AesRx believes Aes-103 is the most promising compound for sickle cell disease currently in human trials. To AesRx's knowledge, Aes-103 is the only agent which is in or near clinical trials that directly blocks cell sickling. It offers the possibility of truly modifying the course of SCD and changing the lives of SCD patients.
AesRx has commenced clinical trials of Aes-103 in collaboration with the NIH. AesRx holds the IND for Aes-103.
Aes-103: Small Molecule That Binds Specifically to Sickle Hemoglobin
Scientists have been working for many years to discover a treatment for sickle cell disease (SCD). Although numerous anti-sickling agents have been studied, hydroxyurea, an agent that induces synthesis of fetal Hb (HbF) is the only drug that is approved for the treatment of SCD. However, hydroxyurea has significant side-effects which limit its use in patients with SCD and the drug is not approved for use in children, an important component of the SCD population.
5-hydroxymethyl-2-furfural (Aes-103) is a small molecule (Da 126) discovered by researchers at Virginia Commonwealth University that increases the affinity of HbS for oxygen. This is important because only red blood cells with no bound oxygen will sickle, and increasing the ability of the sickle red blood cells to bind oxygen reduces the number of cells that can sickle. In addition, Aes-103 is orally bio-available and in animal studies is safe when given in large doses. Thus, it is the first example of a small molecule that binds specifically to sickle hemoglobin (HbS) and can be given in pill form in sufficient quantities to bind to most of the 500 grams of hemoglobin protein in a SCD patient.
The National Institutes of Health, Sickle Cell Disease Reference Laboratory (SCDRF) has screened over 700 compounds sent to them from researchers, universities and companies. The SCDRL has identified Aes-103 as one of the most potent anti-sickling compounds it has tested and uses it as the positive control in their in vitro anti-sickling assay.
AesRx is currently in a collaboration with the NIH to develop Aes-103 through initial proof of principle trials.
Ability of Aes-103 to Prevent Cell Sickling
The potential activity of Aes-103 has been examined in a number of in vitro and in vivo experiments. In one, the anti-sickling effect of Aes-103 was studied by incubating suspensions of SS cells in the absence or presence of three different concentrations (1, 2 and 5 mmol/l) of Aes-103 under 4 percent oxygen at 37°C for five hours. As illustrated in the pictures below, before incubation under air almost all sickle (SS) cells are discocytes with a few irreversibly sickled cells (ISCs). In the absence of Aes-103, more than 90 percent of red blood cells underwent sickling in five hours, while in the presence of Aes-103, the percentage of sickled cells decreased in a dose-dependent manner.
A quantitative analysis of the ability of Aes-103 to decrease sickling of SS cells in the experiment described above is provided in the following graph:
Moreover, when the SS cells were pre-treated with Aes-103 for one hour prior to incubation under low oxygen pressure, inhibition of sickling of SS cells was nearly 100 percent. This suggests that Aes-103 may take as little as one hour to bind with HbS in order to completely inhibit sickling.
Effect of Aes-103 on Sickle Cell Mice
In order to study the potential activity of Aes-103 in animal models, an experiment was undertaken with transgenic sickle mice that are heterozygous for human sickle hemoglobin. Upon exposure of these transgenic sickle mice to hypoxia (low oxygen air), the percentage of sickled cells in the blood increases sharply and most mice die from sickling-dependent acute pulmonary sequestration, a condition similar to the acute chest syndrome which often causes crisis in SCD patients. The experiment was started one hour after oral administration of either saline (untreated mice) or Aes-103 (treated mice). Aes-103 showed a potent anti-sickling effect. The graph below shows the Kaplan–Meier survival plot:
Without treatment, all of the transgenic sickle mice exposed to 5 percent oxygen died within 15 minutes. However, only one of the eight Aes-103-treated mice died under severe hypoxic conditions; the remaining seven mice survived through the full experimental period of 60 minutes. Upon exposure to hypoxia, the percentage of sickled cells in the blood of the untreated mice increased sharply, followed by a sharp decrease before death, while the percentage of sickled cells in the blood of Aes-103-treated mice increased slowly to a level of approximately 25 percent, which was maintained for the full experimental period of 60 minutes in seven mice. The sickled cells found in the blood of drug-treated mice appeared to be the so-called partially oxygenated sickled cells (POSCs) with "blunt" edges. POSCs are flexible and can pass through capillaries without causing vaso-occlusion. This means they are less likely to cause the damage to the vascular system and major organs that characterizes SCD.
