Bleomycin

Bleomycin is a glycopeptide antibiotic complex isolated from Streptomyces verticillus initially by Umezawa.113 At least 13 different fractions of bleomycin have been isolated with the clinically used product (Blenoxane) being a mixture of predominantly A2 (55%-70%) and B2 (25%-32%) fractions (Fig. 10.18). Of these fractions, A2 appears to possess the greatest antineoplastic activity. Copper is found in the naturally occurring material, and its removal is important for the material used clinically because it significantly reduces activity.

Bleomycin binds Fe through multiple interactions with the amino terminal end of the peptide chain (Fig. 10.19).114 Bleomycin may itself initiate the release of iron necessary for this complexation. Interaction with DNA subsequently occurs through the bithiazole portion of the molecule, which intercalates between G-C base pairs with a preference for genes undergoing transcription. Held in proximity to DNA by this interaction, in an aerobic environment, Fe+2 is oxidized to Fe+3 in a one-electron process with the electron being transferred to molecular oxygen.115 This gives the activated form of bleomycin, which has been formulated as HOO~Fe(III)-bleomycin, which is believed to possess square bipyramidal geometry. This then results in the production of ROS in the form of superoxide and hydroxide radical, which initiate single-strand breakage of the phos-phodiester backbone and release of DNA bases by oxidative cleavage of the 3'-4' bond of the deoxyribose moiety.116 This activity may be enhanced in the presence of mercap-tans such as glutathione, which can facilitate the action of reductase enzymes in reducing the Fe+3 that is generated back to Fe+2 so that the process may continue. The agent is most active in the G2 and M phases of the cell cycle.

NMR studies of bleomycin complexed with cobalt have confirmed the intercalation of the bithiazole with adjacent G-C base pairs with the dimethylsulfonium chain (bleomycin A2) projecting into the major groove where the sulfonium cation may interact with the phosphate backbone

Metal Bleomycin
Figure 10.18 • Structure of bleomycins.

or N-7 of a purine base adjacent to the site of intercalation (Fig. 10.20).117,118 Additional stabilization is provided by hydrogen bonding of the amino-pyrimidine ring with a gua-nine residue in the minor groove. This also serves to orient the coordinated metal and the hydroperoxide ligand toward the minor groove.

Bleomycin is notable for its lack of myelotoxicity, and this allows it to be combined with other myelosuppressants without a resulting additive effect. The acute toxicities seen with bleomycin are erythema (reddening of the skin), hyper-pigmentation (skin darkening) found predominately on the extremities, and pulmonary toxicity. The pulmonary toxic-ity may first occur as pneumonitis (inflammation of lung tissue), which normally responds to glucocorticosteroid therapy. Chronic pulmonary toxicity is expressed as pulmonary fibrosis, which is irreversible and limits utility of the agent.

Pneumonitis Bleomycin Toxicity

• Structure of the bleomycin-Fe-O2

The toxicity profile of bleomycin is explained by its route of inactivation. Hydrolysis of the N-terminal amide to the car-boxylic acid increase the pKa of the amine at C-2 from 7.3 to 9.4, resulting in a greater degree of ionization and decreased binding to DNA.119 The enzyme responsible for this conversion is known as bleomycin hydrolase, and it is present in most tissue but found in low concentration in skin and lung tissue. Tumor cells that are resistant to bleomycin may contain high levels of this enzyme.

BLEOMYCIN SULFATE (BLEO, BLM, BLENOXANE)

Bleomycin occurs as a white powder and is available in 15-and 30-U vials for reconstitution in water. It may be given intravenously, intramuscularly, or subcutaneously. It is used

Bleomycin Dna

Figure 10.20 • Representation of the bleomycin-DNA complex. (Reprinted with permission from Zhao, C., et al.: J. Inorgan. Biochem. 91:259, 2002.)

Figure 10.19

complex.

