Purdue researchers develop way to stabilize human calcitonin for better osteoporosis treatment

Researchers have found a way to alter human calcitonin and likely rendering it a safe and effective treatment for osteoporosis and other bone diseases. 

Calcitonin (hCT) is an amino acid peptide hormone that helps protect the human skeleton and promote calcium metabolism. Its use as a treatment for bone diseases has been limited, however, because it is unstable and forms insoluble amyloid fibrils in solution. 

The fibrils are tiny fibers formed when peptide molecules stick together in an ordered way, limiting the hCT drug's effectiveness. Their structure is similar to the amyloid plaques formed in Alzheimer's and Parkinson's diseases.

Salmon calcitonin has been used instead of hCT to treat bone diseases, in part due to the instability of hCT. Although salmon calcitonin is more stable, it can have undesirable side effects and isn't ideal.

Now scientists from Purdue University's College of Pharmacy in Indiana have developed a method to modify hCT to prevent fibrillation. Their work is published in Biophysical Journal, Jan. 5.

Current Science Daily interviewed Elizabeth M. Topp, the Purdue professor who led the study, about its importance. Topp said she initially came at the problem "from a scientific interest, not a clinical one." She wanted to know what makes protein drugs unstable and looked at hCT as a model system to study, using both experiments and computer simulations of how protein molecules interact. 

"One of the things that makes peptide drugs like hCT unstable is that they fibrillate," Topp said. "That's bad. It can change the way they act so that they can be more potent or less potent in fibril form. Also, fibrils are more likely to cause immune responses than the drug itself. A mild immune response might make the drug inactive, while a rare severe immune response might be more like anaphylaxis."

Co-author Harshil Renawala, a graduate student, suggested a way to stop fibrillation by adding a phosphate group to the peptide chain, Topp said. Peptide fibrillation starts when two of the peptide chains interact with one another. 

"When these peptides come together, they do so in a very ordered way," Topp said. "They have a specific structure and they interdigitate in ways that are very particular to that peptide. A phosphate group carries a minus-two charge in its chemistry, so if you put a phosphate group on each of the fibrils, the charges repel each other," and the peptide molecules stay apart." 

Renawala found that "if you put the phosphate group exactly at a site on the fibril called Threonine-13 (Thr-13), it completely shuts down fibril formation, at least in the studies we've done so far," Topp added. "Peptide and protein drugs are fundamentally structured like beads on a chain in a linear polymer of amino acids. When we say Thr-13, it's a way of mapping exactly where we are in that peptide chain." 

Thr-13 turned out to be the best site on the hCT peptide chain to phosphorylate and stop the fibrillation process, Topp said. The phosphorylation allows hCT to act just like natural hCT in the body. 

"We've stabilized the drug in the vial and we've kept it from sticking together by this charge repulsion from the phosphate group," the Purdue scientist said. "Once phosphorylated hCT is given to a patient , enzymes called phosphatases clip off the phosphate group. You're left with the hormone in in its native state."

The result is that "the patient will effectively be receiving the native hormone that is present in the body," Topp said.

Improving osteoporosis treatment

What will this mean for the treatment of osteoporosis? 

"We hope that this new modified form of human calcitonin will give the drug another shot, and give patients another treatment option," Topp said. "Right now, we don’t know whether the side effect profile for hCT will be better than salmon calcitonin. With the new phosphorylated hCT, we should be better able to check the side effect profile and get enough experience to offer it to patients. As our population ages, we need more and better treatments for osteoporosis. We hope this will breathe new life into a drug that's been around for a while and help it find its treatment niche."

As for other treatments, Topp noted, "There are lots of choices for treating osteoporosis. There's a class of compounds called bisphosphonates. They can be really good and they've prevented a lot of fractures."

Fractures are devastating, she emphasized. 

"For people over 60, men and women who have a hip fracture, about 20% of them will die within six to 12 months after the fracture, and 50% will lose their independence. This is a devastating mortality and morbidity issue for older adults, and so if we can start to prevent that, it will be important."

But the bisphosphonates are limited because they are not for everyone, Topp said, adding, "They can be bad for your GI tract, particularly [with the esophagus] and some people can't tolerate them."

The salmon calcitonin that is prescribed is also limited because of its side effects. 

"One of side effects of salmon calcitonin is that there has been a higher incidence of some kinds of cancer in patients who have received it," Topp said. "It's not so high that you'd say that nobody should ever take this stuff, it's carcinogenic, but it's enough to make you use something else if you have another choice."

The glucagon case

Topp's group already has enjoyed success using the phosphorylation approach with another peptide hormone that fibrillates: glucagon, which is used for the treatment of hypoglycemia in diabetic patients. The phosphorylated form of glucagon that the group developed is now in pre-investigational new drug review (PIND) with the U.S. Food and Drug Administration.

"In the past a diabetic patient would have carry a pencil-case sized kit with a powder of glucagon and a syringe filled with an acidic solution," Topp said. "If the patient had severe hypoglycemia and was at risk of going into a diabetic coma, another person would have to reconstitute the powder, swirl it around carefully with solution and draw it back up into the syringe and inject it.

"The reason that people have had to do it this way, is that glucagon does the same thing that human calcitonin does. It fibrillates rapidly, even more quickly than human calcitonin does under some conditions." 

She added, "We think the phosphorylated form of glucagon that we’ve developed may be good for two things down the road. It may enable us to have an Epipen-type device for treating patients with hypoglycemia, and it may also help us develop an artificial pancreas that can deliver both insulin and glucagon. Insulin and glucagon are both peptide hormones, and they change blood glucose levels in opposite directions. With a stable phosphorylated form of glucagon, we could have a device that would be able to both raise and lower blood glucose as needed. It's really exciting stuff"

Topp, in addition to her work at Purdue, is the chief scientific officer at NIBIRT, the National Institute for Bioprocessing Research and Training, in Dublin, Ireland. Phosphorylated glucagon is being developed together with Monon Bioventures, a private company.