Apoptotic Mechanisms of Gallium Nitrate: Basic and Clinical Investigations

ABSTRACT: Gallium nitrate inhibits the growth of various lymphoma cell linesin vitro and exhibits antitumor activity in patients with lymphoma.The mechanism(s) of cytotoxicity is (are) only partly understood butappears to involve a two-step process: (1) targeting of gallium to cells,and (2) acting on multiple, specific intracellular processes. Galliumshares certain chemical properties with iron; therefore, it binds avidlyto the iron transport protein transferrin. Transferrin-gallium complexespreferentially target cells that express transferrin receptors on theirsurface. Expression of transferrin receptors is particularly high onlymphoma cells. Cellular uptake of the gallium-transferrin complexleads to inhibition of cellular proliferation primarily via disruption ofiron transport and homeostasis and blockade of ribonucleotidereductase. Recent studies have shown that cellular uptake of galliumleads to activation of caspases and induction of apoptosis. In phase IItrials in patients with relapsed or refractory lymphoma, the antitumoractivity of gallium nitrate is similar to, or better than, that of othercommonly used chemotherapeutic agents. Gallium nitrate is notmyelosuppressive and may be used in patients with neutropenia orthrombocytopenia. A multicenter trial to evaluate the use of galliumnitrate in patients with relapsed non-Hodgkin's lymphoma is currentlyongoing.

Gallium, like aluminum, indium,and thallium, is a naturallyoccurring group IIIametal.[1] Commercially available fortherapeutic use as the nitrate salt(Ganite), gallium has an oxidationstate of +3; its electric charge, ion diameter,coordination number, andelectronic configuration are similar tothose of iron (Fe⁺⁺⁺).[2] However, atneutral pH, gallium does not transitionbetween divalent and trivalent oxidationstates; therefore, unlike iron, itdoes not participate in biologic redoxreactions.[3]Gallium is the second metal withclinical antitumor activity.[4] Interestin the use of gallium and other metalsas chemotherapeutic agents washeightened after platinum (a groupVIII metal) was discovered to havepotent antineoplastic activity.[4,5]Prior to 1970, the use of gallium wasconfined mainly to the developmentof radiogallium (⁶⁷Ga) as an imagingagent for the diagnosis of malignancy.[4] In 1971, investigators at theNational Cancer Institute studied theantitumor activity of group IIIa metalsalts-including aluminum, thallium,indium, and gallium-in vitro and inanimal tumor models. These studiesshowed that gallium nitrate had thehighest antitumor activity with moderatetoxicity.[6,7]Mechanism of Action inLymphomaThe mechanisms involved in theuptake of radiolabeled gallium (⁶⁷Ga)by malignant tumors in vivo havebeen of interest since its identificationas a tumor-localizing agent. In the circulation,gallium is bound to the irontransport protein transferrin, formingtransferrin-gallium complexes.[8-12]Approximately one-third of circulatingtransferrin binds iron, leaving theremaining two-thirds free to bind gallium.Transferrin-iron complexes andtransferrin-gallium complexes competitivelybind to transferrin receptorsand are incorporated into cells viatransferrin receptor-mediated endocytosis.[13,14]Known and potential mechanismsinvolved in the cytotoxicity of galliumare illustrated in Figure 1. The primarymechanisms appear to include interferencewith iron utilization, inhibitionof ribonucleotide reductase, and inductionof apoptosis. Gallium mayalso have effects on mitochondria.Other evidence indicates that galliumblocks the secretion of interleukin-6in a concentration-dependent mannerin macrophage-like cells.[15] In vitro,gallium also inhibits tyrosine phosphatasein lymphoid cell lines; however,it is not clear how this relates toits antitumor activity.[16]