Mechanism of Action: The Ability of Aes-103
to Increase the Oxygen Affinity
of HbS in Sickle Red Blood Cells
As previously indicated, only sickle red blood cells with no bound oxygen polymerize to form the “sickles” which give SCD its name and cause the damaging occlusions that characterize the clinical pathology of SCD. Increasing the ability of the sickle red blood cells to bind oxygen reduces the number of cells that can sickle. Consequently, gauging the capability of an agent such as Aes-103 to increase the affinity for oxygen of the HbS in sickle red blood cells is an important indicator of its therapeutic potential. It has been proven that Aes-103 shifts the oxygen dissociation curve of sickle cells toward the left (indicating a greater affinity for oxygen) in a dose-dependent manner. This left shift increases the percentage of non-sickling oxy-HbS and inhibits sickling of sickle cells. The more the concentration of Aes-103, the more the degree of left shift.
Chemical and Physical Characteristics of Aes-103
A group of chemicals, known as benzaldehydes, that form Schiff-base adducts with HbS seem to have an advantage over other potential compounds for the treatment of SCD because of their specific stable covalent binding with HbS. However, currently no clinically useful anti-sickling agents of this type that interact with intracellular HbS exist. This is because large doses of such an agent would be required to elicit a significant anti-sickling effect due the presence of a large amount of HbS in the body (approximately 500 grams), coupled with the fact that most of these drug candidates also interact with other proteins. Consequently, efficacious doses tend to be high, possibly resulting in serious adverse effects. Another problem with previously studied benzaldehydes was their lack of bioavailability when given orally, as most are either destroyed or modified in the gastrointestinal tract.
Aes-103 is a five carbon-ring aromatic aldehyde that exists naturally in coffee, honey, dried fruits, fruit juices and flavoring agents. Aes-103 is a common major product of the Maillard reaction and is formed from reactions of reducing sugars and amino acids. The concentration of Aes-103 in food products varies widely: Aes-103 levels in wine, spirits and fruit juices have been found to be as high as 200 mg/l, while prune juice may have Aes-103 concentrations up to 1000 mg/l. The chemical structure of Aes-103 is illustrated below:
As with other chemicals of the aldehyde class, Aes-103 forms a Schiff-base adduct with the amino terminal valine of the hemoglobin protein as shown in the diagram below. The two α-subunits (α1 and α2) are colored magenta and grey, respectively, while the β-subunits (β1 and β2) are colored blue and orange respectively. Aes-103, which binds in a symmetry-related manner at the two N-terminal aVal1 is depicted in red CPK (Corey–Pauling–Koltun) model. Although not shown in the model, each Aes-103 molecule makes a Schiff-base interaction with the aVal1 nitrogen, and the two molecules are joined together by a strong network of six water-mediated hydrogen bonds, through the hydroxyl and the ring oxygen moieties that tie the two α-subunits together.
Conclusion
One of the major challenges to finding effective therapeutic agents for the treatment of SCD has been the lack of agents that would specifically bind with the high concentration of intracellular HbS present in patients without causing adverse effects. Aes-103 appears to have promise in satisfying this condition. Aes-103 specifically binds to intracellular HbS and in pre-clinical models exhibits potent anti-sickling effects. In animal models using transgenic mice with human HbS, Aes-103 showed a dramatic ability to increase survival of the mice under conditions which mimic acute chest syndrome, which is one of the major causes of crisis in SCD patients.
With a molecular weight of only Da 126, Aes-103 is easily absorbed through the gastrointestinal tract into the blood circulation, where it readily passes through the red blood cell membrane and binds covalently with intracellular HbS to form a high-affinity Schiff-base HbS adduct.
AesRx believes Aes-103 is the most promising compound for treatment of SCD in human trials. It offers the possibility of truly modifying the course of SCD and changing the lives of SCD patients.