A nonsense mutation is a type of mutation that causes a genetic disorder by inducing a defect in the production of a critical protein. This type of mutation is an alteration in DNA that, when copied to mRNA, tells the ribosomes (the cellular machinery responsible for translating mRNA to make proteins) to prematurely stop production of that protein. This results in a protein that is too short to perform its necessary function.

PTC Therapeutics discovered PTC124 by looking for a drug that allows the ribosome to read through, or bypass, the premature stop signals in mRNA and continue the translation process to make a full-length and functional protein. PTC is currently analyzing results of the PTC124 Phase 2a clinical trials in patients with cystic fibrosis and in Duchenne muscular dystrophy.

Approximately 5-15% of cases of most inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy, spinal muscular atrophy, hemophilia, neurofibromatosis, retinitis pigmentosa, lysosomal storage diseases, Hurler’s Syndrome and a variety of other genetic disorders, are due to nonsense mutations.

Gene therapy attempts to treat the disease by replacing the defective gene with one that produces the correct protein. For example, genes can be attached to modified versions of viruses that have the ability to penetrate into the nucleus of a cell, become incorporated into the cell's existing DNA, and synthesize new proteins.

Enzyme replacement refers to the administration of purified or synthesized protein to patients in whom that particular enzyme is deficient or absent. Currently this therapy often involves patients receiving periodic intravenous or intramuscular injections of the replacement enzyme.

PTC124 is a small-molecule drug that can be taken by mouth, and so does not suffer from the delivery challenges that have limited gene therapy and enzyme replacement therapy. In addition, it does not necessitate the delivery of foreign genetic material or viruses. It is anticipated that PTC124, by addressing the underlying cause of the disease, might decrease dependence on palliative interventions and ameliorate debilitation and decrease mortality in patients with genetic disorders due to nonsense mutations.

PTC124 may have the potential to treat many genetic disorders in which a nonsense mutation is the basis for the disease. PTC has catalogued over 1,800 distinct genetic disorders where nonsense mutations are the cause of the disease in a significant percentage of patients. Nonsense mutations inactivate gene function and are known to cause anywhere from five to 70 percent of the individual cases of most inherited diseases, such as cystic fibrosis (10%) and Hurler’s syndrome (70%). Other genetic disorders where a percentage of the cases are due to nonsense mutations include spinal muscular atrophy, hemophilia, neurofibromatosis, and retinitis pigmentosa.

We are currently conducting clinical studies in cystic fibrosis and Duchenne muscular dystrophy. If these clinical trials yield positive results we hope eventually to expand development to multiple genetic disorders. In the meantime, we are continuing our preclinical research in order to assess the potential clinical utility of PTC124 in other genetic diseases.

If you are interested in determining if your condition is caused by a nonsense mutation, we recommend that you speak with your treating physician or genetic counselor. This requires gene-sequencing, also called genotyping, determined from a blood sample. Facilities that perform gene sequencing, by condition, can be located through the NIH sponsored website, Gene Tests (www.genetests.org). The Laboratory Directory permits searches by disease and location. For patients with cystic fibrosis, full-length gene sequencing is available through Ambry Genetics (http://www.ambrygen.com). For patients with Duchenne muscular dystrophy, information regarding full-length gene sequencing is available through the University of Utah (http://www.genome.utah.edu/DMD).

No, based on its mechanism of action, PTC124 will not work in patients whose genetic disorder is caused by a mutation other than a nonsense mutation, such as a missense, deletion, or duplication mutation.

Theoretically, inappropriate readthrough of normal stop codons could result in abnormally long proteins being made. For this reason, PTC has carefully analyzed this issue in studies of high doses of PTC124 in tissue culture systems, in animals, and in humans. These studies have demonstrated no evidence of production of abnormal proteins with PTC124 administration. Thus, the information available at this time indicates that PTC124 specifically acts to allow ribosomes to read through nonsense mutations (premature stop codons) but does not induce ribosomes to read through normal stop codons. 
 

Yes, the approach is similar, but PTC124 is not related to gentamicin. Like gentamicin, PTC124 allows ribosomes to read through premature stop codons to produce full-length, functional proteins. However, PTC124 is from a distinct structural class that we believe acts at a different location on the ribosome than gentamicin, and PTC124 does not have antibiotic properties. Although the results involving gentamicin have provided proof of concept for the readthrough of nonsense mutations as a therapeutic approach to treating genetic disorders, gentamicin has serious dose-limiting toxicities and requires intravenous administration, making it an unattractive long-term treatment for genetic disorders. We do not expect PTC124, which is orally administered, to have the serious dose-limiting toxicities associated with gentamicin.  

