Temozolomide and Treatment of Malignant Glioma

By Jeanette Hamilton,2014-06-26 21:30
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Temozolomide and Treatment of Malignant Glioma ...

    Temozolomide and Treatment of Malignant Glioma

Malignant gliomas (glioblastoma multiforme and anaplastic astrocytoma) occur more

    frequently than other types of primary CNS tumors, having a combined incidence of 5

    8/100,000 population. Even with aggressive treatment using surgery, radiation, and

    chemotherapy, median reported survival is less than 1 year (1). Temozolomide, a new

    drug, has shown promise in treating malignant gliomas and other difficult-totreat tumors.

    Temozolomide represents a new class of secondgeneration imidazotetrazine prodrugs

    that undergo spontaneous conversion under physiological conditions to the active

    alkylating agent MTIC.3 Thus, temozolomide does not require hepatic metabolism for

    activation (2).

    Interest in temozolomide as an antitumor agent derives from its broad-spectrum

    antitumor activity in tumor models in mice (3). In vitro, temozolomide has demonstrated

    scheduledependent antitumor activity against a variety of malignancies, including glioma,

    metastatic melanoma, and other difficult-totreat cancers (35). In preclinical studies,

    temozolomide demonstrated distribution to all tissues, including penetration into the CNS;

    relatively low toxicity compared with its parent compound, mitozolomide; and antitumor

    activity against a broad range of tumor types, including glioma, melanoma,

    mesothelioma, sarcoma, lymphoma, leukemia, and carcinoma of the colon and ovary

    (3 8). Its demonstrated ability to cross the bloodbrain barrier is of special interest with respect to its activity in CNS tumors (9). Temozolomide was absorbed rapidly, exhibited

    100% p.o. bioavailability within 12 h of administration,

    and demonstrated antineoplastic activity in recurrent high-grade glioma, melanoma, and

    mycosis fungoides (10 13).

    Results of these trials showed that when temozolomide is administered p.o. once daily

    for 5 days in a 4-week cycle, it is well tolerated, producing mild-to-moderate toxicity that

    is both predictable and easily managed. The results also confirmed the ability of

    temozolomide to penetrate the CNS and indicated that temozolomide has considerable

    potential in treating gliomas and improving the QOL of patients with glioma (1214).

    Additional Phase 1 studies have confirmed these results and have extended these

    observations to pediatric patients (15, 16). Temozolomide was synthesized at Aston

    University in the early 1980s as one of a series of novel imidazotetrazinones (17).

    These agents were structurally unique because they contained three adjacent nitrogen

    atoms that conferred unique physicochemical properties and much greater antitumor

    activity than the previously synthesized bicyclic triazenes, which contained only two

    adjacent nitrogen atoms (17). The most potent antitumor compound of this class of

    compounds, mitozolomide, showed potent antitumor activity against a large panel of

    murine tumors (18). Mitozolomide is a prodrug that spontaneously decomposes to a

    highly reactive DNA-cross-linking metabolite without any need for metabolic activation

    (19 23). Temozolomide, a 3-methyl derivative of mitozolomide, was less toxic than mitozolomide and exhibited comparable antitumor activity against various murine tumors


Mechanism of Action

    The methylation of DNA seems to be the principal mechanism responsible for the

    cytotoxicity of temozolomide to malignant cells. The spontaneous conversion of

    temozolomide to the reactive methylating agent MTIC is initiated by the effect of water at

    the highly electropositive C4 position of temozolomide. This activity opens the ring,

    releases CO2, and generates MTIC . The initial proposal was that this effect of water

    was catalyzed in the close environment of the major groove of DNA (26, 27), but

confirming this mechanism has been difficult, and it is known that temozolomide

    converts readily to MTIC in free solution in the absence of DNA (2). MTIC degrades to

    the methyldiazonium cation, which transfers the methyl group to DNA and to the final

    degradation product, AIC, which is excreted via the kidneys (28, 29). The

    methyldiazonium cation can also react with RNA and with soluble and cellular protein


    However, the methylation of RNA and the methylation or carbamoylation of protein do

    not appear to have any known significant role in the antitumor activity of temozolomide.

