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© 2000 American Society for Clinical Oncology Phase I Clinical and Pharmacokinetic Study of Bcl-2 Antisense Oligonucleotide Therapy in Patients With Non-Hodgkin’s LymphomaFrom the Lymphoma Unit, Royal Marsden Hospital, and Cancer Research Campaign Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, and Institute of Child Health, London, United Kingdom. Address reprint requests to David Cunningham, MD, Department of Medicine, Royal Marsden Hospital, Downs Rd, Sutton, Surrey SM2 5PT, United Kingdom; email dcunn{at}icr.ac.uk
PURPOSE: To evaluate the pharmacokinetics and toxicity of an antisense oligonucleotide targeting bcl-2 in patients with non-Hodgkin’s lymphoma (NHL) and to determine efficacy using clinical and biologic end points. PATIENTS AND METHODS: Twenty-one patients with Bcl-2–positive relapsed NHL received a 14-day subcutaneous infusion of G3139, an 18-mer phosphorothioate oligonucleotide complementary to the first six codons of the bcl-2 open reading frame. Plasma pharmacokinetics were measured by anion exchange high-performance liquid chromatography. Response was assessed by computed tomography. Changes in Bcl-2 expression were measured by fluorescence-activated cell sorting of patients’ tumor samples. RESULTS: Eight cohorts of patients received doses between 4.6 and 195.8 mg/m2/d. No significant systemic toxicity was seen at doses up to 110.4 mg/m2/d. All patients displayed skin inflammation at the subcutaneous infusion site. Dose-limiting toxicities were thrombocytopenia, hypotension, fever, and asthenia. The maximum-tolerated dose was 147.2 mg/m2/d. Plasma levels of G3139 equivalent to the efficacious plasma concentration in in vivo models were produced with doses above 36.8 mg/m2/d. Plasma levels associated with dose-limiting toxicity were greater than 4 µg/mL. By standard criteria, there was one complete response, 2 minor responses, nine cases of stable disease, and nine cases of progressive disease. Bcl-2 protein was reduced in seven of 16 assessable patients. This reduction occurred in tumor cells derived from lymph nodes in two patients and from peripheral blood or bone marrow mononuclear cell populations in the remaining five patients. CONCLUSION: Bcl-2 antisense therapy is feasible and shows potential for antitumor activity in NHL. Downregulation of Bcl-2 protein suggests a specific antisense mechanism.
ANTISENSE OLIGONUCLEOTIDES are chemically modified single-strand DNA molecules of between 13 and 25 nucleotides in length. They have a nucleotide sequence complementary to that of their target mRNA and are capable of inhibiting expression of the target gene. The specificity of this action at the level of gene expression makes antisense oligonucleotide therapy a powerful tool with wide-ranging potential clinical applications. Clinical trials have been initiated in the fields of malignancy and inflammatory and viral diseases, using oligonucleotides targeting a variety of genes. These trials have demonstrated the feasibility of this therapeutic approach, with some evidence of clinical activity. The bcl-2 gene provides a rational target for antisense strategies. Overexpression leads to cellular resistance to programmed cell death (apoptosis),1 resulting in chemoresistance in vitro.2 Bcl-2 is overexpressed in the majority of low-grade non-Hodgkin’s lymphoma (NHL) cases and approximately 50% of high-grade NHL cases. A number of different molecular mechanisms are responsible for the upregulation of the Bcl-2 protein, the most common being a translocation between chromosomes 14 and 18 that brings the bcl-2 gene under the transcriptional control of the immunoglobulin heavy chain promoter. There is evidence for an etiologic role of Bcl-2 overexpression in lymphoma. Transgenic mice with deregulated Bcl-2 expression initially develop lymphoid hyperplasia with extended B-cell survival.3 In some cases, this progresses to diffuse large B-cell lymphoma, often with the accumulation of additional genetic abnormalities, such as rearrangement of the c-myc gene.4 Three recent studies have examined the prognostic significance of Bcl-2 expression in diffuse large B-cell NHL.5-7 In multivariate analyses, Bcl-2 expression was found to be an independent poor prognostic factor in these patients. Antisense oligonucleotides targeting bcl-2 have been demonstrated to reduce bcl-2 mRNA and protein levels in vitro8 and to reverse chemoresistance of Bcl-2–expressing lymphoma cell lines.9 It has also been demonstrated that eradication of lymphoma can be achieved in a severe combined immune deficient mouse model of human NHL by a 14-day subcutaneous infusion of G3139 (Genta Inc, Lexington, MA), an 18-mer phosphorothioate oligonucleotide targeting the first six codons of the bcl-2 mRNA open reading frame, sequence 5'-tctcccagcgtgcgccat.10 These observations provided the rationale for a phase I trial of antisense oligonucleotide G3139 in NHL patients.
