Case Synopsis

LT, a 62 year old female presented to a hospital in Michigan. This particular patient had experienced a broken hip as a result of a fall at home. After looking at an x-ray to determine the patient's bone density, the doctor diagnosed LT with osteoporosis. The doctor discussed treatment options with the pharmacist and they decided on a 10 mg once a day regimen of alendronate, the generic of Fosamax.

The doctor wanted to see the patient in 6 months to look at her bone mineral density as well as calcium, phosphorus, and vitamin D levels. The pharmacist counselled the patient on taking alendronate with a full glass of water and supplementing with calcium and vitamin D. He also talked about possible flu like symptoms such as muscle aches and fever as well as gastrointestinal side effects. On asking the pharmacists why it's important to supplement with calcium and vitamin D and why these side effects occur, the pharmacist said the mechanism of action and off target binding can explain it.

Background

Alendronate, the generic of Fosamax, is a member of the bisphosphonate drug class. This drug class has a unique origin. The first compound, 1-hydroxyethylidene bisphosphonate disodium salt was first synthesized in 1897 by Von Baeyer and Hoffman. Since this agent prevented calcium carbonate precipitation, its use in fertilizers and oil industries was extremely beneficial. However, the usefulness of this compound in medicine was unknown until Procter and Gamble investigated its use in removing dental plaque some sixty years later. With further studies, it was soon proven to prevent bone loss. (1) By the 1990s, the pharmaceutical company Merck & Co. discovered the mechanism of action of non-nitrogen containing bisphosphonates. Soon after, they came out with a more potent bisphosphonate class called nitrogen containing bisphosphonates, which includes alendronate. Today, there are various bisphosphonates on the market approved for different bone diseases ranging from osteoporosis to osteolytic metastasis. (2)
Alendronate has been approved for use in cases of osteoporosis, which is a disease that causes the bones to be weak. The fragility of the bone is due to loss of organic bone matrix resulting in a decrease in bone mineral density. Bone loss is caused by excessive bone resorption which occurs when osteoclasts break down bone and release minerals resulting in a transfer of calcium from bone to the blood. This disease can be very dangerous because of the increased risk for fall injuries and subsequent fractures. (3) Post-menopausal females, such as the patient in the case, are at the highest risk for osteoporosis due to estrogen loss after menopause. Estrogen deficiency seems to accelerate bone loss by shortening the lifespan of osteoblasts while lengthening the lifespan of osteoclasts. When estrogen is low, the cytokine, IL-6 is made in abundance. This cytokine contributes to the recruitment of osteoclasts from the monocyte cell line. (4) Also, T cells are increased during estrogen depletion causing premature apoptosis of osteoblasts through its secretion of the IL-7 cytokine. (5) Alendronate and other bisphosphonates are approved for slowing the progression of this disease through a unique mechanism that inhibits osteoclasts function. (3) In addition to this medication, physical activity helps strengthen bones. When initiated in adults these changes are modest but more active adults could have a reduced risk of falling which would lead to reduced risk of fractures. (6)

The kinetics of alendronate effects how it is administered. Alendronate has a very low oral bioavailability of about 0.6% when fasting. This low bioavailability is lessened by up to 60% if given with food.(7) Alendronate also has a drug-drug interaction with calcium that leads to complexation and reduced bioavailability of alendronate as will be discussed in more detail later.(6)As alendronate already has a very low oral bioavailability, these interactions that lower the bioavailability even more can lead to ineffective doses. (7)
The drug class of bisphosphonates is divided into two drug groups. The first class is called non-nitrogenous bisphosphonates which include Etironate Sodium and Tiludronate Disodium. These medications act by depleting osteoclasts of energy by competing with adenosine triphosphate (ATP). Like all bisphosphonates, the structure contains a (P-C-P) motif that is important in binding divalent cations such as calcium in bone. (8) This is meant to mimic pyrophosphate which is an endogenous regulator of bones. However, pyrophosphates contain a (P-O-P) motif that can be easily hydrolyzed in the body. So by using a P-C-P structure, this drug molecule is chemically stable, allowing it to get to its site of action. As their name suggests, the non-nitrogenous bisphosphonates do not contain a nitrogen atom. (9) On the other hand, nitrogenous bisphosphonates, which include alendronate, are much more specific and potent than the non-nitrogenous bisphosphonates. As can be seen in figure 1, the R2 group on alendronate contains a long flexible chain that ends in a nitrogen atom. As we discuss the mechanism of action for nitrogenous bisphosphonates, the importance of these structural elements will become apparent. (10)

