Mini (Chimeric) Transplants

Clinical research has shown that there is a rift in efficacy between adult and juvenile hematopoietic stem cell transplantation for sickle cell disease treatment.  The procedure is curative when used for children, but so far it has shown to be toxic in the adult. Complications include graft rejection and graft-versus-host disease (GVHD). The treatment is technically termed “myeloablative allogenic hematopoietic stem cell transplantation,” meaning full removal of the patient’s bone marrow, followed by transplantation of blood stem cells from another individual.

Recent research published in the New England Journal of Medicine shows the therapeutic potential of nonmyeloablative stem cell transplantation in ten adults with sickle cell disease. Radiation and lymphocyte antibodies were used to clear out a portion of the patients’ bone marrow and white blood cell population. Hematopoietic stem cells from compatible siblings were then transplanted, and anti-rejection immunosuppressant medication was administered.

After two and a half years, all 10 subjects were alive, and 9 had a successful graft of their siblings’ donor cells. This leads to the dual functioning of the patient’s and donor’s cells with two separate genomes called chimerism. The hematopoietic stem cells are persistent, and produce many other types of cells just as those present normally within the patient’s bone marrow do. As these new cells have the genotype of a healthy donor, they code for normal hemoglobin. This results in the production of healthy red blood cells, and the sickle cell disease is effectively cured, with Hb levels increasing from about 9.0 to 12.6 g per decilitre. There were some side effects seen, such as narcotic withdrawal syndrome, and complications caused by the immunosuppressants. However, there was no GVHD seen.

Sources: nejm.org

Nitric Oxide

Since the cause of pulmonary hypertension is currently unknown and under debate, current treatments for pulmonary hypertension (see Complications and Treatments section) involve managing symptoms, underscoring the need for more research into cause and treatment.

Nitric oxide is the primary regulator (dilator) of blood vessels in the lungs, and in recent years, the hyperhemolytic paradigm was developed to suggest a lack of nitric oxide as the cause of pulmonary hypertension in sickle cell disease. It suggests that hemolysis in sickle cell disease causes an increase in free hemoglobin in plasma, which consumes the nitric oxide, causing a  state of nitric oxide deficiency, which causes the pulmonary arteries to narrow, leading to pulmonary hypertension. It also postulates that there is a hyperhemolytic subgroup of sickle cell patients who have higher rates of hemolysis, predisposing them to the pulmonary hypertension condition. There are some valid criticisms of this paradigm, including the lack of scientific evidence that deficiency in nitric oxide is the cause of or even a major contributor to pulmonary hypertension, but it has led to the possibility of nitric oxide as treatment for pulmonary hypertension.

It has been shown that inhibiting eNOS, the enzyme that makes nitric oxide, leads to endothelial (cells lining the blood vessels) dysfunction and that nitric oxide therapy can partially improve a dysfunctional endothelium. Inhaling nitric oxide has been shown to increase the oxygen affinity of sickled RBCs, suggesting that nitric oxide has anti-sickling properties. Indeed, pulmonary hypertension patients seem to respond to nitric oxide treatment regardless of their cause of pulmonary hypertension. Nitric oxide has also been tested for treatment of painful crises and has not proven useful in reducing the time to resolution of crises.

One criticism of nitric oxide therapy (or arginine therapy) is that soon after administration, these compounds would be degraded in the body, so long-term studies will be required. Another criticism of nitric oxide therapy is the risk of treating sickle cell patients with medications that alter vasoregulation. Sickle mice have been shown to have upregulation of both vasodilating and vasoconstricting regulators, leading to exaggerated responses from various stimuli. This has been recapitulated in sickle cell patients, who have altered vasomotor responsiveness to certain medications.

A possible alternative therapy is treatment with medications that alter nitric oxide levels in the body. Statins are drugs commonly used in cardiovascular disease and can decrease inflammation and improve endothelial function. A pilot study on the statin drug Simvastatin showed that the drug was able to boost nitric oxide levels in sickle cell patients, while being well tolerated and safe.

Another possible treatment for pulmonary hypertension that is currently being investigated in Senicapoc (See Molecules to inhibit sickling section).

Studies concerned with nitric oxide or pulmonary hypertension in sickle cell disease that are currently underway or are recruiting patients include (clinicaltrials.gov):

• nitric oxide therapy for acute chest syndrome in adults and children
• nitric oxide for pediatric painful crises
• natural history of sickle cell disease
• topical sodium nitrite for chronic leg ulcers
• safety and efficacy of sodium nitrite in sickle cell disease
• chronic transfusion for pulmonary hypertension in sickle cell disease
• albuminuria reduction with renin angiotensin system inhibitors
• glutamine therapy for hemolysis-associated pulmonary hypertension
• evaluation of toxicity from stem cell transplant

Source: clinicaltrials.gov


If the HHP was correct, three other therapies should have worked for the treatment of pulmonary hypertension. Hydroxyurea treatment over 16 weeks decreased the rate of hemolysis (RBC bursting), but did not improve pulmonary pressure. Sildenafil, used to improve the availability of nitric oxide in the body, did not improve pulmonary pressure either and actually increased pain. However, treatment with both hydroxyurea and sildenafil resulted in a small decrease in pulmonary pressure and a significant improvement in the six minute walk. Arginine should also have been an effective treatment for pulmonary hypertension according to the HHP.

