgkin’s disease. From these studies, their side effect profile appears to be good and it is hoped that, at the lower doses used to stimulate Hb F synthesis, the drugs will be well tolerated and safe, an essential pre-requisite for any sickle cell disease treatment.

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About rogerjamos

I am a consultant haematologist who has worked in Hackney, London, UK with patients who have sickle cell disease for many years. Knowledge is power; the hope is that this blog will empower patients by putting them in touch with contemporary research into sickle cell disease and facilitating informed discussion on the issues raised. Dr Roger Amos MA, MD, FRCPath
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