Methods for this trial have been described previously.11 Eligible patients were men or women with B-cell NHL of any histologic subtype, provided there was immunohistochemical evidence of Bcl-2 expression by tumor cells in the biopsy material. Patients were required to have received at least one previous line of chemotherapy and to have progressive disease. Informed consent was obtained from all patients before entry onto the study, which was approved by the Royal Marsden Hospital Ethics Committee. Antisense oligonucleotide G3139 was delivered as a continuous subcutaneous infusion for 14 days by a portable infusion pump. Toxicity was graded according to the common toxicity criteria and assessed during the 2-week treatment period and during the subsequent 4 weeks. One course of treatment was planned per patient, but additional courses of treatment were considered in the event of a tumor response. A second course was administered to patients no. 2, 17, and 21. The initial dose was 4.6 mg/m2/d, calculated as one tenth of the dose that would kill 10% of mice in preclinical toxicity studies. Planned dose escalation was in 100% increments. One patient was enrolled at each of the first three dose levels, after which three patients per dose level were included. The following were considered dose-limiting toxicities (DLTs): grade 4 leukopenia or neutropenia, grade 3 or 4 thrombocytopenia, any complicated grade 3 or 4 myelosuppression, and any grade 3 or 4 nonhematologic systemic toxicity. In the event of a DLT in one of three patients at a single dose level, the subsequent dose escalation was reduced to 33%. In the event of DLTs in more than one patient at a single dose level, the dose level below was expanded to a maximum of six patients. Plasma concentrations of G3139 were measured by anion exchange high-performance liquid chromatography, as described previously.12 Briefly, duplicate standard curves were produced in control plasma at the levels of G3139 0.25, 0.5, 1, 2, 5, 10, and 20 µg/mL. Plasma from standard curves and patient samples was extracted with phenol:chloroform:isoamyl alcohol. Chromatographic separation was achieved on a GenPak-fax column (Waters, Watford, United Kingdom). G3139 eluted with a gradient of LiCl 2 mol/L in LiOH 20 mmol/L. Detection was achieved by spectroscopy at 254 nm. Pharmacokinetics were evaluated by compartmental and noncompartmental analysis using WinNonlin Pro Edition software model 202 (infusion model) (Pharsight, Mountain View, CA). Statistical analysis was performed with Minitab statistical software (Minitab Inc, State College, PA). Relationships between variables were assessed by linear regression and analysis of variance with the general linear model. Tumor response was assessed primarily by computed tomography (CT) scanning using World Health Organization response criteria, as described previously.11 Additional parameters of response included tumor-related symptoms, numbers of circulating lymphoma cells in the peripheral blood (assessed morphologically and quantified by an experienced hematologist who counted cells on a blood film or by fluorescence-activated cell sorting [FACS] analysis for CD19 expression), and serum concentrations of lactate dehydrogenase (normalization of a previously elevated level was considered relevant). Bcl-2 protein levels were measured by FACS analysis of mononuclear cells obtained from peripheral-blood and bone marrow aspirates and tumor cells obtained from fine-needle aspirates of palpable lymph nodes collected before, during, and after treatment.11 To examine the effect of treatment on the target lymphoma cells, this analysis was confined to Bcl-2–expressing cells by gating on the appropriate population.
Pretreatment Characteristics Twenty-one patients were treated at doses ranging from 4.6 mg/m2/d to 195.8 mg/m2/d. Eleven patients were male, and the median age was 54 years (range, 41 to 73 years). The majority had a diagnosis of low-grade NHL (nine had follicular NHL and eight had small lymphocytic NHL), but three patients had diffuse large B-cell lymphoma and one had mantle-cell lymphoma. Patients had received a median of four previous chemotherapy regimens (range, one to eight). Twenty-six different regimens had been used, the most common being chlorambucil (15 patients), cyclophosphamide, doxorubicin, vincristine, and prednisolone (10 patients), and fludarabine (10 patients). Four patients had received high-dose chemotherapy with autologous peripheral stem-cell support, and three patients had received prior radiotherapy (Table 1).
