ACAPI - Ismafron

THERE ARE TWO TYPES OF INTERFERONS that are naturally produced by the body. Type II interferon is involved with controlling the immune functions of the body—there is only one form of Type II interferon, and it is called gamma interferon.
Type I interferon is known as alpha or beta and is made up of proteins produced by the body in response to a viral infection. For instance, when an individual becomes infected with the flu virus, the immune system immediately produces various interferons to protect other cells from the invading virus. The symptoms people experience from the flu, such as muscle pain, fever and headaches, are due to the body’s natural production of interferon.
In more detail, interferon works by binding to cell surface receptors and triggering the production of intracellular second messengers, including 2’5’ oligo-adenylate synthetase, protein kinases, cell surface proteins, and nuclear proteins.
Interferon also works in other ways by increasing the body’s immune response against viruses and infected cells:

Antiviral action – Interferon prevents the entry of a virus into a cell, which limits the amount of new cells that become infected. Interferon also inhibits the replication of viruses by preventing the virus from uncoating within the cell and preventing the viral replication process within the cell by interfering with the viral protein synthesis.

Immunomodulatory effect – Interferon stimulates the production of cytokines (chemical messengers) that activate macrophages, natural killer (NK) cells, and cytotoxic T-lymphocytes (CTLs, or killer T-cells) which then kill the virus and the infected cell.

Antitumor effects – Interferon reduces the production of both normal and malignant (cancerous) cells and inhibit oncogene expression. Interferon also enhances direct T-cell-mediated cytotoxicity against tumor cells. This kills cancer and tumor cells.
Enhanced cell surface expression of MHC – Interferon enhances the expression of class 1 major histocompatibility (MHC) antigens on the surface of infected cells. Expression of these proteins on the cell surface allows virus infected cells to be targeted and destroyed by CTLs.

Sometimes the body does not make enough natural interferon to effectively fight an infection and the addition of interferon helps the body to fight off a viral infection such as hepatitis C.
Scientists discovered the antiviral properties of interferon, a naturally occurring substance, in 1957. Further research developed 10 purified interferon proteins in 1978.It was approved by the FDA five years later. Interferon is also approved to treat many other diseases, such as cancer, hepatitis B, Kaposi’s sarcoma, and hepatitis C.

Standard interferon plus ribavirin , this combination interferon plus ribavirin was approved by the FDA for treatment of hepatitis C. Standard interferons are administered by subcutaneous or intramuscular injection three times a week. Standard interferon’s half life (the time required by the body to metabolize or inactivate half the amount of the substance) is two to five hours.

The future use of interferon to treat a variety of diseases is unlimited. It is estimated that there are over 180 clinical trials using interferon to treat cancer. The on-going evolution of technology is producing refined forms of interferon that scientists believe will have the ability to treat many more forms of cancer and viral infections.

Injection form,

Interferons are a family of pleiotropic cytokines with antiviral, anti-proliferative and immuno-modulatoryproperties. The interferon family is subdivided into two subfamilies: Type I and Type II. Type I interferons are a family of monomeric cytokines with an aminoacid similarity of 30-80%, very similar three-dimensionalstructure (5-alpha helix-bundle), that use the same receptor (IFNAR) to initiate a signalling response.
The 12 interferon alpha protein subtypes exhibit very high amino-acid similarity (over 75%). However, the
functional role for the existence of so many distinct proteins is unknown. Their relative antiviral andante-proliferative activities are different depending on the target cell involved.
The alpha and B interferons bind to the same membrane receptor (IFNAR).