Transfusion

One complication of transfusions is the possibility for alloimmunization. Alloimmunization occurs when the patient’s white blood cells (leukocytes) attack the donor’s red blood cells because they recognize them as a foreign intruder. There are now blood preparations that are reduced in donor leukocytes in order to avoid alloimmunization. Alloimmunization can also be avoided by matching minor blood group antigens between the donor and the recipient. These blood group antigens include Rh, C, E, and D antigens, Kell, Duffy, Kidd, Lutheran, P and MNS groups.

A serious complication of chronic transfusions is the possibility for iron overload (See Complications Section for more details). Magnetic resonance imaging (MRI) is increasingly being utilized as a non-invasive method for measuring liver iron concentration as well as cardiac iron concentration. As this technology becomes available, it will gradually replace the biopsy. Current guidelines recommend that every patient that has had or currently undergoes transfusions be regularly tested for LIC with MRI.

Iron overload is usually treated with iron chelators that remove iron from the blood (See Complications Section for more details). There are established parenteral and oral chelators that are currently used clinically, and there are also newer chelators undergoing clinical trials for efficacy and safety. The oral iron chelator deferiprone is only available in some countries outside North America. It is taken three times daily and is only used in patients with thalassemia major when parental therapy with desferoxamine (DFO) cannot be used. Since only a small number of short-term studies have been done, larger-scale longer-term studies are required to determine efficacy and safety. When these studies are completed, and if they are successful, deferiprone could be licensed for use in patients with sickle cell disease and in North America.

Chemotherapeutic Treatments

Chemotherapeutic drugs are molecules that alter the function of cellular and molecular processes in the body, usually to combat the progression of disease in a patient. Combinations of these drugs may be used to form a standardized treatment regimen, commonly referred to as chemotherapy. While there is no specific drug that can explicitly cure sickle cell anemia currently available, there are a number of drugs that exist to alleviate the discomfort experienced by patients suffering from the disease. These drugs generally aim to treat the resulting symptoms of the disease, such as pain, low haemoglobin levels, sickle cell crises and acute chest syndrome.

Source: nhlbi.nih.gov

Hydroxyurea

In 1995, a study was conducted by NHLBI to find out whether the drug hydroxyurea was clinically useful to individuals suffering from sickle cell disease. Initially shown to be effective in patients suffering from certain types of cancer, its health benefits were thought to extend to sickle cell patients. The study showed that hydroxyurea was effective in the prevention of vaso-occlusive pain crises and acute chest syndrome, both of which are directly caused by sickle cell anemia. The drug was effective in reducing the number of patient hospitalizations due to sickle cell crises by half with minimal toxicity effects. Hydroxyurea is now a popular drug administered to patients suffering from sickle cell anemia and was the first chemical agent shown to effectively prevent the aforementioned symptoms of sickle cell disease as well as reduce the mortality rate of its patients.

Function

Hydroxyurea functions to increase the concentration of healthy fetal haemoglobin in patients. This is facilitated by the increased production of nitric oxide, which in turn allows for the synthesis of fetal haemoglobin instead of its sickle haemoglobin counterpart. Hydroxyurea additionally functions to remove the cells that preferentially differentiate into sickle haemoglobin cells, reducing their levels in the SCD patient and contributing to a decreased mortality rate.

Side Effects

However, while hydroxyurea is an effective chemotherapeutic agent to combat the effects of sickle cell disease, its use is not without negative side effects. Reported side effects include drowsiness, nausea, vomiting, diarrhea or constipation, anorexia and bone marrow toxicity. Due to its effects on bone marrow, the drug was initially used to treat individuals suffering from myeloproliferative diseases prior to being administered to sickle cell anemia patients. Its effect on bone marrow results in suppressed blood cell counts, more specifically those of white blood cells (a condition called neutropenia), and platelets (a condition called thrombocytopenia). Reduced numbers of these two types of blood cells result in increased risks for infection and bleeding respectively. As a result, full blood cell counts are regularly monitored in patients being administered the drug, and uric acid, electrolyte and liver enzyme counts and renal function are monitored. While  these effects on the bone marrow of sickle cell patients confer a slight risk for the development of acute myeloid leukemia, many studies have shown that this risk is mostly absent or minute. Nonetheless, this risk is a key factor in the prevention of the wider use of hydroxyurea in patients. While hydroxyurea has been shown to be effective in the treatment of many sickle cell symptoms, there is an ongoing search for the ‘next hydroxyurea’ in the hope that a new treatment regimen may be developed with minimal negative side effects.

