Multiple Myeloma Research Center

RITUXAN™ IN THE MANAGEMENT OF MULTIPLE MYELOMA

1.0 OBJECTIVES

1.1 Primary Objectives

1.1.1 To evaluate the role of Rituxan in inducing apoptosis of malignant plasma cells.

1.1.2 To evaluate the role of Rituxan in improving the response rate to melphalan, and prednisone.

1.1.3 To determine if Rituxan therapy decreases residual disease and consequently prolongs plateau phase Multiple myeloma patients.

1.2 Secondary Objectives

1. Safety: To evaluate the toxicity of Rituxan in Multiple Myeloma patients.

2.0 BACKGROUND AND RATIONALE

2.1 Multiple Myeloma

2.1.1 Disease and Induction Therapy:

Multiple myeloma is a fatal neoplasm of Plasma cells with a median patient survival of about 30 months^1,2. The disease is regarded as responsive to alkylating agents, corticosteroids and irradiation although few patients achieve true and complete remissions. The disease is not considered curable with standard therapy. Thirty percent of patients with multiple myeloma show chemotherapy resistance to initial treatment, and all of those who initially respond will ultimately relapse after a median of 15 months³. Treatment with more aggressive Multi-agent chemotherapy has not significantly improved results over conventional therapy with an alkylating agent and corticosteroids.^4,5 Patients with relapsed disease after one or more first-time therapies are a particularly difficult group to treat. Thirty to fifty percent of relapsed patients will not respond to subsequent alkylator-based chemotherapy. Occasional remissions are seen in these patients, but the duration is brief and overall survival is poor.

2.1.2 Melphalan and Prednisone:

Multiple myeloma (MM) is a neoplastic disease characterized by the expansion of monoclonal plasma cells that seed throughout bone marrow, causing lytic bone lesions, and introduce monoclonal immunoglobulins, hence the term monoclonal gammopathy.

The incidence of monoclonal gammopathies in the healthy population is very high. It is rare in persons under 40 years of age, with incidence increasing to 7% to 8% in persons over 65 years of age.^6,7 The majority of MM patients had a prior asymptomatic gammopathy⁸, however, most persons with monoclonal gammopathy of undetermined significance (MGUS) will not develop MM. It is, therefore, probable that there are two distinct types of monoclonal gammopathies, benign and malignant.

There is currently no test that distinguishes between benign and malignant monoclonal gammopathy. Clinically, patients with MGUS can be divided into two groups, with most requiring only routine follow-up, and the remainder requiring immediate chemotherapeutic intervention owing to evidence of overt disease progression. Helpful tests, such as measures of plasma cells in S phase,^9-11 b₂-microglobulin serum levels,^12,13 and IL-6 serum levels,^14-17 may contribute to determining a diagnosis; however, disease progression is clearly defined only after precipitous increase in monoclonal immunoglobulins or appearance of lytic bone lesions.

The choice of appropriate therapy regimen remains controversial. The efficacy of melphalan and prednisone (MP), first introduced for MM more than 25 years ago, has been compared with several alternative regimens. Other regimens are equivalent to MP, but should be employed only in special cases such as renal failure, or to prevent stem cell damage.¹⁸

2.1.3 Multiple Myeloma and biologic therapy for maintenance:

While many induction regimens have been well studied and some are quite effective, a long-standing problem in myeloma treatment has been what to do to maintain remissions. A variety of studies have compared chemotherapy maintenance to unmaintained remission and have failed to demonstrate any added benefit from maintenance chemotherapy as long as patients were retreated at the time they relapsed. Unmaintained remissions in stage III and Bence-Jones myeloma patients are short (6-12 months). Several studies have examined the role of alpha-interferon as maintenance therapy. An Italian study reported an improved remission duration and survival as compared to no maintenance therapy.¹⁹ Other randomized trials, including a large Southwest Oncology Group study, confirmed a prolonged remission but failed to demonstrate any survival advantage.²⁰

Biologic Modifier therapy may improve immune reconstitution and reduce the risk of hematologic malignancy relapse in the setting of minimal residual disease by augmenting cytotoxic effect mechanism directed at residual malignant cells. However this model has not resulted in an improvement of survival.

2.1.4 The Role of anti CD-20 antibodies.

Multiple myeloma is a disease characterized by the accumulation of neoplastic plasma cells in the bone marrow. Despite the predominance of myeloma cells in the bone marrow, mitotic figures are rarely observed, and kinetic studies of myeloma cells indicate that they have a low labeling index during the early and the plateau phases of the disease^21,22.

The BM is widely affected from the earliest recognizable stage of the disease, while there are very few circulating plasma cells except during the terminal phase. Thus, a proliferating compartment which is at an earlier stage of B-cell differentiation than plasma cells has been postulated to exist and searches for the clonogenic precursors which feed the non-proliferative myeloma-cell compartment have been undertaken²³.

Somatic mutations, which result in amino acid substitutes, are observed frequently in the Ig variable region genes in multiple myeloma, but there is no intraclonal variation. This fact suggests that the target cell of malignant transformation in multiple myeloma is a B-lineage cell, which already has undergone antigenic selection. This B-lineage cell probably corresponds to a pre-plasma cell or a plasma cell rather than a memory B cell. Tumor cells which share an identical third-complementary-determining -region (CDR3) sequence with the myeloma cells can be detected from the various fractions representing different stages of B-cell differentiation, such as CD34^+, CD20⁺ CD10⁺, CD20⁺ CD21⁺, CD20⁺ CD19⁺ cells from the peripheral blood. Thus the tumor cell in multiple myeloma are composed of immunophenotypically heterogeneous subpopulations at various stages of differentiation, similar the normal B-lineage cells²⁴.

The use of Rituxan after achieving a plateau phase, could theoretically prevent the proliferative B-cell compartment from feeding the non-proliferative myeloma cell compartment, and thus relapse.

