Protease Inhibitors
From Günter Scheuerbrandt Report of the PPMD Conference 2006:
Inhibition of proteasesThe degradation of muscle proteins in Duchenne dystrophy is caused by several different proteases, protein-destroying enzymes, such as the enzyme calpain, which is activated by calcium, and a large protease complex, called the proteasome. When, as in Duchenne dystrophy, muscle cell membranes become leaky because dystrophin is absent, calcium ions, charged atoms, from outside of the cells activate calpain and indirectly also the proteasome. This increased enzymatic activity leads to widespread destruction of important cellular proteins that are required for muscle cell function and survival. Specially designed inhibitors allow researchers to block the activity of calpain and other proteases which can delay muscle cell degradation.
The modified tripeptide leupeptin was the first inhibitor identified that could reduce calpain activity in mdx mice. This first-generation inhibitor consists of three amino acids, two leucines and one arginine, with the arginine containing a chemically reactive aldehyde group that is essential for the inhibitory activity. Leupeptin, however, can also inhibit other proteases including those of the blood plasma coagulation cascade, causing intolerable side effects. On the other hand it might be desirable to inhibit not only the calpain enzyme but also the proteasome.
C101. Theresa Michele, Vice President of Clinical Research of the CepTor Corporation in Baltimore
described that by combining leupeptin with carnitine, an inhibitor, called C101, was obtained which only enters skeletal and heart muscle cells. The reason for this specificity is that there exists a protein, the carnitine transporter OCTN2, which binds C101 at its carnitine end and carries it to a receptor protein on the muscle cell membrane, which then that transports the inhibitor C101 across the membrane into the cells. In order to measure precisely the effect of a potential drug like C101, a new quantitative test method had to be developed that allowed analysis of the calpain-induced breakdown of muscle cell proteins in living animals and possibly in humans, too. In this test, the ability of calpain to cleave the muscle cell protein alpha-II-spectrin is measured. Calpain uses two unique steps to break the spectrin protein down to the two smaller proteins, SBDP150 and 145, which leak out into the blood and then can be analyzed in serum with specially designed antibodies.
This new test is designed to follow the progress of muscle degradation and thus will probably become important for clinical trials with Duchenne patients. With this new test, it was shown that C101 can inhibit calpain 50 to 100 times more effectively than leupeptin. It also preserves the structure of the muscles and increases the diameter of the muscle fibers in mdx mice significantly. C101, which can be administered orally, thus is a potentially effective drug for treating Duchenne patients.
BBIC
In his overview of the different therapeutic approaches, Lee Sweeney mentioned the Bowman-Birk inhibitor concentrate, BBIC, that blocks other proteases than calpain which also participate in the destruction of muscular proteins, The active substance in this raw concentrate is a natural protein composed of 71 amino acids that can be isolated in pure form from soybeans.
Long-term treatment with BBIC increases the muscle mass and strength in mdx mice. CK activities are reduced considerably and fibrosis also. And from other applications in cancer patients it is known that BBIC is a very safe drug which can be applied orally.
SNT 198'438. Thomas Meier, Chief Scientific Officer at Santhera Pharmaceuticals in Liestal near Basel in Switzerland,
described their preclinical studies for the development of a dual-specificity inhibitor which simultaneously can block the activity of calpain and the proteasome enzyme complexes. Starting with a known calpain inhibitor scientists at Santhera have synthesized over 800 chemical variants and tested these inhibitors in biochemical and cell culture experiments as well as in mdx mice. Several compounds with the desired properties were identified.
One of them, SNT 198'438 was further optimized: It can be administered subcutaneously, i.e., by injection under the skin. Thus, it acts systemically and reaches and enters all muscles. In mdx mice, where it is well tolerated, 10 this inhibitor normalizes histological parameters of the muscles and improves the exercise performance of adult animals. This function test is performed in a large “mouse gym” where up to 40 mice can run voluntarily for several weeks in computer-monitored running wheels.