    Additional studies are required to clarify the role of these targets in the biochemical

    mechanism of action of temozolomide. The spontaneous conversion of temozolomide to

    MTIC is dependent on pH. Comparison of the half-life of temozolomide in phosphate

    buffer [(pH 7.4) t1/2 5 1.83 h; Refs. 28, 29] with the mean plasma half-life observed in

    patients after i.v. and p.o. dosing (t1/2 5 1.81 h; Refs. 10, 29) indicates that the conversion of temozolomide to MTIC is a chemically controlled reaction with little or no

    enzymatic component. The nonenzymatic conversion of temozolomide to MTIC may

    contribute to its highly reproducible PK in comparison with that of other alkylating agents

    such as DTIC and procarbazine, which must undergo metabolic conversion in the liver

    and are, thus, subject to interpatient variation in rates of conversion (27, 29). Among the

    lesions produced in DNA after treatment of cells with temozolomide, the most common is

    methylation at the N7 position of guanine, followed by methylation at the O3 position of

    adenine and the O6 position of guanine (29). Although both the N7-methylguanine and

    O3-methyladenine adducts probably contribute to the antitumor activity of temozolomide

    in some if not all sensitive cells, their role is controversial (30 32). The O6-MG adduct

    (which accounts for 5% of the total adducts formed by temozolomide; Ref. 29) probably

    plays a critical role in the antitumor activity of the agent. This is supported by the

    correlation between the sensitivity of tumor cell lines to temozolomide and the activity of


    DNA repair protein O6-alkylguanine alkyltransferase, which specifically removes alkyl

    groups at the O6 position of guanine. Cell lines that have low levels of AGT are sensitive

    to the cytotoxicity of temozolomide, whereas cell lines that have high levels of this repair

    protein are much more resistant to it (33 35). This correlation has also been observed in human glioblastoma xenograft models (4, 5, 8). The preferential alkylation of guanine

    and adenine and the correlation of sensitivity to the drug with the ability to repair the O6-

    alkylguanine lesion also have been seen with triazine, DTIC, and the nitrosourea

    alkylating agents BCNU and CCNU (3537). The cytotoxic mechanism of temozolomide appears to be related to the failure of the DNA MMR system to find a complementary

    base for methylated guanine. This system involves the formation of a complex of

    proteins that recognize, bind to, and remove methylated guanine (3840). The proposed

    hypothesis is that when this repair process is targeted to the DNA strand opposite the

    O6-MG, it cannot find a correct partner, thus resulting in long-lived nicks in the DNA (41).


    nicks accumulate and persist into the subsequent cell cycle, where they ultimately inhibit

    initiation of replication in the

    daughter cells, blocking the cell cycle at the G2-M boundary (41 44). In both murine (42)

    and human (45) leukemia cells, sensitivity to temozolomide correlates with increased

    fragmentation of DNA and apoptotic cell death. In addition to causing cell death, there is

    evidence from preclinical studies that DNA adducts formed by temozolomide and the

    subsequent alteration of specific genes and their cognate protein products may reduce

    the metastatic potential of tumor cells by altering the immunogenicity of the tumor cells

    (4648). It has also been postulated that temozolomide-induced DNA damage and

subsequent cellcycle arrest may reduce the metastatic properties of some tumor cells

    (49 51).

Mechanisms of Resistance

AGT. The two primary mechanisms of resistance for temozolomide and other alkylating

    agents are the enzyme AGT

    (52, 53) and a deficiency in the MMR pathway. Of these two mechanisms, AGT plays a

    primary role in resistance to temozolomide by removing the alkyl groups from the O6

    position of guanine, in effect reversing the cytotoxic lesion of temozolomide (54). The

    sensitivity of tumor cell lines to temozolomide and the alkylating agents BCNU and DTIC

    can be correlated with AGT levels (37, 45, 5558). Furthermore, retrovirusmediated

    transfer of human AGT gene to cells that are devoid of endogenous AGT activity confers

    a high level of resistance on temozolomide and other methylating and chloroethylating

    agents (59).

