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COOH

COOH

Light chain

Variable (V) _ regions

Constant (C) region

Heavy chain

COOH

COOH

Heavy chain

Constant (C) region

1

= Hypervariable region

= Hinge region

o

= Carbohydrate

= Disulfide bridge

FIGURE 22.4 This image represents the polypeptide chain structure of a molecule of IgG. The numbers indicate the number of amino acids. In the actual molecule, the chains are folded so that each cysteine is brought close to the partner with which it forms a disulfide (S-S) bridge.

FIGURE 22.4 This image represents the polypeptide chain structure of a molecule of IgG. The numbers indicate the number of amino acids. In the actual molecule, the chains are folded so that each cysteine is brought close to the partner with which it forms a disulfide (S-S) bridge.

intracellular pathogens. NK cells are actors in antibody dependant cell-mediated cytotoxicity (ADCC). ADCC is the killing of antibody coated target cells by immune cells with FC receptors that recognize the C-region of the bound antibody. NK cells have the Fc receptor CD16a or FCyIIIa on their surface to facilitate the recognition.

22.3 DISEASES OF THE IMMUNE SYSTEM

Malfunction of the immune system will result in pathological consequences such as transplant rejection, autoimmune disease, or allergy. These situations are accompanied by inflammation that leads to tissue damage. Individuals may also be immunodeficient (lacking a component of the immune system) and have an increased susceptibility to infections and cancer; one such example is HIV (decrease in the number of CD4+ T cells).

22.3.1 Autoimmune Diseases

Adaptive immune responses are sometimes elicited by antigens that are not associated with infectious agents and this causes serious disease and tissue damage. Autoimmunity is the failure of an organism to recognize its own constituent parts as "self," which results in an immune response against its own cells and tissues. Any disease that results from such an immune response is termed as an autoimmune disease. Prominent examples include insulin-dependent diabetes mellitus type 1 (IDDM), multiple sclerosis (MS), and rheumatoid arthritis (RA).

In IDDM, the immune system attacks the beta cells in the Islets of Langerhans of the pancreas, destroying them, or damaging them sufficiently to reduce and eventually eliminate insulin production leading to hyperglycemia. The autoimmune attack may be triggered by reaction to an infection.

In MS, the inflammatory responses are launched in the absence of a pathogen. It seems that certain T cells by mistake recognize the insulating sheaths around nerves (myelin) as a foreign invader resulting in the formation of auto antibodies against the myelin sheaths leading to destruction of the insulating layer surrounding neurons in the brain and spinal cord. When the myelin sheath is destroyed, nerve messages are sent less efficiently. Patches of scar tissue, called plaques, form over the affected areas, further disrupting nerve communication. The symptoms of MS occur when the brain and spinal cord nerves no longer communicate properly with other parts of the body. MS causes a wide variety of symptoms and can affect vision, balance, strength, sensation, coordination, and bodily functions.

RA is a chronic autoimmune disease that causes inflammation and deformity of the joints. In RA, the underlying event that promotes RA in a person is unknown. Given the known genetic factors involved in RA, some researchers have suggested that an outside event occurs that triggers the disease cycle in a person with a particular genetic profile. The body's normal response to such an organism is to produce cells that can attack and kill the organism, protecting the body from the foreign invader. In an autoimmune disease like RA, this immune cycle spins out of control. The body produces misdirected immune cells, which accidentally identify parts of the person's body as foreign. These immune cells then produce a variety of chemicals that injure and destroy parts of the body. Autoantibodies (antibodies against self protein) are observed in 80% of the patients.

IDMM and MS are examples of organ-specific autoimmune diseases as only one organ is affected whereas RA is an example of a systemic autoimmune disease involving many tissues.

22.3.2 Inflammation

An inflammation is a manifestation of the immune system's response to invading organisms or substances. At the site of the infection, there are a number of physiological changes that take place to assist the destruction of the invaders. These include

• Increased blood flow to the area to maximize the number of leukocytes that can get to the infection site.

• A thinning of the cells in local blood capillary walls (endothelial cells) to allow the leukocytes to squeeze through.

• An increase in local temperature that has an antibiotic effect.

• A large number of immune system signaling molecules (chemokines) are released by leukocytes to coordinate the immune response and to call more leukocytes to the site.

Once the invader has been dealt with, the body terminates the immune response by killing off the leukocytes in the locality. This is done by depriving them of nutrients (necrosis) and by apoptosis.

22.3.3 Cancer Immunology

Recently, we have learned that the immune system may play a central role in protecting the body against cancer and in combating cancer that has already developed. This latter role is not well understood, but there is evidence that in many cancer patients the immune system slows down the growth and spread of tumors. On the other hand, when the immune system is weakened by old age or environmental factors, it can be more easily overwhelmed by cancerous cells.

One immediate goal of research in cancer immunology is the development of methods to harness and enhance the body's natural tendency to defend itself against malignant tumors. Immunotherapy represents a new and powerful weapon in the arsenal of anticancer treatments. Immunotherapies involving certain cytokines or antibodies that recognize cancer cells specifically and deplete the cells via ADCC have now become part of the standard cancer treatment.

22.4 IMMUNOSUPPRESSIVE AGENTS

The proliferative nature of the immune response may be controlled with immunosuppressive drugs. These drugs work by inhibiting the division of cells and, therefore, also suppress nonimmune cells leading to side effects such as anemia, neurotxicity, hepatotoxicity, nephrotoxicity, and diabetes. In addition the same immune system that we are suppressing in order to avoid graft rejection in transplantation or reactivity against auto antigens in autoimmune diseases is responsible for pathogen infections and tumor surveillance; thus, the immunosupressed patient is liable to come down with opportunistic infections and malignancies.

The most used immunosuppressive drugs are azathioprine, cyclosporine A, leflunomide, cyclo-phosphamide, glucocorticoids, and methotrexate. The mechanism of action of these drugs on the immune system is briefly described in the following.

Azathioprine is rapidly hydrolyzed in the blood to 6-mercaptopurine (Figure 22.5). In this form (as a purine analog), it incorporates into the DNA, inhibiting nucleotide synthesis by causing feedback inhibition in the early stages of purine metabolism. This ultimately prevents mitosis and proliferation of rapidly dividing cells, such as activated B- and T-lymphocytes. Through this action, Azathioprine is able to block most T cell functions and inhibit primary antibody synthesis.