2.1.5 Rituxan and enhancing the effects of chemotherapy:

Sections 2.1.5.1, and 2.1.5.2, will describe the rationale for using Rituxan in enhancing the initial response to chemotherapy. In summary, the role of bcl-2 in the resistance of multiple myeloma patients to chemotherapy has been described, and hence the possible role for Rituxan in enhancing the effect of MP. Also, Rituxan induces apoptosis in lymphoid cells where the drug could work synergisticaly with chemotherapy.

2.1.5.1 Rituxan monoclonal antibody sensitizes a b-cell lymphoma cell line to cell killing by cytotoxic drugs

More than 50% of patients with aggressive B lymphomas and the majority of patients with low-grade lymphomas are not cured by current therapeutic strategies. The lymphomas express the B cell antigen CD20 on the cell surface and this antigen serves as target for antibody-directed therapies. Clinical studies with encouraging results have been underway with the use of a chimeric anti-CD20 antibody (IDEC-C2B8), consisting of human IgGI-6 constant regions and variable regions from the murine monoclonal anti-CD20 antibody IDEC-2B8. This study investigated the potential anti-tumor therapeutic value of combination treatment with anti-Rituxan and cytotoxic drugs. The in vitro study examined the sensitizing effect of Rituxan antibody on the DHL-4 B lymphoma line to various cytotoxic agents. Cytotoxicity was determined by the MTT assay. Surface and cytoplasmic proteins were determined by flow cytometry. Pretreatment of DHL-4 with Rituxan resulted in inhibition of cell proliferation and cell death and a fraction of the cells underwent apoptosis. While the DHL-4 tumor cells were relatively resistant to several cytotoxic drugs, pretreatment with Rituxan rendered the cells sensitive to TNF-a, ricin, diphtheria toxin (DTX), adriamycin and cisplatin but not to VP16. Chemosensitization of DHL-4 tumor cells was not due to down modulation of either the MDR-1 or bcl-2 gene products. However, treatment of DHL-4 with Rituxan inhibited TNF-a secretion. These findings demonstrate that Rituxan antibody potentiates the sensitivity of DHL-4 tumor cells to several cytotoxic agents. Further, the findings suggest that combination treatments with Rituxan antibody and drugs may be of clinical benefit in the treatment of patients with resistant aggressive B lymphomas.^25,26

2.1.5.2 Overexpression of Bcl-2 family proteins and chemoresistance:

Bcl-2 overexpression can render cells markedly more resistant to the cytotoxic effects of essentially all currently available anticancer drugs.²⁸ While cytotoxic drugs damage tumor cells by a variety of mechanisms (e.g., DNA cleavage, DNA alkylation, microtubule aggregation, nucleotide precursor inhibition), ultimately the damage induced must somehow be translated into signals for apoptosis. In this regard, overexpression of Bcl-2 or its antiapoptotic homolog Bcl-X_L has been shown to prevent proteolytic processing and activation of caspases in response to chemotherapy-induced damage.^29-32 The predicted consequence of the cytoprotection provided by Bcl-2 is that damaged malignant cells remain viable and may be able to repair drug-induced injury and resume proliferation. Overexpression of Bcl-2 or Bcl-X_L has been shown to create a cellular environment that is permissive for accumulation of mutations and aberrant chromosomal segregation.^33-34 This presumably occurs because cells with genetic defects and chromosomal damage would otherwise be eliminated by apoptosis. Thus, damaged tumor cells that overexpress Bcl-2 or Bcl-X_L may survive and acquire additional secondary mutations that can lead to the emergence of other mechanisms of drug resistance (e.g., overexpression of MDR-1; amplification of dihydrofolate reductase, loss of glucocorticoid receptors) and tumor progression via oncogene activation or tumor suppressor gene inactivation. Bcl-2 has been experimentally shown to render cells more resistant to killing by dexamethasone, cytosine arabinoside (Ara-C), methotrexate, cyclophosphamide, Adriamycin, daunomycin, 5-fluoro-deoxyuridine, 2-chlorodeoxyadenosine, fludarabine, taxol, etoposide (VP-16), camptothecin, nitrogen mustards, mitoxantrone, cisplatin, vincristine, and some retinoids. The extent to which gene transfer-mediated elevations in Bcl-2 protein levels provide protection from the cytotoxic effects of these drugs varies, depending on the particular drug and the cell line, but it can be as much as 4 or more logs (10,000 x) or as little as half a log (5 X).2.1.5.2 Overexpression of Bcl-2 family proteins and chemoresistance:

Bcl-2 overexpression can render cells markedly more resistant to the cytotoxic effects of essentially all currently available anticancer drugs.²⁸ While cytotoxic drugs damage tumor cells by a variety of mechanisms (e.g., DNA cleavage, DNA alkylation, microtubule aggregation, nucleotide precursor inhibition), ultimately the damage induced must somehow be translated into signals for apoptosis. In this regard, overexpression of Bcl-2 or its antiapoptotic homolog Bcl-X_L has been shown to prevent proteolytic processing and activation of caspases in response to chemotherapy-induced damage.^29-32 The predicted consequence of the cytoprotection provided by Bcl-2 is that damaged malignant cells remain viable and may be able to repair drug-induced injury and resume proliferation. Overexpression of Bcl-2 or Bcl-X_L has been shown to create a cellular environment that is permissive for accumulation of mutations and aberrant chromosomal segregation.^33-34 This presumably occurs because cells with genetic defects and chromosomal damage would otherwise be eliminated by apoptosis. Thus, damaged tumor cells that overexpress Bcl-2 or Bcl-X_L may survive and acquire additional secondary mutations that can lead to the emergence of other mechanisms of drug resistance (e.g., overexpression of MDR-1; amplification of dihydrofolate reductase, loss of glucocorticoid receptors) and tumor progression via oncogene activation or tumor suppressor gene inactivation. Bcl-2 has been experimentally shown to render cells more resistant to killing by dexamethasone, cytosine arabinoside (Ara-C), methotrexate, cyclophosphamide, Adriamycin, daunomycin, 5-fluoro-deoxyuridine, 2-chlorodeoxyadenosine, fludarabine, taxol, etoposide (VP-16), camptothecin, nitrogen mustards, mitoxantrone, cisplatin, vincristine, and some retinoids. The extent to which gene transfer-mediated elevations in Bcl-2 protein levels provide protection from the cytotoxic effects of these drugs varies, depending on the particular drug and the cell line, but it can be as much as 4 or more logs (10,000 x) or as little as half a log (5 X).