    MMR Pathway. Although AGT is clearly important in the resistance of cells to temozolomide, some cell lines that

    express low levels of AGT are nevertheless resistant, which indicates that other

    mechanisms for resistance may be involved (60, 61). A deficiency in the MMR pathway

    resulting from mutations in any one or more of the five or six protein complexes that

    recognize and repair DNA can render cells tolerant to methylation and to the cytotoxic

    effects of temozolomide. This deficiency in the MMR pathway results in a failure to

    recognize and repair the O6-MG adducts produced by temozolomide and other

    methylating agents (33, 62, 63). DNA replication continues past the O6-MG adducts

    without cell cycle arrest or apoptosis. Resistance in tumor cells that are deficient in MMR

    is unrelated to the level of AGT and is, therefore, unaffected by AGT inhibitors.

PARP. Another possible mechanism of resistance for temozolomide is the base excision

    repair pathway. Studies have

    shown that treatment of human tumor cells with temozolomide induced an increase in

    the activity of PARP, which is believed to be involved in nucleotide excision repair (64,

    65), and the inhibition of PARP has been reported to enhance the cytotoxicity of

    methylating agents (6668). Several studies with inhibitors of PARP and with cell lines

    deficient in either MMR or excision repair have indicated a role of the repair of N7-

    methylguanine and O3-methyladenine adducts in the resistance to the antitumor activity

    of temozolomide and other alkylating agents (30, 33, 66, 67). However, the importance

    of these adducts in the antitumor activity of the drug may be secondary to that of the O6-

    MG adduct, except in those tumors that are deficient in base excision repair (31, 32, 69).

Primary Brain Tumors.

    O’Reilly et al. (12) treated 28 patients with primary brain tumors. The initial dosage of 150 mg/m2/day of temozolomide was given p.o. once daily for 5 days; this dosage was

    increased to 200 mg/m2/day once daily for

    5 days and repeated at 28-day intervals if the patient did not experience significant

    myelosuppression at day 22 of the first cycle. Treatment was well tolerated. Grade 3

    leukopenia occurred in only 3 (5%) of 57 courses, and grade 3 thrombocytopenia was

    reported in only 4 (7%) of 57 courses. Of the 10 evaluable patients with recurrent

    astrocytoma after radiation therapy, 5 showed major improvement on CT and complete

    resolution of clinical signs and symptoms that persisted for 36 months; 3 other patients

    showed a slight reduction or no change on CT, although their neurological condition improved (12). Major improvement on CT also was reported for two of nine

    evaluable patients treated with two courses of temozolomide before cranial irradiation for newly diagnosed high-grade astrocytomas; two others showed slight improvement. Three additional evaluable patients with primary brain tumors, including one with recurrent medulloblastoma after chemotherapy and radiation therapy, experienced major improvement on CT that was maintained for 6 months (12).This study was extended to 75 patients48 with recurrent disease and 27 with new diagnoses (90). Improvements

    on CT were seen in 12 (25%) of the patients with recurrent disease and in eight (30%) of the patients with new diagnoses. Twenty-two % of patients with recurrences and 43% of those with newly diagnosed tumors survived to 1 year. Although there was a clear improvement in the QOL in responders who used the 5-day schedule, no conclusions could be reached about the effect on extended survival benefit in comparison with the overall survival data established by historical results (90). This study confirmed, however, the activity of temozolomide against gliomas in patients who have failed to respond to intensive radiation therapy.

    Similar results were reported in a multicenter Phase 2 study conducted by the CRC that evaluated temozolomide in patients diagnosed with anaplastic astrocytoma, glioblastoma multiforme (grade 4), and unclassified high-grade astrocytoma (grades 34;