The Interferon Receptor (IFNAR)
The Interferon Receptor has two components (IFNARI and IFNAR2). The first receptor component
(IFNAR1) was cloned in 1990. It has an apparent molecular weight of 110-130kD and encodes for a trans-membrane protein with a large extra-cellular portion with a duplication of a structural domain composed of repeated fibronectin III motif (class II receptor family) and a very short intracellular region (100 amino acids) with no well-defined motifs. IFNAR1 has a very low affinity for interferon on its own and it is presumed to complement and enhance binding only when accompanied by its companion receptor chain (IFNAR2).
A splice variant of IFNAR1 has been discovered with a deletion in the extra-cellular region, with a preferential sensitivity to interferon alpha-2 relative to alpha-8. The tissue specific expression of this variant form and its physiological role are still unknown. Phosphatases (SHP-1 and SHP-2) also associate with IFNAR1, supposedly playing a negative regulatory role in the activation of the JAK-STAT pathway.
The second component of the Type I interferon receptor (IFNAR2) was isolated in 1994 and again shown to be composed of both an extra-cellular and an intracellular region with a molecular weight of 55 –95 kD. It was discovered that IFNAR2 exists in three splice variant forms depending on the length of the intracellular region:
IFNAR2a (short), IFNAR2b (soluble) and IFNAR2c (long). The IFNAR2c variant is necessary and sufficient for JAK-STAT activation and an antiviral response, although both IFNAR1 and IFNAR2c are required for interferon signaling. The role of the short and soluble variant forms are not yet known, but the presence of the short form in some human and murine cells might suggested a modulatory role.

The interferon ligand is presumed to bind to IFNAR2 first and then to IFNAR1, which stabilizes the complex.
There is no direct evidence for this model, however, it is a logical hypothesis based on the calculated binding constants for each receptor component. While IFNAR2 binds interferon with moderate affinity (KD E2-10nM for interferon alpha-2), the intrinsic affinity of IFNAR1 is low (KD>100nM), stabilizing the complex by approximately 10-20 fold.

The interferons regulate a variety of important biological functions, through interaction with the specific receptors on the surface of cells. These receptors are responsible for carrying the signal through the cell membrane and re-directing it to various cytoplasmic and nuclear compartments. Most of the nuclear signals lead to the induction of specific genes. Upon stimulation with interferon, protein complexes are formed that are translocated to the nucleus, bind to DNA regulatory elements and activate specific genes. These cytoplasmic factors are called STATs (Signal Transducers and Activators of Transcription).

The Signal Transduction System (Jak-Stat):
Binding of interferon œ / ?? ?initiates the signaling cascade by causing dimerization of the receptor subunits,IFNAR1 and IFNAR2. It is almost certain that this initial step triggers a conformation change that is propagated through the cell membrane and is responsible for the initiation of the phosphorylation cascade.
The first intracellular component of the signaling pathway that receives the “transduction pulse” is the JAK kinase, Tyk2, which is pre-associated with IFNAR1. Upon interferon stimulation, Tyk2 is immediately phosphorylated by JAK1, another JAK kinase, which is bound to IFNAR2. Activated Tyk2 then in turn phosphorylates JAK1.
The activated JAK kinases, Tyk2 and JAK1, are responsible for the subsequent phosphorylation of IFNAR1 and IFNAR2 at specific tyrosine residues. STAT2 then binds to specific phosphorylated residues on IFNAR1.
Upon docking, STAT2 is phosphorylated at a conserved tyrosine residue (Y701) by the JAK kinases thereby creating an additional docking port for STAT1, which is also subsequently phosphorylated at Y690. The phosphorylated STATs then dissociate from the receptor heterodimer and bind to p48 (IRF9), a member of the interferon regulatory factor (IRF) family, forming the major interferon transcription factor, known as ISGF-3.
ISGF-3 translocates to the nucleus and binds to specific regulatory DNA sequences (ISRE-Interferon stimulated response elements) and initiates transcription of several interferon-inducible genes. STAT1:2 heterodimers as well as STAT1:1 homodimers also form and are capable of driving the expression of a minority of interferon stimulated genes (ISGs), independently of p48. In this case, they bind to different DNA regulatory sequences than ISRE called GAS (gamma activated sequence elements), which are usually found
in the promoters of interferon gamma stimulated genes.

Functions of Interferons
Interferon are pleiotropic proteins, able to initiate and regulate a variety of responses, either directly or by stimulating the induction/activation of additional proteins. Different interferon subtypes intrinsically have the ability to stimulate different but overlapping sets of genes. However, the overall phenotypic responses appear to be similar.

