Phase III Committee III December 2013 Immunomodulators Phase III Committee III December 2013

Immunomodulators The development of agents that modulate the immune response rather than suppress it has become an important area of pharmacology. The rationale underlying this approach is that such drugs may increase the immune responsiveness of patients who have either selective or generalized immunodeficiency. The major potential uses are in immunodeficiency disorders, chronic infectious diseases, and cancer. The AIDS epidemic has greatly increased interest in developing more effective immunomodulating drugs.

Immunomodulators Cytokines INF-a Colony-stimulating factors INF-b INF-g Interleukins TNF-a TNF-b Colony-stimulating factors Granulocyte colony-stimulating factor G-CSF Erythropoietin (epoetin, EPO) Thrombopoietin (TPO)

Cytokines The cytokines are a large and heterogeneous group of proteins with diverse functions. Some are immunoregulatory proteins synthesized within lymphoreticular cells and play numerous interacting roles in the function of the immune system and in the control of hematopoiesis. In most instances, cytokines mediate their effects through receptors on relevant target cells and appear to act in a manner similar to the mechanism of action of hormones. In other instances, cytokines may have antiproliferative, antimicrobial, and antitumor effects.

Cytokines (2) The first group of cytokines discovered, were the interferons (IFNs), followed by the colony-stimulating factors. Colony-stimulating factors. regulate the proliferation and differentiation of bone marrow progenitor cells. Most of the more recently discovered cytokines have been classified as interleukins (ILs) and numbered in the order of their discovery. Cytokines are produced using gene cloning techniques. Most cytokines (including TNF-a, IFN-g, IL-2, granulocyte colony-stimulating factor [G-CSF], and granulocyte-macrophage colony-stimulating factor [GM-CSF]) have very short serum half-lives (minutes). The usual subcutaneous route of administration provides slower release into the circulation and a longer duration of action. Each cytokine has its own unique toxicity, but some toxicities are shared. For example, IFN-a, IFN-b, IFN-g, IL-2, and TNF-a all induce fever, flulike symptoms, anorexia, fatigue, and malaise.

Interferons (IFNs) Interferons are proteins that are currently grouped into three families: IFN-a, IFN-b, and IFN-g. The IFN-a and IFN-b families comprise type I IFNs, ie, acid-stable proteins that act on the same receptor on target cells. IFN-g, a type II IFN, is acid-labile and acts on a separate receptor on target cells. Type I IFNs are usually induced by virus infections, with leukocytes producing IFN-a. Fibroblasts and epithelial cells produce IFN-b. IFN-g is usually the product of activated T lymphocytes.

Interferons (IFNs) 2 IFNs interact with cell receptors to produce a wide variety of effects that depend on the cell and IFN types. IFNs, particularly IFN-g, display immune-enhancing properties, which include; increased antigen presentation and macrophage, NK cell, and cytotoxic T-lymphocyte activation. IFNs also inhibit cell proliferation. In this respect, IFN-a and IFN-b are more potent than IFN-g. Another striking IFN action is increased expression of MHC molecules on cell surfaces. While all three types of IFN induce MHC class I molecules, only IFN-g induces class II expression.

Interferons (IFNs) 3 IFN-a is approved for the treatment of several neoplasms, including; hairy cell leukemia, chronic myelogenous leukemia, malignant melanoma, and Kaposi's sarcoma, IFN-a is approved for the treatment of hepatitis B and C infections. It has also shown activity as an anticancer agent in renal cell carcinoma, carcinoid syndrome, and T cell leukemia.

Interferons (IFNs) 4 IFN-b is approved for use in relapsing-type multiple sclerosis. IFN-g is approved for the treatment of chronic granulomatous disease. IL-2 is approved for the treatment of metastatic renal cell carcinoma and malignant melanoma. Numerous clinical investigations of the other cytokines, including IL-1, -3, -4, -6, -11, and -12, are still in progress.

Interferons (IFNs) 5 Toxicities of IFNs include; fever, chills, malaise, myalgias, myelosuppression, headache, and depression These side effects can severely restrict their clinical use.