• Structure of the bleomycin-Fe-O2

Figure 10.20 • Representation of the bleomycin-DNA complex. (Reprinted with permission from Zhao, C., et al.: J. Inorgan. Biochem. 91:259, 2002.)

in the treatment of squamous cell carcinoma of the head neck, cervix, penis, and vulva. It is also used in Hodgkin's and non-Hodgkin's lymphoma as well as testicular carcinoma. Unlabeled uses include the treatment of mycosis fun-goides, osteosarcoma, and AIDS-related Kaposi sarcoma.

Bleomycin given intravenously has a distribution halflife of 10 to 20 minutes with a small volume of distribution of 20 L. The major metabolite results from hydrolysis of the amide at C-1 to give the inactive carboxylic acid. The drug is primarily eliminated in the kidneys with 60% to 70% of the drug recovered in the urine as unchanged drug. The serum elimination is related to glomerular filtration rate, such that those patients with a creatinine clearance greater than 35 mL/min have a serum total elimination half-life of 115 minutes, and in those with a creatinine clearance less than 35 mL/min, the serum total elimination half-life exponentially increases with decreasing creatinine clearance.

Dermatological reactions are the most commonly seen adverse effect with erythema, hyperpigmentation, rash, and tenderness occurring in 50% of the patients taking the drug. The most serious adverse effect is the pulmonary fibrosis previously mentioned. Treatment with bleomycin can increase the risk of pulmonary toxicity that may occur if the patient is subsequently administered oxygen during surgery.

Vinca Alkaloids

The vinca alkaloids (Fig. 10.21) are extracted from the leaves of Catharanthus roseus (periwinkle), and were originally investigated for their hypoglycemic properties but latter found to possess antineoplastic actions.120 The alkaloids are composed of a catharanthine moiety containing the indole subunit and the vindoline moiety containing the dihydroindole subunit joined by a carbon-carbon bond. Vincristine and vin-

blastine differ only in the group attached to the dihydroindole nitrogen, which is a methyl group in vinblastine and a formyl group in vincristine. Vinorelbine is a semisynthetic material resulting from loss of water across the 3',4' bond and first prepared by the use of a modified Polonovski reaction of vinblastine followed by hydrolysis.121

The vinca alkaloids were initially believed to gain entry into the cell by an energy-dependent process, but more recent work suggests that entry occurs by an energy- and temperature-independent mechanism similar to passive diffusion. The agents then begin to accumulate in cells with intracellu-lar concentrations 5- to 500-fold higher than extracellular concentrations. Once in the cell, the vincas bind to tubulin disrupting formation and function of the mitotic spindle.

The mitotic spindle is composed of the microtubules, which function as part of the cell's cytoskeleton and are important in maintaining cellular shape. They are also involved in transport within the cell and cell signaling as well as playing a pivotal role in the movement of chromosomes during mitosis. The microtubules are composed of heterodimers of a-tubulin and ^-tubulin, which may arrange as alternating heterodimers around a hollow axis to form the protofilaments of the microtubule. The behavior of the microtubules are regulated in part by microtubule-associated proteins (MAPs), which may bind to soluble tubulin or the microtubules themselves.122 As part of the mitotic process, the length of the microtubule will change because of polymerization and depolymerization of the associated tubulin. Tubulin is capable of binding guanosine-5'-triphosphate (GTP) and hydrolyzing it to guanosine diphosphate (GDP) and inorganic phosphate (Pi). When this hydrolysis occurs and Pi dissociates another tubulin-GTP complex adds to the microtubule. This forms the GTP cap and as long as tubulin-GTP or tubulin-GDP with associated Pi is present at the end

Navelbin Caps

Vinorelbine Figure 10.21 • Structures of vinca alkaloids.

of the microtubule, shortening or depolymerization is prevented. The ends of the microtubules are differentiated by the activity that is occurring and are designated as plus and minus. When the microtubule elongates and shortens, it does so primarily at the plus end. The minus end undergoes less pronounced changes in length and is normally attached to a microtubule-organizing center. In a process known as "dynamic instability," the ends go through phases of slow prolonged growth and rapid shortening, such that the overall length changes. In the process of treadmilling, there is growth at one end counterbalanced by shortening at the other, such that the overall length does not change appreciably. Both of these processes are important for the cell to successfully complete mitosis.123