Toxicity The only hematologic toxicity that seemed to be unequivocally related to G3139 therapy was thrombocytopenia (Table 2). Of the five patients treated at a dose of 147.2 mg/m2/d, two patients developed grade 2 and one patient developed grade 3 thrombocytopenia, and two patients treated at a dose of 195.8 mg/m2/d developed grade 3 and grade 4 thrombocytopenia. In all of these cases, the platelet count fell progressively during the course of the G3139 infusion and recovered after discontinuation of treatment. Furthermore, a correlation existed between plasma concentration of G3139 and nadir platelet count (Fig 1), suggesting a dose-dependent effect.
Some hematologic abnormalities observed in this trial seemed to be unrelated to G3139 treatment. Patient no. 8 had grade 3 leukopenia and neutropenia, grade 4 lymphopenia, and grade 2 thrombocytopenia within the first 4 days of therapy. These observations coincided with a Haemophilus influenzae pneumonia, and all abnormalities resolved on antibiotic treatment, despite continuation of the G3139 infusion. Grade 2 leukopenia was also observed in patient no. 13, in the absence of neutropenia; this patient had long-standing lymphopenia that did not worsen during treatment. Grade 3 neutropenia occurred in patient no. 2, but this was a pre-existent abnormality resulting from bone marrow infiltration by lymphoma. Grade 3 or 4 lymphopenia was observed in 10 patients (patients no. 6, 7, 8, 12, 13, 15, 18, 19, 20, and 21) and grade 1 or 2 lymphopenia was seen in a further three patients (patients no. 3, 9, and 10). This predated the start of therapy in most cases but seemed to worsen transiently during treatment in some patients (patients no. 6, 10, 12, 18, 20, and 21), suggesting a possible oligonucleotide-related effect. However, the lymphopenia was not clinically significant. Grade 1 or 2 anemia occurred in 15 of the 21 patients, but this was coincidental with lymphomatous bone marrow infiltration, with no obvious temporal relationship to treatment. No clotting abnormalities or changes in the CD4 to CD8 ratio were observed. A repeat bone marrow examination demonstrated no evidence of hypoplasia or G3139-induced toxicity to bone marrow precursors. Nineteen patients developed transient grade 1 or 2 nonfasting hyperglycemia during G3139 infusion. There was no apparent relationship with drug dose. Transient increases in concentrations of hepatic aminotransferases or alkaline phosphatase occurred in seven patients. Slight transient increases in urea and creatinine were also documented in four patients. These abnormalities were more common at higher doses, with the majority occurring in patients treated at 147.2 mg/m2/d and above. Intervention was not required, and all biochemical abnormalities resolved after completion of the treatment course. All patients experienced local skin inflammation at the sites of subcutaneous infusion. This was manageable in most cases by rotation of the infusion site. However, two patients had a more severe reaction. Patient no. 4 had grade 2 local inflammation that was intolerably painful despite a reduction in drug concentration; the infusion was discontinued after 4 days. Patient no. 13 developed a grade 3 local reaction with inflammation and ulceration at one infusion site. This site was slightly edematous before commencement of the infusion as a result of lymphomatous involvement of the draining lymph nodes. Other infusion sites were less severely affected, and the complete course of treatment was delivered. Skin biopsy samples from affected sites in these two patients showed a mild perivascular lymphocytic infiltrate in the superficial dermis. Features were nonspecific and there was no evidence of vasculitis. Five patients (patients no. 11, 13, 16, 17, and 19) developed tender enlarged lymph nodes during the G3139 infusion in lymph node regions draining the skin at the infusion site. The skin and lymph node changes resolved after completion of the infusion. In a number of patients, nonhematologic abnormalities occurred that were considered unlikely to be related to G3139 treatment. Four patients developed chest infections while on treatment (patients no. 6, 8, 11, and 20). All of these patients had advanced NHL with impaired bone marrow function. Three cases were confirmed microbiologically to be H influenzae (patient no. 8) or Haemophilus parainfluenzae (patients no. 6 and 20) infections and responded to appropriate antibiotic therapy. A microbiologic diagnosis was not possible in patient no. 11, but the symptoms resolved with empirical oral antibiotic treatment. Three patients developed circulatory abnormalities secondary to progressive lymphoma during the course of treatment. Patient no. 3 had swelling of the arms, neck, and scrotum and developed enlarging