Apoptotic Mechanisms of Gallium Nitrate: Basic and Clinical Investigations

Gallium, like aluminum, indium, and thallium, is a naturally occurring group IIIa metal.[1] Commercially available for therapeutic use as the nitrate salt (Ganite), gallium has an oxidation state of +3; its electric charge, ion diameter, coordination number, and electronic configuration are similar to those of iron (Fe⁺⁺⁺).[2] However, at neutral pH, gallium does not transition between divalent and trivalent oxidation states; therefore, unlike iron, it does not participate in biologic redox reactions.[3] Gallium is the second metal with clinical antitumor activity.[4] Interest in the use of gallium and other metals as chemotherapeutic agents was heightened after platinum (a group VIII metal) was discovered to have potent antineoplastic activity.[4,5] Prior to 1970, the use of gallium was confined mainly to the development of radiogallium (⁶⁷Ga) as an imaging agent for the diagnosis of malignancy.[ 4] In 1971, investigators at the National Cancer Institute studied the antitumor activity of group IIIa metal salts-including aluminum, thallium, indium, and gallium-in vitro and in animal tumor models. These studies showed that gallium nitrate had the highest antitumor activity with moderate toxicity.[6,7] Mechanism of Action in Lymphoma The mechanisms involved in the uptake of radiolabeled gallium (⁶⁷Ga) by malignant tumors in vivo have been of interest since its identification as a tumor-localizing agent. In the circulation, gallium is bound to the iron transport protein transferrin, forming transferrin-gallium complexes.[8-12] Approximately one-third of circulating transferrin binds iron, leaving the remaining two-thirds free to bind gallium. Transferrin-iron complexes and transferrin-gallium complexes competitively bind to transferrin receptors and are incorporated into cells via transferrin receptor-mediated endocytosis.[ 13,14] Known and potential mechanisms involved in the cytotoxicity of gallium are illustrated in Figure 1. The primary mechanisms appear to include interference with iron utilization, inhibition of ribonucleotide reductase, and induction of apoptosis. Gallium may also have effects on mitochondria. Other evidence indicates that gallium blocks the secretion of interleukin-6 in a concentration-dependent manner in macrophage-like cells.[15] In vitro, gallium also inhibits tyrosine phosphatase in lymphoid cell lines; however, it is not clear how this relates to its antitumor activity.[16] Findings from an in vitro study using HL60 cells suggested that two mechanisms are involved in the cellular uptake of ⁶⁷Ga: one transferrinreceptor- dependent and the other transferrin-receptor-independent, with the independent mechanism accounting for less than 1% of the uptake.[ 14] Increasing concentrations of transferrin are associated with a progressive and marked increase in the cellular uptake of ⁶⁷Ga. This transferrin- mediated uptake of ⁶⁷Ga can be blocked by a monoclonal antibody to the transferrin receptor. These data suggest that iron and gallium share a common transport mechanism-transferrin and the transferrin receptor. Exposure of HL60 cells to transferrin-gallium complexes results in a decreased cellular uptake of iron and a subsequent arrest in cell growth.[17] The cytotoxicity of gallium is increased by its binding to transferrin; this cytotoxicity can be reversed by transferrin-iron but not by other transferrin forms.[17] Gallium may also impair the intracellular release of iron from transferrin by interfering with processes responsible for intracellular acidification, such as ATP-dependent endosomal acidification.[ 17] The inhibition of cellular proliferation by gallium is due, in part, to the inhibition of ribonucleotide reductase, an iron-containing enzyme responsible for the reduction of ribonucleotides to deoxyribonucleotides, a ratelimiting step in DNA synthesis.[18,19] Iron is required for the activity of ribonucleotide reductase.[20,21] Gallium inhibits ribonucleotide reductase by at least two mechanisms.[22] First, a gallium-induced decrease in cellular iron uptake at the transferrin receptor results in lower amounts of intracellular iron available for the irondependent activity of the M2 (R2) subunit of ribonucleotide reductase.Exposure of cells to transferrin-gallium complexes results in a diminution of the M2 subunit tyrosyl radical ESR (electron spin resonance) signal on ESR spectroscopy.[18] The markedly diminished ESR signal can be fully restored if ferrous ammonium sulfate is added to cell lysates, indicating that gallium interferes with the incorporation of iron into the R2 subunit.[19,23] Furthermore, cells exposed to transferrin-gallium complexes incorporate significantly less ¹⁴C-adenosine into DNA and contain significantly smaller deoxyribonucleotide pools than control cells.[18] Second, gallium appears to inhibit ribonucleotide reductase directly via competitive inhibition of substrate interaction with the enzyme.