Gene sequencing, also called genotyping, can determine if a patient has the disease because of a nonsense mutation. This is one of the first instances where knowledge of genetic sequence may prove useful in determining if a patient may benefit from a drug. Patients who wish to determine what type of mutation is responsible for their disease should consult with their physician about the possibility of having the relevant gene sequenced. Usually, a small amount of blood is required to perform gene sequencing. The blood sample will be sent to a specialized laboratory, sometimes at a university hospital that has expertise in studying patients with a particular disease. For patients with cystic fibrosis, full-length gene sequencing is available through Ambry Genetics (http://www.ambrygen.com). For patients with Duchenne muscular dystrophy, information regarding full-length gene sequencing is available through the University of Utah (http://www.genome.utah.edu/DMD).


For other genetic disorders, we recommend that you speak with your treating physician or genetic counselor. Facilities that perform gene-sequencing can be located through the NIH-sponsored website, Gene Tests (www.genetests.org). The Laboratory Directory at this site permits searches by disease and location.

No new Phase 2a trials are currently planned in Duchenne muscular dystrophy. We have initiated a new Phase 2a study in cystic fibrosis at Hôpital Necker-Enfants Malades, in Paris, France. An additional site in Belgium may join the French study. The study is similar in design to the recently completed Phase 2 cystic fibrosis studies and will enroll patients 6 years of age and older. Please visit www.clinicaltrials.gov and type”PTC124” in the search area for more information on the locations and enrollment criteria for these studies.

In addition, we have completed enrollment and treatment in a Phase 2 extension study in cystic fibrosis ongoing in Israel at the Hadassah Medical Center, Jerusalem and are currently analyzing the results.

We are continually engaged in discussions with regulatory authorities regarding PTC124. With the completion of the Phase 2a studies and data analysis, we hope to begin Phase 2b/3 studies by the end of the year.

After completing two Phase 1 clinical trials in healthy volunteers to characterize the general safety, palatability and effects of food on PTC124 as well as its pharmacokinetics in order to understand its absorption into the bloodstream, we initiated the Phase 2a trials in late 2005.

Three Phase 2a trials were initiated in late 2005. In cystic fibrosis, we sponsored two studies, one study at five sites in the United States and a similar study at one site in Israel. In Duchenne muscular dystrophy, we sponsored one study in three sites in the United States. The study centers were primarily university hospitals selected for their expertise in performing specialized clinical trials in cystic fibrosis or Duchenne muscular dystrophy.

The main goals in Phase 2a were to obtain indications of pharmacological activity and to assess dose response, safety, and pharmacokinetics in patients.
  

To confirm the diagnosis of a nonsense mutation for the clinical trials in cystic fibrosis, PTC Therapeutics employed the gene sequencing test offered by Ambry Genetics (www.ambrygen.com).

For the clinical trial in Duchenne muscular dystrophy, we used the test offered by the University of Utah (http://www.genome.utah.edu/DMD/clinical_test.shtml).

Phase 2a studies are typically conducted on a relatively small number of patients, with the goal of determining drug activity, evaluating short-term side effects, and assessing pharmacokinetics in patients. The duration of several of the initial Phase 2a studies has been based on knowledge that PTC124 is safe when given for up to 28 days to animals and because it has been expected that early drug effects in patients might be observed within this duration of treatment.

In the Phase 2a studies the drug has been supplied as a vanilla-flavored powder. The powder is mixed in water, apple juice or milk to form a suspension that the patient drinks. PTC124 is being dosed based on patient body weight (i.e., milligrams of drug per kilograms of patient body weight) in order to accommodate the varying size range of the children, adolescents, and young adults who will be treated.

In November 2006, we presented data from the PTC124 cystic fibrosis clinical trials in Israel and the U.S. Cystic fibrosis is caused by a lack of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. To determine whether PTC124 was inducing production of active CFTR protein in patients, we measured nasal transepithelial potential difference (TEPD). TEPD is assessed by means of a standardized procedure in which a small plastic catheter is used to assess electrical differences across the cell membrane of the skin in each nostril. Cystic fibrosis patients with nonsense mutations have abnormalities in the electrical difference measured by the TEPD test because they lack sufficient CFTR protein and thus have a defect in the movement of chloride ions across the cells (the chloride conductance). A change in the chloride conductance towards normal during PTC124 treatment would suggest that PTC124 is inducing the cells to make full-length, functional CFTR protein. Also assessed were circulating blood neutrophil and liver enzyme values, lung function, and body weight, as well as safety, compliance, and pharmacokinetics.

