Targeting of Antineoplastic Drug Action (A)

When degenerating neoplastic cells display special metabolic properties which are different from those of normal cells, targeted pharmacotherapeutic intervention becomes possible.

Imatinib. Chronic myelogenous leukemia (CML) results from a genetic defect in the hematopoietic stem cells of the bone marrow. Nearly all CML patients possess the

Philadelphia chromosome. It results from translocation between chromosomes 9 and 22 of the c-abl protooncogene, leading to the hybrid bcr-abl fusion gene on chromosome 22. The recombinant gene encodes a tyrosine kinase mutant with unregulated (constitutive), enhanced activity that promotes cell proliferation. Imatinib is a tyrosine kinase inhibitor that specifically affects this mutant but also interacts with some other kinases. It can be used orally in Philadelphia chromo- some-positive CML.

Asparaginase cleaves the amino acid asparagine into aspartate and ammonia. Certain cells, in particular the tumor cells in acute lymphatic leukemia, require asparagine for protein synthesis and must take it up from the extracellular space, whereas many other cell types are themselves able to synthesize asparagine. Supply of the amino acid can be disrupted by administration of the asparagine-hydrolyzing enzyme. Consequently, protein synthesis and proliferation of neoplastic cells are inhibited. Asparaginase is obtained from E. coli bacteria or may be of plant origin (Erwinia chrysanthemi), when it is also named crisantaspase. Allergic reactions against the exogenous protein occur after parenteral administration.

Trastuzumab exemplifies a growing number of monoclonal antibodies that have become available for antineoplastic therapy. These are directed against cell surface proteins that are strongly expressed by cancer cells. Trastuzumab binds to HER2, the recep-

tor for epidermal growth factor. The density of this receptor is greatly increased in some types of breast cancer. When the tumor cells have bound antibody, immune cells can recognize them as elements to be eliminated. Trastuzumab is indicated in advanced cases under certain conditions. The antibody is cardiotoxic; it is likely that cardiomyocytes also express HER2.

Mechanisms of Resistance to Cytostatics (B)

Initial success can be followed by loss of effect because of the emergence of resistant tumor cells. Mechanisms of resistance are multifactorial.

Diminished cellular uptake may result from reduced synthesis of a transport protein that may be needed for membrane penetration (e.g., methotrexate).

Augmented drug extrusion: increased synthesis of the P-glycoprotein that extrudes drugs from the cell (e.g., anthracyclines, vinca alkaloids, epipodophyllotoxins, and paclitaxel) is responsible for multidrug resistance (mdr1 gene amplification).

Diminished bioactivation of a prodrug, e.g., cytarabine, which requires intracellular phosphorylation to become cytotoxic.

Change in site of action: e.g., increased synthesis of dihydrofolate reductase may occur as a compensatory response to methotrexate.

Damage repair: DNA repair enzymes may become more ef cient in repairing defects caused by cisplatin. Inhibition of apoptosis due to activation of antiapoptotic cellular mechanisms.

Inhibition of Immune Responses

Both the prevention of transplant rejection and the treatment of autoimmune disorders call for a suppression of immune responses. However, immune suppression also entails weakened defenses against infectious pathogens and a long-term increase in the risk of neoplasms.

A specific immune response begins with the binding of antigen by lymphocytes carrying specific receptors with the appropriate antigen-binding site. B-lymphocytes “recognize” antigen surface structures by means of membrane receptors that resemble the antibodies formed subsequently. T-lymphocytes (and naive B cells) require the antigen to be presented on the surface of macrophages or other cells in conjunction with the major histocompatibility complex (MHC); the latter permits recognition of antigenic structures by means of the T-cell receptor. T-help- er (TH) cells carry adjacent CD3 and CD4 complexes, cytotoxic T cells a CD8 complex. The CD proteins assist in docking to the MHC. Besides recognition of antigen, stimulation by cytokines plays an essential part in the activation of lymphocytes. Interleukin-1 is formed by macrophages, and various interleukins (IL), including IL-2, are made by T- helper cells. As antigen-specific lymphocytes proliferate, immune defenses are set into motion.

I. Interference with antigen recognition.

Muromonab CD3 is a monoclonal antibody directed against mouse CD3 that blocks antigen recognition by T-lymphocytes (use in graft rejection).

Glatirameracetate consists of peptides of varying lengths, polymerized in random sequence from the amino acids glutamine, lysine, alanine, and tyrosine. It can be used in the treatment of multiple sclerosis besides β- interferon. This disease is caused by a T-lym- phocyte-mediated autoaggression directed against oligodendrocytes that form myelin sheaths of CNS axons. The culprit antigen

appears to be myelin basic protein. Glatiramer resembles the latter; by blocking antigen receptors, it interferes with antigen recognition by lymphocytes.