The hematopoietic growth factors The hematopoietic growth factors are glycoprotein hormones that regulate the proliferation and differentiation of hematopoietic progenitor cells in the bone marrow. The first growth factors to be identified were called colony-stimulating factors because they could stimulate the growth of colonies of various bone marrow progenitor cells in vitro. Many of these growth factors have been purified and cloned, and their effects on hematopoiesis have been extensively studied. These growth factors are produced by recombinant DNA technology. Of the known hematopoietic growth factors, erythropoietin (epoetin alfa), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-11 (IL-11) are currently in clinical use. Thrombopoietin and other potentially useful hematopoietic factors are still in development.

Erythropoietin Erythropoietin, a 34-39 kDa glycoprotein, was the first human hematopoietic growth factor to be isolated. It was originally purified from the urine of patients with severe anemia. Recombinant human erythropoietin (rHuEPO, epoetin alfa) is produced in a mammalian cell expression system. After intravenous administration, erythropoietin has a serum half-life of 4-13 hours in patients with chronic renal failure. It is not cleared by dialysis. It is measured in international units (IU). Darbepoetin alfa is a glycosylated form of erythropoietin and differs from it functionally only in having a twofold to threefold longer half-life.

Erythropoietin Erythropoietin stimulates erythroid proliferation and differentiation by interacting with specific erythropoietin receptors on red cell progenitors. The erythropoietin receptor is a member of the JAK/STAT superfamily of cytokine receptors that use protein phosphorylation and transcription factor activation to regulate cellular function. Erythropoietin also induces release of reticulocytes from the bone marrow. Endogenous erythropoietin is primarily produced in the kidney. In response to tissue hypoxia, more erythropoietin is produced through an increased rate of transcription of the erythropoietin gene. Normally, an inverse relationship exists between the hematocrit or hemoglobin level and the serum erythropoietin level.

Erythropoietin Nonanemic individuals have serum erythropoietin levels of less than 20 IU/L. As the hematocrit and hemoglobin levels fall and anemia becomes more severe, the serum erythropoietin level rises exponentially. Patients with moderately severe anemias usually have erythropoietin levels in the 100-500 IU/L range, and patients with severe anemias may have levels of thousands of IU/L. The most important exception to this inverse relationship is in the anemia of chronic renal failure. In patients with renal disease, erythropoietin levels are usually low because the kidneys cannot produce the growth factor. These are the patients most likely to respond to treatment with exogenous erythropoietin.

Erythropoietin Anemia can result from any of a large number of underlying conditions that either interrupt the normal process of erythropoiesis or result in the premature loss or destruction of mature erythrocytes. One common indication for erythropoietin therapy is chronic kidney disease, in which the loss of functional kidney tissue results in loss of the cells responsible for erythropoietin production. Another potential indication for erythropoietin is cancer, which can induce a state of relative resistance to endogenous erythropoietin by mechanisms that may involve proinflammatory cytokines, oxidative stress, and antierythropoietin antibodies. Cancer can also cause anemia through bleeding, poor nutrition, and infiltration of the bone marrow by tumor cells; these causes can often be diagnosed and treated directly. Often, cancer-related anemia results from the bone marrow toxicity of the chemotherapeutic agents used to treat the cancer.

Erythropoietin Patients with endogenous erythropoietin levels of less than 100 IU/L have the best chance of response, although patients with erythropoietin levels between 100 and 500 IU/L respond occasionally. These patients generally require higher erythropoietin doses (150-300 IU/kg three times a week) to achieve a response, and responses are often incomplete. Erythropoietin has been used successfully to offset the anemia produced by zidovudine treatment in patients with HIV infection and in the treatment of the anemia of prematurity. Erythropoietin is one of the drugs banned by the International Olympic Committee. The use of erythropoietin by athletes is based on their hope that increased red blood cell concentration will increase oxygen delivery and improve performance. The most common adverse effects of erythropoietin are associated with a rapid increase in hematocrit and hemoglobin and include hypertension and thrombotic complications. These difficulties can be minimized by raising the hematocrit and hemoglobin slowly and by adequately monitoring and treating hypertension. Allergic reactions have been infrequent and mild.