Antiviral properties
The ability of interferon to establish an “antiviral state” is the distinctive fundamental property of interferons, essential for the survival of higher vertebrates against viral infection (Figure 3).

There are multiple redundant antiviral pathways that allow interferons to combat multiple different viruses in every vulnerable step in their replication cycle, starting from entry/uncoating (SV40, retroviruses), transcription (influenza, VSV), RNA stability (picornaviruses), translation initiation (reoviruses, adenovirus, vaccinia), maturation and assembly/release (retroviruses, VSV).

Time course of immune response Interferons are the first line of defence against viral infections. They act very rapidly, usually within hours or a few days after infection against a variety of viruses.

Interferon a/b JAK-STAT signalling pathway
Multiple interactions among the JAKs and STATs are responsible for a variety of signaling responses, with possible positive or negative regulatory roles on the transcriptional activation of interferon stimulated genes (ISGs)

The vital antiviral role of interferons has been demonstrated in interferon alpha/beta-deficient mice that are extremely susceptible to viral infections. It should be noted that interferons have no antiviral activity of their own but rather induce the expression of potent antiviral genes.
*The Protein Kinase R (PKR) system:
This is perhaps the most well characterized interferon-induced antiviral pathway. PKR is a 551 amino-acid serine-threonine kinase mainly found in the cytoplasm of most cells and partly in the nucleus, where it normally lies inactive. The expression of PKR is rapidly stimulated by interferons and reaches levels approximately 5- 10 fold higher that in resting cells. This action of interferon is mediated through an ISRE (interferon stimulated regulatory element) and GAS (gamma interferon-activated site) in the promoter region of the PKR gene.
PKR contains two conserved dsRNA-binding motifs at the amino-terminus, RI (amino-acids 55-75) and RII (amino-acids 145-166). Both regions possess a similar core sequence, but RI alone appears to be necessary and sufficient to mediate dsRNA binding. It is believed that a combination of both domains creates a single binding site where the different domains bind to different regions of the dsRNA molecule. No RNA sequence specificity has been identified.
PKR is activated by double stranded RNA (viral RNA or secondary RNA structures), which results in a conformational change that uncovers the carboxy-terminal catalytic domain of PKR. This activates the kinase activity of PKR, which then undergoes auto-phosphorylation/activation in several serine and threonine residues. The activated kinase phosphorylates in turn the translation initiation factor eIF2- a ?? t Ser51. eIF2--
G ? TP is required for initiation of translation. It is recycled from eIF2-?-GDP, which is produced after each round of initiation, by eIF-2B, which mediates the guanine nucleotide exchange step. However, eIF-2B binds preferentially to the phosphorylated form of eIF-2 ?. Therefore, the cellular stock of eIF-2B is sequestered in
the inactive complex eIF2 ?-GDP-eIF-2B resulting in rapid inhibition of translation.
PKR has also been involved in regulating cell proliferation, playing a role as tumor suppressor and in signal transduction by regulating the serine phosphorylation of STAT1 and by phosphorylating I kB, which in turn results in activation of NF-k B-dependent genes.

Interferon “priming” Interferons are produced in response to a viral infection and spread to the nearby cells where they activate interferon-stimulated genes (ISGs), which are responsible for the establishment of an “antiviral state” by preventing viral replication and also alert the immune system.

** The 2-5A oligosynthetase/RNAse L system
This multi-enzyme system is composed of three separate components: 2-5A synthetase, endo-ribo-nuclease RNAse L and 2-5A phosphodiesterase. The different 2-5A synthetases (40, 46, 69 and 100kD) are encoded by multiple genes and reside in different subcellular compartments. The components of the 2-5A system are
present at low levels in resting cells but are strongly induced by interferons.
The system is activated by dsRNAs, probably of viral origin. Initially, 2-5A synthetase is stimulated by dsRNA
and produces a series of short 2’-5’-oligoadenylates that bind to inactive, monomeric RNAse L converting it into a dimeric, active enzyme. RNAse L degrades all single-stranded RNA inhibiting, in theory, all viral replication that uses an RNA intermediate step. 2-5A phosphodiesterase regulates the entire process by catalysing the degradation of the 2’-5’ oligoadenylates thereby “switching off” the system.
The efficiency of the 2-5A system has been shown for EMC virus as well as vaccinia and HIV-1, but not for VSV or HSV. Since the 2-5A system operates mostly in the cytoplasm it is possible that viruses that replicate in the nucleus, such as VSV and HSV, might escape the antiviral actions is the system.