II. Inhibition of cytokine production and action. Glucocorticoids modulate the expression of numerous genes; thus, the production of IL-1 and IL-2 is inhibited, which explains the suppression of T-cell-depend- ent immune responses. In addition, glucocorticoids interfere with inflammatory cytokines and signaling molecules at various other sites. Glucocorticoids are used in organ transplantations, autoimmune diseases, and allergic disorders. Systemic use carries the risk of iatrogenic Cushing syndrome (p.244).

Ciclosporin and related substances inhibit the production of cytokines, in particular interleukin-2. In contrast to glucocorticoids, the plethora of accompanying metabolic effects is absent (see p.306 for more details).

Daclizumab and basiliximab are monoclonal antibodies against the receptor for IL- 2. They consist of murine Fab fragments and a human Fc-segment. They are used to suppress transplant rejection reactions.

Anakinra is a recombinant form of an endogenous antagonist at the interleukin-1 receptor; it is used in rheumatoid arthritis (p.332).

III. Disruption of cell metabolism with inhibition of proliferation. At dosages below those needed to treat malignancies, some cytostatics are also employed for immunosuppression; e.g., azathioprine, methotrexate, and cyclophosphamide. The antiproliferative effect is not specific for lymphocytes and involves both T and B cells.

Mycophenolate mofetil has a more specific effect on lymphocytes than on other cells. It inhibits inosine monophosphate dehydrogenase, which catalyzes purine synthesis in lymphocytes. It is used in acute tissue rejection responses.

IV. Anti-T-cell immune serum is obtained from animals immunized with human T- lymphocytes. The antibodies bind to and damage T cells and can thus be used to attenuate tissue rejection.

Ciclosporin is of fungal origin; it is a cyclic peptide composed of 11, in part atypical, amino acids. Therefore, orally administered ciclosporin is not degraded by gastrointestinal proteases. In T-helper cells, it inhibits the production of interleukin-2 by interfering at the level of transcriptional regulation. Normally, “nuclear factor of activated T cells,” (NFAT) promotes the expression of interleu- kin-2.Thisrequiresdephosphorylationofthe precursor,phosphorylatedNFAT,bythephosphatase calcineurin, enabling NFAT to enter the cell nucleus from the cytosol. Ciclosporin binds to the protein cyclophilin in the cell interior; the complex inhibits calcineurin, hence the production of interleukin-2.

The breakthroughs in modern transplantation medicine are largely attributable to the introduction of ciclosporin. It is now also employed in certain autoimmune diseases, atopic dermatitis, and other disorders.

The predominant adverse effect of ciclosporin is nephrotoxicity. Its dosage must be titrated so that blood levels are neither too high (risk of renal injury) nor too low (rejection reaction). To complicate the problem, ciclosporin is a substance dif cult to manage therapeutically. Oral bioavailability is incomplete. Back-transport of the drug into the gut lumen occurs via the P-glycoprotein ef ux pump, in addition to metabolization by cytochrome oxidases of the 3A subfamily. Hepatic CYP3A4 enzymes contribute to presystemic elimination and are responsible for elimination of systemically available ciclosporin. Diverse drug interactions may occur by interference with CYP3A and P-glycopro- tein. For optimal dosage adjustment, monitoring of plasma levels is mandatory.

Drug-mediated suppression of transplant rejection entails long-term treatment. Protracted immunosuppression carries an in-

creased risk of malignomas. Risk factors for cardiovascular diseases may be adversely af- fected—a critical and important concern in long-term prognosis.

Tacrolimus is a macrolide antibiotic from

Streptomyces tsukubaensis. In principle, it acts like ciclosporin. At the molecular level, however, its “receptor” is not cyclophilin but a so-called FK-binding protein. Tacrolimus is likewise used to prevent allograft rejection. Its epithelial penetrability is superior to that of ciclosporin, allowing topical application in atopic dermatitis.

Sirolimus (rapamycin) is another macrolide, produced by Streptomyces hydroscopicus. Its immunosuppressant action, evidently, does not appear to involve inhibition of calcineurin. It forms a complex with the FK protein, imparting a special conformation on it; and the complex then inhibits the mTOR (mammalian target of rapamycin) phosphatase. The latter operates in the signaling path leading from the interleukin-2 receptor to activation of mitosis in lymphocytes. Thus, sirolimus inhibits lymphocyte proliferation. It is approved for the prevention of transplant rejection.

NFAT Immunophilin/drug

complex

P

Calcineurin

P

NFAT

DNA

Synthesis

FK-binding protein

mTOR

Sirolimus

Lymphocyte proliferation

Inhibition of transplant

rejection

Nephrotoxicity

Long-term adverse effects Neoplasia, hypertension, hyperlipidemia, hyperglycemia

FK-binding protein

Tacrolimus