As the cell prepares for mitosis, the microtubules that make up the cytoskeleton are degraded and reassembled into the mitotic spindle. During prometaphase, the microtubules grow out from the spindle poles to connect to the kineto-chores of the individual chromosomes. This involves both polymerization of tubulin and depolymerization as the mi-crotubule seeks out the chromosome representing a period of dynamic instability. These processes continue as the chromosomes are aligned at the metaphase plate, and during this alignment, treadmilling occurs.124 Chromosomes are subsequently moved to the spindle poles during anaphase as microtubules attached to the spindle poles shorten and other microtubules known as intepolar microtubules lengthen.

The vincas bind to tubulin in a reversible manner at sites different from those at which other inhibitors of spindle function bind including the podophyllotoxins and the taxanes. Combinations of these agents may give synergistic effects because of their unique binding sites. X-ray studies indicate that vinblastine binds between the a- and ^-tubulin heterodimers and other studies have shown that there are both high-affinity binding sites located at the end of the spindle and low-affinity sites located along the intact spin-dle.125,126 Binding at the high-affinity sites prevents both lengthening and shortening of the spindle and thereby disrupts its function. Both dynamic instability and treadmilling are inhibited by the vinca alkaloids. Binding at low-affinity sites, which occurs at higher drug concentration, leads to breakdown of the spindle as tubulin depolymerizes. Initial binding at these low-affinity sites leads to an alteration in structure that exposes additional vinca-binding sites. The in-tracellular concentration of the drug then determines which of these effects is seen. As a result of these actions, the mi-totic spindle fails to form properly, chromosomes do not move to the metaphase plate, anaphase fails to occur, and the cell undergoes apoptosis.127 The agents are considered specific for the M phase of the cell cycle. Other activities have been observed including antimetabolite activity, inhibition of protein synthesis, and altered lipid metabolism, but these are only seen at very high concentrations of the drugs. Inhibition of angiogenesis has also been associated with the vinca alkaloids.128

Microtubules also play important roles in axons or nerve fibers, and disruption of this function is thought to be responsible for the neuropathies seen with this group of compounds. This seems to be most pronounced for vincristine and presumably represents a greater affinity for this type of microtubule. The specific effects the vincas have on axonal microtubules is not as well defined as it is for the mitotic spindle.

Resistance to the vinca alkaloids occurs by several different mechanisms that are also associated with resistance to several high-molecular-weight molecules with diverse mechanisms of action also used in treating cancer.129 This multidrug resistance (MDR) is also seen with the taxanes, epipodophyllotoxins, and the anthracyclines although the resistance is usually greatest to the principal agent to which the patient was exposed. The MDR has been associated with several proteins including permeability glycoprotein (Pgp) and multidrug resistance protein (MRP1), which function to actively secrete the molecules from the cell. There are several inhibitors of Pgp such as calcium channel blockers and cyclosporine, which have been investigated to decrease drug efflux; however, these first-generation inhibitors were not successful in reducing drug efflux. There are several newer agents that have proven to be more potent inhibitors of Pgp, but none is currently approved. An additional mechanism of resistance involves altered forms of tubulin to which the vincas fail to bind.

VINBLASTINE SULFATE (VLB, VELBAN, VELSAR)