Cyclosporine A is a small (11 amino acids) fungal (nonribosomal) cyclic peptide that is a calcineurine inhibitor (Figure 22.6). Cyclosporine works by binding to a protein found in the cytosol: cyclophilin. This complex inhibits calcineurin and ultimately leading to the inhibition of IL-2 production and secretion. The interaction between IL-2 and the IL-2 receptor is crucial in the activation and differentiation of B and T cells. Cyclosporine is therefore a highly effective immunosuppressant. The clinical introduction of cyclosporine has significantly increased graft survival and significantly reduced the occurrence of acute rejection in transplant patients.

Leflunomide (Figure 22.5) interferes with an enzyme called dihydroorotate dehydrogenase, an enzyme involved in the de novo pyrimidine synthesis. Thus leflunomide inhibits the synthesis of pyrimidines and thereby inhibits lymphocyte proliferation, and reduces adhesion molecules that allow the immune cells to home in to the area of inflammation. As a result the immune process is slowed. The drug is developed for RA and is also used in combination with methotrexate.

Cyclophosphamide is an inactive cyclic phosphamide ester of mechlorethamine. It is transformed via hepatic and intracellular enzymes to active alkylating metabolites, 4-hydroxycyclophophosph-amide, aldophosphamide, acrolein, and phosphoramide mustard (Figure 22.7). Cyclophosphamide causes the prevention of cell division primarily by cross-linking DNA strands. It is therefore referred

Azathioprine

6-Mercaptopurine

Azathioprine

6-Mercaptopurine

Leflunomide

Leflunomide

FIGURE 22.5 Azathioprine is an immunosuppressant and it is a prodrug, converted in the body to active metabolites 6-mercaptopurine and 6-thioinosinic acid, which is a purine synthesis inhibitor. Leflunomide is used in moderate to severe RA and psoriatic arthritis and is a pyrimidine synthesis inhibitor.

Acrolein From Cyclosporine
7 Ala

6 MeLeu 5 Val

Cyclosporine A

FIGURE 22.6 Structure of cyclosporine A, which is a cyclic nonribosomal peptide of 11 amino acids (undecapeptide) produced by the fungus Tolypocladium inflatum Gams, and contains unnatural amino acids, including D-amino acids.

Cyclophosphamide

4-Hydroxy-cyclophosphamide

O NH

Aldophosphamide

Phosphoramide mustard

Acrolein

FIGURE 22.7 Cyclophosphamide is a nitrogen mustard alkylating agent, which is used to treat various types of cancer and some autoimmune disorders. It is a prodrug and is converted in the liver to active forms such as phosphoramide mustard that have chemotherapeutic activity.

Cl to as a cytotoxic drug. Unfortunately, normal cells also are affected, and this results in serious side effects. It is used in cancer treatment and to treat severe cases of RA and other autoimmune diseases.

Glucocorticoids such as prednisone (Figure 22.8) works principally to block T cell and APC derived cytokine and cytokine-receptor expression. The major elements blocked are IL-1 and

Prednisone Dexamethasone

FIGURE 22.8 Glucocorticoids are steroid hormones characterized by an ability to bind with the glucocorticoid receptor. Synthetic derivatives of the natural corticoids, including prednisone and dexamthasone, which are used as effective immunosuppressants.

Methotrexate o^oh o ^

Methotrexate

FIGURE 22.9 Structure of methotrexate, which is a structural analog of folic acid, and is used in the treatment of severe autoimmune and inflammatory diseases.

IL-6. Secondary effects of corticosteroids include the blocking of IL-2, IFN-y, and tumor necrosis factor alpha (TNF-a). These elements, notably IL-1, are essential for lymphocyte and APC communication. A decrease in production of these cytokines effectively obstructs an APC's capacity to activate antigen-specific lymphocytes. Glucocorticoids have a hydrophobic structure that allows them to easily diffuse into cells and bind to specific cytoplasmic receptors. The resulting complexes progress to the nucleus, where they are able to inhibit the transcription of the cytokine genes. Corticosteroids are also able to inhibit cytokine production in macrophages. This subsequently inhibits the macrophage phagocytosis and chemotaxis properties. Corticosteroids are potent nonspecific anti-inflammatory agents-administration of corticosteroids results in an acute reduction of circulating lymphocytes and monocytes.

Methotrexate (Figure 22.9), a structural analog of folic acid, is involved in the first line treatment of severe autoimmune and inflammatory diseases. It is classified as an antimetabolite drug, which means it is capable of blocking the metabolism of cells. As a result of this effect, it is used in treating diseases associated with abnormally rapid cell growth, such as cancer, RA, and psoriasis. Methotrexate and its active metabolites compete for the folate binding site of the enzyme dihydro-folate reductase (DHFR). Folic acid is reduced to tetrahydrofolic acid by DHFR for DNA synthesis and cellular replication to occur (see Chapter 20). Competitive inhibition of the enzyme leads to blockage of tetrahydrofolate synthesis, depletion of nucleotide precursors, and inhibition of DNA, RNA, and protein synthesis. Methotrexate also inhibits thymidylate synthase and the transport of reduced folates into the cell. Methotrexate is believed to diminish inflammation by diminishing cytokine production. It has been shown that it has direct effect on T cell function in vitro and in vivo. Due to the nonspecific effects on cell proliferation methotrexate treatment is accompanied by serious side effects and more specific alternatives are required. The new immunomodulating bio-logics (see below) may offer alternative possibilities for less toxic treatments.

22.5 IMMUNOMODULATING BIOLOGICS 22.5.1 Recombinant Protein and Engineered Proteins

Biologics cover peptides or proteins that are used as therapeutic modalities for medical treatment. Immunomodulating biologics are proteins such as cytokines, interferons (IFNs), or interleukins or it could be monoclonal antibodies (mAbs) blocking their action. It has so far not been successful to make small molecules or peptide mimetics of the large extra cellular protein-protein interactions through which these molecules exert their action and we therefore see a lot of new biologics coming to the market in this area. Many proteins that may be used for medical treatment are normally expressed at very low concentrations; however, through recombinant DNA technology a large quantity of proteins can be produced.

Protein engineering can be used to stabilize proteins and improve in vivo half lives either by introduction of mutations or by introducing chemical modifications such as PEGylation, the latter meaning that a poly ethylene glycol (PEG) moiety is specifically attached to the protein such that it does not interfere with the protein interactions (see also Chapter 4). The resulting molecule has an increased size and renal clearance of the molecule is decreased, thereby extending the half-life of the molecule.