right and left pleural effusions. Patient no. 7 had superior vena cava obstruction with episodes of transient syncope. Patient no. 15 had peripheral edema and ascites associated with hypercalcemia and acute renal failure. DLTs were observed in patients treated at doses of 147.2 mg/m2/d and above. Of the five patients treated at a dose of 147.2 mg/m2/d, two experienced adverse events considered to be DLTs (Table 2). These comprised grade 3 thrombocytopenia (patient no. 12) and grade 3 fever associated with grade 3 hypotension (patient no. 17). The latter event occurred within a few hours of the patient’s starting the G3139 infusion and resolved rapidly once the infusion was discontinued, 48 hours after commencement. It is of note that the plasma concentration of G3139 in this patient reached an unusually high level (Table 3). He subsequently completed a second course of treatment at a reduced dose of 36.8 mg/m2/d, without systemic toxicity. None of the three patients treated at a dose of 195.8 mg/m2/d were able to complete the scheduled 14-day infusion. Patient no. 14 had treatment discontinued on day 8 because of a combination of fatigue, fever, and grade 4 thrombocytopenia. Patient no. 15 developed rapid disease progression associated with hypercalcemia and acute renal failure on the first day of treatment; treatment was therefore discontinued immediately. Patient no. 16 continued treatment until day 12, when the combination of grade 3 thrombocytopenia, fever, fatigue, and a generalized maculopapular rash made further treatment intolerable. The maximum-tolerated dose was therefore considered to be 147.2 mg/m2/d (approximately 4.1 mg/kg/d).
Pharmacokinetics Detectable levels of G3139 were observed in plasma at doses of 36.8 mg/m2/d and above. Steady-state plasma levels were observed in the majority of patients 48 hours after the beginning of the infusion, although minor fluctuations in the steady-state level were observed, corresponding to changes in the infusion site. Table 3 shows the pharmacokinetic parameters derived from noncompartmental analysis. The mean plasma steady-state concentration (Css) was 0.45 µg/mL (range, undetectable to 0.96 µg/mL) for the 36.8-mg/m2 group, 1.03 µg/mL (range, 0.47 to 1.59 µg/mL) for the 73.6-mg/m2 group, 2.43 µg/mL (range, 2.11 to 2.78 µg/mL) for the 110.4-mg/m2 group, 3.16 µg/mL (range, 0.96 to 6.67 µg/mL) for the 147.2-mg/m2 group, and 5.63 µg/mL (range, 4.17 to 7.37 µg/mL) for the 195.8-mg/m2 group. There was a linear correlation between Css and dose (P = .002) (Fig 2). Apart from the dose delivered, the only factor identified in multivariate analysis to significantly affect Css was pretreatment renal function (P = .044, general linear model). Representative concentration-versus-time curves are shown in Fig 3 (with patients treated at the 110.4-mg/m2/d dose level as an example). The mean plasma half-life for elimination (t1/2) was 7.46 hours (SD, ± 4.32 hours; SE, ± 1.15 hours). There was no difference in t1/2 between dose levels (t test for unpaired samples), nor was there any correlation between renal function and t1/2.
Response All 21 patients were assessable for response on an intention-to-treat basis (Table 4). Patient no. 8 had a complete response to treatment, with resolution of all lymph node masses to less than 1 cm in diameter, and clearance of bone marrow involvement. This was accompanied by resolution of lymphoma-related alcohol intolerance, which allowed him to drink alcohol for the first time in 2 years. His remission has been maintained without further antilymphoma therapy and confirmed on repeat CT scans up to 3 years after he completed treatment (Fig 4). Two patients had a minor response to treatment. Patient no. 6 had a near partial response in her right axillary and mediastinal nodes but stable disease in her more bulky lymphadenopathy below the diaphragm. Patient no. 21 had a good partial response at nodal sites in the retrocrural area and the celiac axis and resolution of a pulmonary lymphoma deposit. However, other disease sites showed slight enlargement over the course of treatment. Eight patients had stable disease on CT scan over the 6-week assessment period (patients no. 1, 2, 11, 13, 16, 18, 19, and 20), and one patient was lost to follow-up after 3 weeks (patient no. 10), at which point he had stable disease. The remaining nine patients showed disease progression. In several cases, CT scans at the end of the 2-week G3139 infusion demonstrated enlargement of lymph nodes that subsequently returned to baseline by the 6-week assessment CT scan. In view of the observation of painful lymphadenopathy in some patients during the G3139 infusion, it is more likely that this transient nodal enlargement resulted from an inflammatory process rather than disease progression.
