INTERFERONS

--Dr. Varun GoelDr. Varun GoelMEDICAL ONCOLOGISTMEDICAL ONCOLOGISTRAJIV GANDHI CANCER INSTITUTE, DELHIRAJIV GANDHI CANCER INSTITUTE, DELHI

INTRODUCTION

Among the most powerful immunomodulatory substances available for therapeutic use.

Highly pleiotropic properties : • immunomodulatory• direct cytostatic • direct cytotoxic. • antiproliferative activity and apoptotic effects

DISCOVERY OF INTERFERONS 1957 Isaacs and Lindenmann Did an experiment using chicken cell cultures Found a substance that interfered with viral

replication and was therefore named interferon (“Interference factors”)

Nagano and Kojima also independently discovered this soluble antiviral protein

WHAT ARE INTERFERONS?

• Naturally occurring proteins and glycoproteins• Secreted by eukaryotic cells in response to viral infections, tumors, and other biological inducers

• Strucurally, they are part of the helical cytokine family which are characterized by an amino acid chain that is 145-166 amino acids long

•

WHEN A TISSUE CELL IS INFECTED BY A VIRUS, IT RELEASES INTERFERON. INTERFERON WILL DIFFUSE TO THE SURROUNDING CELLS. WHEN IT BINDS TO RECEPTORS ON THE SURFACE OF THOSE ADJACENT CELLS, THEY BEGIN THE PRODUCTION OF A PROTEIN THAT PREVENTS THE SYNTHESIS OF VIRAL PROTEINS. THIS PREVENTS THE SPREAD OF THE VIRUS THROUGHOUT THE BODY.

General Action ofInterferons

Induce a state in which viral replication is impaired by the synthesis of a number of enzymes that interfere with cellular and viral processes

Anti-viral activity induces antiproliferative as well as immunomodulatory activities.

It is these additional functions induced by interferons that resulted in their evaluation as anticancer agents

Humans -2 classes Type I family - α, β, δ, ε, κ, τ and ω subtypes.

Type II interferon family - γ interferon subtype.

Subtypes further subdivided for pharmaceutical preparations (e.g., interferon alfa-2a, interferon gamma-1b).

Type I interferons

- chromosome 9p - all bind to the same receptor - induced primarily in response to a viral

infection of a cell.

- Interferon α, ω - produced predominantly from leukocytes,

- Interferon β - can be produced by most cell types, esp. fibroblasts.

v@

Viral ds RNA

@

protein kinase R (PKR),

Iκ B)

@

NFκ B

@

NFκ B

(TRANSCRIPTION(TRANSCRIPTION))INTERFERON -INTERFERON -

ββ

INTERFERON β induction by fibroblasts during viral infection

Viral induction of interferon- α in leukocytes is less well understood

Induction stimuli

- Classical -Viral infection

- proteins or DNA constructs of microbial derivation, (bacterial endotoxins, CpG dinucleotide repeats,) also stimulates production of type I interferons.

Type II interferons (Interferon γγ )

- composed of a single subtype.

- binds to its own unique receptor

- produced by T lymphocytes and

natural killer (NK) cells

- in response to immune and inflammatory stimuli.

NK cellsNK cells-part of the innate immune response

-activation not dependent on antigen recognition.

T cells- part of the adaptive immune response - activated by stimulation of antigen- specific

T-cell receptor

NK cells are stimulated to produce interferon- γ when exposed to inflammatory cytokines (Tumor necrosis factor-α and interleukin-12 (IL-12)) from macrophages.

In a positive amplification loop, the interferon- γ then stimulates the secretion of TNF- α and IL-12 from macrophages.

TNF –α IL-12

NKNK

++

Interferon- γ++

MM

The distinct receptors and the downstream signaling that is initiated by ligand-receptor interactions are responsible for the biologic effects of the individual type I and type II interferons.

BIOLOGIC EFFECTS OF INTERFERONS

Interferons ά and β bind to the same receptor, which is composed of two subunits

The binding of either interferon- ά or interferon- β to this receptor results in the activation of Janus tyrosine kinases Jak1 and Tyk2, which results in the phosphorylation of signal transducers and activators of transcription 1 and 2 (STAT1 and STAT2).