Patients received two sequential two-week courses of treatment, first at a lower and then at a higher dose level. Across the two studies, at both PTC124 dose levels tested, we observed statistically significant improvements of mean CFTR-dependent chloride secretion in the airways. By the end of the first cycle of treatment, 18 out of 42 patients had responded with a change of at least -5 mV in chloride secretion TEPD and 15 out of 42 patients had chloride secretion TEPD values in the generally accepted normal range (more electrically negative than -5 mV). CFTR chloride secretion responses were observed in both the U.S. and Israel among several of the most common nonsense mutation genotypes affecting patients with cystic fibrosis. In evaluating the studies separately, the results from the Israeli study demonstrated statistical significance for chloride secretion TEPD response for the population as a whole, while the interim results from the U.S. study demonstrated such responses in several patients but the trends did not reach statistical significance. The potential causes of these differences are now being studied.

Blood neutrophil counts were also monitored before and during PTC124 treatment because cystic fibrosis is a neutrophil-mediated disease, and reductions in blood neutrophil counts may be consistent with PTC124 activity. Statistically significant reductions in blood neutrophil counts were observed in both the U.S. and Israeli studies. Furthermore, improvements in circulating levels of liver enzymes in the blood were seen in both trials, supporting the hypotheses that PTC124 would offer systemic benefits to patients with multiorgan compromise due to cystic fibrosis. Trends toward improved pulmonary function and body weight were also observed in patients participating in the Phase 2a program. Although a formal symptom assessment was not a component of the Phase 2a program, a number of patients described decreased sputum volume and thickness, decreased frequency and severity of coughing, and a better sense of well-being during PTC124 therapy. PTC124 was well tolerated, resulting in excellent compliance with the treatment regimen.

The pharmacokinetic data from adult patients in the interim analysis indicated that PTC124 was readily absorbed from the gastrointestinal tract. The desired blood levels were achieved and maintained at the first and fourteenth days of both the lower-dose and higher-dose treatment regimens.

PTC124 was generally well-tolerated among the forty-two patients included in the studies. No serious drug-related adverse events were reported. All adverse events that were potentially drug-related were mild or moderate in severity. There were no safety concerns identified in patients’ physical examinations, vital sign measurements, or electrocardiograms. We did not observe any serious changes in laboratory safety tests. There were no dosing interruptions or trial discontinuations due to toxicity. Treatment compliance was very good, with patients taking more than 95% of the intended total drug treatment at both the lower and higher dose levels.

In May of 2007, we presented positive preliminary data from a Phase 2a clinical trial of PTC124 in patients with Duchenne muscular dystrophy due to a nonsense mutation. This Phase 2a multi-site, open-label, dose-ranging clinical trial has evaluated muscle dystrophin expression in patients with nonsense-mutation-mediated Duchenne muscular dystrophy. Blood levels of muscle-derived creatine kinase have been measured as assessments of muscle integrity. PTC124 safety, compliance, and pharmacokinetics are also being evaluated.

The Phase 2a clinical trial has been conducted at three sites in the United States: Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; and the University of Utah, Salt Lake City, Utah. In the study, patients have received 28 days of PTC124 treatment at one of three dose levels. All clinical trial participants are boys with a nonsense mutation in the dystrophin gene, substantially elevated serum creatine kinase levels, and symptoms associated with Duchenne muscular dystrophy. The analysis presented includes data from 26 patients with Duchenne muscular dystrophy who received PTC124 at the low-dose and medium-dose levels. Completion of analysis of data from a higher dose level is ongoing.

The primary endpoint of the trial has been the proportion of patients having an increase in dystrophin expression in muscle during 28 days of treatment with PTC124. Pre- and post-treatment muscle biopsies were available from all 26 patients for analysis. In vitro treatment of patient muscle cells with PTC124 showed evidence of a dose-dependent increase in dystrophin expression in all of the evaluable patients. Preliminary review of the data indicates that, at both dose levels evaluated in this analysis, approximately half of the patients demonstrated visible improvement in the staining for muscle dystrophin in vivo. Overall, four of the six, or 67 percent, of patients treated at the lower dose level and 10 of the 20, or 50 percent, of patients treated at the medium dose level demonstrated an increase in the expression of dystrophin post-treatment.

Additionally, statistically significant reductions in the concentrations of muscle-derived creatine kinase levels in the blood were observed during PTC124 treatment. Several parents and teachers reported that boys participating in the study had improvements in terms of greater activity level and increased endurance during treatment. Individual subjects at both dose levels demonstrated some improvements in upper and lower muscle strength; however, in the overall analysis the magnitude of change was not statistically significant.
PTC124 was generally well tolerated among the 26 patients included in the study. Adverse events were infrequent, mild to moderate in severity, and did not result in therapy interruptions or discontinuations. There were no safety concerns based on physical examinations, vital sign measurements, electrocardiograms, or laboratory parameters. Compliance with PTC124 treatment was excellent (greater than 98%) at both dose levels.