3.0 TREATMENT PLAN

3.1 Patients will be treated with Rituxan, intravenously every week for a total of 4 weeks. This will be repeated every 6 months, for a total of 6 cycles. If uncontrollable recurrent infectious process occurs, the drug will be discontinued after 4 cycles.

3.2 Melphalan, will be administered in the standard fashion . This will be repeated every 4-6 weeks as allowed by counts for a minimum of 9 cycles, and two cycles after best response.

3.3 Prednisone will be given 1-4. This will be repeated every 4-6 weeks with the melphalan, for a minimum of 9 cycles, and two cycles after best response.

3.4 The combination of chemotherapy will start after the first 4 treatments of Rituxan have been administered, i.e., day 35 of therapy.

4.0 PATIENT SELECTION

4.1 Inclusion Criteria:


Each patient must meet these criteria to be considered for enrollment.

4.1.1 Newly diagnosed Patients with multiple myeloma

4.1.2 Patients with pancytopenia related to multiple myeloma will be eligible for treatment i.e. patients with > 50% plasma cells in the BM, or have splenomegaly, or have plasma cell leukemia.

4.1.3 Patients must have an Eastern Cooperative Group (ECOG) Performance Status of 0-2.

4.1.4 Organ function permitted:

4.1.4.1 If bone marrow is occupied by <50% plasma cells: WBC > 2500/µl or Absolute Neutrophil Count > 1000/µl, however if the platelet count is > 75,000/m l, a neutrophil count of > 500/m l will be allowed.

4.1.4.2 Platelets > 45,000/µl. Patients with Thrombocytopenia related to ITP, B12 or folate deficiency will be eligible.

4.1.4.3 Bilirubin < 2x institutional upper limits of normal

4.1.4.4 Liver enzymes (ALT or AST) < 2 x normal (unless >1/3 of liver is involved by tumor, in which case ALT or AST must be < 5 x normal)

4.1.4.5 Creatinine £ 2.0 mg/dl

4.2 Ineligibility Criteria:

4.2.1 The patient will be evaluated within three weeks prior to entry. Any one of the following conditions eliminates a patient from participating in this protocol.

4.2.2 Concurrent involvement in any other clinical trial using an investigational drug or device, or participation in any investigational drug study within 4 weeks prior to study registration. Exceptions may be made by the study investigators for participation in certain studies (e.g. antimicrobial studies) on a case by case basis.

4.2.3 Severe infection requiring intravenous antibiotic treatment.

4.2.4 Severe hepatic disease (e.g. SGOT or SGPT, bilirubin, alkaline phosphatase more than 2.5 times the normal laboratory range).

4.2.5 Patients with previous bone marrow transplantation as part of their previous treatment regimen.

4.2.6 Patients with a life expectancy of less than 3 months will be ineligible.

4.2.7 Pregnant or lactating patients will be ineligible. Men or women of reproductive potential may not participate unless they have agreed to use an effective contraceptive method.

4.2.8 No prior malignancy is allowed, except for adequately treated basal cell or squamous cell skin cancer, in-situ cervical cancer, or other cancer from which the patient has been disease-free for at least 5 years.

4.2.9 Patients with solitary bone or solitary Extramedullary plasmacytoma as the only evidence of Plasma cell dyscrasia.

 5.0 STUDY PARAMETERS

5.1 Allowable Concomitant Therapy

• Erythropoietin for anemia, GM-CSF for severe, symptomatic neutropenia.
• Aredia and immunoglobulin therapy will be allowed at any stage of therapy.
• Plasmapheresis at the initiation of therapy to treat renal failure will be allowed.

5.2 Treatment and Dose Modifications

5.2.1Melphalan:
The dose of melphalan will be reduced by 25% if the neutrophil count persists at < 1000/m l after a two-week delay. This dose reduction will not apply if the neutrophil count at the initiation of therapy was < 1,500/m l, and the platelet count at the initiation of the chemotherapy cycle is > 100,000/m l. Melphalan will be given every 4-6 weeks if the neutrophil count is > 1,000/m l and/or platelet count > 75,000/m l, or at least 75% of baseline WBC and platelets. Therapy can be delayed up to 4 weeks for counts to recover. 5.2.1Melphalan:
The dose of melphalan will be reduced by 25% if the neutrophil count persists at < 1000/m l after a two-week delay. This dose reduction will not apply if the neutrophil count at the initiation of therapy was < 1,500/m l, and the platelet count at the initiation of the chemotherapy cycle is > 100,000/m l. Melphalan will be given every 4-6 weeks if the neutrophil count is > 1,000/m l and/or platelet count > 75,000/m l, or at least 75% of baseline WBC and platelets. Therapy can be delayed up to 4 weeks for counts to recover.

5.2.2Prednisone:
The dose of prednisone will be reduced by up to 50% if uncontrollable diabetes mellitus results from the therapy.5.2.2Prednisone:
The dose of prednisone will be reduced by up to 50% if uncontrollable diabetes mellitus results from the therapy.

5.2.3 Rituxan:

No dose modifications are necessary. However, in-patients who experience a gram positive infectious process requiring IV antibiotics following the use of Rituxan, therapy with IVIG will be instituted at a loading dose of 1.0gm/kg, then 0.4gm/kgm every 4-6 weeks or oral Bactrim DS TIW at the discretion of the PI, and the infectious disease faculty. Patients who experience at least two episodes of infections in areas traditionally caused by gram positive organisms, will also, be prophylaxed in the same manner as listed above. If the prophylaxis procedure does not result in a change in the infectious trend, Rituxan will be discontinued after only 4 cycles.