Findings from an in vitro study usingHL60 cells suggested that twomechanisms are involved in the cellularuptake of ⁶⁷Ga: one transferrinreceptor-dependent and the othertransferrin-receptor-independent,with the independent mechanism accountingfor less than 1% of the uptake.[14] Increasing concentrations oftransferrin are associated with a progressiveand marked increase in thecellular uptake of ⁶⁷Ga. This transferrin-mediated uptake of ⁶⁷Ga can beblocked by a monoclonal antibody tothe transferrin receptor.These data suggest that iron andgallium share a common transportmechanism-transferrin and the transferrinreceptor. Exposure of HL60cells to transferrin-gallium complexesresults in a decreased cellular uptakeof iron and a subsequent arrest in cellgrowth.[17] The cytotoxicity of galliumis increased by its binding totransferrin; this cytotoxicity can bereversed by transferrin-iron but not byother transferrin forms.[17] Galliummay also impair the intracellular releaseof iron from transferrin by interferingwith processes responsible forintracellular acidification, such asATP-dependent endosomal acidification.[17]The inhibition of cellular proliferationby gallium is due, in part, to theinhibition of ribonucleotide reductase,an iron-containing enzyme responsiblefor the reduction of ribonucleotidesto deoxyribonucleotides, a ratelimitingstep in DNA synthesis.[18,19]Iron is required for the activity ofribonucleotide reductase.[20,21]Gallium inhibits ribonucleotide reductaseby at least two mechanisms.[22]First, a gallium-induced decrease incellular iron uptake at the transferrinreceptor results in lower amounts ofintracellular iron available for the irondependentactivity of the M2 (R2) subunitof ribonucleotide reductase.Exposureof cells to transferrin-galliumcomplexes results in a diminution ofthe M2 subunit tyrosyl radical ESR(electron spin resonance) signal onESR spectroscopy.[18] The markedlydiminished ESR signal can be fullyrestored if ferrous ammonium sulfateis added to cell lysates, indicating thatgallium interferes with the incorporationof iron into the R2 subunit.[19,23]Furthermore, cells exposed totransferrin-gallium complexes incorporatesignificantly less ¹⁴C-adenosineinto DNA and contain significantlysmaller deoxyribonucleotide poolsthan control cells.[18] Second, galliumappears to inhibit ribonucleotide reductasedirectly via competitive inhibitionof substrate interaction with theenzyme.[22]Following exposure to gallium, themorphologic appearance of CCRFCEMcells displays features characteristicof apoptosis, including chromatincondensation and nuclear fragmentation.[24] Apoptotic cell deathincreased directly with increasingconcentrations of gallium. In addition,DNA cleavage into oligonucleosomalfragments was observed after 48 hourswhen cells were incubated withgallium and analyzed for DNA fragmentationvia electrophoresis. Thegallium-induced apoptosis could beprevented by the addition of ferrousammonium sulfate, showing that irondeprivation played a role in triggeringthe apoptosis.More recent experiments indicatethat exposure of cells to gallium leadsto the release of cytochrome c fromthe mitochondria and the activation ofcaspase-3, leading to apoptosis.[25]Preliminary evidence suggests thatgallium may act on the mitochondria.Findings from recent studies that usedan RNA differential display techniqueand Northern blotting to identifypossible genetic differences betweenCCRF-CEM cells sensitive or resistantto growth inhibition by gallium nitratesuggested that gallium-resistant cellsdisplayed an increase in nucleotidesequences sharing homology withthe mitochondrial DNA controlregion.[26] Ongoing studies areproviding additional insights into themechanism of action of galliumrelating to the induction of apoptosis.Targeting of Lymphoma CellsIn vitro, gallium nitrate inhibits thegrowth of lymphoma cell lines in aconcentration-dependent manner. Targetingof gallium (as transferrin-gallium)to lymphoma cells is most likelyrelated to the high densities of transferrinreceptors known to exist on cellsin non-Hodgkin's lymphoma.[27]Transferrin receptors are present inincreased numbers on proliferatingcells.[17,28-31] Furthermore, thedensity of transferrin receptors isincreased in more aggressive lymphomas.[32-35] This process may berelated to additional growth demandsthat require more iron or to transferrinreceptors conferring a type of selectivegrowth advantage in these cells.[27]Antitumor Activity inLymphomaMonotherapy at Various DosingSchedules