[22] Following exposure to gallium, the morphologic appearance of CCRFCEM cells displays features characteristic of apoptosis, including chromatin condensation and nuclear fragmentation.[ 24] Apoptotic cell death increased directly with increasing concentrations of gallium. In addition, DNA cleavage into oligonucleosomal fragments was observed after 48 hours when cells were incubated with gallium and analyzed for DNA fragmentation via electrophoresis. The gallium-induced apoptosis could be prevented by the addition of ferrous ammonium sulfate, showing that iron deprivation played a role in triggering the apoptosis. More recent experiments indicate that exposure of cells to gallium leads to the release of cytochrome c from the mitochondria and the activation of caspase-3, leading to apoptosis.[25] Preliminary evidence suggests that gallium may act on the mitochondria. Findings from recent studies that used an RNA differential display technique and Northern blotting to identify possible genetic differences between CCRF-CEM cells sensitive or resistant to growth inhibition by gallium nitrate suggested that gallium-resistant cells displayed an increase in nucleotide sequences sharing homology with the mitochondrial DNA control region.[26] Ongoing studies are providing additional insights into the mechanism of action of gallium relating to the induction of apoptosis. Targeting of Lymphoma Cells In vitro, gallium nitrate inhibits the growth of lymphoma cell lines in a concentration-dependent manner. Targeting of gallium (as transferrin-gallium) to lymphoma cells is most likely related to the high densities of transferrin receptors known to exist on cells in non-Hodgkin's lymphoma.[27] Transferrin receptors are present in increased numbers on proliferating cells.[17,28-31] Furthermore, the density of transferrin receptors is increased in more aggressive lymphomas.[ 32-35] This process may be related to additional growth demands that require more iron or to transferrin receptors conferring a type of selective growth advantage in these cells.[27] Antitumor Activity in Lymphoma Monotherapy at Various Dosing Schedules
In phase I studies of gallium nitrate in patients with advanced cancer, antitumor activity was noted in patients with lymphoma, soft-tissue sarcoma, or small-cell lung cancer.[36,37] Phase I studies of gallium nitrate investigated escalating doses and various dosing schedules, including single brief (15- to 30-minute) IV infusions administered every 2 to 3 weeks, daily brief infusions for 3 days administered every 2 weeks, and continuous IV infusions for 7 consecutive days administered every 3 to 5 weeks.[36- 40] Brief IV infusions and higher doses (> 700 mg/m²) were associated with adverse gastrointestinal effects, hypocalcemia, pulmonary edema, and renal insufficiency. Consequently, gallium nitrate doses recommended for phase II study were 300 mg/m²/d as a 7-day continuous IV infusion every 3 to 5 weeks or 700 mg/m² by brief IV infusion every 2 to 3 weeks.[4] In phase II studies, the continuous 7-day IV infusion of gallium nitrate administered via a peripheral indwelling catheter was shown to have improved tolerability, including a lower and acceptable incidence of renal toxicity.[41-43] Several phase II studies of gallium nitrate demonstrated significant activity in lymphoma and bladder cancer and minor activity in small-cell lung cancer.[37,41,44-47] The Southwest Oncology Group evaluated gallium nitrate in 38 patients with malignant lymphoma, including diffuse and nodular non-Hodgkin's lymphoma and Hodgkin's disease.[47] Patients received gallium nitrate (700 mg/m²) by IV infusion over 30 minutes every 2 weeks. To minimize renal toxicity, patients were given 2,000 mL of fluid IV or orally within 12 hours prior to gallium nitrate infusion, with an additional 500 mL of normal saline IV within 2 hours of gallium nitrate infusion. All patients were to receive a minimum of two cycles. Of the 38 patients, 33 were fully evaluable; the remaining 5 patients died 4 to 13 days after treatment began. The median age of patients was 49 years (range: 18 to 76 years), and the median number of prior regimens was 3 (range: 1 to 7 regimens). Response was defined as at least a 50% reduction in the sum of the diameters of measured lesions, lasting for at least 1 month. Two of 10 patients with diffuse histiocytic, 2 of 6 patients with diffuse poorly differentiated, and 2 of 6 patients with diffuse mixed non-Hodgkin's lymphoma had a partial response. The duration of response lasted from 3 to 11 months. No responses were observed in patients with diffuse well-differentiated (n = 2), diffuse undifferentiated (n = 2), nodular poorly differentiated (n = 3), or nodular histiocytic non-Hodgkin's lymphoma (n = 1). One of seven patients (14%) with Hodgkin's disease had a partial response. Of the seven patients who responded, three had not responded to prior chemotherapy regimens, suggesting that gallium nitrate was not cross-resistant with other chemotherapeutic drugs. Sites of disease response included lymph nodes (n = 7), liver (n = 2), lungs (n = 1), and skin (n = 1). Toxicity was acceptable and included leukopenia (n = 4), thrombocytopenia (n = 4), gastrointestinal effects (n = 9), and renal toxicity (n = 5) in some patients; most toxicities were mild or moderate. Investigators at Memorial Sloan- Kettering Cancer Center evaluated gallium nitrate administered as a continuous IV infusion.