6.0 Drug Information:

6.1 MELPHALAN

6.1.1 GENERAL

The alkylating agents are antitumor drugs that act through the covalent bonding of alkyl groups (one or more saturated carbon atoms) to cellular molecules. Historically, the alkylating agents have played an important role in the development of cancer chemotherapy. The nitrogen mustards, mechlorethamine (HN₂, nitrogen mustard) and tris (b -chlorethyl) amine (HN₃), were the first non hormonal agents to show significant antitumor activity in humans.^35-37 The clinical trials of nitrogen mustards in patients with lymphomas evolved from clinical observations of the effects of sulfur mustard gas used in World War I. This compound was found to produce lymphoid aplasia in addition to the expected irritation of the lungs and mucous membranes and was evaluated as an antitumor agent.³⁸ The related, but less reactive, bischloroethylamines (nitrogen mustards) were found to be less toxic and to cause regressions of lymphoid tumors in mice. The first clinical studies produced some dramatic tumor regressions in lymphoma patients, and the antitumor effects were confirmed by an organized multi-institution study.^35-37 The demonstration of the clinical utility of the nitrogen mustards encouraged further efforts to find chemical agents with antitumor activity, leading to the wide variety of antitumor agents in use today. At present, alkylating agents occupy a central position in cancer chemotherapy, both in conventional combination regimens and in high-dose protocols with bone marrow transplantation. Because of their linear dose-response curve in cell culture experiments, these drugs [particularly cyclophosphamide, melphalan, and carmustine (BCNU)] have become the primary tools used in allogeneic transplantation protocols for acute leukemia and in autologous transplantation for lymphomas and breast cancer.

6.1.2 CHEMISTRY

Mechanisms of Alkylating Reactions

Traditionally, alkylating reactions have been classified as S_N1 (nucleophilic substitution, first-order) or S_N2 (nucleophilic substitution, second-order). In the S_N1 reaction there is an initial formation of a highly reactive intermediate, followed by the rapid reaction of this intermediate with a nucleophile to produce the alkylated product. In this reaction, the rate-limiting step is the initial formation of the reactive intermediate. Thus the reaction exhibits first-order kinetics with regard to the concentration of the original alkylating agent, and the rate is essentially independent of the concentration of the substrate, hence the designation S_N1.

The S_N2 alkylation reaction represents a bimolecular nucleophilic displacement. The rate of this reaction is dependent on the concentration of both the alkylating agent and the target nucleophile. Therefore, the reaction follows second-order kinetics. The terms S_N1 and S_N2 are defined kinetically but normally are used in reference to the mechanism of action.

Melphalan is transported into several cell types by at least two active transport systems, which also carry leucine and other neutral amino acids across the cell membrane.^39-41 High levels of leucine in the medium will protect cells from the cytotoxic effects of melphalan by competing with melphalan for transport into the target cells.⁴² Because appreciable levels of leucine are present in plasma and extracellular fluid, this competition may have pharmacologic significance. Although murine leukemia cells contain at least two transport systems for melphalan and L-leucine, one of these systems is lacking in murine granulocyte precursors (CFU-Cs).⁴³ This system, missing in CFU-Cs but present in leukemia cells, is identified by its capacity to transport the amino acid analogue 2-amino-bicyclo [2,2,] heptane-2-carboxylic acid (BCH). These unexpected findings have prompted a search for cytotoxic analogues of BCH that might be taken up by tumor cells but not by normal granulocyte precursors. In contrast to the active transport systems for mechlorethamine and melphalan, the highly lipid-soluble nitrosoureas BCNU and CCNU enter cells by passive diffusion.⁴⁴

6.1.3 TUMOR RESISTANCE

The emergence of alkylating agent-resistant tumor cells is a major problem that limits the clinical effectiveness of these drugs. One mechanism for drug resistance is that of decreased drug entry into the cell. Numerous studies have shown that L5178Y lymphoblast cells resistant to mechlorethamine may have decreased uptake of the drug.^45-49 Murine L1210 leukemia cells that are resistant to melphalan have a specific mutation in the lower-affinity, higher-velocity L-transport system, which results in a decreased affinity of the carrier protein for leucine and melphalan.

6.1.4 Melphalan Clinical Pharmacology

The clinical pharmacology of melphalan has been examined by several groups. Alberts and colleagues⁵⁰ studied the pharmacokinetics of melphalan in patients who received 0.6 mg of the drug per kilogram intravenously. the peak levels of melphalan, as measured by HPLC, were 4.5 to 13 mM (1.4 to 4.1 mg/ml), and the mean half-life (t1/2b) of the drug in the plasma was 1.8 hours. The 24-hour urinary excretion of the parent drug averaged 13% of the administered dose. Inactive mono- and dihydroxy metabolites appear in plasma within minutes of drug administration.

Other studies have shown that there is low and variable systemic availability of the drug after oral dosing.^51-52 Food slows its absorption. After oral administration of melphalan, 0.6 mg/kg, much lower peak levels of drug of about 1 mM (0.3 mg/ml) were seen. The time to achieve peak plasma levels varies considerably and occurs as late as 6 hours after dosing. The low bioavailability was due to incomplete absorption of the drug from the gastrointestinal tract, because 20% to 50% of an oral dose could be recovered in the feces.⁵² No drug or drug products were found in the feces after intravenous administration. In addition to its unpredictable bioavailability, oral melphalan AUC is reduced one-third by concomitant cimetidine administration.⁵³

After conventional oral doses of 0.15 to 0.25 mg/kg,⁵⁴ peak plasma levels of 0.16 to 0.625 mM (50 to 190 ng/ml) occurred 0.7 to 2.3 hours after drug administration. The same plasma levels were found after the initial dose of drug or after the second dose in a 5-day schedule, indicating that no accumulation of plasma levels of the drug occurs with daily administration. In this study the magnitude and time of peak plasma levels appear to be more consistent than was seen with the higher doses reported by Alberts et al.⁵⁰ Cornwell and colleagues⁵⁵ pointed out that for patients receiving intravenous melphalan, the incidence of severe myelosuppression is increased in patients with a blood urea nitrogren (BUN) greater than 30 mg/dl, suggesting that these patients have altered drug excretion. The half-life of melphalan in plasma is significantly prolonged in anephric dogs.⁵⁶ Thus, as an approximation, intravenous doses of this agent should be reduced by 50% in patients with an elevated BUN.