Vinblastine sulfate is available as a powder in 10-mg vials and as a solution in 10- and 25-mL vials for IV administration in the treatment of various cancers including Hodgkin's disease, lymphocytic lymphoma, histiocytic lymphoma, advanced mycosis fungoides, advanced testicular carcinoma, and Kaposi sarcoma. It has also been used in treating chorio-carcinoma and breast cancer when other therapies have failed. Vinblastine is part of the ABVD (adriamycin, bleomycin, vinblastine, and dacarbazine) regimen used in the treatment of Hodgkin's lymphoma. This may be alternated with the MOPP regimen. Extravasation is a concern with the vincas and is treated by administration of hyaluronidase and application of heat. Hyaluronidase hy-drolyzes hyaluronic acid, a polysaccharide component of the connective tissue. In so doing, the drug may increase diffusion out of the area of extravasation more quickly, preventing the buildup of toxic levels. Vinblastine is highly protein bound and metabolized by CYP to the 4-O-desacetyl metabolite, which has been shown to be active although only small amounts have been recovered in the bile and feces. The metabolism of the vinca alkaloids has not been well characterized. Vinblastine is eliminated primarily in the feces; however, little of the unchanged drug has been recovered. Metabolism appears to involve CYP3A but other than the desacetyl derivative, other metabolites have not been characterized. The elimination half-life for vinblastine is 25 hours. Like vinorelbine, myelosuppression is commonly seen with vinblastine and is dose limiting. Inflammation of the GI tract is more commonly seen with vinblastine than vincristine. Nausea and vomiting may also occur. Other adverse effects include alopecia, secretion of antidiuretic hormone, headache, and depression. Neurotoxicity is mild compared with vincristine, but peripheral neuropathy may be seen. An additional manifestation of this neurotoxicity is hypertension related to disruption of autonomic function.

VINCRISTINE SULFATE (LCR, VCR, LEUROCRISTINE, ONCOVIN)

Vincristine sulfate is available as a 1-mg/mL solution in 1-, 2-, and 5-mL vials for IV administration in acute leukemia and as part of a multidrug regime for Hodgkin's and

Figure 10.22 • Structures of the taxanes.

non-Hodgkin's lymphoma as well as rhabdomyosarcoma, neuroblastoma, Ewing sarcoma, Wilms tumor, soft tissue sarcoma, testicular cancer, liver cancer, and head and neck cancers. It has also been utilized in treating pediatric cancer. Vincristine is highly protein bound (75%) and may also bind to platelets that contain large amounts of tubulin. Numerous metabolites have been detected and several have been identified, one of which is the 4-O-desacetyl derivative. The metabolism that does occur is believed to largely be mediated by CYP3A. Elimination occurs primarily in the bile with a terminal elimination half-life of 23 to 85 hours. The most commonly seen toxicity for vincristine is a dose-limiting neurotoxicity caused by effects on axonal microtubules. Symptoms are variable and include peripheral neuropathy, ataxia, seizure, bone pain, and coma. Constipation is also a commonly seen toxicity, and laxatives may be used prophy-lactically. Other toxicities include alopecia, skin rash, mild myelosuppression, secretion of antidiuretic hormone, azospermia, and amenorrhea.

VINORELBINE DITARTRATE (VNB, VRL, NAVELBINE)

Vinorelbine ditartrate is available in 1- and 5-mL vials at a concentration of 10 mg/mL for IV use. It is FDA approved for the treatment of NSCLC. The agent has also been used in treating metastatic breast cancer, cervical cancer, uterine cancer, and lung cancer especially in older patients or those with physical difficulties. Vinorelbine is the most lipophilic of the vinca alkaloids because of modifications of the catharanthine ring system and dehydration of the piperidine ring. This allows the agent to be quickly taken up into cells including lung tissue where concentrations are 300-fold higher than plasma concentrations. This is 3 to 13 times higher than the lung concentrations seen with vincristine. The agent is highly protein bound (80%-91%) and metabolized by CYP3A. The major metabolite seen is the 4-O-desacetyl derivative, which is equally active with the parent but only formed in small quantities. The agent is eliminated primarily (33%-88%) in the bile with some appearing in the urine (16%-30%). The elimination half-life is 27 to 43 hours. The toxicities seen for vinorelbine include myelosuppression, which is dose limiting but ceases upon discontinuation of drug. This is most commonly seen as a neutropenia, and patient's neutrophil count should be monitored prior to and during therapy to decrease the chance of infection. Additional toxicities include nausea/vomiting, elevation of liver function tests, alopecia, generalized fa tigue, and inappropriate secretion of antidiuretic hormone. Neurotoxicity is seen with vinorelbine but occurs to a lesser degree compared with other vinca alkaloids because of its decreased affinity for axonal microtubules.