In addition to objective measurement of tumor response by CT scan, additional supplementary markers of response were also assessed (Table 4). Lymphoma-related symptoms were present in 10 patients before the initiation of treatment and improved in six cases (patients no. 1, 8, 11, 18, 19, and 20). Fourteen patients had circulating lymphoma cells present in the peripheral blood, and 10 of them showed a reduction after treatment (patients no. 2, 6, 7, 10, 11, 12, 14, 18, 19, and 20). Two patients had normalization of an elevated serum lactate dehydrogenase level (patients no. 6 and 8). Three patients received a second course of G3139. Patient no. 2 had a further symptomatic response with the second course after the recurrence of his pruritus and night sweats between treatments. Patient no. 17 was re-treated at a reduced dose after the occurrence of DLT after 48 hours of his initial course. This was well tolerated with no systemic toxicity, but his disease progressed. Patient no. 21 received the second course by intravenous infusion because of poor tolerance of the local skin inflammation at the subcutaneous infusion sites. This was well tolerated without systemic toxicity, but again disease progressed.
Survival
Molecular Response
This study has demonstrated the feasibility of bcl-2 antisense oligonucleotide therapy by 14-day subcutaneous infusion in patients with advanced NHL. Treatment was universally well tolerated at doses up to 110.4 mg/m2/d in this population. Other phase I/II studies are ongoing with G3139 administered by intravenous infusion to patients with breast, prostate, or renal cell cancers, melanoma, or other epithelial malignancies. Initial reports from these trials indicate that G3139 is tolerated with doses exceeding the maximum-tolerated dose from our study, even when combined with cytotoxic chemotherapy.13,14 The generally advanced stage of disease, multiple prior courses of chemotherapy, and compromised bone marrow function in the patients enrolled may account for the greater degree of toxicity observed in our study. On the basis of our results, we would recommend a dose of 110.4 mg/m2/d for further evaluation in phase II studies in patients with advanced NHL. Although this was primarily a safety study, there was evidence for clinical antitumor activity and for specific downregulation of Bcl-2 protein in target tissues. Three patients with low-grade lymphoma had an objective reduction in overall tumor bulk after treatment, and one of them had a complete remission that has been sustained for 36 months. Although spontaneous remissions are known to occur in low-grade lymphoma, it is extremely unlikely that this would happen by chance within a few weeks of antisense therapy, particularly in heavily pretreated patients. The clinical relevance of disease stabilization over a 6-week period in patients with low-grade lymphoma is less clear. This is an indolent disease with a low proliferation rate. However, all patients had disease progression before study entry, and the median progression-free survival of 3.6 months associated with a single 2-week course of treatment is encouraging. Other trials of antisense oligonucleotides in malignant diseases have investigated repeated courses of therapy over protracted periods. In these studies, tumor responses were often not observed until several months after the initiation of treatment.15 In the three patients with diffuse large B-cell lymphoma included in this study, the disease progressed rapidly during treatment. Although this is a small sample on which to base any firm conclusions, it is possible that the high proliferation rate of the malignant cells in this disease renders apoptosis (and therefore Bcl-2 expression) a less important factor in determining the rate of tumor growth. One aim of this study was to demonstrate a specific antisense oligonucleotide effect on the molecular target. Preclinical studies both in vitro and in vivo have established that bcl-2 mRNA and protein are specifically downregulated by G3139.8,10,16 The more limited availability of tumor cells from clinical material restricted the scope of the laboratory investigations we were able to perform. FACS was selected to evaluate Bcl-2 protein levels because of its ability to provide data on individual cells. We were therefore able to restrict the analysis to Bcl-2–expressing cells; thus, we predominantly examined tumor cells. Normal mature memory B lymphocytes and T lymphocytes (CD4 and CD8) also express Bcl-2 protein. These cells may, therefore, also have been included in the analysis in some cases, particularly in the PBMC samples. This raises the question of whether observed reductions in Bcl-2 protein resulted from