6.2 GLUCOCORTICOIDS

The physiologic effects of the glucocorticoids are many and protean. Insight into the role of glucocorticoid hormones in maintaining homeostasis was gained early with recognition of the pathologic conditions of glucocorticoid deficiency and excess. In 1855, Addison⁵⁷ provided the classic description of the wasting disease associated with the destruction of the suprarenal glands and shortly thereafter, Brown-Sequard⁵⁸ demonstrated the essential role of the adrenal gland in sustaining life in dogs. The syndrome of glucocorticoid excess was characterized in 1932 by Cushing.⁵⁹

It is now known that adrenal function is regulated in a circadian fashion by the pituitary gland. In 1926, Foster and Smith⁶⁰ established that hypophysectomy resulted in adrenal atrophy, which, in 1932, was shown by several groups ^61-63 to be reversed by treatment with extracts of the pituitary. The agent in these extracts, adrenocorticotropic hormone (ACTH) was purified by 1943,^64-65 chemically and structurally identified by 1956,⁶⁶ and synthesized by 1963.⁶⁷

Under the negative-feedback mechanisms governing the release of ACTH, the glucocorticoids are synthesized in the fascinculata zone of the adrenal cortex from cholesterol upon binding of ACTH to the steroid synthesizing cells. The rate-limiting step is generally considered to be the conversion of cholesterol to 5-pregnenolone by a mechanism mediated by cyclic adenosine monophosphate (AMP) and calcium ions, possibly requiring the synthesis of a short-lived protein.⁶⁸ The capability of the adrenal gland for upregulating steroidogenesis is rapid (taking place within minutes) and great; the daily production of cortisol can be increased up to 10-fold in periods of stress.

Once synthesized in the adrenal gland, cortisol enters the circulation and reaches the periphery mostly bound to plasma proteins including albumin, orosomucoid (a-acidic glycoprotein), and CBG or transcortin.⁶⁹ These proteins differ in their binding affinities for the corticoids (CBG having the highest and albumin the lowest) and in their plasma concentrations. At physiologic concentrations of cortisol in human plasma (approximately 10mg/dl), 76% of cortisol is bound to CBG, 13.5% is bound to albumin, 10.5% is unbound, and negligible amounts are bound to orosomucoid. Certain changes in endocrine status can alter the binding capacity of CBG and therefore the total blood levels of the steroids (although the amount of free hormone does not change drastically because of normal feedback control of pituitary ACTH secretion). Estrogens stimulate CBG synthesis in the liver, and as a result, total cortisol levels are markedly elevated during pregnancy. However, hypercortisolism is not evident clinically because the transcortin-bound steroid is not biologically active. ^70,71 Corticosteroids also affect CBG levels; adrenalectomy causes a decrease in plasma CBG that is reversible with cortisol replacement.⁷² Finally, thyroidectomy decreases and thyroxine administration increases CBG activity in rats.⁷³

Although it was thought initially that steroid binding to plasma proteins served a transport function, it now seems that protein binding provides a storage or buffer function. Thus large quantities of hormone circulate in a biologically inert reservoir, from which the active agent is readily available by dissociation. An additional advantage of protein binding is protection from degradation and excretion, which decreases the metabolic clearance rate. Furthermore, protein binding decreases the accumulation of highly lipophilic steroids in adipose tissue, where they would be relatively inaccessible to the bloodstream and ultimately to the target tissues.

Free cortisol, upon dissociation from the plasma binders, exerts its effects by entering cells and binding to an intracellular receptor. Although glucocorticoid-responsive tissues may respond in a highly tissue-specific fashion, these actions all seem to be mediated by the glucorcorticoid receptor. The specificity of the response is probably accounted for by differences in specific gene activation within each tissue. The physiologic actions of glucocorticoids include the following: (1) metabolic effects - a permissive role in epinephrine and glucagon-stimulated lipolysis, gluconeogenesis, and glycogenolysis; (2) catabolic effects - increased protein degradation and decreased protein synthesis in muscle, adipose, lymphoid, and connective tissue to provide increased amino acids for hepatic protein synthesis and gluconeogenesis; (3) cardiac effects - increased contractility, cardiac output, and sensitivity to catecholamines; and (4) musculoskeletal effects - increased capacity for muscular work.⁷⁴ Another major glucocorticoid effect is the anti-inflammatory action that provides the basis for much of the therapeutic usefulness of the glucocorticoids.

Glucocorticoids in pharmacologic concentrations produce marked lymphocytopenia and thymic atrophy in experimental animals.⁷⁵ Thus these steroids were used initially with great enthusiasm when it was discovered that they also could kill some leukemic lymphoblasts in humans.⁷⁶ Glucocorticoid receptors can be demonstrated in normal peripheral blood lymphocytes as well as in partially purified subpopulations of lymphocytes as well as in partially purified subpopulations of lymphocytes and monocytes ^77,78 and in leukemia cells.^79-81 These receptor proteins are similar to the glucocorticoid receptors more extensively characterized in liver and rat thymocytes.

Many protocols utilize vastly suprapharamcologic concentrations of glucorcorticoid such as 1 g of prednisolone per square meter of body surface area. Plasma concentrations with these doses approach 1000 times those required to saturate receptor and induce killing of sensitive cells in vitro. However, on a once-daily dosage, 20 or more half-lives may elapse before the next dose of drug is administered, so there is a rationale for using such large doses. Also, it is possible that at least some effects of glucocorticoids may not require receptor. There are no data, however, to suggest that such massive doses have any benefit over conventional regimens.