[41] The results of this study indicated that a 7-day infusion of gallium nitrate is active and well tolerated in patients with relapsed or refractory malignant lymphoma. This study was performed in two parts: the phase I component was a doseseeking study conducted to determine the appropriate dose, and the phase II component was conducted to evaluate the efficacy and tolerability of the chosen dose. A total of 64 patients participated in the study: 27 patients in the phase I and 37 patients in the phase II part of the study. The median age of patients was 42 years (range: 17 to 70 years), and the median Karnofsky performance status was 60 (range: 50 to 80). All patients had received extensive prior chemotherapy; the mean number of prior cytotoxic drugs used was 9 (range: 4 to 15 drugs), and the mean number of prior regimens was 3 (range: 1 to 8 regimens). The phase I study examined 4 dose levels of gallium nitrate (200, 250, 300, and 400 mg/m²/d) administered as a continuous IV infusion for 7 days. The incidence of gastrointestinal and renal toxicities increased with the higher dose. At the 400-mg/m² dose, 3 of 10 patients developed an increase in serum creatinine levels of 1.5 mg/dL or higher; however, this increase was observed in only 1 of 7 patients receiving the 300-mg/m² dose. Also, at the 400-mg/m² dose, 4 of 10 patients experienced mild nausea that significantly impaired oral fluid intake. Therefore, the 300-mg/m² dose was selected for evaluation in the phase II study. Forty-seven patients were evaluable for response to treatment in the phase II study. Overall, 34% (16/47) of patients had objective responses. Response rates in histologic subtypes of non-Hodgkin's lymphoma ranged from 40% to 50% (Table 1). Overall, treatment was well tolerated. Although renal toxicity was the most serious adverse event (Table 2), it was reversible in all instances; two patients needed short-term hemodialysis before renal function recovered. Three patients with serum creatinine concentrations 4.0 mg/dL or higher had received gentamicin(Drug information on gentamicin) during treatment with gallium nitrate. Based on this observation, the concurrent use of nephrotoxic drugs, including aminoglycosides, during gallium nitrate therapy is not advised. Hypocalcemia was observed within 3 to 4 days of treatment initiation in two-thirds of all patients and lasted several weeks. Most patients were asymptomatic; however, a few patients developed symptoms and required oral or parenteral calcium supplements. Hypomagnesemia also occurred occasionally but less frequently than hypocalcemia. Myelosuppression was somewhat difficult to evaluate because of confounding factors, such as prior treatment and varying degrees of myelophthisis or splenomegaly. However, of 39 patients with normal blood counts at baseline, only 3 developed a leukocyte count less than 2,500/μL and only 1 patient developed a platelet count less than 50,000/μL at any point during the study. Pulmonary complications (n = 9) included pleural effusions and interstitial lung infiltrates. Infectious organisms were identified in three cases, and all but one case resolved after empirical treatment. Mild, transient, asymptomatic hyperchloremic respiratory alkalosis was also observed in some patients. Combination Therapy With Hydroxyurea
In vitro, the cytotoxic effects of gallium nitrate are synergistic with those of hydroxyurea, fludarabine (Fludara), interferon-alpha, and gemcitabine(Drug information on gemcitabine) (Gemzar).[18,48-50] The combination of gallium nitrate and hydroxyurea, both of which inhibit ribonucleotide reductase, is synergistic in vitro against both lymphoid and myeloid leukemic cell lines.[18,48] Thus, a clinical trial was undertaken to evaluate the efficacy and toxicity of gallium nitrate plus hydroxyurea in patients with refractory non-Hodgkin's lymphoma.