Jmol Analysis

Alendronate's mechanism of action occurs inside of osteoclasts. In order for alendronate to get to its site of action, it must first bind to bone. As previously explained, alendronate binds divalent cations such as calcium with its P-C-P motif. Bone is made up of hydroxyapatite (HAP) which consists of calcium, phosphate, and hydroxide groups. The oxygen atoms on the phosphonate groups of alendronate interact with the calcium ions in HAP. The affinity of this interaction is further increased with a hydroxyl group at the R1 position which also forms a bond with calcium. (2) Recently, structure activity relationships have shown that a hydrogen bond donor at the R2 amino group of alendronate can increase this drug's affinity for HAP. This is because the amino group is long and flexible allowing it to bind a hydroxyl group on HAP at a precise angle of 132 degrees and a distance of 2.7 Angstroms. This hydrogen bond along with the P-C-P motif and hydroxyl group allow alendronate to find its site of action and bind to it with strong affinity. (11) These binding interactions can be seen in figure 2.
Alendronate has two phosphonate groups that have an overall negative charge of two and an R2 amino group with a positive charge. When alendronate binds HAP, it confers an overall positive charge because the phosphonate group's negative charge is cancelled by its interaction with calcium ions through an ionic interaction. The amino group on alendronate remains positively charged which changes the overall zeta potential at the HAP surface. The zeta potential is the electrical potential at the shear plane around the HAP surface. It is believed that this change in zeta potential on the bone surface may allow for free negatively charged alendronate molecules to become attracted to the positively charged bone. This causes an accumulation of alendronate at the bone surface so that more drug can be at the site of action. Nichollonas was able to prove this by showing that the zeta potential of bone became increasingly positive with higher concentrations of alendronate. (12)

In order for alendronate to get inside osteoclasts, it first has to break its strong connection with HAP. When osteoclasts are resorbing bone, the area becomes acidic. This allows for protonation of the phosphonate groups and subsequent release of alendronate from HAP. Through a process of pinocytosis, also known as fluid phase endocytosis, alendronate is taken up into the osteoclast. Once inside the cytosol of osteoclasts, alendronate has reached its site of action and can now inhibit bone resorption. (13)

Farnesyl pyrophosphate synthase (FPPS) is a key enzyme in the mevalonate pathway, which is involved in cholesterol synthesis. Specifically, FPPS is responsible for synthesizing farnesyl pyrophosphate in a two-step pathway from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). (5) The mevalonate pathway is vital to osteoclast function in the bone because the isoprenoid metabolites that come from this pathway are required for prenylation of certain enzymes called GTPases. These enzymes play many important roles in a cell such as signal transduction, protein biosynthesis, and transport of vesicles within a cell. Without prenylation, these GTPases cannot function normally. (14) This results in the osteoclasts going through apoptosis so that bone resorption can no longer occur. (15)
The enzyme FPPS has three conformations: open, partially closed, and fully closed as depicted in figure 3. Alendronate binds FPPS in the DMAPP binding site. When this occurs, the DDXXD motifs come closer together so that two loops form a gate. This partially closed confirmation allows for the IPP binding site to open. The second step occurs when IPP binds this partially closed confirmation causing Lys350, Arg351, and Arg352 to clamp down. Normally when DMAPP is present, it will interact with IPP in the fully closed conformation and then revert back to the open conformation with subsequent product release. However with alendronate present, this interaction cannot occur and the enzyme is effectively inhibited. Alendronate stabilizes this conformation by interacting with both DDXXD motifs and Lys257. It has been proposed that alendronate acts as a transition state analogue by mimicking the carbocation intermediate formed during catalysis. (16)

New research has found that once the enzyme is fully closed, an intracellular accumulation of IPP results. High amounts of IPP lead to production of the metabolite ApppI, an ATP analogue. This metabolite inhibits adenine nucleotide translocase, which is responsible for exporting ATP from the mitochondrial matrix and importing ADP into the matrix. Without the translocator working, the osteoclast does not have any source of energy. It therefore goes through apoptosis. (17)
A key characteristic of alendronate is its affinity for FPPS. The orientation of the R2 group, which contains a nitrogen atom, relative to the phosphonate groups affects alendronates ability to inhibit FPPS. The R2 nitrogen in alendronate binds with the hydroxyl group of Thr201 through a hydrogen bond and to varying degrees Tyr204. This hydrogen bond leads to a tight binding affinity. (18) The R1 group contains a hydroxyl group that along with the P-C-P group binds bone. However, the hydroxyl group also helps bind FPPS by making a hydrogen bond to Asp243. In addition, the FPPS enzyme is made up of two aspartic-rich structures with the novel DDXXD sequence. This particular sequence is responsible for properly organizing three metal ions within the FPPS active site. Studies have shown that these divalent cations can be magnesium, manganese, or zinc. The alendronate backbone P-C-P, binds to these divalent metal ions which are bound to the carboxylate portions of the aspartate 103 and 107 side chains in the first aspartic rich DDXXD motif and then the aspartate 243 in the other DDXXD motif. (19)