Glucocorticoid effects at normal physiologic levels are short-lived, with a rapid metabolic clearance of cortisol (plasma half-life of about 60 minutes). Other steroids are cleared from the plasma at rates of about 2000 liters/day, which correspond to a plasma half-life of 20 minutes.⁷⁴ The half-lives of several synthetic steroids in dog plasma are as follows: prednisone, 33 minutes; dexamethasone, 60 minutes; prednisolone, 60 to 71 minutes; 6a -methylprednisolone, 81 minutes; and triamcinolone, 116 minutes. ⁸² Cortisol is extensively metabolized to inactive glucuronides, sulfates, and other forms in a number of tissues such that only 1% to 2% of the unaltered steroid actually ends up in the urine. ⁸³ By far the most important organ for metabolism is the liver. The liver also plays a crucial role in activating certain synthetic 11-keto glucocorticoids such as cortisone and prednisone, which must be converted to 11-hydroxymetabolites to exert activity.

Thus patients with compromised hepatic function may not respond to these agents because of decreased ability to convert to the 11b -OH steroid by the 11-keto reductase system. Hyperthyroidism markedly shifts the equilibrium of this reaction in favor of the inactive oxidized forms, whereas hypothyroidism does the reverse.⁷³ Anorexia nervosa and other malnourished states favor the 11b -OH steroids in a manner similar to the effect of hypothyroidism.⁸⁴ Drugs that induce hepatic enzymes may increase metabolism of glucocorticoids. These include barbiturates, phenytoin, and rifampin.⁸⁵

At least seven enzymatic reactions that occur predominantly in the liver contribute to the metabolism of cortisol; of these, tetrahydroreduction of a ring alone provides nearly half the total urinary metabolites. ^86,87 Synthetic glucocorticoids, having a ketone group at C-11 (cortisone, prednisone, prednisolone), must be reduced to their hydroxy analogues to become active. One example is prednisone, which is rapidly converted to prednisolone.⁸⁵ In children, a considerable proportion of the urinary steroids are excreated unconjugated or free, whereas in adults, most of the steroids are conjugated to form glucuronides and, to a lesser degree, sulfates. These modifications either decrease or abolish glucocorticoid activity. Inactive metabolites are excreted by the kidney with small amounts of unmetabolized drug. Only negligible amounts are excreted in the bile, with no enterohepatic circulation.⁸⁵ Because the metabolic clearance rate of cortisol is quite similar among most people, the replacement dose of hydrocortisone (12 to 15 mg/m²/day) is fairly uniform. There is systemic absorption from the skin and mucous membranes; therefore, topical preparations containing triamcinolone or other potent fluorinated glucocorticoids analogues also may have cushinoid consequences.

The dosages of drugs used can be described ad physiologic or that which is normally secreted by the adrenal gland (equivalent to 20 mg daily of hydrocortisone). Pharmacological doses are greater than physiologic doses. Approximate equivalent oral dosages are cortisone, 25 mg; hydrocortisone, 20 mg; prednisolone, 5 mg; prednisone, 5 mg; methylprednisone, 4 mg; triamcinolone, 4mg; dexamethasone, 0.75 mg; and betamethasone, 0.6 mg.⁸⁵

The duration of hypothalamic-pituitary axis suppression with a single oral dose of glucocorticoids is 1.25 to 1.5 days with 250 mg of hydrocortisone, 250 mg of cortisone, 50 mg of prednisolone, 40 mg of methylprednisolone, and 50 mg of prednisone.⁸⁵ Suppression is 2.25 days with 40 mg of triamcinolone, 2.75 days with 5 mg of dexamethasone, and 3.25 days with 6 mg of betamethasone.⁸⁵

Deleterious effects produced by pharmacological amounts of glucocorticoids are listed in Table 5-3. These include immunosuppression with concomitant nosocomial infections, Cushing syndrome, diabetes mellitus, poor wound healing, psychosis, posterior subcapsular cataracts, osteoporosis, and alterations in mentation including euphoria and psychosis.

Utilization of glucocorticoids with no mineralocorticoid activity may reduce toxicity. Synthetic steroids with very little mineralocorticoid activity include dexamethasone, methylprednisone, prednisolone, prednisone, and triamcinolone.

  6.3 Rituxan^TM

6.3.1 Rituxan

DESCRIPTION

The Rituxan (Rituximab) antibody is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD2O antigen found on the surface of normal and malignant B lymphocytes. The antibody is an IgG₁ kappa immunoglobulin containing murine light- and heavy-chain variable region sequences and human constant region sequences. Rituximab is composed of two heavy chains of 451 amino acids and two light chains of 213 amino acids (based on cDNA analysis) and has an approximate molecular weight of 145 kD. Rituximab has a binding affinity for the CD2O antigen of approximately 8.0 nM.

The chimeric anti-CD2O antibody is produced by mammalian cell (Chinese Hamster ovary) suspension culture in a nutrient medium containing the antibiotic gentamicin. Gentamicin is not detectable in the final product. The anti-CD2O antibody is purified by affinity and ion exchange chromatography. The purification process includes specific viral inactivation and removal procedures.

Rituxan is a sterile, clear, colorless, preservative-free liquid concentrate for intravenous (IV) administration. Rituxan is supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for intravenous administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5.