PTC124 is being dosed based on patient body weight (i.e., milligrams of drug per kilograms of patient body weight). Some healthy volunteers in Phase 1 trials received very high single doses of the drug (150 or 200 mg/kg) that are above those expected to be given for therapeutic purposes. The Phase 1 subjects experienced transient effects of nausea, vomiting, diarrhea, headache, and dizziness. These effects were mild and disappeared rapidly (generally within minutes to hours). Further evaluation of the safety of the drug in healthy volunteers receiving treatment for up to 14 days at doses up to 50 mg/kg twice-per-day revealed no symptomatic drug-related adverse events at any dose level. Modest elevations of liver enzymes were observed in some subjects. These elevated enzyme levels did not require cessation of PTC124 administration, and enzyme levels typically returned to normal after completion of the treatment phase.

PTC124 was generally well-tolerated among the forty-two patients included in the Phase 2a cystic fibrosis studies. No serious drug-related adverse events were reported. All adverse events that were potentially drug-related were mild or moderate in severity. There were no safety concerns identified in patients’ physical examinations, vital sign measurements, or electrocardiograms. We did not observe any serious changes in laboratory safety tests. There were no dosing interruptions or trial discontinuations due to toxicity. Treatment compliance was very good, with patients taking more than 95% of the intended total drug treatment at both the lower and higher dose levels.

PTC124 was generally well tolerated among the 26 patients included in the Phase 2a Duchenne muscular dystrophy study. Adverse events were infrequent, mild to moderate in severity, and did not result in therapy interruptions or discontinuations. There were no safety concerns based on physical examinations, vital sign measurements, electrocardiograms or laboratory parameters. Compliance with PTC124 treatment was excellent (greater than 98%) at both dose levels.

We believe the data are consistent with our hypothesis that treatment with PTC124 can restore the production and function of CFTR in some patients with cystic fibrosis and of dystrophin in some patients with Duchenne muscular dystrophy when these disorders are caused by a nonsense mutation. We feel that the improvements in clinical parameters observed in these studies are encouraging and the safety results show that the drug is well tolerated over two to four weeks of treatment in children and in adult patients. However, these results are preliminary and were obtained from a small number of patients. In order to improve certainty about these results, we must finalize the ongoing studies and the full analyses of all data. In order to determine whether PTC124 treatment will lead to long-term clinical benefit and be safe for patients, we will need to perform additional studies of longer duration. 

Our early analysis of the preliminary data from the cystic fibrosis and Duchenne muscular dystrophy studies is encouraging. However, full analyses are still ongoing. Once the final Phase 2a results are available and regulatory authorities concur, PTC hopes to advance PTC124 into Phase 2b/Phase 3 studies in patients with cystic fibrosis and Duchenne muscular dystrophy due to nonsense mutations. We are also assessing additional genetic disorders that are characterized by nonsense mutations to determine whether to initiate clinical trials of PTC124 for the treatment of those diseases.

The Phase 2a trials are not designed to assess the efficacy of PTC124 or to compare the efficacy to other therapies. Because they are short in duration, they also cannot examine the safety of longer-term administration of PTC124. The Phase 2a studies form the basis for the development of longer-term Phase 2b/Phase 3 trials. We expect that conduct of Phase 2b/Phase 3 clinical trials will be required to evaluate the efficacy and long-term safety of PTC124 and to support registration of the drug with regulatory authorities. We expect that primary endpoints for future trials in cystic fibrosis would include clinical measures of lung function and that primary endpoints for future trials in Duchenne muscular dystrophy would include clinical measures of muscle integrity or endurance.

Is an expanded-access program planned for PTC124?

At this time, an expanded access program would be premature. Although the early data in cystic fibrosis and Duchenne muscular dystrophy are promising, the results are very preliminary, and were obtained from a small number of patients receiving PTC124 for a short period of time. Conduct of an expanded access program at this time might result in unacceptable risks for patients and might jeopardize the development of PTC124 so that it cannot become available for all patients who might benefit if its efficacy and safety are eventually proven.

Based on the results of the evolving Phase 2a data and data from the long-term safety studies in animals, we are in discussions with investigators, patient advocacy organizations, regulatory authorities, and potential collaborators regarding the conduct of additional trials in several regions of the world.

Once a clinical trial is initiated, important information is made available at www.clinicaltrials.gov. PTC sends updates via email about our trials and other news about PTC. To join the mailing list, visit the “Contact Us” portion of our website (www.ptcbio.com/contact_form). We also recommend that patients work closely with their treating physician and genetic counselor, as well as communicating with patient advocacy groups, in order to understand their options.

There are a few sources for this information. We recommend you visit our website (www.ptcbio.com), which has an extensive section on PTC124. Additionally, in May 2007 the journal Nature featured an article on PTC124. You can read this article online at www.ptcbio.com.

Additional details about PTC124 clinical trials can be found on www.clinicaltrials.gov and by contacting Ms. Diane Goetz, Director, Patient and Professional Advocacy at PTC Therapeutics (908-222-7000 ext.9256, PatientInfo@ptcbio.com).