    13). In this study, objective responses, measured by improvement in neurological status, were seen in 11 (11%) of 103 patien ts who received temozolomide; 5 of these patients had improvement on CT or magnetic resonance imaging scans (13). An additional 47% of the patients in the study had stable disease. The median survival of all patients with measurable response was 5.8 months, and 22% of the patients had no neurological or radiological evidence of progressive disease at 6 months. The results of this study further confirmed the activity of temozolomide in patients with recurrent and progressive high-grade glioma. Recently, three open-label, multi-institutional studies have evaluated the use of temozolomide in 525 patients with malignant glioma. These studies represent the largest evaluation of a single agent in patients with recurrent malignant gliomas that were rigorously controlled with strict prospectively defined criteria for assessment of tumor response, central review of histology, and validated instruments to assess health-related QOL. The first two trials evaluated the safety, efficacy, and healthrelated QOL effects of temozolomide in patients with glioblastoma multiforme at first relapse and were as follows: (a) a pivotal multicenter Phase 2 study that compared

    the PFS at 6 months and the safety in patients treated with temozolomide with those in patients treated with procarbazine (91); and (b) a second supportive trial in patients with glioblastoma multiforme

    to further examine the efficacy and health-related QOL aspects of temozolomide. In the pivotal Phase 2 study, 225 patients were randomized to receive either temozolomide (n

    5 112) or procarbazine (n 5 113; Ref. 91).

Malignant Metastatic Melanoma. The efficacy of temozolomide was evaluated in a

    study of patients with advanced

    metastatic melanoma, including patients with brain metastases (11). Fifty-six patients were given p.o. temozolomide 150 mg/m2 once daily for 5 days. If grade 2 or greater myelosuppression did not occur by day 22, subsequent 5-day courses (200 mg/m2/day) were administered at 28-day intervals. Among the 56 patients (49 with evaluable lesions), CRs occurred in 3, all with lung metastases only, and PRs occurred in 9, which yielded a response rate of 21%. Stable disease was observed in an additional eight patients. The mean duration of response was 6 months (range, 2.518 months), and the median

    survival times were 14.5 months in responding patients and 4.5 months in

    nonresponders. Leukopenia was the major toxicity; five cases of grade 4 leukopenia, two cases of grade 4 thrombocytopenia, and

    no other grade 4 toxic effects occurred in the 55 evaluable patients over 217 courses of treatment. These results confirmed the safety and efficacy of temozolomide in malignant metastatic melanoma that were observed in the Phase 1 study (11). A second Phase 2 trial in advanced malignant melanoma evaluated the relationship between pretreatment AGT levels in biopsies of cutaneous tumors and involved lymph nodes and clinical response to treatment with temozolomide (94). Among the 50 evaluable patients, there were 3 CRs and 4 PRs for an overall response rate of 14%. Stable disease was observed in an additional six patients. Lymphocytopenia was the major toxicity, however, with only eight and nine cases of grade 3 or higher neutropenia and thrombocytopenia, respectively. Analysis of the pretreatment AGT levels and clinical response to temozolomide in 33 patients revealed that pretreatment levels of AGT are not predictive of response to temozolomide in melanoma (94).

Overcoming Resistance

    Combining two or more drugs that have different cytotoxic mechanisms or are subject to different mechanisms of resistance can produce additive or synergistic effects. The favorable safety profile of temozolomide allows it to be coadministered with various agents. The antitumor activity of temozolomide is dependent on the level of AGT within the tumor cell. Results from preclinical studies indicate that inhibition of AGT potentiates the activity of temozolomide in several human tumor cell lines (5). Several studies have investigated methods to deplete AGT levels further and increase the antitumor effect of temozolomide in combination therapy with BCNU and different dosing schedules.

Combination with Cisplatin. Preclinical evidence indicates that cisplatin enhances the

    antitumor activity of temozolomide (96). On the basis of these data and complementary toxicity profiles, a Phase 1 trial of the combination was conducted in 15 patients with advanced cancer (97). In this study, cohorts of three patients received temozolomide daily for 5 days together with cisplatin on day 1 for 4 weeks at the following temozolomide (mg/m2/day) and cisplatin (mg/m2) dose levels: 100/75; 120/75; 200/75; and 200/100. The DLT observed at the highest temozolomide/cisplatin dose level was myelosuppression (neutropenia and thrombocytopenia) and vomiting. The MTDs established in this trial were 200 mg/m2/day for temozolomide and 75 mg/m2 for cisplatin. This combination did not alter the PK or the MTD of temozolomide. The principal nonhematological toxicities consisted of nausea, vomiting, and hearing loss. PR was achieved in 2 of the 14 evaluable patients, one with untreated non-small cell lung cancer and the other with

    squamous cell carcinoma.