6.3.2 CLINICAL PHARMACOLOGY

6.3.2.1 General

Rituximab binds specifically to the antigen CD2O (human B-lymphocyte-restricted differentiation antigen, Bp3 5), a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B and mature B lymphocytes. ^{88, 89} The antigen is also expressed on> 90% of B-cell non-Hodgkin’s lymphomas (NHL)⁹⁰ but is not found on hematopoietic stem cells, pro-B cells, normal plasma cells or other normal tissues.⁹¹ CD2O regulates an early step(s) in the activation process for cell cycle initiation and differentiation,⁹¹ and possibly functions as a calcium ion channel.⁹² CD2O is not shed from the cell surface and does not internalize upon antibody binding.⁹³ Free CD2O antigen is not found in the circulation.⁸⁹

 

 6.3.2.2 Pre-clinical Pharmacology and Toxicology

Mechanism of Action: The Fab domain of Rituximab binds to the CD2O antigen on B-lymphocytes and the Fc domain recruits immune effector functions to mediate B-cell lysis in vitro. Possible mechanisms of cell lysis include complement-dependent cytotoxicity (CDC)⁹⁴ and antibody-dependent cellular cytotoxicity (ADCC). The antibody has been shown to induce apoptosis in the DHL-4 human B-cell lymphoma line.⁹⁵

Normal Tissue Cross-reactivity: Rituximab binding was observed on lymphoid cells in the thymus, the white pulp of the spleen, and a majority of B-lymphocytes in peripheral blood and lymph nodes. Little or no binding was observed in non-lymphoid tissues examined.

Human Pharmacokinetics/Pharmacodynamics

In patients given single doses at 10, 50, 100, 250 or 500 mg/m² as an IV infusion, serum levels and the half-life of Rituximab were proportional to dose. In 9 patients given 375 mg/m² as an IV infusion for four doses, the mean serum half-life was 59.8 hours (range 11.1 to 104.6 hours) after the first infusion and 174 hours (range 26 to 442 hours) after the fourth infusion. The wide range of half-lives may reflect the variable tumor burden among patients and the changes in CD2O positive (normal and malignant) B-cell populations upon repeated administrations.

Rituximab at a dose of 375 mg/m² was administered as an IV infusion at weekly intervals for four doses to 166 patients. The peak and trough serum levels of Rituximab were inversely correlated with baseline values for the number of circulating CD2O positive B cells and measures of disease burden. Median steady-state serum levels were higher for responders compared to nonresponders; however, no difference was found in the rate of elimination as measured by serum half-life. Serum levels were higher in patients with International Working Formulation (IWF) subtypes B, C, and D as compared to those with subtype A. Rituximab was detectable in the serum of patients three to six months after completion of treatment.

The pharmacokinetic profile of Rituximab when administered as six infusions of 375 mg/m² in combination with six cycles of CHOP chemotherapy was similar to that seen with Rituximab alone.

Administration of Rituxan™ resulted in a rapid and sustained depletion of circulating and tissue-based B cells. Lymph node biopsies performed 14 days after therapy showed a decrease in the percentage of B-cells in seven of eight patients who had received single²⁹ doses of Rituximab ³ 100 mg/in . Among the 166 patients in the pivotal study, circulating B-cells (measured as CD 19+ cells) were depleted within the first three doses with sustained depletion for up to 6 to 9 months post-treatment in 83% of patients. One of the responding patients (1%), failed to show significant depletion of CDl9+ cells after the third infusion of Rituximab as compared to 19% of the nonresponding patients. B-cell recovery began at approximately six months following completion of treatment. Median B-cell levels returned to normal by twelve months following completion of treatment.

There were sustained and statistically significant reductions in both 1gM and IgG serum levels observed from 5 through 11 months following Rituximab administration. However, only 14% of patients had reductions in IgG and/or 1gM serum levels, resulting in values below the normal range.

the full course of therapy. (See DOSAGE and ADMINISTRATION.) Medications for the treatment of hypersensitivity reactions, e.g., epinephrine, antihistamines and corticosteroids should be available for immediate use in the event of a reaction during administration.

Infusions should be discontinued in the event of serious or life-threatening cardiac arrhythmias. Patients who develop clinically significant arrhythmias should undergo cardiac monitoring during and after subsequent infusions of Rituxan™. Patients with preexisting cardiac conditions including arrhythmias and angina have had recurrences of these events during Rituxan™ therapy and should be monitored throughout the infusion and immediate post-infusion period.

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6.3.5.1 Infusion-Related Events: An infusion-related symptom complex consisting of fever and chills/rigors occurred in the majority of patients during the first Rituxan infusion. Other frequent infusion-related symptoms included nausea, urticaria, fatigue, headache, pruritus, bronchospasm, dyspnea, sensation of tongue or throat swelling (angioedema), rhinitis, vomiting, hypotension, flushing, and pain at disease sites. These reactions generally occurred within 30 minutes to 2 hours of beginning the first infusion, and resolved with slowing or interruption of the Rituxan infusion and with supportive care (IV saline, diphenhydramine, and acetaminophen). The incidence of infusion-related events decreased from 80% (7% Grade 3/4) during the first infusion to approximately 40% (5% to 10% Grade 3/4) with subsequent infusions. Mild to moderate hypotension requiring interruption of Rituxan infusion with or without the administration of IV saline occurred in 32 (10%) patients. Isolated occurrences of severe reactions requiring epinephrine have been reported in patients receiving Rituxan for other indications. Angioedema was reported in 41(13%) patients and was serious in one patient. Bronchospasm occurred in 25 (8%) patients; one-quarter of these patients were treated with bronchodilators. A single report of bronchiolitis obliterans was noted. 6.3.5.1 Infusion-Related Events: An infusion-related symptom complex consisting of fever and chills/rigors occurred in the majority of patients during the first Rituxan infusion. Other frequent infusion-related symptoms included nausea, urticaria, fatigue, headache, pruritus, bronchospasm, dyspnea, sensation of tongue or throat swelling (angioedema), rhinitis, vomiting, hypotension, flushing, and pain at disease sites. These reactions generally occurred within 30 minutes to 2 hours of beginning the first infusion, and resolved with slowing or interruption of the Rituxan infusion and with supportive care (IV saline, diphenhydramine, and acetaminophen). The incidence of infusion-related events decreased from 80% (7% Grade 3/4) during the first infusion to approximately 40% (5% to 10% Grade 3/4) with subsequent infusions. Mild to moderate hypotension requiring interruption of Rituxan infusion with or without the administration of IV saline occurred in 32 (10%) patients. Isolated occurrences of severe reactions requiring epinephrine have been reported in patients receiving Rituxan for other indications. Angioedema was reported in 41(13%) patients and was serious in one patient. Bronchospasm occurred in 25 (8%) patients; one-quarter of these patients were treated with bronchodilators. A single report of bronchiolitis obliterans was noted.