STAT1 and STAT2 phosphorylation results in their heterodimerization, dissociation from the interferon receptor, and translocation to the nucleus.

In nucleus, the STAT complex associates with DNA-binding protein p48 (interferon-stimulated gene factor 3) (ISGF3),which binds to the interferon-stimulated response element of ά- and β-responsive genes.

induction of interferon target genes, responsible for the biologic effects of interferons ά and β .

INTERFERON- γγ Interferon-γγ binds as a

homodimer to the specific interferon-γγ receptor

Dimerization of the receptor activates the Janus tyrosine kinases Jak1 and Jak2, which ultimately results in the phosphorylation of STAT proteins.

INTERFERON- INTERFERON- γγ Instead of activating STAT1

and STAT2( as with type I interferon receptor) interferon-γγ activates two separate STAT1 molecules.

form a homodimer known

as the - γ γ activated factor (GAF)

translocates to the nucleus and binds to γγ -activating sequences (GAS), elements of interferon- γγ inducible genes

The antiviral activity of interferons is mediated by three pathways.

- 2'-5' oligoadenylate synthetases

-dsRNA-dependent PKR

-Mx pathways.

ATP2'-5' oligoadenylate synthetases2'-5' oligoadenylate synthetases

Adenosine oligomers (2'-5’A)

RIBONUCLEASE- L (RNase L)

@@

cleaves single-stranded RNA

inhibition of protein synthesis and hence cellular proliferation

dsRNA-dependent PKR

PKR is activated by binding to dsRNA.

1) Once activated, PKR phosphorylates the translation initiation factor eIF2, which inhibits messenger RNA translation.

2)

3) PKR also can activate NFκ B, which can lead to increased cytokine and chemokine levels.

4) Increased PKR activity can also induce apoptosis by a Bcl2- and caspase-dependent mechanism.

dsRNA-dependent PKR

dsRNAdsRNA

1.eIF21.eIF2

PPKRPKR

_ mRNAtranslation

2.NFκ B2.NFκ B@

CHEMOKINES +

CYTOKINES

Eif2-Translation initiation factor

• Another group of proteins induced by interferon that exhibits antiviral properties is the Mx family of glutamyl transpeptidases.

• MxA is produced during viral infections and inhibits viral replication at the level of transcriptiontranscription by binding to susceptible viral nucleocapsids in the cytoplasm and preventing their movement into the nucleus.

EFFECTS OF INTERFERONS

IMMUNOLOGIC EFFECTS

Cytokines regulate the innate immune system natural killer (NK) cells, macrophages and neutrophils.

They also regulate the adaptive immune system, the T and B cell immune responses.

Direct effects on tumor cells

Increased MHC class I antigen expression, increased expression of tumor-associated antigens, increased expression of adhesion molecules

Nonimmunologic effects

Antiangiogenesis effects*-Following therapyendothelial cells damagecoagulative necrosisRegulates expression of angiogenic basic fibroblast growt factor (bFGF).

Direct cytostatic and cytotoxic effects on tumor cells

Antimetastatic effects on tumor cells

Interferons induce hundreds of specific gene products, most of which are important in regulation of cell proliferation and apoptosis.

Type I interferons- up-regulate cdk inhibitor p21 inhibition of the G1 to S phase transition.

Interferon-γγ - down-regulates c-myc transcription (essential in driving

cell-cycle progression.) - induces Fas and Fas ligand, which can increase cell

sensitivity to apoptosis.

Interferon-ά +anti-CD3 induces the surface expression of tumor necrosis–related apoptosis-inducing ligand on peripheral T cells.

Interferons induce apoptosis by activation of several

members of the caspase pathway

T1/2 of recombinant interferon alfa-2 varies between 2 and 8 hours in the blood after s.c. or I.m. administration,& somewhat shorter after I.v. administration.

Oral delivery- impractical due to proteolytic degradation.

Peak plasma concentrations are highest after iv administration.