Combination with BCNU. In a Phase 1 study evaluating the combination of BCNU (75

    mg/m2) given before or

    after a 5-day course of temozolomide, no differences between the regimens were seen in the PK of temozolomide or the

    toxicity of the drugs at the doses used (98). One patient with glioblastoma had a PR that has been maintained for 1 year,

    and two other patients (one with osteosarcoma and one with uterine carcinosarcoma) have had minor responses. This

    study is continuing so that the MTD for this combination can be established (98).

Combination with IFN-a-2b. Both temozolomide and IFN-a-2b have demonstrated

    antitumor activity against melanoma. IFN-a-2b is approved in the United States for postsurgical adjuvant treatment of melanoma with high-risk metastases. It is approved in some European countries for use as monotherapy for the palliative treatment of melanoma. In a Phase 1 study to determine the MTD and DLT, patients with histologically confirmed, surgically incurable metastatic melanoma were treated with 5-day courses of p.o. temozolomide in dosages of 100 or

    200 mg/m2/day with continuous s.c. injections of IFN-a-2b three times a week at escalating doses starting at 5 mU/m2 (99). In the cohort treated with 1000 mg/m2 of temozolomide and 5 mU/m2 of IFN-a-2b, two patients developed dose-limiting thrombocytopenia, and one patient developed grade 4 neutropenia. When higher doses of IFN-a-2b (7.5 and 10.0 mU/m2) were combined with 150 mg/m2 of temozolomide, grade 4 hematological toxicity was observed in one of six and one of three patients, respectively. No DLT occurred in patients treated with 750 mg/m2 of temozolomide plus 5 mU/m2 of IFN-a-2b. The MTD was determined as temozolomide 150 mg/m2 and IFNa- 2b 7.5 mU/m2 (99). Antitumor responses were seen in 3 of 12 patients, and stable disease in 4 of 12 patients. These results indicate that this combination when administered at the MTD is well tolerated, and the antitumor activity observed provides the

    basis for additional studies.

Continuous Dosing Schedule. Because AGT levels may recover within the 24-h

    interval between individual temozolomide doses in each 5-day cycle, dosing more frequently than once a day for 5 days may improve the response to treatment. A Phase 1 study of 24 patients with recurrent tumors, 17 of which were malignant gliomas (81), examined continuous dosing of temozolomide over a 6- to 7-week period with dosages ranging from 50 to 100 mg/m2/day. This schedule produced a higher cumulative dose of drug than the indicated 5-day schedule and increased the AUC by a factor of 2.1 without producing any hematological DLT. No major toxicity was observed at the dosage level of 75 mg/m2/day (81). Objective responses were reported in patients with high-grade glioma and melanoma, and the overall response rate for the prolonged schedule was 33%. Seven (41%) of 17 glioma patients demonstrated tumor responses.

    Six of the 17 glioma patients maintained stable disease. (Reference: Henry S. Friedman,2 Tracy Kerby, and Hilary Calvert. Clinical Cancer Research 2000; 6: 25852597)

Abbreviations used: MTIC, 5-(3-methyltriazen-1-yl)imidazole-4-

    carboximide; DTIC, 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide, or dacarbazine; CNS, central nervous system; CRC, Cancer Research

    Campaign; BCNU, carmustine; AIC, 5-aminoimidazole-4-

    carboxamide; O6-MG, O6-methylguanine; AGT, alkylguanine

    alkyltransferase; CCNU, lomustine; PARP, poly(ADP)-ribose polymerase;

    O6-BG, O6-benzylguanine; AUC, area under the concentration-time

    curve; MTD, maximum tolerated dose/dosage; CT, computerized tomography; PK, pharmacokinetic(s); CR, complete response; PR, partial

    response; QOL, quality of life. FDA, United States Food and Drug

    Administration; MMR, mismatch repair; DLT, dose-limiting toxicity;

    PFS, progression-free survival; CI, confidence interval.

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