22.5.2 Monoclonal Antibodies

Monoclonal antibodies (mAbs) are artificial antibodies against a particular target (the "antigen") and are produced in the laboratory. The original method involved hybridoma cells (a fusion of two different types of cells) that acted as factories for antibody production. A major advance in this field was the ability to convert these antibodies, which originally were made from mouse hybridomas, to "humanized" antibodies that more closely resemble our natural antibodies. mAbs have been widely used in scientific studies of cancer, as well as in cancer diagnosis. They are now used as a very successful molecular format in treatment of several diseases where there is a high unmet medical need and where targets are the so-called nondruggable, i.e., it has not been successful to make small molecules against the target. The format is highly attractive due to the relatively long in vivo half-life of the antibodies and the fact that we have high levels of antibodies circulating so the body does not see them as foreign entities and the risk of immunogenic effects is therefore reduced. The primary drawback of using antibodies is that so far have to be delivered as injectibles either by the intravenous or subcutaneous administration.

22.5.3 Cytokines

Cytokines are relatively small proteins that play a major role in modulating the immune system. Among the large number of cytokines are the IFNs, TNF, and the interleukins. Cytokines are manufactured and released by cells of the immune system and perform a variety of functions including cell activation, inflammation, tissue breakdown, and repair as well as cell death. Various different cytokines send different complex signals to other cells including: calling immune system cells to the site of the infection, telling endothelial (blood vessel "lining") cells to let these cells through and telling immune system cells to activate themselves.

Using the body's own immunomodulators is becoming an exciting possibility to target inefficient or misdirected immune responses that result in diseases. The potential benefits in terms of treatment of human diseases are enormous and still largely unexplored. Thus, using cytokines and their antagonists as therapeutic agents is an emerging and growing area of research.

22.5.3.1 Interferons

IFNs belong to the cytokine protein family. They are produced by white blood cells in the body (or in the laboratory) in response to infection, inflammation, or stimulation. They have been used as a treatment for certain viral diseases, including hepatitis B and C as well as MS. IFNs can be divided into three groups, IFN-a, IFN-P, and IFN-y, respectively. Sub-variants of each group have been developed as therapeutic agents in the form of recombinant proteins for injection.

IFNs regulate cell function in the immune system by blocking cell growth and differentiation and stimulating monocytes and macrophages. In MS, IFN-P works indirectly on the central nervous system (CNS) by reducing the inflammatory reaction. The exact mechanism is unknown but it is believed that IFN-P can improve the activity of regulatory T cells, reduce the production of proinflammatory cytokines such as, TNF-a, IL-6, IL-1, and IL-8. They have been shown to downregu-late antigen presentation and the mobility of the activated T cells in the CNS.

IFN-a was one of the first cytokines to show an antitumor effect, and it is able to slow tumor growth directly, as well as help to activate the immune system. IFN- a has been approved by the FDA and is now commonly used for the treatment of a number of cancers, including multiple myeloma, chronic myelogenous leukemia, hairy cell leukemia, and malignant melanoma.

Some of the problems with these cytokines, including many of the IFNs and interleukins, are their side effects, which include flu-like syndromes when given at a high dose.

22.5.3.2 Tumor Necrosis Factor

TNF-a is a proinflammatory cytokine that is produced by macrophages, NK cells as well as B and T cells. Its action is to promote local inflammation, and to activate endothelial cells. TNF-a, in soluble or membrane form, binds to two types of receptors that will cause the activation of the target cell.

TNF-a plays a predominant part in the inflammatory process of RA and is highly up regulated in the synovial fluid of the joints in RA patients. TNF-a causes release of intra-articular metallo-proteases, which destroy bone cartilage, and via activation of transcription factors (NF-kB), TNF-a causes the production of proinflammatory and immunomodulating cytokines.

The implication of TNF-a in the inflammatory process and in the destruction of bone cartilage (RA) and in inflammatory intestinal lesions (Crohn's disease) has enabled the development of two approaches to treatment using TNF-a inhibitor agents, two mAbs and a soluble receptor, which are used in combination with methotrexate.

There are two mAbs directed at TNF-a, one chimeric, Infliximab (Remicade®); the other entirely humanized, Adalimumab (Humira®). A soluble receptor of TNF-a, Etanercept (Enbrel®), which limits the biological activity of TNF-a, acts by binding to it and preventing it from interacting with its receptors. The TNF-a inhibitors are successfully used for treatment of RA, psoriasis, and Chron's disease.

22.5.3.3 Interleukins

The role of interleukins is to mediate and control the immunologic and inflammatory response (Table 22.1). The list of known ILs is still increasing most of which have only been discovered in the last few years. Their role within the immune system is only beginning to be understood and they are just starting to be utilized in the treatment of a wide variety of diseases including cancer, AIDS, and autoimmune diseases. The following table briefly describes the role of each of the interleukins where the action is well understood.

TABLE 22.1

The Role of Interleukins

Interleukin

Secreting Cells

Action

IL-1

Macrophages

Stimulates T cells to secrete interleukin-2 and activate the inflammatory response. It also causes the hypothalamus to increase the body temperature.

Causes activated T- and B cells to proliferate themselves. It also induces antibody synthesis.

Causes other leukocytes to be proliferated—it does this by making certain types of stem cell in the bone marrow to differentiate and grow.

Causes T- and B cells to grow. It is also a factor in the production of IgE antibodies.

Stimulates B cells, and eosinophils. It causes B cells that produce IgA antibodies to proliferate

Works in combination with alpha interferon to induce B cell differentiation. It also causes the production of acute phase proteins in the liver and stimulates T cells and other leukocytes.

Causes lymphoid stem cells to differentiate into progenitor T and B cells.

IL-2

Helper T cells

IL-3

T cells

IL-4

Helper T cells

IL-5

Helper T cells

IL-6

T cells and macrophages

IL-7

Stromal cells

TABLE 22.1 (continued) The Role of Interleukins

Interleukin Secreting Cells

IL-8 Macrophages and endothelial cells

IL-9

IL-10 T cells, B cells, monocytes, and macrophages

IL-11

IL-12 Macrophages and DCs

IL-13 T cells

IL-14 DCs and T cells

IL-15 Monocytes and macrophages

IL-16 T cells

IL-17 T cells

IL-18 Leukocytes and nonleukocytes

IL-19 Monocytes

IL-20 Unknown

IL-21 Activated T cells

IL22 T cells and mast cells

IL-23 Activated DCs

IL-24 T cells

IL-25 Not known

IL-26 Activated memory T cells

IL-27 Activated monocytes, macrophages, and DCs

IL-28,29 Various cells

Action

IL-8 is "sticky" for T cells and neutrophils and helps to bring them to the site of an inflammation.

