Giovanna Ababioh's CIAP Aptamer Project (2016)

Treating Paget's Disease with CIAP Aptamer- Drug Conjugation

Introduction/Background

Paget’s disease is a rare chronic bone disorder that is characterized by increased but disorganized bone remodeling.⁹ Paget’s disease is the second rarest bone disorder – osteoporosis being the first uncommon bone disorder – and this disease is usually seen in the elderly. In America, the prevalence of Paget’s disease in people over 40 years old is about 3%, and the prevalence in people over 80 years old is 10%. ¹ Paget’s disease cannot be cured, and there is no known concrete cause of this disease but research has shown that, Paget’s disease could be linked to increased levels of alkaline phosphatase, and genetic factors (Figure 1)⁹. It is currently treated by bisphosphonates which are drugs that inhibit mineralization or resorption of the bone by blocking the action of osteoclasts.³ Bisphosphonates can prevent or treat Paget’s disease but they can also have adverse effects. By conjugating a CIAP Aptamer to this drug, it is can reduce the inflammatory side effects of bisphosphonates.

Calf Intestinal Alkaline Phosphatase known as CIAP or CIP is a type of alkaline phosphatase whose primary function is to catalyze the dephosphorylation of 5’ and 3’ ends of DNA and RNA phosphomonoesters. Alkaline phosphatase is found in many tissues of the human body especially the bone, liver, kidney, and bile. Elevated levels of alkaline phosphatase have been seen in diseases like Paget’s disease, lymphoma, osteomalacia, hepatitis and others. Lowered levels of alkaline phosphatase have been seen in. CIAP is a dimer consisting of two subunits of 69000 molecular weight, and it is a zinc-containing enzyme.⁶ CIAP uses a very simple active site involving two zinc ions to carry out most of its rate acceleration: magnesium also plays an indirect but important role in stabilizing this enzyme in its most active conformation.³ Figure 2 shows the active site region of alkaline phosphatase.⁵

An aptamer is an oligonucleotide that has a high binding affinity to a specific target. An Aptamer is found through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Aptamers are similar to antibodies but research has shown that aptamers display a tight binding to cognate analytes, and display high affinity. Aptamers can be used for diagnostics, therapeutics, and drug delivery. In this situation when an aptamer is found against CIAP, it will be used as a therapeutic by covalently binding it to the bisphosphonates through a linker. A hydrazone linker will be used to connect the aptamer to the drug. This linker allows “bond cleavage and subsequent drug release in acidic environment.” This will then allow the aptamer covalently bonded to the bisphosphonates attach to the hydroxyapatite binding site. After binding to the hydroxyapatite binding site, the bisphosphonates will “inhibit hydroxyapatite breakdown, thereby effectively suppressing bone resorption,”¹⁰ and with the aptamer bonded to it, minimize inflammation.

A CIAP aptamer is being covalently bonded to bisphosphonates because it has been shown that CIAP has anti – inflammatory effects. This was seen in a research conducted by Beumer et al, when it was found that CIAP potentially could be a new drug for treating Lipopolysaccharide (LPS)-Mediated Diseases. Lipopolysaccharide diseases include sepsis, and ulcerative colitis.² In this research it was found that CIAP could detoxify LPS, and this was seen when mice survived a lethal Escherichia coli after being injected with CIAP.

Aptamers can be covalently conjugated with chemotherapeutics to overcome complications.⁹ Chemotherapy has many adverse effects so the ability to selectively target cancer tissues without the adverse complications is promising with an Aptamer.¹⁰ Aptamers bind to proteins, DNA/RNA, and other nucleic acids. By finding an Aptamer against CIAP that can be conjugated to bisphosphonates, it is promising that the inflammatory effects of these bisphosphonates will be reduced, and bisphosphonates conjugated with a CIAP Aptamer will make them better drugs for treating Paget’s disease. Currently one round of selection has been performed, and round 2 of selection is underway to develop an aptamer against CIAP. Round 2 of selection is just now underway because during Round 1, there were some contamination issues with cycle course PCR (ccPCR) and large scale PCR (LsPCR).

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References