[43] Fourteen patients with advanced lowor intermediate-grade lymphoma were treated with one of the following four doses for 7 days every 3 to 4 weeks (at least three patients were treated at each level): (1) gallium nitrate (200 mg/m2/d) by continuous IV infusion plus oral hydroxyurea (500 mg/m²/d); (2) gallium nitrate (250 mg/m²/d) plus hydroxyurea (1,000 mg/d); (3) gallium nitrate (300 mg/m²/d) plus hydroxyurea (1,000 mg/d); or (4) gallium nitrate (350 mg/m²/d) plus hydroxyurea (1,000 mg/d). The median age of the patients was 64 years (range: 53 to 89 years). All patients had been heavily pretreated. The patients completed a median of 2 (range: 1 to 6) treatment cycles (one cycle = 7 days). Tumor regression was observed in 10 of 14 patients (1 complete response, 1 near-complete response, 4 partial responses, and 4 minor responses). Excluding patients with minor responses, the overall response rate was 43% (6/14 patients). The median duration of response was 7 weeks (range: 3 to 38 weeks). Responses were not confined to a particular histologic subtype; cytotoxic activity was observed in both low- and intermediate-grade lymphomas. The response to treatment can be rapid; a dramatic shrinkage in an abdominal nodal mass was observed in one patient after just one treatment cycle. Although it was hoped that ⁶⁷Ga scanning would predict treatment responsiveness, no correlation was identified between tumor localization of ⁶⁷Ga and tumor response to gallium nitrate. However, two patients with negative ⁶⁷Ga scans did not respond to gallium nitrate. Overall, toxicities were mild, and minimal myelosuppression occurred. As expected, the most common toxicities were hypocalcemia and diarrhea. The most serious toxicities were anemia and reversible nephrotoxicity. Of four patients with nephrotoxicity, one patient had received prior treatment with cisplatin(Drug information on cisplatin) and one patient had a long history of diabetes mellitus that may have caused occult diabetic nephropathy. Another patient was unable to maintain adequate fluid intake because of anorexia. Transient, decreased visual acuity was observed in two patients. General Toxicity
In general, the toxicity of gallium nitrate does not overlap with that of other drugs commonly used for the treatment of malignant lymphoma. In particular, myelosuppression is minimal. Mild to moderate anemia has been observed; however, its direct relationship to gallium nitrate is not entirely clear because all patients treated with gallium nitrate have received myelosuppressive agents previously. No evidence of cumulative nephrotoxicity was observed in patients who received the drug for more than 14 months with adequate hydration.[ 41] Nephrotoxicity can be ameliorated or prevented with adequate hydration during treatment and by avoiding concomitant use with other nephrotoxic agents. Transient, mild to moderate hypophosphatemia may also occur and may require oral phosphorus supplements. Optic neuritis has occurred rarely in patients receiving gallium nitrate.[ 42,45,51] However, when the drug is administered at the recommended dose and frequency, its incidence is similar to that observed with other chemotherapeutic agents, such as cisplatin, carboplatin(Drug information on carboplatin) (Paraplatin), paclitaxel(Drug information on paclitaxel), and etoposide(Drug information on etoposide). Conclusion Gallium nitrate is a promising agent in the treatment of non-Hodgkin's lymphoma. Its mechanism of action involves drug delivery via transferrin and the transferrin receptor. Galliumtransferrin complexes target lymphoma cells because they express high numbers of transferrin receptors on their surfaces. Following cellular uptake of gallium, ribonucleotide reductase is inhibited indirectly via cellular iron depletion and also directly via competitive inhibition of substrate interaction with the enzyme. During a single-agent phase II study in patients with relapsed lymphoma, response rates of 40% to 50% in various histologic subtypes of non- Hodgkin's lymphoma were noted with continuous-infusion gallium nitrate administered over 7 days. Continuousinfusion gallium nitrate is well tolerated and is not associated with significant myelosuppression. The response rates to gallium nitrate in non-Hodgkin's lymphoma compare favorably with other single agents used in the treatment of patients with relapsed or refractory disease, including bleomycin(Drug information on bleomycin),[52] cyclophosphamide(Drug information on cyclophosphamide) (Cytoxan, Neosar),[53] doxorubicin(Drug information on doxorubicin),[ 54] and vincristine.[55] A multicenter US phase II trial is currently under way to study further the effects of gallium nitrate in patients with refractory low- or intermediategrade non-Hodgkin's lymphoma. This study is evaluating gallium nitrate (200 to 300 mg/m²/d) for 7 days every 3 weeks by continuous IV infusion using a portable infusion pump. Future studies may include evaluating the efficacy and toxicity of gallium nitrate in combination with fludarabine, rituximab(Drug information on rituximab) (Rituxan), and gemcitabine; gallium nitrate may serve as a replacement for platinum in some salvage regimens. Because of its apparent lack of cross-resistance with other drugs and its nonoverlapping toxicity profile, gallium nitrate is well suited for use in combination chemotherapy regimens. Ongoing investigations are studying other possible mechanisms of action of gallium nitrate, including the identification of additional molecular targets and the possibility of predicting treatment response through the use of complementary DNA microarrays. Further knowledge of the mechanisms of action of this drug may help to determine its optimal use in patients with lymphoma.