Induces growth in Helper T cells.

Acts to inhibit some aspects of the immune system while stimulating others. It represses the production of other cytokines within the immune system, especially INF-y, TNF-a, IL-1, and IL-6. It inhibits antigen presentation but activates B cells.

Causes plasmacytoma cells to proliferate.

Causes T cells and NK cells to proliferate. Promotes Th17 lineage.

Promotes B cell differentiation but inhibits inflammatory cytokine production.

Enhances memory B cell production and proliferation.

Enhances T cell proliferation in the blood and NK cell activation.

Acts as a chemo attractant and adhesion molecule and activator for T cells. Plays a part in both asthma and autoimmune diseases.

Activates neutrophils.

Stimulates the release of Th1 cytokines.

May be involved in regulation of proinflammatory cytokines.

May regulate inflammation in the skin.

Stimulates the proliferation of activated T cells.

Production of acute phase proteins, increases the number of basophils and platelets.

Acts on memory CD4+ T cells to support their differentiation. IL-23 sustains differentiated Th17 cells.

Can promote induction of apoptosis in cancer cells.

Cytokine production.

May act as autocrine growth factor.

Expression of IL12RRE2 making T cells responsive to IL-12.

Induce an antiviral state in infected cells.

IL-1Ra, an antagonist of the IL-1 receptor, is a natural inhibitor of IL-1, a proinflammatory cytokine that is involved in inflammation and joint destruction. It has been shown that mice deficient in IL-1Ra develop a type of inflammatory joint disease similar to RA. In the animal arthritis models, IL-1Ra improves the clinical symptoms and slows bone and joint destruction. Anakinra (Kineret®) is a recombinant nonglycosylated IL-1Ra. It is administered subcutaneously in combination with methotrexate. This product received regulatory approval for the treatment of active RA insufficiently controlled by methotrexate.

ILs with antitumor activity includes IL-2 (Figure 22.10). IL-2 is frequently used to treat kidney cancer and melanoma. IL-2 is the major growth and differentiation factor of immunocompetent killer cells, including the CTLs, NK cells, and monocytes. In metastatic renal cell cancer and melanoma, IL-2-based treatments have induced therapeutic responses. Efficacy of IL-2 as a single agent has been reported in 15%-25% of cases in these diseases and could possibly be increased by the addition of other agents such as IFN or chemotherapy. There are many side effects associated with IL-2 treatment; a serious, but very uncommon side effect of IL-2 in high doses is "capillary leak syndrome." Capillary leak syndrome is a potentially serious disease in which fluids within the vascular system (veins and capillaries) leak into the tissue outside the bloodstream. Due to the serious side effects, low dose IL-2 treatment is now applied.

Free Printable Heart Borders

FIGURE 22.10 IL-2 is a soluble protein of 133 amino acids. It is characterized by four a-helixes that are bundled together and belong to the structural family of cytokines called the four a-helix bundle family. Other interleukins in this subfamily are IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-13, IL-15, IL-21, and IL-23.

FIGURE 22.10 IL-2 is a soluble protein of 133 amino acids. It is characterized by four a-helixes that are bundled together and belong to the structural family of cytokines called the four a-helix bundle family. Other interleukins in this subfamily are IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-13, IL-15, IL-21, and IL-23.

Basiliximab (Simulect®) is a chimeric (70% human and 30% murine) mAb utilized in the prevention of acute organ rejection. This mAb has specificity and high affinity for the subunit of the IL-2 receptor (IL-2Ra, also known as CD25 or Tac) preventing IL-2 from binding to the receptor on the surface of activated T cells. By acting as an IL-2 antagonist, basiliximab inhibits IL-2-mediated activation and proliferation of T cells, the critical step in the cascade of cellular immune response of allograft rejection. Therefore, basiliximab has a long half-life of ~7-12 days and saturates the IL-2 receptor for up to 59 days. Other ILs or IL receptor antagonists in clinical development are IL-6, IL-10, IL-12/23, IL-15, IL 20, and IL-21.

22.5.4 B Cell Depletion

As a therapy for cancer, mAbs can be injected into patients to seek out the cancer cells, potentially leading to disruption of cancer cell activities or to enhancement of the immune response against the cancer. This strategy has been of great interest since the original invention of mAbs in the 1970s. After many years of clinical testing, researchers have proven that improved mAbs can be used effectively to help treat certain cancers. An antibody called rituximab (Rituxan®) can be useful in the treatment of leukemias and other new mAbs are undergoing active testing.

Rituximab is a chimeric mAbs directed against the CD20 antigen specific to B-lymphocytes. Once the antibody recognizes the surface antigen, the Fc part of the antibody recognizes Fc receptors on the NK cells and induces ADCC. It has been used for a number of years in the treatment of B cell lymphomas. Various data now suggest an important role for B-lymphocytes in the inflammatory cascade of RA that causes the destruction of cartilage and erosion of bone. Rituximab may intervene by destroying the B cells that produce auto antigens (rheumatoid factor). Systemic lupus erythematosus (SLE) is a disease that is driven by B cells that produce antibodies directed against self-antigens to form immune complexes that deposit in the tissues and instigate an inflammatory process. Rituximab has shown very promising clinical results in the treatment of SLE. Antibodies against other surface antigens are being pursued for B cell depletion therapy; these include CD19, CD22, CD32, CD38, and CD138.

22.5.5 Targeting T Cells

Agents that can interfere with T cell functions open up an important new avenue to the treatment of various autoimmune diseases as well as transplantation. There are several mechanisms that can be exploited: depletion of the T cells, interfering with TCR-mediated signaling and targeting costimu-latory signaling to induce anergy (immune unresponsiveness), and finally targeting several cytok-ines indirectly modulates the T cell function (see Section 22.5.3). Below are examples of mAbs developed for depletion of T cells as well as mAbs interfering with the TCR described.

Anti-CD3 (Muromomab-CD3) is the first type of murine mAb directed against the epsilon chain of the CD3 molecule (an integral part of the TCR complex). It thereby inactivates T-cell function blocking both naive T cells and CTLs. This results in rapid depletion of T cells from circulation and cytokine release. This antibody is used to treat acute graft rejection in transplantation. Several severe adverse effects as a result of Muromonab-CD3 are thought to be a product of the cytokine release (also known as cytokine storm). Humanized versions of this antibody are being pursued that are also engineered in the Fc part of the molecule to be nondepleting and to decrease the cytokine storm. The antibodies are believed to induce tolerance by up regulation of regulatory T cells. These antibodies are being investigated in clinical trials for treatment of Type I diabetes and other autoimmune and inflammatory diseases such as RA, psoriasis, and inflammatory bowel disease.