Discussion

As discussed, alendronate slows the progression of osteoporosis through a unique mechanism that inhibits osteoclasts from breaking down the bone. Off target effects along with low bioavailability can be attributed towards the noncompliance that occurs with this medication. In the case study above, the pharmacist discussed some of the side effects that can occur with alendronate such as symptoms of a fever and GI pain. The importance of supplementing with calcium and vitamin D when taking this medication for optimal benefit was also explained and is revisited below.
Bone consists of calcium, so in order to help reverse the effects of osteoporosis, calcium supplementation is needed to repair degraded bone structure. While the surplus of calcium aids in restoring bone, alendronate prevents it from being broken down. Despite the somewhat synergistic action of calcium and alendronate, it is important for patients to take calcium supplementation two hours before or after taking alendronate. This must be done because the negative-charged phosphonate groups present in alendronate cause the drug to bind divalent cations, such as the positively charged calcium ions. As a result of this, if a patient takes alendronate simultaneously with calcium, the negatively charged phosphonate groups will interact with the calcium cations in the stomach and reduce its absorption. Therefore, this is why it is recommended that patients take calcium at least two hours before or after their dose of alendronate to ensure that the GI tract does not contain both oppositely charged ions at the same time. (6)
Besides binding to calcium, alendronate can also induce fever or flu like symptoms such as muscle aches and cold sweats. It is believed that monocytes in the blood may take up alendronate in the plasma. When this occurs, FPP synthase in the monocyte is inhibited leading to an accumulation of IPP. Interestingly enough, IPP is a ligand for certain gamma delta T cells. Activation of these T cell receptor leads to release of TNF alpha which results in fever like symptoms. After this discovery, researchers are now trying to use this side effect as a way of treating cancer. If gamma delta T cells can be stimulated by bisphosphonates perhaps it can be used to up regulate the immune system to target tumor growth. This could offer a new and exciting addition to the use of this drug class. (20)
Another side effect of alendronate is gastrointestinal pain and ulceration. There is a theory that these gastrointestinal side effects are caused by alendronate inhibiting FPPS in keratinocytes. By inducing apoptosis in cells that line the GI tract, the protective barrier is destroyed and ulcer formation can occur much more easily. For this reason it is important for patients to take alendronate with a glass of water to make sure that the medication goes all the way down into the stomach. It is also important for patients to avoid concomitant medications, such as NSAIDs, that may add to the ulceration of the GI tract. (21)
Alendronate is far from perfect and several changes may be made in order to increase its effectiveness. For instance, this drug could be taken with a monoclonal antibody that binds to gamma delta T cell's IPP receptor blocking its activation. Another approach would be making a structural change that prevents its uptake into monocytes. Both of these options would eliminate the flu-like symptoms that occur upon initiation of this medication.
It would also be clinically desirable to prevent the action that alendronate has on esophageal keratinocytes that lead to the bothersome gastrointestinal symptoms. A possible solution to this problem would be finding a different route of administration that bypasses the stomach such as intravenous administration. However, many patients do not want to go to the hospital every day to get their daily dose of alendronate; an alternative to this could be the use of a dendrimer, which is a nanoparticle that encapsulates drugs. If this is used with alendronate, the dendrimer could hide the drug from the keratinocytes and release it once absorbed into the bloodstream. (22) Another option that has recently been explored is turning alendronate into an effervescent tablet, which may increase patient compliance by decreasing adverse GI side effects. The tablet would be dissolved in water before administration. With this formulation the bisphosphonate spends less time in contact with esophageal tissue compared to oral tablet. Additional potential benefits are faster absorption and increased bioavailability of alendronate. (23)

One last change that could dramatically increase compliance and effectiveness would be making a change to the structure of the drug to increase bioavailability. The negative charge of the phosphonate groups along with their ability to chelate divalent cations makes this a tough challenge. One possible solution would be making alendronate a prodrug. Possible solutions would be adding a cyclosaligenyl or S-Acyl-2-thioethyl group onto the phosphonate groups so that they can better cross the GI tract. These groups can then be cleaved before reaching their site of action. (24) It is, however, important for alendronate to keep its pharmacophore, such as the P-C-P motif and nitrogen atom, because they are especially important for binding to bone and FPPS. However, structurally the nitrogen can be placed in a ring so that it is not floppy. This rigid structure would assure that the drug molecule binds at the exact angle that is needed. (15)
Bisphosphonates, such as alendronate, have been around for many years. They have helped patients with osteoporosis by slowing the progression of this disease. Nonetheless, compliance with this medication can be low because of the side effects that some patients experience. These adverse effects along with the low bioavailability need to be addressed in order for this medication to see its full potential. In spite of this, alendronate has been shown to reduce the relative risk of vertebral fractures by up to 55% and a higher increase in bone mineral density than other bisphosphonates (25). Therefore, the patient should continue her alendronate therapy to prevent the progression of osteoporosis and support bone strength. Ideally, this medication or medication class will be improved upon to increase efficacy and reduce limitations. In the meantime, pharmacists play a pivotal role in addressing the expectations and concerns that patients have with alendronate along with advocating alendronate as a first line agent in osteoporosis treatment. It is their duty to make sure that their patients are knowledgeable with this medication in order to see its maximum clinical benefit.

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