I.V. administration of interferon alfa is performed daily (5 days per week in the most commonly used regimen for adjuvant therapy of interferon)

S.c or I.m. administration is performed thrice weekly. Other dose schedules have been explored in the hopes

of maximizing certain properties of interferons while minimizing toxicities (e.g., low-dose, twice-daily schedules of subcutaneous interferon alfa in "antiangiogenesis" trials).

EFFECTS OF DOSE AND SCHEDULE ON EFFICACY AND TOXICITY

A variety of doses, schedules, and routes of administration of interferon have been tested in clinical trials, particularly in the setting of adjuvant therapy for melanoma patients

No consistent observation that responses are linked to a specific dose or schedule of administration.

Responses are more common in patients with small, generally soft tissue or lung nodules.

Clinical trials have clearly established a greater incidence of grade 3 and 4 toxicity for high-dose regimens than for lower doses.

ONCOLOGIC APPLICATIONS OF INTERFERONS

Pharmacology and Dosage Available in both natural and recombinant forms, Commercially available interferon formulations include

- human leukocyte–derived interferon alfa-n3 (Alferon N);

- recombinant "consensus" interferon, alfacon-1 (Infergen);

- recombinant interferon alfa-2a (Roferon-A);

- recombinant interferon alfa-2b (Intron A);

- recombinant interferon beta-1a (Avonex, Rebif);

- recombinant interferon beta-1b (Betaseron);

-recombinant interferon gamma-1b (Actimmune,InterMune).

Most of the oncologic experience has been accumulated with interferon alfa-2a and alfa-2b, which are approved by the U.S. FDA for use in

-Interferon alfa-2a- hairy cell leukemia,AIDS – related Kaposi's sarcoma,Philadelphia chromosome–positive CML

-Interferon alfa-2b- hairy cell leukemia,AIDS– related Kaposi's sarcoma,follicular lymphoma, and malignant melanoma

Interferon alfa-n3 (Alferon-N)

genital and perianal warts 

Interferon beta-1b (Betaseron) and beta-1a(Avonex)

multiple sclerosis

Interferon gamma-1B (Actimmune)

chronic granulomatous disease and severe, malignant osteopetrosis.

Interferon alfa-2b condylomata acuminata, chronic hepatitis C, and chronic hepatitis B. 

Oncologic Applications of Interferons

Tumor types with established indications

Chronic myelogenous leukemia

Hairy cell leukemia

Non-Hodgkin's lymphoma

Acquired immunodeficiency–related Kaposi's sarcoma

Malignant melanoma

Tumor types for which interferons are commonly used

Renal cell carcinoma

Bladder carcinoma (intravesical therapy)

Other tumor types for which interferons have shown some evidence of activity

Colorectal carcinoma (with 5-fluorouracil)

Carcinoid tumor

Desmoid tumor (aggressive fibromatosis

Human cancer targets of interferon therapy

"palliative" treatment for CML only rarely results in a prolonged complete

cytogenetic response.

A patient who cannot tolerate tyrosine kinase inhibitors might be offered IFNa with or without another chemotherapy medication, cytarabine.

In chronic phase CML, with use of interferon-α (dose 5 x 106 U/m2/day or three times weekly)

Complete hematological response in ~70%Complete cytogenetic response in ~ 25% cases.

o Its effects on remission duration and survival are controversial.

Silver R et al. An evidence-based analysis of the effect of busulfan, hydroxyurea, interferon, and allogeneic bone marrow transplantation in treating the chronic phase of chronic myeloid leukemia: developed for the American Society of Hematology. Blood. 1999;94:1517-1536.

In HCL interferon- α became first effective non-surgical treatment.

75% patients achieved partial or complete response. Median duration of response=1-2 yr but virtually all

patients eventually relapse. Quesada J, Hersh EM, Manning J, et al. Treatment of hairycell

leukemia with recombinant alpha interferon. Blood. 1985;1986:493-497.

Now cladribine is treatment of choice.

Interferon-alpha is a very effective salvage therapy for patients with hairy cell leukemia relapsing after cladribine.

Effective against some common forms of NHL, particularly low-grade, follicular NHL in advanced stages. sometimes combined with chemotherapy

In NHL, as a single agent, interferon-α modest activity but significant toxicities.