In phase I studies of gallium nitratein patients with advanced cancer, antitumoractivity was noted in patientswith lymphoma, soft-tissue sarcoma,or small-cell lung cancer.[36,37] PhaseI studies of gallium nitrate investigatedescalating doses and various dosingschedules, including single brief (15-to 30-minute) IV infusions administeredevery 2 to 3 weeks, daily briefinfusions for 3 days administeredevery 2 weeks, and continuous IVinfusions for 7 consecutive daysadministered every 3 to 5 weeks.[36-40] Brief IV infusions and higherdoses (> 700 mg/m²) were associatedwith adverse gastrointestinal effects,hypocalcemia, pulmonary edema, andrenal insufficiency.Consequently, gallium nitrate dosesrecommended for phase II study were300 mg/m²/d as a 7-day continuous IVinfusion every 3 to 5 weeks or700 mg/m² by brief IV infusion every2 to 3 weeks.[4] In phase II studies,the continuous 7-day IV infusion ofgallium nitrate administered via a peripheralindwelling catheter wasshown to have improved tolerability,including a lower and acceptable incidenceof renal toxicity.[41-43]

Several phase II studies of galliumnitrate demonstrated significant activityin lymphoma and bladder cancerand minor activity in small-cell lungcancer.[37,41,44-47] The SouthwestOncology Group evaluated galliumnitrate in 38 patients with malignantlymphoma, including diffuse andnodular non-Hodgkin's lymphomaand Hodgkin's disease.[47] Patientsreceived gallium nitrate (700 mg/m²)by IV infusion over 30 minutes every2 weeks. To minimize renal toxicity,patients were given 2,000 mL of fluidIV or orally within 12 hours prior togallium nitrate infusion, with an additional500 mL of normal saline IVwithin 2 hours of gallium nitrate infusion.All patients were to receive a minimumof two cycles. Of the 38 patients,33 were fully evaluable; the remaining5 patients died 4 to 13 days after treatmentbegan. The median age of patientswas 49 years (range: 18 to 76 years),and the median number of prior regimenswas 3 (range: 1 to 7 regimens).Response was defined as at least a50% reduction in the sum of the diametersof measured lesions, lastingfor at least 1 month. Two of 10 patientswith diffuse histiocytic, 2 of 6patients with diffuse poorly differentiated,and 2 of 6 patients with diffusemixed non-Hodgkin's lymphoma hada partial response. The duration of responselasted from 3 to 11 months. Noresponses were observed in patientswith diffuse well-differentiated (n = 2),diffuse undifferentiated (n = 2), nodularpoorly differentiated (n = 3), ornodular histiocytic non-Hodgkin'slymphoma (n = 1). One of seven patients(14%) with Hodgkin's diseasehad a partial response. Of the sevenpatients who responded, three had notresponded to prior chemotherapy regimens,suggesting that gallium nitratewas not cross-resistant with other chemotherapeuticdrugs.Sites of disease response includedlymph nodes (n = 7), liver (n = 2),lungs (n = 1), and skin (n = 1). Toxicitywas 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 ormoderate.Investigators at Memorial Sloan-Kettering Cancer Center evaluatedgallium nitrate administered as a continuousIV infusion.[41] The results ofthis study indicated that a 7-day infusionof gallium nitrate is active andwell tolerated in patients with relapsedor refractory malignant lymphoma.This study was performed in two parts:the phase I component was a doseseekingstudy conducted to determinethe appropriate dose, and the phase IIcomponent was conducted to evaluatethe efficacy and tolerability of thechosen dose.