Source: nhlbi.nih.gov

Decitabine

With the extensive use of hydroxyurea in sickle cell disease patients, it was inevitable that certain patients would not respond to treatments involving the drug. As a result, a search was enlisted to find an alternative drug that could be used in its stead. A study performed in 1982 showed that the drug 5-Azacytidine, with its analog 5-aza-2’-deoxycytidine (also known as Decitabine), functioned similarly to hydroxyurea. A study performed at the University of Illinois at Chicago in 2002 showed that the administration of Decitabine to patients resulted in fewer hospitalizations due to symptoms and crises directly resulting from sickle cell disease. While the results show that Decitabine is a viable alternative treatment for those who do not respond to hydroxyurea treatments, it may in fact prove to be an improvement upon hydroxyurea due to the fewer resulting side effects.
 

Function

It is known that sickle cell disease symptoms can be alleviated by excess production of fetal hemoglobin. Hemoglobin production can be altered via extracellular or intercellular molecular mechanisms, the latter being how Decitabine functions. The drug prevents the genes that produce fetal haemoglobin from turning off in the patient, as they usually do in healthy individuals. By doing this, more fetal haemoglobin is produced upon extended exposure to the drug without any risk of cell toxicity.
 

Side Effects

Other than slight neutropenia (lack of white blood cells), few other hematologic toxicities were noted in the use of Decitabine. Fetal haemoglobin levels remained elevated in patients, even after the treatment was finished. This suggests that prolonged administration of the drug is not necessary to treat patients suffering from sickle cell disease. Current information suggests that the side effects produced by Decitabine administration are far less than those produced by hydroxyurea treatments. However, more research needs to be done to reconfirm these assertions.
 
Source: ncbi.nlm.nih.gov, bloodjournal.hematologylibrary.org

Adenosine A2A/B receptor antagonists

Recently, it has been found that the inactivation or destruction of certain cells in the body, called invariant Natural Killer T (iNKT) cells, improves pulmonary function and blood oxygen saturation in sickle cell mammals. Research has shown that adenosine A2A receptors are vital in the production of these cells and the receptors in sickle cell patients are 6-9 times more active than in healthy individuals, resulting in the overproduction of iNKT cells. Since the A2A receptor is also responsible for blood flow regulation throughout the body, overexpression of the receptor (like in those of sickle cell patients) can result in more painful SCD crises. A study performed in 2010 by researchers in the University of Virginia showed that treating sickle cell mammals  with A2A receptor antagonists reversed the effects of A2A receptor overexpression, resulting in improved pulmonary function and healthier sickle cell individuals. The A2B receptor is also known to be involved in the sickling of red blood cells, and is highly elevated in SCD patients, leading to increased cell sickling, hemolysis and multiple tissue damage. A study performed in 2010 by researchers at the University of Texas Health Science Centre showed that treating sickle cell mammals with A2B receptor antagonists reduces the sickling of their red blood cells, leading to healthier mammals.
 

Function

Adenosine A2A receptor antagonists function by blocking adenosine A2A receptor function. This is done by blocking the binding of A2A receptors to their  natural ligands, which are most likely overproduced in sickle cell patients. When the antagonist binds to the A2A receptor as a ligand, the natural ligand is then blocked from interacting with the receptor, thereby halting the receptor’s overexpression.

The A2B receptor antagonist functions by biochemically blocking the production of a molecule called 2,3-diphosphoglycerate, a small molecule involved in metabolism, that decreases the ability of haemoglobin to bind to oxygen. Since adenosine A2B receptors are overexpressed in sickle cell patients, the resulting lack of the haemoglobin protein’s ability to bind to oxygen leads to the sickling of the red blood cell where it resides. Therefore, blocking this process restores the health and oxygen affinity of red blood cells in sickle cell patients.
 

Side Effects

As this treatment has not yet been tested extensively on humans, adverse side effects are currently unknown.
 
Sources: ncbi.nlm.nih.gov, nature.com, onlinelibrary.wiley.com

Mouse Model

Recent research published in the journal Science builds on new breakthroughs in stem cell research. Certain cells in human tissue are capable of being transformed into a state not unlike that seen in embryonic stem (ES) cells. This regression is induced by using a viral vector to introduce certain factors into this cell (in this study, a fibroblast, or connective tissue cell). The result is an induced pluripotent stem (iPS) cell, which has the capability of differentiating into any cell type found in the body, including the hematopoietic progenitor cells (HPC) that go on to form red blood cells (RBC), platelets and white blood cells.