Alemtuzumab (Campath®) is a humanized anti-CD52 mAb that has emerged as an effective lymphocyte-depleting agent for organ transplantation, RA, MS, and vasculitis. Most of the depleted T cells return to near normal in 3 months after dosing. There is a risk that anti-CD52 treatment elicits autoimmune diseases; this is believed to be due to depletion of the regulatory T cells.

Abatacept (Orencia®) is a fusion protein of CTLA-4 (a surface protein of CTLs) and the Fc fragment of the human immunoglobulin IgG. It acts at the level of the T-lymphocytes where it blocks activation by interference with the path of costimulation between the APC and the T-lymphocyte. Abatacept thus modulates the activity of the B cells, DCs, macrophages, and other cells implicated in the inflammatory cascade and it is currently being developed for treatment of RA.

22.6 PROSPECTS OF IMMUNE MODULATING AGENTS

The immune system is highly complex and new immunological mechanisms and how they relate to human disease is still being identified. In this chapter, focus has been on the more established therapeutic approaches, general immune suppression that is still the first line treatment in autoimmune disease, depleting antibodies against cell surface ligands, the messenger proteins of the immune system—the cytokines and finally the targeting of the T cells. There are several other molecular avenues that can be explored, the first inhibitory antibody to the complement system has just reached the market; some are in clinical development including inhibitory antibodies to the innate immune system and blocking of the Th17 pathway. We expect to see many more immunological pathways to be explored over the years to come as we learn more about this highly fascinating biological system with plenty of therapeutic opportunities.

FURTHER READINGS

E. S. L. Chan and B. N. Cronstein, Molecular action of methotrexate in inflammatory diseases, Arthritis Research, 4, 266-274, 2001.

J. C. W. Edwards, B cell targeting in rheumatoid arthritis and other autoimmune disease, Nature Reviews Immunology,

M. Feldmann and R. N. Maini, Anti TNFa therapy of rheumatoid arthritis: What have we learned? Annual Review of Immunology, 19, 163-196, 2001.

C. Janeway, P. Travers, M. Walport, M. Shlomchik, Immuno Biology, 6th edition, Earland Science Publishing, New York, 848, 2005.

E. H. Liu, R. M. Siegel, D. M. Harlan, and J. J. O'Shea, T cell-directed therapies: Lessons learned and future prospects, Nature Immunology, 8, 25-30, 2007.

T. W. Mak and M. E. Saunders, The immune response, Basic and Clinical Principles, Elsevier Academic Press, San Diego, CA, 1194, 2006.

Anticancer Agents

Fredrik Björkling and Lars H. Jensen contents

23.1 The Disease 375

23.1.1 The Hallmarks of Malignant Cancer 375

23.1.2 Acquired Capabilities of Cancer Cells 378

23.1.3 Enabling Characteristics of Cancer Cells 379

23.1.4 Anticancer Agents 380

23.2 Anticancer Agents Currently Used 380

23.2.1 Xeloda 381

23.2.2 Alimta 382

23.2.3 Taxol 384

23.2.4 Zolinza 385

23.2.5 Gleevec 388

23.2.6 Herceptin 390

23.3 Concluding Remarks 391

Abbreviations 391

Further Readings 392

23.1 THE DISEASE

Cancer is the second most common cause of death in the United States, and the rest of the western world, exceeded only by heart diseases. Thirty years ago, 50% of the Americans diagnosed with cancer died due to the disease within 5 years; today's 5-year survival rate is 65%. The FDA has approved 43 new cancer drugs over the last 10 years, compared with 27 in the previous 10 years. Table 23.1 gives an overview of some of the anticancer agents approved today indicating their primary target/mechanism of action and the indications they are used for. Despite this improvement in anticancer therapy, more and more people are being diagnosed with cancer; the primary reason for this being the prolongation of mean life span as a result of more effective treatments against deadly infections and cardiovascular diseases. The need for effective anticancer treatments is therefore still urgent. In this chapter, we will give an overview of the molecular and cellular alterations associated with the development of cancer, and the challenges to be overcome for the effective treatment of this disease. Finally, we will focus on the development and characterization of some anticancer agents used in today's praxis.

23.1.1 The Hallmarks of Malignant Cancer

Metastatic cancer, responsible for 90% of all human cancer deaths, is characterized by a number of molecular, cellular, and morphologic characteristics that has been described as the six hallmarks, or acquired capabilities, of cancer. These hallmarks relate to different levels of dysregulation required

TABLE 23.1

Examples of Currently Used Anticancer Agents with Indication of (1) Their Site of Interaction, (2) Their Interactions and Compound Classes, (3) Their Name/Indications/Approval Year, and (4) Their Specific Target/Mode of Interaction

Examples of Launched and Experimental Drugs, Trade Name (Generic Drug Name, FDA Approval Year, Indication)

Site of Interaction

Nucleus

Tumor cell DNA

Nucleus

Tumor cell DNA

Nucleus

Nuclear receptors

Interaction/Compound Class

Nonspecific DNA break

Nitrogen mustards

Nitrosoureas

Triazenes

Platinum compounds

DNA-related proteins

Epipodophyllotoxines

Topo I inhibitors

Antimetabolites

Antifolates

Anthracyclines

Hormone receptor Retinoids Anti hormones Steroids

Cytoxan, Neosar (cyclophosphamide, 1959, broad; ovary, lung breast)

Temodar (temozolomide, 1999, anaplastic astrocytoma)

Blenoxane (bleomycin, 1973, testicular, ovarian)

Platinol (cisplatin, 1978, testicular)

Eloxatin (oxaliplatin, 2002, colorectal)

Adriamycin, Rubex (doxorubicin, 1974, ovary)

Idamycin (idarabicin, 1990, AML)

Camptosar (irinotecan, 1996, colon)

Hycamtin (topotecan, 1996, ovary)

Vepesid (Etoposide, VP16, 1983, lung, testicular)

Cerabidine (daunorabicin, daunomycin, 1987, ovary)

Methotrexate (methotrexate, 1953, osteosarcoma)

Adracil (fluorouracil, 5-FU, 1962, broad colon, rectum, breast)