*** The Mx system
Mx proteins are interferon-inducible, high-abudance 70-80kD GTPases of the dynamin superfamily. Human MxA proteins assemble into oligomeric complexes in cell-free systems. They interfere with replication at the transcriptional and other levels in influenza and other negative-stranded viruses replication.
Initially the Mx system was studied in mice, in which Mx1 was found to be responsible for resistance to orthomyxovirus infection. Two homologues of Mx1 were found in humans, MxA and MxB, which appear to be functionally different from Mx1 and are also localized in the cytoplasm as opposed to Mx1, which is mainly a
nuclear protein. The exact mode of action of Mx protein is not completely known, but they are believed to interfere with trafficking or activity of viral polymerases.
**** Other anti-viral systems
Several proteins are been known to interfere with virus replication. Guanylate Binding Protein (GBP) is an interferon-stimulated protein with demonstrates antiviral properties, although the mechanism of action is unknown. Interferon inducible protein 9-27 inhibits VSV replication and nitric oxide synthase (iNOS) protects
macrophages from infection by several viruses. A protein with very high homology to 9-27 has been identified to bind to the rev-response element of the human immunodeficiency virus HIV-1 and inhibits rev-mediated transcription. Additionally, experiments in yeast have shown proteins to bind to viral mRNAs and prevent their translation.

Antiproliferative properties
Interferons have the ability to arrest cell growth, which is why they are used as treatment for cancer. It seems that both direct and indirect (via the immune system) inhibition of the tumor cells might be involved, however, the exact mechanism of action is unclear. No specific genes have been linked to the anti-proliferative activity of interferons, however, STAT1 is believed to be involved since it is often deficient in tumors. The mechanism of cellular arrest in not known, but it is likely to target components of the cell-cycle control apparatus, including induction of CDK inhibitors such as p15/16 and p21WAF1/Cip or the decrease in levels of cyclin D and cdc25A. PKR is also suspected to play a role in the regulation of cell growth.

Control of apoptosis
Regulation of apoptosis and control of cell growth is very important for host responses against viruses.
Interferons in conjunction with dsRNA, TNF or LPS are a potent inducer of cell death. Over expression of PKR induces apoptosis through a mechanism dependent on bcl-2 and ICE. Additionally, PKR-deficient mice resist apoptotic death through a mechanism linked to a defect in activating the DNA-binding activity of IRF-1.
Novel interferon regulated genes have been identified that encode for death associated proteins (DAPs) and are necessary for interferon-induced apoptosis in HeLa