Remission duration are generally short.

Low dose interferon-α with standard chemo Reduction in rate of transformation to higher grade lymphoma Moderate prolongation of remission duration BUT no effect on overall survival

High-dose IFN is the only treatment showing activity against melanoma in the adjuvant setting. 

only medication for people with high-risk malignant melanoma that has been shown to improve both relapse-free survival and overall survival. 

Simone Mocellin et al.Interferon Alpha Adjuvant Therapy in Patients With High-Risk Melanoma: A Systematic Review and Meta-analysis. J Natl Cancer Inst 2010;102:1–9

Response rates for metastatic melanoma with IFN-α2 as a single agent ~15%, comparable to cytotoxic agents used alone.

When combined with chemotherapy and IL-2, response rates can >45%, but with increased toxicity and with no marked prolongation in progression-free

survival or overall survival.Tawbi HA, Kirkwood JM. Management of metastatic

melanoma. Semin Oncol. 2007;34:532-545

5-year estimated RFS (relapse-free survival) rates for the high-dose interferon, low-dose interferon, and observation groups were 44%, 40%, and 35%, respectively. Neither high-dose interferon nor low-dose interferon yielded an OS benefit when compared with observation (hazard ratio [HR] = 1.0; P = .995).

Kirkwood JM, Ibrahim JG, Sondak VK, et al.: High- and low-dose interferon alfa-2b in high-risk melanoma: first analysis of intergroup trial E1690/S9111/C9190. J Clin Oncol 18 (12): 2444-58, 2000

IFN-a2a and IFN-a2b were among the first agents to be used therapeutically for AIDS-associated KS

yet a role for IFN-a in combination with agents that target angiogenesis

recently, a case report described how ocular adnexal and conjunctival KS was successfully treated with intra-lesional IFN-a2b, leading to a dramatic decrease in tumor mass

Qureshi YA et al. Intralesional interferon alpha-2b therapy for adnexal Kaposi sarcoma. Cornea 28(8):941–943.

Response rates from 4% to 26% have been reported in trials IFN-α2 in metastatic renal carcinoma, with a mean response of 15% in cumulative summary of several trials.

Bukowski RM. Cytokine therapy for metastatic renal cell carcinoma. Semin Urol Oncol. 2001;19:148-154.

In two randomized trials comparing IFN-α alone or in combination with other treatments, a statistically significant increases in survival resulted.

Members of the MRC Renal Cancer Collaborators. Interferon-á and survival in metastatic renal carcinoma: early results of a randomized controlled trial. Lancet. 1999:14-17.

IFN-α in midgut carcinoid tumors, a mean response rate of ~50% resulted.

Moertel C et al. J Clin Oncol. 1989;7:865-868.

Objective tumor regression occurs less frequently but can include both primary tumors and hepatic metastases.

Other solid tumors, such as ovarian, bladder, and basal cell carcinomas, have responded to IFN-α administered regionally, particularly in patients with lesser tumor bulk

Greenway H, Cornell RC, Tanner DJ, et al. Treatment of basal cell carcinoma with intralesional interferon. J Acad Dermatol. 1986;15:437-443.

Belldegrun A, Franklin JR, O'Donnell MA, et al. Superficial bladder carcinoma: the role of interferon alpha. J Urol. 1998;159:1793-1801.

Berek J, Markman M, Stonebraker B, et al. Intraperitoneal interferon-alpha in residual ovarian carcinoma: a Phase II Gynecologic Oncology Group study. Gynecol Oncol.

CLINICAL TOXICITY OF INTERFERON ADMINISTRATION

Constitutional Toxicities Hematologic Toxicities Organ Toxicities Neuropsychiatric Toxicities Endocrine and Metabolic Toxicities Potential Drug Interactions

CONSTITUTIONAL TOXICITYCONSTITUTIONAL TOXICITY

Most common toxicities Schedule and dose dependent. Acute administrationAcute administration can result in

-fever, chills, myalgias, arthralgias, headache, nausea, vomiting, and fatigue.