FUDR (floxuridine, 1970, metastatic gastrointestinal)

Xeloda (capecitabine, 2001, breast, colon)

Cytosar-U (cytarabine, 1969, leukemia)

Gemzar (gemcitabine, 1996, pancreas, lung)

Alimta (pemetrexed disodium, 2004, mesothelioma, lung)

Nolvadex (tamoxifen, 1977, breast)

Vesanoid (ATRA, 1995, APL)

Femara (letrozole, 1997, breast)

Aromasin (exemestane, 1999, breast)

Specific Targets/Mode of Action

DNA cross linking

DNA methylation

DNA fragmentation

DNA cross-linking

DNA cross-linking

Topo II-induced breaks

Topo II-induced breaks

Topo I-induced breaks

Topo I-induced breaks

Topo II-induced breaks

Topo II-induced breaks

Dihydrofolic reductase—DHFR

Thymidylate synthase—TS

DNA polymerase DNA polymerase GRAFT, TS, and others Estrogen-receptor antagonist Retinoic acid receptor Arometase/ CYP-19 inhibitor Arometase/CYP-19 inhibitor

Microsomes

Nucleus Histones, DNA

Cytoplasm Acetylated proteins Cell-membrane

Membrane-bound tumor proteins

Oxidoreductases Small molecules Epigenetics Hydroxamic acids Cyclic peptides Benzamide

Short chain fatty acids Membrane receptors MoAb

Small molecule antagonists

Cytoplasm Signal transduction kinase inhibitors Small molecule antagonists Protein degradation Proteasome inhibitor Tubulin modulator Taxanes and vinca alkaloids

Endothelial cells

Cell membrane

Cells of the immune system

Endothelial receptors of VEGF, bFGF, and TGF-a Monoclonal antibodies Small molecules antagonists Lymphocytes, macrophages, and dendritic cells Interferons Interleukin2

Cancer vaccines (DNA- or protein-based)

Zolinza (vorinostat, 2006, CTCL) Belinostat (pxdlOl, phase II, various indications) (valporic acid, phase II, various indications) Decogen (decitabine, 2006, MDS)

Rituxan (rituximab, 1997, non-Hodgkin's lymphoma)

Tarceva (erlotinib, 2004, lung, pancreas)

Iressa (gefitinib, 2003, lung)

Herceptin (trastuzumab, 1998, breast)

Sutent (sunitinib maleate, 2006, gastrointestinal, renal)

Nexavar (sorafenib, 2005, renal)

Gleevec (imatinib mesylate, CML, gastorintestinal, 2001)

Nexavar (sorafenib, 2005, renal)

Velcade (bortezomib, 2006, multiple myeloma)

Oncovin (vincristine, 1963, broad leukemia, brain, breast) Taxol (paclitaxel, 1992, Kaposi's sarcoma, breast, ovary) Taxotere (docetaxel, 1996, breast, lung) Avastin (bevacizumab, 2006, lung, colon)

Histone deacetylases Histone deacetylases Histone deacetylases DNA methyl transferases

CD20 antagonist EGFR antagonist EGFR antagonist Her-2 antagonist

Multiple tyrosine kinase inhibitor Receptor tyrosine kinase inhibitor Bcr/alb and c-kit

MAPK kinase pathway Proteasome inhibitor

Hyperstabilization of microtubules Hyperstabilization of microtubules Vascular endothelial growth factor receptor (VEGFR) antagonist

Roferon A (interferon alfa 2a, 1986, leukemias)

Stimulates the ability of immune cells to attack cancer cells for cancer cells to develop into and maintain malignancy. These include (1) self-sufficiency in growth signals, (2) insensitivity to antigrowth signals, (3) evading apoptosis, (4) limitless replicative potential, (5) sustained angiogenesis, and (6) tissue invasion and metastasis.

23.1.2 Acquired Capabilities of Cancer Cells

1. Self-sufficiency in growth signals can be achieved directly by the production of growth-promoting signaling molecules by the cancer cell itself. This phenomenon is known as autocrine stimulation, thus creating a positive growth feed back loop. The production of platelet derived growth factor (PDGF), transforming growth factor a (TGF-a) by cancer cells are examples. Another way of achieving self-sufficiency in growth signals is to constitutively activate downstream signal transduction pathways normally activated by progrowth signals. The SOS-Ras-Raf-MAPK signal (Figure 23.1) transduction pathway is a good example, and the Ras proteins are indeed altered in 25% of all cancers leading them to produce mitogenic signals in the absence of progrowth signals (Figure 23.1).

Trans Membrane Signalling Cartioons
FIGURE 23.1 Cartoon depicting two signal transduction cascades often deregulated in cancer, namely, the PI3K-AKT and the SOS-Ras-Raf-Mek cascades. One way of activating these pathways is by signaling through the HER2 transmembrane tyrosine kinase receptor.

2. At the G1 cell cycle checkpoint, also termed the restriction point, normal cells detect the composition of growth/antigrowth signals including cytokines and nutrients in their environment, and then decide whether or not to enter another round of cell division. One key effector halting the cell cycle at G1 is the retinoblastoma protein (pRb). Hypophosphorylated pRb blocks cell proliferation by sequestering E2f family transcription factors, thereby inhibiting the binding of these to DNA so that the genes required for entry into the S phase of the cell cycle are not expressed. Deletion of pRb is frequent in cancers and thus represents one of the most common means by which cancer cells obtains resistance to antigrowth signals.

3. Evading apoptosis, or programmed cell death, is also a key acquired capacity of cancer cells. In normal cells, apoptosis targets cells for self-destruction when their DNA is damaged beyond repair, or when the cells do not receive the proper prosurvival signals from their environment. A key family of regulatory proteins involved in regulating apop-tosis is the Bcl-2 family containing proapoptotic (Bax, Bak, Bid, and Bim) as well as antiapoptotic (Bcl-2, Bcl-XL, and Bcl-W) regulatory proteins. The Bcl-2 protein itself is frequently unregulated in cancer cells leading directly to resistance toward drug-induced apoptosis. Furthermore, deletion or mutational inactivation of the tumor suppressor gene p53 represents another way of evading apoptosis, because p53 normally induces up regulation of proapoptotic Bax protein upon DNA damage. p53 is inactivated in 50% of all human tumors.