Myeloid Growth Factors G-CSF and GM-CSF, the two myeloid growth factors currently available for clinical use, were originally purified from cultured human cell lines. Recombinant human G-CSF (rHuG-CSF; filgrastim) is produced in a bacterial expression system. It is a nonglycosylated peptide of 175 amino acids, with a molecular weight of 18 kDa. Recombinant human GM-CSF (rHuGM-CSF; sargramostim) is produced in a yeast expression system. It is a partially glycosylated peptide of 127 amino acids, with three molecular species with molecular weights of 15,500; 15,800; and 19,500. These preparations have serum half-lives of 2-7 hours after intravenous or subcutaneous administration. Pegfilgrastim, a covalent conjugation product of filgrastim and a form of polyethylene glycol, has a much longer serum half-life than recombinant G-CSF, and so it can be injected once per myelosuppressive chemotherapy cycle instead of daily for several days.

Myeloid Growth Factors The myeloid growth factors stimulate proliferation and differentiation by interacting with specific receptors found on various myeloid progenitor cells. Like the erythropoietin receptor, these receptors are members of the JAK/STAT superfamily. G-CSF stimulates proliferation and differentiation of progenitors already committed to the neutrophil lineage. It also activates the phagocytic activity of mature neutrophils and prolongs their survival in the circulation. G-CSF also has a remarkable ability to mobilize hematopoietic stem cells, to increase their concentration in peripheral blood. GM-CSF has broader biologic actions than G-CSF. It is a multipotential hematopoietic growth factor that stimulates proliferation and differentiation of early and late granulocytic progenitor cells as well as erythroid and megakaryocyte progenitors. Like G-CSF, GM-CSF also stimulates the function of mature neutrophils. GM-CSF acts together with interleukin-2 to stimulate T-cell proliferation and appears to be a locally active factor at the site of inflammation. GM-CSF mobilizes peripheral blood stem cells, but it is significantly less efficacious than G-CSF.

Myeloid Growth Factors CANCER CHEMOTHERAPY-INDUCED NEUTROPENIA Neutropenia is a common adverse effect of the cytotoxic drugs used to treat cancer and increases the risk of serious infection in patients receiving chemotherapy. Unlike the treatment of anemia and thrombocytopenia, transfusion of neutropenic patients with granulocytes collected from donors is performed rarely and with limited success. The introduction of G-CSF in 1991 represented a milestone in the treatment of chemotherapy-induced neutropenia. This growth factor dramatically accelerates the rate of neutrophil recovery after dose-intensive myelosuppressive chemotherapy. It reduces the duration of neutropenia and usually raises the low neutrophil count seen following a cycle of chemotherapy. The ability of G-CSF to increase neutrophil counts after myelosuppressive chemotherapy is nearly universal, but its impact on clinical outcomes is more variable. Some clinical trials have shown that G-CSF reduces episodes of febrile neutropenia, requirements for broad-spectrum antibiotics, and days of hospitalization; however, other trials failed to find these favorable outcomes. To date, no clinical trial has shown improved survival in cancer patients treated with G-CSF.

Myeloid Growth Factors Clinical guidelines for the use of G-CSF after cytotoxic chemotherapy recommend reserving G-CSF for patients with a prior episode of febrile neutropenia after cytotoxic chemotherapy, Pegfilgrastim is an alternative to G-CSF for prevention of chemotherapy-induced febrile neutropenia. Although the two growth factors have similar effects on neutrophil counts, G-CSF is used more frequently because it is better tolerated. G-CSF can cause bone pain, which clears when the drug is discontinued. GM-CSF can cause more severe side effects, particularly at higher doses. These include fever, malaise, arthralgias, myalgias, and a capillary leak syndrome characterized by peripheral edema and pleural or pericardial effusions. Allergic reactions may occur but are infrequent. Splenic rupture is a rare but serious complication of the use of G-CSF.

Megacaryocyte Growth Factors A low platelet count, or thrombocytopenia, is an important adverse effect of many cancer chemotherapeutic agents, occasionally limiting the doses that can be delivered with acceptable safety and tolerability. The complications of thrombocytopenia include increased bleeding risk and platelet transfusion requirement; in turn, platelet transfusion is associated with an increased risk of infection, febrile reaction, and, rarely, graft-versus-host disease. Research into the pharmacologic management of chemotherapy-induced thrombocytopenia has focused on the thrombopoietin analogues recombinant human thrombopoietin (rhTPO) and pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF).