A total of 64 patients participatedin the study: 27 patients in the phase Iand 37 patients in the phase II part ofthe study. The median age of patientswas 42 years (range: 17 to 70 years),and the median Karnofsky performancestatus was 60 (range: 50 to 80).All patients had received extensiveprior chemotherapy; the mean numberof prior cytotoxic drugs used was9 (range: 4 to 15 drugs), and the meannumber of prior regimens was 3(range: 1 to 8 regimens).The phase I study examined 4 doselevels of gallium nitrate (200, 250,300, and 400 mg/m²/d) administeredas a continuous IV infusion for 7 days.The incidence of gastrointestinaland renal toxicities increased withthe higher dose. At the 400-mg/m²dose, 3 of 10 patients developed anincrease in serum creatinine levels of1.5 mg/dL or higher; however, thisincrease was observed in only 1 of7 patients receiving the 300-mg/m²dose. Also, at the 400-mg/m² dose, 4 of10 patients experienced mild nausea thatsignificantly impaired oral fluid intake.Therefore, the 300-mg/m² dosewas selected for evaluation in thephase II study. Forty-seven patientswere evaluable for response to treatmentin the phase II study. Overall,34% (16/47) of patients had objectiveresponses. Response rates in histologicsubtypes of non-Hodgkin's lymphomaranged from 40% to 50% (Table 1).Overall, treatment was well tolerated.Although renal toxicity was themost serious adverse event (Table 2),it was reversible in all instances; twopatients needed short-term hemodialysisbefore renal function recovered.Three patients with serum creatinineconcentrations 4.0 mg/dL or higherhad received gentamicin during treatmentwith gallium nitrate. Basedon this observation, the concurrent useof nephrotoxic drugs, includingaminoglycosides, during gallium nitratetherapy is not advised. Hypocalcemiawas observed within 3 to 4 daysof treatment initiation in two-thirds ofall patients and lasted several weeks.Most patients were asymptomatic;however, a few patients developedsymptoms and required oral orparenteral calcium supplements.Hypomagnesemia also occurredoccasionally but less frequently thanhypocalcemia.Myelosuppression was somewhatdifficult to evaluate because of confoundingfactors, such as prior treatmentand varying degrees of myelophthisisor splenomegaly. However, of39 patients with normal blood countsat baseline, only 3 developed a leukocytecount less than 2,500/μL and only1 patient developed a platelet countless than 50,000/μL at any point duringthe study.Pulmonary complications (n = 9)included pleural effusions and interstitiallung infiltrates. Infectious organismswere identified in three cases,and all but one case resolved afterempirical treatment. Mild, transient,asymptomatic hyperchloremic respiratoryalkalosis was also observed insome patients.Combination Therapy WithHydroxyurea
In vitro, the cytotoxic effects ofgallium nitrate are synergistic withthose of hydroxyurea, fludarabine(Fludara), interferon-alpha, andgemcitabine (Gemzar).[18,48-50] Thecombination of gallium nitrate andhydroxyurea, both of which inhibit ribonucleotidereductase, is synergisticin vitro against both lymphoid andmyeloid leukemic cell lines.[18,48]Thus, a clinical trial was undertakento evaluate the efficacy andtoxicity of gallium nitrate plushydroxyurea in patients with refractorynon-Hodgkin's lymphoma.[43]Fourteen patients with advanced loworintermediate-grade lymphoma weretreated with one of the following fourdoses for 7 days every 3 to 4 weeks(at least three patients were treated ateach level): (1) gallium nitrate (200mg/m2/d) by continuous IV infusionplus oral hydroxyurea (500 mg/m²/d);(2) gallium nitrate (250 mg/m²/d) plushydroxyurea (1,000 mg/d); (3) galliumnitrate (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 was64 years (range: 53 to 89 years). Allpatients had been heavily pretreated.The patients completed a median of2 (range: 1 to 6) treatment cycles (onecycle = 7 days).Tumor regression was observed in10 of 14 patients (1 complete response,1 near-complete response, 4 partialresponses, and 4 minor responses).Excluding patients with minor responses,the overall response rate was43% (6/14 patients). The median durationof response was 7 weeks (range:3 to 38 weeks). Responses were notconfined to a particular histologic subtype;cytotoxic activity was observedin both low- and intermediate-gradelymphomas. The response