Taxanes

The taxanes, specifically, taxol (or paclitaxel) was discovered in the 1960s as part of a large-scale screening program conducted by the National Cancer Institute on plant ex-tracts.130 Taxol (Fig. 10.22), isolated from the bark of the pacific yew tree, proved to be active against various cancer models; however, interest in the material waned because of the lack of available material and formulation problems. Interest was renewed as the mechanism of action was elucidated, new sources of material were identified, and the formulation problems were resolved.

The formulation problems seen in the early development of paclitaxel were caused by poor water solubility, which was addressed by the use of Cremophor EL and ethanol as vehicles. Cremophor EL is a polyoxyethylated castor oil, and early experience with this formulation resulted in a high percentage of hypersensitivity reactions including rash, bronchospasm, and hypotension, which were associated with the ability of Cremophor EL to cause histamine release. Administration of antihistamines and corticosteroids prior to paclitaxel administration reduces the percentage of patients experiencing hypersensitivity reactions to 1% to 3%.131

The taxanes bind to tubulin at a site distinct from the vinca alkaloids. In the absence of x-ray crystal structures of bound drug, photoaffinity probes of paclitaxel have identified two sites at which binding may occur to jS-tubulin. The first site was located at the N-terminus and involved residues 1 to 31. The second site involved residues 217 to 231 of j-tubulin and although these two sites are widely separated in the primary structure, they are close to each other in the tertiary structure.132 The binding site is located on the luminal side of the microtubule and located in the middle of the S-tubulin subunit. Docetaxel binds to the same site with greater affinity. Binding of taxanes at low concentration results in stabilization of the microtubule and prevents depolymerization. At higher concentrations, polymerization is enhanced, and the normal equilibrium between free tubulin and polymer is modified. The taxanes inhibit both treadmilling and dynamic instability, and cells are most affected in the M phase when microtubule dynamics are undergoing the greatest change. Mitosis is blocked at the metaphase anaphase boundary, and cells undergo apopto-sis.133 Paclitaxel has also been shown to enhance phosphorylation of a serine residue of Bcl-2, an antiapoptotic protein resulting in inhibition of Bcl-2's ability to block apoptosis. The proapoptotic proteins Bad and Bax are stimulated and in a similar manner, docetaxel has been shown to induce apoptosis by activation of caspase enzymes.134

Resistance to the taxanes is like that seen for the vinca alkaloids and other agents and involves Pgp-mediated efflux. Alterations in the structure of ^-tubulin may also occur and result in decreased binding of taxanes to the microtubules and therefore reduced cytotoxicity.

PACLITAXEL (TAX, TAXOL)

Paclitaxel is available in single-dose vials of 30 mg/5 mL and 100 mg/16.7 mL for IV administration in the treatment of breast, ovarian, NSCLC, and AIDS-related Kaposi sarcoma. Other uses have included treatment of head, neck, esophageal, cervical, prostate, and bladder cancers.

Paclitaxel is highly plasma protein bound (>90%) and does not penetrate the CNS. Metabolism involves CYP-mediated oxidation to give 6a-hydroxypaclitaxel (CYP2C8) and para hydroxylation of the phenyl group attached to the 3'-position (CYP3A4). The 6a-hydroxy metabolite normally predominates, but the para hydroxy metabolite may occur to a greater degree in those patients with liver disease or when CYP3A4 has been induced. Both metabolites are less active than the parent and do not undergo phase II conjugation reactions. Elimination occurs primarily in the feces, and the elimination half-life is 9 to 50 hours depending on the infusion period.

The major toxicity seen with paclitaxel is a dose-limiting myelosuppression that normally presents as neutropenia. The previously mentioned hypersensitivity reactions occur but are greatly reduced by antihistamine pretreatment. Interaction with the axonal microtubules such as that seen for the vincas also occurs and leads to numbness and paresthesias (abnormal touch sensations including burning and prickling). The agent is also available as an albumin-bound formulation (Abraxane) to eliminate the need for the solubilizing agents associated with the hypersensitivity reactions. Other adverse effects include bradycardia, which may progress to heart block, alopecia, mucositis, and/or diarrhea. Paclitaxel produces moderate nausea and vomiting that is short-lived.