Megacaryocyte Growth Factors A small-molecule oral drug that directly stimulates the TPO receptor is also in clinical trials. To date, only recombinant human IL-11 (rhIL-11 or oprelvekin) has been approved by the FDA for clinical use. These agents all have the potential to increase megakaryocytopoiesis (platelet production) in a dose-dependent fashion; although these drugs stimulate some multipotent as well as committed precursor cells, they do not significantly increase the hematocrit or white blood cell count. Importantly, these agents must all be administered prophylactically because their activity is delayed in onset, with a 1- to 3-week before the platelet count reaches its peak value.

Megacaryocyte Growth Factors Interleukin-11 (IL-11) is a 65-85 kDa protein produced by fibroblasts and stromal cells in the bone marrow. Oprelvekin, the recombinant form of interleukin-11 approved for clinical use, is produced by expression in E coli. The half-life of IL-11 is 7-8 hours when the drug is injected subcutaneously. Thrombopoietin, a 65-85 kDa glycosylated protein, is constitutively expressed by a variety of organs and cell types. Hepatocytes appear to be the major source of human thrombopoietin, and patients with cirrhosis and thrombocytopenia have low serum thrombopoietin levels. Recombinant thrombopoietin is produced by expression in human cells.

Megacaryocyte Growth Factors Interleukin-11 acts through a specific cell surface cytokine receptor to stimulate the growth of multiple lymphoid and myeloid cells. It acts synergistically with other growth factors to stimulate the growth of primitive megakaryocytic progenitors and, most importantly, increases the number of peripheral platelets and neutrophils. Acting through its own cytokine receptor, thrombopoietin also independently stimulates the growth of primitive megakaryocytic progenitors. In addition, it stimulates mature megakaryocytes and even activates mature platelets to respond to aggregation-inducing stimuli. The critical in vivo role of thrombopoietin has been demonstrated in genetically engineered knockout mice who lack either thrombopoietin or its receptor. These mice have marked thrombocytopenia but do not display anemia or leukopenia.

Megacaryocyte Growth Factors The most common adverse effects of interleukin-11 are fatigue, headache, dizziness, and cardiovascular effects. The cardiovascular effects include anemia (due to hemodilution), dyspnea (due to fluid accumulation in the lungs), and transient atrial arrhythmias. Hypokalemia has also been seen in some patients. All of these adverse effects appear to be reversible. Recombinant thrombopoietin and some other potentially useful hematopoietic factors are still in development. In the limited clinical trial data available so far, recombinant thrombopoietin appears to be well tolerated.

Bone Marrow Toxicity of Drugs Phase III Committee III December 2010

Development of cells of the hematopoietic system BFU, burst forming unit; CFU, colony forming unit; CSF, colony stimulating factor; IL, interleukin; SCF, stem cell factor; TPO, thrombopoietin.

Development of cells of the hematopoietic system. Mature cells of the hematopoietic system all develop from pluripotent stem cells that reside in the bone marrow. The type of mature cell that develops is dependent on the extracellular factors and the exposure of stem cells and progenitor cells to specific growth factors. The pluripotent stem cell differentiates into a trilineage myeloid stem cell (CFU-S) or a lymphoid stem cell. Depending on the growth factors that are present, CFU-S cells differentiate into granulocytes (eosinophils, monocyte/macrophages, neutrophils), platelets, or erythrocytes. Lymphoid stem cells differentiate into B cells, natural killer (NK) cells, or T cells. Except for the terminal differentiation of pro-T cells to mature T cells, which takes place in the thymus, the differentiation of all hematopoietic stem cells, progenitor cells, and precursor cells occurs in the bone marrow. Of the growth factors, G-CSF, GM-CSF, erythropoietin (EPO), and IL-11 are currently used as therapeutic agents. BFU, burst forming unit; CFU, colony forming unit; CSF, colony stimulating factor; IL, interleukin; SCF, stem cell factor; TPO, thrombopoietin.

Cancer chemotherapy can cause deficiencies on the bone marrow Cause anemia, neutropenia, and/or thrombocytopenia in these cell populations. Thus, the erythropoietin analogues rhEPO and darbepoetin treat anemia; the G-CSF and GM-CSF analogues filgrastim and sargramostim treat neutropenia; and rhIL-11 treat thrombocytopenia.