It is now well accepted that type 1 interferons (IFNs), IFN-alpha and IFN-beta, in addition to being molecules with powerful antiviral activity, play a critical role in modulating immune responses to foreign and self-antigens. This review of the literature documents the immunomodulatory effects of IFN-alpha and discusses its position and importance in the cytokine cascade. In addition, this review attempts to organize the literature describing local and systemic immunomodulatory effects of orally administered low doses of IFN-alpha, and provide a physiological explanation for the mechanism of action. Evidence suggests that, early in the process of antigen presentation to T helper (Th) cells, IFN-alpha derived principally from the antigen-presenting cells (APC) provides an important signal for Th precursor differentiation in favor of a Th1 immune response. IFN-alpha, perhaps via upregulation of the high-affinity interleukin-12 beta1/beta2 (IL-12beta1/beta2) receptor, renders Th1 cells responsive to IL-12 resulting in production of high levels of IFN-gamma crucial to the development of Th1 immune responses. In addition to being instrumental in the development of Th1 immune responses, IFN-alpha appears to be the major cytokine responsible for the amplification of the CD8+ effector's cell response and resistance to viral infections. Orally administered IFN-alpha induces similar Th1 cytokine responses in buccal mucosal lymph nodes (LN), including up-regulation of IFN-gamma expression and down regulation of IL-4. Moreover, reports of systemic immune effects such as decreased autoimmune responses, increased antiviral and antibacterial responses, and generalized immune function changes after oral IFN-alpha administration are consistent with the known immunomodulatory role of IFN-alpha in a physiological setting. Responses to orally administered low doses of IFN-alpha also adhere to the principle of low-dose priming and high-dose anergy that dictates the cellular and cytokine responses to exogenously added cytokines both in vivo and in vitro. These observations collectively suggest that IFN-alpha administered to mucosal-associated immune tissue replicates the known physiological role of IFN-alpha, including regulation of CD4+ Th1 immunomodulatory cells and activation of CD8+ effector cells, which are both crucial to development of protective immune responses. What remains to be determined is how local mucosal immune responses to IFN-alpha given orally are translated into systemic immune responses and resistance to disease. This important question, the answer to which will have profound implications for new immune-therapies for immune-based diseases, is the focus of current research.

Ismafron is a highly purified, multi-subtype, natural human alpha interferon derived from human white blood cells, a central component of the body’s immune system. It has been studied in more than 1600 patients in clinical and supporting studies, including hepatitis C, malignant melanoma and other cancers.
Ismafron is expected to offer a safe and effective first-line or second-line therapy for many viral and malignant indications including chronic hepatitis C and certain cancers.
Ismafron is approved for the second-line treatment of any and all diseases in which recombinant (synthetic) interferon therapy failed or the patient was unable to tolerate the regimen, probably due to the formation of neutralizing antibodies.
Ismafron is also approved as a second-line therapy for the treatment of Hairy Cell Leukemia (HCL) and Chronic Myelogenou Leukemia (CML)
It is estimated that a large portion of patients treated with recombinant interferon fail to respond to standard therapy. It is this market that ACAPI is focusing on. This substantial percentage of patients whose standard hepatitis C treatments failed or could not be tolerated represents a critical need for an alternative strategy since no other secondary treatment is currently approved.

ACAPI manufactures Ismafron in its facilities in EGYPTand has filed five patent approvals related to the product including production techniques and processes which ACAPI believes will allow the Company to offer natural human alpha interferon at a price competitive with recombinant interferons.
What is Natural Interferon?
Produced by leukocytes (white blood cells), natural interferon helps improve the body’s natural resistance against disease. It is one of the body’s natural defensive responses to foreign substances such as viruses, and is so named because it “interferes” with viral growth.
Recombinant Interferon VS Ismafron
Therapy resistance is not unusual in recombinant interferon treatments. This may be caused by the development of antibodies to recombinant interferon which often leads to a loss of clinical efficacy. Therefore, it seems more advantageous for a patient to be treated with a natural product that potentially elicits an effective immune response and may carry a superior side-effect profile. ACAPI believes that Ismafron should be regarded as an important therapeutic product against a variety of viral and malignant diseases.

Cytokines that mediate and regulate innate immunity
Type I interferons
Tumor necrosis factor-a
Interleukins 1, 6, 10, 12, and 15
Chemokines
Cytokines that mediate and regulate specific immunity
Interleukins 2, 4, 5, 13, 16, and 17
Interferon-g
TGF-?
Lymphotoxin
Cytokines that stimulate hematopoiesis
c-kit ligand
Interleukins 3, 7, 9, 11
Colony stimulating factors
Another way is to group them according to the sequence homology of the receptors to which they bind. Receptors for the various cytokines belong to one of four or five (depends on what you read) families of receptor proteins.

Ismafron contains cytokines including:
IFN?, IL1, IL2, IL3, IL6, IL7, IL12, TNF?, GM-CSF.

So the indication of Ismafron are
The injection form (3MIU, 1MIU)
Viral infection