4. Normal cells have the capacity to divide only a finite number of times, typically 60-70 doublings before they die due to senescence, a cell fate different from apoptosis. This is because the telomeres, DNA sequences at the end of the chromosome arms, shorten a little at each mitotic cell division. Loss of telomeres results in DNA recombination and end to end fusions of chromosomes leading to cell senescence. More than 90% of human cancers avoid telomere shortening by expressing the catalytic subunit of telom-erase hTERT, thereby avoiding senescence. The remaining use DNA recombination for this purpose.

5. Angiogenesis is the process of developing new blood vessels. In order to grow beyond the size of a few 100 |im, tumors must develop neovascularization through the process of angiogenesis. However, the tumor cells do not directly participate in neovasculariza-tion, which is mediated only by endothelial cells. Instead tumor cells produce the factors required for endothelial cells to perform angiogenesis in that they secrete growth factors like vascular endothelial growth factor (VEGF) or fibroblast growth factor (FGF1/2), which can be produced by different mechanisms as shown for VEGF in Figure 23.1. Angiogenesis is attractive in anticancer therapy because its inhibition is expected to have no implications for normal cells in the body regardless of their proliferation rate.

6. Primary tumor growth is seldom the direct cause of death in cancer patients. Instead, the spreading of tumors known as metastasis is the most frequent cause of death (90%). Invasion is penetration of tumor tissue through basal membranes allowing a tumor to grow out from its original site into the surrounding tissue; whereas metastasis is the spreading of cancer cells to other sites in the body, and the subsequent establishment of secondary tumors in other tissues. Both processes involve changing the interaction of cancer cells with the extracellular environment/matrix.

23.1.3 Enabling Characteristics of Cancer Cells

In addition to these hallmarks, cancer cells display a number of additional alterations enabling cancer growth. One of the most important alterations is genome instability. The number of individual mutations required to induce all six hallmarks of cancer would not normally accumulate in a single cell if not for genome instability. Genome instability is caused by mutations of DNA repair/ checkpoint surveillance mechanisms that normally results in cell cycle arrest until damaged DNA has been repaired, or in case DNA cannot be efficiently repaired results in elimination of the cell via apoptosis. The genome guarding protein p53 as well as proteins sensing DNA damage such as ATR, ATM, and their downstream signal kinases Chk1 and Chk2 play important roles in such checkpoints, and their loss often precedes genome instability.

One of the main obstacles in cancer therapy is tumor heterogeneity that is closely related to genome instability. Within one tumor several different subpopulations of tumor stem cells often exist, due to accelerated chromosomal aberrations driven by genome instability resulting in chromosomal deletions, duplications and translocations. Consequently, while first line chemotherapy does often lead to good responses and tumor regression, tumor heterogeneity makes it very difficult to kill all tumor cells within a tumor. Consequently, some cancer cells may be inherently resistant toward a given drug due to mutation of its primary target/receptor or due to dysregulation of downstream signal transduction cascades. Such cells will often continue to divide and will eventually overgrow the drug sensitive cells resulting in a new tumor that is now resistant. As a result in modern anticancer therapy, combinations of several (five or more) different drugs targeting different receptors/pathways are often used.

23.1.4 Anticancer Agents

Despite intensive research aimed at understanding the molecular pathology of cancer, a great deal of anticancer agents currently in clinical use were discovered and even entered the clinic before their exact mechanism of action was clarified. These drugs were often discovered in cellular screens of extracts from natural sources, or in in vivo screens using a leukemic P388 mouse model. The drugs discovered typically inhibit DNA synthesis (antimetabolites), damage DNA (DNA alky-lating agents and topoisomerase poisons), or inhibit the function of the mitotic microtubule-based spindle apparatus (taxanes) (Table 23.1). The reason for these agents still being in clinical use relates to the fact that they are often highly effective although they have toxic properties toward normal fast proliferating cells as the intestinal and gut lining hair follicles, and the bone marrow cells, leading to the well-known effects of classical chemotherapy including nausea, vomiting, hair loss, and myleosuppression. The cytotoxics stands in contrast to the so-called targeted therapies that are developed in a totally different fashion by applying knowledge concerning the structure of a primary target with molecular in silico screening as well as high-throughput compound library screening, or by designing protein-based medications, which in the case of cancer treatment are often in the form of cell surface tyrosine kinase antagonistic antibodies. Examples of targeted therapeutic anticancer agents are kinase inhibitors (Gleevec, Iressa), proteasome inhibitors (Velcade), histone deacetylase (HDAC) inhibitors (Zolinza, belinostat), and antibodies against cell surface receptors (Herceptin) (Table 23.1).

23.2 ANTICANCER AGENTS CURRENTLY USED

Reviewing the multitude of anticancer agents in current use is an overwhelming task and not within the scope of this chapter. Instead, we will describe the development and mechanism of action of three classic cytotoxics. Two antimetabolites Xeloda and Alimta inhibiting the production of precursors for DNA synthesis in the cell, as well as an agent targeting the structural protein P-tubulin (Paclitaxel) involved in microtubule functioning. We will likewise review the development and mechanism of three new classes of anticancer therapeutics, the HDAC inhibitors exemplified by Zolinza, the kinase inhibitors exemplified by the BCR-ABL tyrosine kinase inhibitor Gleevec, and finally the HER2/Neu tyrosine kinase antagonist monoclonal antibody Herceptin (Table 23.1).

23.2.1 Xeloda

The classic antineoblastic agent 5-Fluorouracil, 5-FU (Figure 23.2A), is a fluorinated analog of uracil. 5-FU exhibits its main activity via two known biochemical mechanisms originating from the 5-FU metabolites 5-fluoro-2'-deoxyuridine monophosphate (FdUMP) and 5-fluorouridine triphosphate (FUTP) (Figure 23.2A). FdUMP and the folate cofactor N5,N10-methylene-tetrahydrofolate bind to thymidylate synthase (TS) to form a covalently bound ternary complex. This binding inhibits the formation of thymidylate from 2'-deoxyuridylate. Since thymidylate is a necessary precursor of thymidine triphosphate, which is essential for the synthesis of DNA, its inhibition causes inhibition of cancer cell division. Second, nuclear transcriptional enzymes can mistakenly incorporate

HO R2

HO R2

5-FU

5-FU

II II

HO OH Xeloda

HO OH Xeloda

Xeloda

Intestine

Xeloda

CE 5'-DFCR

CvD I

5'-DFUR Liver

CvDJ

5'-DFUR TP

5-FU

Cancer cell

FIGURE 23.2 (A) Chemical structures of 5-FU and its bioactive metabolites. (B) Bioreactive pathways leading to the generation of 5-FU from Xeloda. The relevant enzymes and their compartments are shown.