to treatmentcan be rapid; a dramatic shrinkage inan abdominal nodal mass was observedin one patient after just onetreatment cycle.Although it was hoped that ⁶⁷Gascanning would predict treatment responsiveness,no correlation was identifiedbetween tumor localization of⁶⁷Ga and tumor response to galliumnitrate. However, two patients withnegative ⁶⁷Ga scans did not respond togallium nitrate.Overall, toxicities were mild, andminimal myelosuppression occurred.As expected, the most common toxicitieswere hypocalcemia and diarrhea.The most serious toxicities wereanemia and reversible nephrotoxicity.Of four patients with nephrotoxicity,one patient had received prior treatmentwith cisplatin and one patient hada long history of diabetes mellitus thatmay have caused occult diabetic nephropathy.Another patient was unableto maintain adequate fluid intake becauseof anorexia. Transient, decreasedvisual acuity was observed intwo patients.General Toxicity
In general, the toxicity of galliumnitrate does not overlap with that ofother drugs commonly used for thetreatment of malignant lymphoma. Inparticular, myelosuppression is minimal.Mild to moderate anemia hasbeen observed; however, its direct relationshipto gallium nitrate is not entirelyclear because all patients treatedwith gallium nitrate have receivedmyelosuppressive agents previously.No evidence of cumulative nephrotoxicitywas observed in patients whoreceived the drug for more than14 months with adequate hydration.[41] Nephrotoxicity can be amelioratedor prevented with adequatehydration during treatment and byavoiding concomitant use with othernephrotoxic agents.Transient, mild to moderate hypophosphatemiamay also occur and mayrequire oral phosphorus supplements.Optic neuritis has occurred rarely inpatients receiving gallium nitrate.[42,45,51] However, when thedrug is administered at the recommendeddose and frequency, its incidenceis similar to that observed withother chemotherapeutic agents, suchas cisplatin, carboplatin (Paraplatin),paclitaxel, and etoposide.ConclusionGallium nitrate is a promising agentin the treatment of non-Hodgkin'slymphoma. Its mechanism of actioninvolves drug delivery via transferrinand the transferrin receptor. Galliumtransferrincomplexes target lymphomacells because they express highnumbers of transferrin receptors ontheir surfaces. Following cellular uptakeof gallium, ribonucleotide reductaseis inhibited indirectly via cellulariron depletion and also directly viacompetitive inhibition of substrate interactionwith the enzyme.During a single-agent phase IIstudy in patients with relapsed lymphoma,response rates of 40% to 50%in various histologic subtypes of non-Hodgkin's lymphoma were noted withcontinuous-infusion gallium nitrateadministered over 7 days. Continuousinfusiongallium nitrate is welltolerated and is not associated withsignificant myelosuppression. Theresponse rates to gallium nitrate innon-Hodgkin's lymphoma comparefavorably with other single agents usedin the treatment of patients withrelapsed or refractory disease, includingbleomycin,[52] cyclophosphamide(Cytoxan, Neosar),[53] doxorubicin,[54] and vincristine.[55]A multicenter US phase II trial iscurrently under way to study furtherthe effects of gallium nitrate in patientswith refractory low- or intermediategradenon-Hodgkin's lymphoma. Thisstudy is evaluating gallium nitrate (200to 300 mg/m²/d) for 7 days every 3weeks by continuous IV infusion usinga portable infusion pump.Future studies may include evaluatingthe efficacy and toxicity ofgallium nitrate in combination withfludarabine, rituximab (Rituxan), andgemcitabine; gallium nitrate may serveas a replacement for platinum in somesalvage regimens. Because of itsapparent lack of cross-resistance withother drugs and its nonoverlappingtoxicity profile, gallium nitrate is wellsuited for use in combination chemotherapyregimens. Ongoing investigationsare studying other possiblemechanisms of action of gallium nitrate,including the identification ofadditional molecular targets and thepossibility of predicting treatmentresponse through the use of complementaryDNA microarrays. Furtherknowledge of the mechanisms ofaction of this drug may help to determineits optimal use in patients withlymphoma.

Disclosures:

Dr. Chitambar is aconsultant for Genta Incorporated.

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