Using an approach called gene therapy, iPS cells were created using a mouse’s own fibroblasts. Using molecular genetic techniques in vitro, the SS gene (causing sickle cell disease) is altered to the normal genotype AA. These cells are transformed into HPCs, which are subsequently reintroduced into the mouse. This provides a population of normal HPCs (genotype AA) that can develop into normal red cells. The implications of this research are very important to the future of sickle cell treatment.

• It avoids the use of controversial ES cells.
• Fibroblasts are easily obtained from skin tissue.
• It generates healthy autologous (from one’s own body) HPC cells that are not rejected by the patient.
• HPC cells derived from the iPS cells can persist in the bone marrow, producing a constant supply of normal RBCs.
• This treatment would require personalized medicine, rather than the distribution of a drug.

(Figures from original Science article included here, with permission)

Unfortunately, treatment using autologous iPS cells is not a treatment option expected to be available anytime soon. The theory is good, and there have been proven results in mice. Further research is needed to determine the clinical safety of this technique in human subjects, as there is always the danger of the modified cells becoming cancerous because of genetic modificiations, or the viral vector reverting to a harmful state. Autologous stem cell and gene therapy is at the cutting edge in research on the treatment of genetic conditions, and may yield the ultimate “cure” for sickle cell in the future, but at present the risks of such procedures far outweigh any possible benefit to a patient.

Why is there a variation in severity of Sickle Cell Disease?

The main determinant of the severity of sickle cell is the rate and extent of HbS polymerization. The following are protective and result in less severe sickle cell disease:

1. Co-inheriting the alpha-thalessemia allele
2. Hereditary persistence of the fetal variant of hemoglobin (HbF).

The concentration of HbF varies from patient to patient and can be as low as 1% or as high as 30%. The concentration is inherited as a quantitative genetic trait, meaning that multiple genes determine it. So far, 3 major genes have been identified that contribute to HbF concentration.

Molecules to Inhibit Sickling

Cation transport channels on the surface of red blood cells can be blocked to prevent cell dehydration. Blocking transport channels also results in a lower concentration of HbS and reduces hemolysis.

Senicapoc

Although hydroxyurea treatment increases survival, many patients continue to suffer from acute vaso-occlusive crises, which occur when the spindle-shaped RBCs in sickle cell get stuck on the inside of blood vessels and form a clump blocking normal blood flow. The spindle shape is caused by the polymerization (linking) of multiple mutated hemoglobin (HbS) molecules. This polymerization is influenced by the concentration of HbS or the number of HbS per water molecule. Therefore the amount of water in the RBC or its hydration affects HbS polymerization. Polymerization is increased when the RBCs become dehydrated. In order to prevent painful crises, scientists are trying to prevent RBC dehydration by blocking protein channels found on the surfaces of RBCs that allow small molecules and ions to pass through. One such channel is the Gardos channel, which allows potassium ions to exit RBCs when it is activated by calcium ions. This exit of potassium ions causes an electrolyte imbalance between the inside and outside of the RBCs, which causes water molecules to exit the cells leading to cell dehydration. The dehydration then leads to increased HbS polymerization and more painful crises. Senicapoc specifically blocks potassium ion exit from the Gardos channel and prevents RBC dehydration. In clinical trials, senicapoc reduced RBC lysis, increased hemoglobin levels and was well tolerated by patients, however it did not reduce the incidence of painful crises, so the Phase III trials were stopped early. The authors explain this result by noting that painful crises are multi-factorial events. Senicapoc remains a possible treatment for other aspects of sickle cell due to its ability to improve hemoglobin levels, reduce dehydration, increase RBC longevity and positively affect other cell types. Although patients taking senicapoc had a higher frequency of nausea and urinary tract infections, serious side effects were no different from the placebo group. One specific complication that senicapoc may be used to treat in the future could be pulmonary hypertension (high blood pressure in the lungs) because it is thought to be partially due to hemolysis (RBC bursting), which is reduced by treatment with senicapoc.

Arginine as Treatment for Pulmonary Hypertension

During states of inflammation that can occur in sickle cell patients, a certain enzyme called endothelial arginase can become overactive and deplete the body of the amino acid arginine. The lack of arginine can limit the production of nitric oxide, the primary blood vessel regulator. As noted in the Nitric Oxide section, the lack of nitric oxide may lead to pulmonary hypertension. Scientists have postulated that arginine therapy could relieve the limitation on the production of nitric oxide, thereby preventing pulmonary hypertension. In one study, arginine therapy reduced pressure elevations, but not baseline pressure, and another study showed that chronic arginine therapy increased pulmonary pressure. In two other studies, arginine did not improve pulmonary hypertension.

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