6.3.5.2 Immunologic Events: Rituxan™ induced B-cell depletion in 70 to 80% of patients and was associated with decreased serum immunoglobulins in a minority of patients. The incidence of infection does not appear to be increased. During the treatment period, 50 patients in the pivotal trial developed 68 infectious events; 6 (9%) were Grade 3 in severity and none were Grade 4 events. Of the 6 serious infectious events, none were associated with neutropenia. The serious bacterial events included sepsis due to Listeria (n= 1), Staphylococcal bacteremia (n1) and polymicrobial sepsis (n1). In the post-treatment period (30 days to 11 months following the last dose), bacterial infections included sepsis (n1); significant viral infections included herpes simplex infections (n=2) and herpes zoster (n3). Rituxan™ induced B-cell depletion in 70 to 80% of patients and was associated with decreased serum immunoglobulins in a minority of patients. The incidence of infection does not appear to be increased. During the treatment period, 50 patients in the pivotal trial developed 68 infectious events; 6 (9%) were Grade 3 in severity and none were Grade 4 events. Of the 6 serious infectious events, none were associated with neutropenia. The serious bacterial events included sepsis due to Listeria (n= 1), Staphylococcal bacteremia (n1) and polymicrobial sepsis (n1). In the post-treatment period (30 days to 11 months following the last dose), bacterial infections included sepsis (n1); significant viral infections included herpes simplex infections (n=2) and herpes zoster (n3).

6.3.5.3 Retreatment Events: Twenty-one patients have received more than one course of Rituxan™. The percentage of patients reporting any adverse event upon retreatment was similar to the percentage of patients reporting adverse events upon initial exposure. The following adverse events were reported more frequently in retreated subjects: asthenia, throat irritation, flushing, tachycardia, anorexia, leukopenia, thrombocytopenia, anemia, peripheral edema, dizziness, depression, respiratory symptoms, night sweats, and pruritus. Twenty-one patients have received more than one course of Rituxan™. The percentage of patients reporting any adverse event upon retreatment was similar to the percentage of patients reporting adverse events upon initial exposure. The following adverse events were reported more frequently in retreated subjects: asthenia, throat irritation, flushing, tachycardia, anorexia, leukopenia, thrombocytopenia, anemia, peripheral edema, dizziness, depression, respiratory symptoms, night sweats, and pruritus.

6.3.5.4 Hematologic Events: During the treatment period (up to 30 days following last dose)

Severe thrombocytopenia occurred in 1.3% of patients, severe neutropenia occurred in 1.9% of patients, and severe anemia occurred in 1.0% of patients. A single occurrence of transient aplastic anemia (pure red cell aplasia) and two occurrences of hemolytic anemia following Rituxan therapy were reported.

6.3.5.5 Cardiac Events: Four patients developed arrhythmias during Rituxan infusion. One of the four discontinued treatment because of ventricular tachycardia and supraventricular tachycardias. The other three patients experienced trigeminy (1) and irregular pulse (2) and did not require discontinuation of therapy. Angina was reported during infusion and myocardial infarction occurred 4 days post-infusion in one subject with a prior history of myocardial infarction. 6.3.5.5 Cardiac Events: Four patients developed arrhythmias during Rituxan infusion. One of the four discontinued treatment because of ventricular tachycardia and supraventricular tachycardias. The other three patients experienced trigeminy (1) and irregular pulse (2) and did not require discontinuation of therapy. Angina was reported during infusion and myocardial infarction occurred 4 days post-infusion in one subject with a prior history of myocardial infarction.

 

Adverse Events ³ 5% of Patients (N=315)

Severe and life-threatening (Grade 3 and 4) events were reported in 10% (32/315) of patients. The following Grade 3 and 4 adverse events were reported: neutropenia (1.9%), chills (1.6%), leukopenia and thrombocytopenia (1.3% for each), hypotension, anemia, bronchospasin, and urticaria (1.0% for each), headache, abdominal pain, arrhythmia (0.6% for each), and asthenia, hypertension, nausea, vomiting, coagulation disorder, angioedema, arthralgia, pain, rhinitis, increased cough, dyspnea, bronchiolitis obliterans, hypoxia, asthma, pruritus, and rash (one patient each, 0.3%).

The following adverse events occurred in ³ 1.0% but < 5.0% of patients, in order of decreasing incidence: flushing, arthralgia, diarrhea, anemia, cough increase, hypertension, lacrimation disorder, pain, hyperglycemia, back pain, peripheral edema, paresthesia, dyspepsia, chest pain, anorexia, anxiety, malaise, tachycardia, agitation, insomnia, sinusitis, conjuctivitis, abdominal enlargement, postural hypotension, LDH increase, hypocalcemia, hypesthesia, respiratory disorder, tumor pain, pain at injection site, bradycardia, hypertonia, nervousness, bronchitis, and taste perversion.

The proportion of patients reporting any adverse event was similar in patients with bulky disease and those with lesions <10 cm in diameter. However, the incidence of dizziness, neutropenia, thrombocytopenia, myalgia, anemia and chest pain was higher in patients with lesions >10 cm. The incidence of any Grade 3 and 4 event was higher (31% vs. 13%) and the incidence of Grade 3 or 4 neutropenia, anemia, hypotension, and dyspnea was also higher in patients with bulky disease compared with patients with lesions <10 cm.

6.3.5.6 OVERDOSAGE

There has been no experience with overdosage in human clinical trials. Single doses higher than 500 mg/m² have not been tested.

7.0 REFERENCES:

 

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