FUTP in place of uridine triphosphate (UTP) during the synthesis of RNA. This metabolic error can interfere with RNA processing and protein synthesis and consequently with cell growth. It has been suggested that while inhibition of TS is primarily responsible for the anticancer activity of 5-FU, its effect on RNA synthesis may be the main cause of its toxicity.

Even though 5-FU is an efficient drug that has been used for many years in the treatment of solid tumors, such as breast and colorectal cancers, there has been a wish to develop an orally available fluoropyrimidine with improved efficacy and safety profile. The goal was to design derivatives of 5-FU that could specifically be converted to the parent drug (5-FU) by enzymes preferentially located in tumor tissue. Several attempts have been made to make orally active prodrugs of 5-FU, the most advanced version being a 5'-deoxy-5-fluorouridine (5'-DFUR) derivative (Figure 23.2B), which is transformed by pyrimidine nucleoside phosphorylase (PyNPase) enzymes, which are preferentially found not only in tumor tissue but unfortunately also in the intestine. Consequently, even though there is some selective targeting of cancer cell with this compound, 5'-DFUR also releases 5-FU in the intestine causing dose limiting toxicological effects there.

To get around the unwanted effects in the intestine, another strategy was developed. The chemical starting point for 5-FU prodrugs was the 5'-deoxy-5-fluorocytidine (5'-DFCR) described above (Figure 23.2B), which was known to be effectively transformed to 5-FU by cytidine deaminase (CyD), particularly in tumor tissue where it is highly expressed. This in combination with a low activity of the same enzyme in human bone marrow cells indicated that selective killing of tumor cells could be obtained. The strategy was now to find a fluoropyrimidine carbamate that was stable in the intestinal tract but efficiently hydrolyzed to 5'-DFCR by carboxylesterase (CE) located in the liver, where 5'-DFCR could be further transformed to 5'-deoxy-5-fluorouridine (5'-DFUR), and finally to 5-FU by thymidine phosphorylase (TP) (Figure 23.2B).

Among the many prodrugs prepared to achieve this activity pattern, through extensively testing for (1) selectivity for hepatic CE, (2) oral bioavailability, and (3) activity in human cancer xenograft models in vivo, Capecitabine (V4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine, Xeloda™), developed and marketed by Roche, was found to have the most favorable characteristics resulting in substantially higher 5-FU concentrations within tumors than observed in plasma and in normal tissue (muscle) (Figure 23.2B). Additionally, the tumor 5-FU levels were much higher than those that could be achieved by the intraperitoneal administration of 5-FU at equitoxic doses. This tumor selective delivery of 5-FU ensures a greater efficacy and a more favorable safety profile than can be obtained by other fluoropyrimidines. Xeloda is now approved in the United States, Canada, and other countries for the treatment of metastatic breast cancer.

23.2.2 Alimta

The discovery of Alimta has its chemical origin in the early findings of the antimetabolites amin-opteridines and thereafter methotrexate and both inhibit folate metabolism (Figure 23.3C). The impressive anticancer effects found for methotrexate validated folate antimetabolites early on as antiproliferative agents. For decades, researchers have worked on the task to find inhibitors of folate-dependent enzymes such as TS, dihydrofolate reductase (DHFR), and glycinamide ribonucle-otide formyltransferase (GARFT), which take part in the folic acid activation (Figure 23.3A). The active form of folate is the reduced form tetrahydrofolate (Figure 23.3B), which plays an important role in the biochemical pathways to donate one carbon unit in the form of methyl, methylene, or formyl groups. These metabolic reactions are essential for the formation of DNA, RNA, ATP, and the catabolism of certain amino acids. Consequently, inhibiting this metabolic pathway abrogates cancer cell proliferation because cancer cells have high demands for ATP, and because they require high levels of nucleic acid precursors for DNA synthesis.

The pathway leading to the formation of tetrahydrofolate (THF) begins when folate (F) is reduced to dihydrofolate (DHF), which is then reduced to THF, DHFR catalyses both steps (Figure 23.4). Methylene tetrahydrofolate (CH2THF) is formed from tetrahydrofolate by the addition of o ovoh

H2N N N Pterin ring

Folic acid

H2N N N Pterin ring

Folic acid

h2n n

R3 = H; aminopterin R3 = Me; methotrexate

NN H

NN H

H2N N

R2 = H; tetrahydrofolic acid R2 = Formyl; natural cofactor for GAR FTase

Sites of modification R1

Chirality

H2N N

R3 = H; aminopterin R3 = Me; methotrexate

Alimta

FIGURE 23.3 Chemical structures of various antimetabolites preceding and founding the development of Alimta.

Alimta

FIGURE 23.3 Chemical structures of various antimetabolites preceding and founding the development of Alimta.

methylene groups forming N5, N10-methylene tetrahydrofolate from one of the three carbon donors: formaldehyde, serine, or glycine (Figure 23.4). The key reaction is the TS-catalyzed methylation of deoxyuridine monophosphate (dUMP) to generate thymidylate (dTMP), which is needed for DNA synthesis. Methyl tetrahydrofolate (CH3THF) is formed from methylene tetrahydrofolate by reduction of the methylene group and formyl tetrahydrofolate (CHOTHF, folinic acid) results from the oxidation of the same precursor (Figure 23.4).

Inspired by the active pterine structures (Figure 23.3A), many modifications were made in this ring system including modifications in ring A, such as substitution of NH2 with methyl or hydrogen as well as exchange of the fused ring B for a fused phenyl ring. This resulted in compounds having high biological activity as TS inhibitors with concomitant antiproliferative activity. Some of these analogs were indeed taken into early clinical testing, but were stopped due to pharmacokinetic or toxicological problems.

An important new class of potent folate antimetabolites that are active as antitumor agents are represented by 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF, Lometrexol) in which the two nitrogens in positions 5 and 10 is exchanged for carbon and the B ring is reduced and as such mimic the structure of THF (Figure 23.3D). The target enzyme for DDATHF was shown to be glycinamide ribonucleotide formyltransferase (GARFT) (Figure 23.4) catalyzing the first folate cofactor-dependent formyl transfer step in the de novo purine biosynthetic pathway instead of the DHFR enzyme, which was the target for earlier folate inhibitors described above.

The two diastereomeric forms of DDATHF were separated and their biological activity examined. Interestingly, they did not show any significant difference in activity and further work was therefore undertaken to remove this chiral center so as to obtain a stereochemically pure compound.

dUMP

DHFR

dUMP

D

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