Pegasys improves things for patients with chronic hepatitis C

Envelope improves resistance to enzymatic breakdown

Pegylated interferon alfa-2a (PEG-INF, Pegasys) shows better characteristics in the treatment of chronic hepatitis C than does the unmodified interferon (IFN) used to date. In order to produce the second generation of this drug, a branched polyethylene glycol (PEG) molecule with an average molecular weight of around 43 kilodaltons was attached to it. In animal models this pegylation produced a 12 to 135-fold increase in effect against viruses and an 18-fold increase in effect against tumor cells. The modified drug has a biological half-life around ten times greater than the 8.5-hour half-life of the unmodified drug and therefore needs to be injected only once weekly as compared with three times weekly. An improvement in response rate is also favored by absence of the fluctuations in serum level that occur with the unmodified drug.

1. After the initial infection viruses are unable to harm the cells

Interferon was discovered in 1957 by two London-based scientists, the Briton Alick Isaacs and the Swiss Jean Lindemann. They came across the substance when analyzing the effects of viral infection on cells in a tissue culture. They noticed that cells already infected with a virus appeared to be resistant to infection by other viruses for a certain period of time. The first infection was said to "interfere" with (inhibit) the second. The protein isolated from these cell cultures that was absent from uninfected cells was therefore given the name interferon (IFN).

It is now known that these substances belong to a class of proteins that are produced by white blood cells as part of the body's natural immune response as soon as the body is exposed to attack by viruses, other microorganisms, or tumor cells.

They can be classified on the basis of their structure into three groups, namely interferon alpha, beta, and gamma. The alpha group alone consists of at least 15 subtypes that differ in terms of their amino acid sequence and are maintained in their folded shape by disulfide bonds.

Interferon alfa-2a is a protein consisting of 165 amino acids without a glucose unit that is maintained in its three-dimensional loop structure by two disulfide bridges.

2. Interferons initiate a cascade of reactions

As a result of binding of interferon alpha to a specific receptor on the cell surface, the concentrations of fifty to a hundred proteins inside the cell can be altered. Some of these proteins act as a signal or target structure for mechanisms of the human immune system. Interferons inhibit cell division and the formation of new vessels. They also influence cell communication and are involved in the breakdown of unstable cellular messenger RNA, which plays an important role in the replication of tumor cells. By inducing cell differentiation, they can alter the cell cycle and retard uncontrolled division of tumor cells.

Interferon alpha is therefore used against malignant cells of the skin and immune system such as hairy cell leukemia, chronic myeloid leukemia, non-Hodgkin's lymphomas, malignant melanoma, renal cell carcinoma, and HIV-related Kaposi's sarcoma.

The drug combats not only tumor cells, but also viruses. For example, one of the first proteins to appear after addition of interferon alfa-2a is the enzyme oligoadenylate synthetase. This enzyme catalyzes the synthesis of small oligonucleotides. This is followed by activation of another enzyme that breaks down single-stranded viral RNA and thus prevents replication of viruses. As soon as IFN attaches to a cell receptor, various other cells of the immune system are stimulated to attach themselves to viral fragments on the surface of infected cells and thus to induce an immune response. In this way infecting viruses are destroyed and uninfected cells are protected from attack. Because of these mechanisms of action, IFN is also suitable for the treatment of chronic hepatitis B and C.

3. Bacteria can produce single products

For a long time about 60,000 liters of human blood were required in order to produce one gram of interferon. In order to increase this low yield, the substance is now produced by recombinant DNA technology. This method utilizes cells that can be cultivated in large amounts and that also produce high concentrations of interferon. In order to achieve this, the DNA sequence that codes for the desired protein is isolated and inserted into the genetic material of a foreign organism.

The genetic information that is required to be mass-produced in the case of interferon is a human leukocyte interferon gene. The rapidly replicating microorganism used for production is a nonpathogenic Escherichia coli bacterium. The bacteria that have been rendered "recombinant" by insertion of the human genetic information now produce not only their own proteins, but also human interferon. In this way a purified product containing only the desired interferon subtype alfa-2a can be obtained. With the aid of a number of chromatographic and filtration steps, the purity of the product is brought up to 99 percent.

In June 1986 the first recombinant interferon alfa-2a produced by Roche (Roferon A®) was licensed in the USA and in Switzerland for the treatment of hairy cell leukemia. The actual manufacturing process was developed by Sidney Pestka and his coworkers at the Roche Institute for Molecular Biology.

4. The liver can often be protected from further damage

For the treatment of chronic hepatitis C the relevant Swiss professional associations recommend a dose of 3-6 million International Units (3-6 MIU) of interferon alpha three times weekly by subcutaneous (s.c.) injection for a total period of 12 months, while the dose for chronic hepatitis B is 6-8 MIU three times weekly s.c. for four months.

The extent of the response to interferon is determined largely by the infecting virus, but also by the type of cells infected. A response is defined as normalization of the concentration of the enzyme alanine aminotransferase, which in simplified terms can be regarded as an indicator of the degree of liver cell damage, and a fall in the concentration of the RNA of the hepatitis C virus to below the limit of detection. Interferon sometimes has side effects. These range from nausea and loss of appetite through to flu-like symptoms including fever, headache, chills, and muscle pain. Blood changes and hair loss are common, though the hair loss ceases as soon as treatment is stopped. Symptoms generally become less pronounced with continued treatment. Nevertheless, autoimmune diseases and depression may occur and in rare cases depression has led to suicide.

The ideal patient in terms of likelihood of recovery is young and thin, was infected only recently, has a low concentration of virus in the body, shows little or no cell damage in the liver biopsy, and is infected with virus of a favorable genotype. Unfortunately, these criteria are met by only 10 to 20 percent of patients.

5. Pegylation improves molecular properties

In order to improve the efficacy of the drug, interferon alfa-2a has now been pegylated. Rearrangement of molecular structure with the aid of polyethylene glycols (PEGs) allows the molecular properties of a drug to be altered. PEGs are nontoxic and amphophilic, i.e. soluble both in water and in most organic solvents.

Protein pegylation is generally achieved via stable covalent bonds between an amino or sulfhydryl group on the protein and a chemically reactive group (carbonate, ester, aldehyde, or tresylate) on the PEG. The resulting structures can be linear or branched. The reaction can be controlled via factors such as protein type and concentration, reaction time, temperature, and pH value. Environmental factors such as these likewise influence electrostatic binding properties and protein charge, form, and size. Suitable PEGs must therefore be found for each therapeutic molecule.

Pegylation is a technique via which intravenously administered therapeutic proteins, in particular, can be altered so as to improve their stability, biological half-life, water solubility, and immunologic characteristics. A PEG mass of 40 to 50 kilodaltons is sufficient to increase the size of a small molecule to such an extent that it is less readily excreted through the kidneys and therefore persists in the body for longer. In addition, as they are more or less surrounded by the attached PEGs, pegylated proteins are less rapidly broken down by the body's enzymes than are unmodified proteins.

By increasing the biological half-life and improving the efficacy of drugs in the body, these modifications can reduce the frequency of injections a patient requires. They also reduce the rapidity and intensity of the body's immune reaction against the interferon molecules.

Almost any molecule can be modified in this way. Not only are therapeutic proteins and peptides such as growth factors and blood products now being pegylated for medicinal purposes, but, because of the safety of the method, substances such as liposomes used in foods and cosmetics are also being pegylated. Human beings could thus find themselves coming into daily contact with PEGs.

3-D Structure Interferon