DOCETAXEL (TXT, TAXOTERE)

Docetaxel is available in single-dose vials of 20 mg/0.5 mL and 80 mg/2 mL for IV administration in the treatment of breast, NSCLC, and prostate cancers. It has also been utilized in non-FDA-approved treatment of head, neck, gastric, bladder, and refractory ovarian cancers.

Docetaxel is highly plasma protein bound (80%) and widely distributed with the highest concentration in the he-patobiliary system, but it does not appear to cross the blood-brain barrier. The metabolism of docetaxel has been less well studied than that of paclitaxel. The use of human liver microsomes has indicated that metabolism involves oxidation of one of the íerí-butyl methyl groups of the C-13 side chain to initially give the alcohol. Further oxidation to the aldehyde and carboxylic acid both of which may cyclize with the carbamate nitrogen to give stereoisomeric hydrox-yoxazolidinones and an oxazolidinedione, respectively. No active metabolites have been identified. The major enzyme involved is CYP3A4. The drug is primarily eliminated in the feces with a terminal half-life of 11 hours.

The adverse effects profile for docetaxel is similar to that of paclitaxel but also includes reversible fluid retention that is dose related. This has been associated with an initial increase in capillary permeability followed by a decrease in lymphatic drainage later in the therapy. Restriction of sodium intake and pretreatment with corticosteroids is usually successful in minimizing this adverse effect. Peripheral neuropathy is seen with docetaxel but occurs less often than with paclitaxel. Fatigue and muscle pain are commonly seen, and fever may occur in up to 30% of patients who are infection free.

IXABEPILONE (AZAEPOTHILONE B, IXEMPRA)

The epothilones are macrocyclic lactones that have a mechanism of action similar to that of the taxanes but offer several advantages (Fig. 10.23).135 Ixabepilone is the semisynthetic amide analog of epothilone B that is isolated from the myxobacterium Sorangium cellulosum. The epothilones showed potent in vitro activity but greatly decreased activity in vivo caused by metabolic instability via hydrolysis of the macrocyclic lactone. Conversion to the lactam increased stability and maintained in vivo activity. Ixabepilone has been recently (2007) approved for the treatment of metasta-tic breast cancer that is resistant to the taxanes. The agent is believed to bind to the same site occupied by the taxanes. Molecular modeling studies have been utilized to identify a common pharmacophore between the taxanes and epothilones.136 Key structural components that assume comparable relative position are indicated in Table 10.2. Like the taxanes, ixabepilone binds to S-tubulin and stabilizes microtubules resulting in cell death. The agent is useful in cancers that have become resistant to the taxanes, because it is not removed by Pgp and is still capable of binding

Epothilone B Ixabepilone

Figure 10.23 • Structures of epithilones.

TABLE 10.2 Functional Groups Yielding Analogous Microtubule Binding in Paclitaxel and Epothilone B

to altered beta tubulin to which the taxanes no longer bind. Increased water solubility also allows the agent to be administered without the need for Cremophor EL, reducing the chance of hypersensitivity reactions. The current indications for the agent are in metastatic breast cancer in combination with capecitabine after the failure of an anthracycline and a taxane and as monotherapy in metastatic breast cancer after failure of an anthracycline, a taxane, and capecitabine. The agent is extensively metabolized in the liver primarily by CYP3A4 to give over 30 different metabolites. Elimination occurs primarily in the feces (65%) with a smaller amount (21%) occurring in the urine. The terminal elimination halflife is 52 hours. Major toxicities associated with the use of ixabepilone have included peripheral neuropathy and myelosuppression occurring as neutropenia. Occurring less frequently are alopecia, nausea, vomiting, mucositis, diarrhea, and muscle pain.

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