- Rigors - uncommon. - Fatigue usually increases with repetitive dosing until a

baseline level of fatigue is reached

Appropriate timing of administration (e.g., at or just before bedtime) can limit the impact of symptoms.

Anorexia and weight loss -commonly seen with higher-dose regimens

HEMATOLOGIC TOXICITIES

Hematologic toxicities -anemia, neutropenia, and thrombocytopenia. Appear to be dose related, rarely reported in lower-dose

regimens.

Neutropenia requiring dosage reduction reported in 26% to 60% of patients receiving high-dose interferon-α.

Neutropenic fevers or infections requiring antibiotic administration or hospitalization are quite rare.

Thrombocytopenia -rarely severe enough to warrant dosage reductions.

ORGAN TOXICITIES CVS-CVS-

-SVT esp AF- risk increased in elderly patients & with preexisting cardiac disease. -reversible cardiomyopathy – rare - Hypotension

o Kidneys -Reversible proteinuria - 15% to 20% of pts. -Nephrotic syndrome rare -Interstitial nephritis.

Thrombotic microangiopathy in patients with CML treated for several years.

Skin -macular rashes , -psoriatic-type skin reactions –resolve with discontinuation of therapy.

ORGAN TOXICITIES-CONTD

Acute hepatic toxicity High-dose interferon regimens

- manifested as increase in serum levels of SGOT,SGPT - can be fatal - fatal complications can be avoided with

careful monitoring and appropriate dosage modification.

RETINOPATHY

Associated with type I interferon therapy

- includes retinal hemorrhages, cotton-wool spots

- can be unilateral or bilateral.

- Incidence of interferon retinopathy-18% to 86% in different

series.

- DM may be an associated risk factor

- rarely results in any visual disturbance and disappears

spontaneously during treatment or resolves after interferon is

discontinued.

NEUROPSYCHIATRIC TOXICITIES

The neuropsychiatric or neurocognitive toxicities

- subtle changes detected only by formal

testing - overt with depression, hypomania, or

suicidal ideation requiring discontinuation of interferon and active intervention.

ENDOCRINE AND METABOLIC TOXICITIES

Thyroid abnormalities -in 5% to 31% of patients - 70% to 80% with thyroid disorders while on interferon

have detectable thyroid autoantibodies, Exact mechanism unknown. - may be a manifestation of increased risk of autoimmune

disorders that has been seen in patients taking interferon. Metabolic alterations in the blood lipid profile - hypertriglyceridemia - elevated levels of LDL, secondary to inhibition of

lipoprotein lipase. - plasma cholesterol level in 15% to 40% of patients.

OTHERSAutoimmune phenomena - Thyroid disorders - Rheumatoid arthritis - Raynaud's and Sjögren's syndromes. Common in females and longer

duration of therapy Rhabdomyolysis Sarcoidosis

PHARMACOLOGIC MODIFICATION OF PHARMACOLOGIC MODIFICATION OF INTERFERONSINTERFERONS

The relatively short half-life of interferons requires repetitive dosing to maintain exposure to biologically effective concentrations.

A pharmacologic approach to improving interferon efficacy and decreasing toxicity has been to couple the recombinant interferon molecule with a polyethylene glycol moiety ("pegylation").

This slows metabolism of the interferon, providing more sustained levels of exposure, but also diminishes the specific activity of the interferon, because steric interference decreases binding affinity with its receptor.

2 pegylated interferons commercially available, each using different form of PEG: -branched-chain pegylated interferon alfa-2a

(Pegasys) straight-chain pegylated) interferon alfa-2b (PEG-

Intron).

In comparable concentrations,each pegylated interferon formulation has biologic activity identical to unmodified recombinant molecule.

POTENTIAL DRUG INTERACTIONS

Not been extensively examined, Inhibition of the activity of cytochrome P-450

(isozymes CYP1A2 and CYP2D) within 24 hours of the first intravenous dose.

After 26 days of treatment, significant inhibition of CYP2C19 was found.

Implication - -Activity of drugs metabolized by these enzymes may

be diminished after interferon therapy. Such drugs include tricyclic antidepressants, SSRIs,

theophylline, phenytoin, and many others However, clinically significant drug-interferon

interactions have not been reported.