a change in the proportion of tumor cells before and after treatment rather than from reduced Bcl-2 expression by tumor cells. However, one patient with significant tumor infiltration of the blood had a reduction in Bcl-2 protein in the PBMC samples (patient no. 2). Morphologic criteria and immunocytochemistry showed that the proportion of lymphoma cells in the samples analyzed did not change between time points, and these were the predominant species of cell present. Two patients had a reduction in Bcl-2 protein levels in lymph node fine-needle aspirate samples. Both patients had a diagnosis of follicular lymphoma, in which the vast majority of Bcl-2–positive cells in the involved lymph nodes are lymphoma cells. It is likely, therefore, that these observations represent a true antisense-mediated reduction in Bcl-2 protein expression. Although numbers are small, it is of interest that of the seven patients in whom a reduction in Bcl-2 expression was observed, one patient had a minor response and five had stable disease, whereas only one had progressive disease. In contrast, four of the nine patients in whom we observed no reduction in Bcl-2 expression had progressive disease by CT criteria. A further patient with unchanged Bcl-2 expression had a dramatic increase in numbers of circulating lymphoma cells, although his nodal disease remained stable. Using a dose range and schedule similar to those used in this study, another phase I/II study of G3139 by intravenous infusion, combined with dacarbazine, has demonstrated decreased Bcl-2 expression by Western blots from serial biopsies of melanoma lesions, corresponding with durable antitumor activity in some patients with drug-resistant disease.13 A reduction in c-raf mRNA levels in PBMC samples, determined by reverse transcriptase-polymerase chain reaction, was reported in patients with a variety of solid tissue malignancies treated with a c-raf antisense oligonucleotide.17 Intestinal expression of intercellular adhesion molecule-1 (ICAM-1), assessed qualitatively by immunohistochemistry, was reported to be reduced in patients with Crohn’s disease treated with an antisense oligonucleotide targeting ICAM-1 mRNA.18 Patients treated with a placebo in this study showed no reduction in ICAM-1 expression. Thus, there are now four clinical trials supporting an antisense mechanism of action of phosphorothioate oligonucleotides. Phosphorothioate oligonucleotides may also have immunologic effects,19 and it could be speculated that the G3139 antitumor activity was related to this property rather than reduction of target protein expression. Localized activation of natural killer (NK) cell lytic activity in the draining lymph nodes was reported in mice injected subcutaneously with oligonucleotides containing the CpG dinucleotide sequence motif.20 Activation of NK cells was dependent on the production of interleukin-12, interferon-alfa/beta, and tumor necrosis factor alpha by accessory cells. CpG-containing oligonucleotides have been shown to activate dendritic cells in the skin of BALB/c mice after local injection, which leads to a local TH1 response.21 Our observation of skin inflammation at the subcutaneous injection site, together with painful enlargement of local lymph nodes, may represent a similar phenomenon because G3139 contains two CpG motifs. We aimed to address this point. G3139 was administered to a human lymphoma xenograft in nonobese, diabetic, severe combined immune deficient (NOD-SCID) mice that lack B or T lymphocytes or NK cells, and showed eradication of disease (F. Cotter, personal communication, June 1998). In addition, we examined intracellular expression of interleukin-2, interferon-gamma, interleukin-4, and perforin in PBMCs and serum levels of immunoglobulin E in four patients before and at the end of the G3139 infusion. There was no evidence for a systemic TH1 response or activation of NK cells as a result of treatment in these patients (unpublished data). These observations do not support an immunologic mechanism of action for G3139. The toxicity of G3139 observed in this study seemed to be related primarily to the phosphorothioate backbone chemistry and not to a bcl-2 sequence-specific effect. Clinical trials investigating phosphorothioate antisense oligonucleotides targeting a number of different genes have reported a spectrum of toxicity similar to that observed in this study. DLTs have included fatigue,15,17,22,23 fever,17 fever and hypotension,23 and thrombocytopenia.15,22,23 Other reported treatment-related toxicities include clotting abnormalities,15,17,18,24 elevation of complement components,15,17,24 elevation of hepatic enzymes,25 and hyperglycemia.25 It is likely, therefore, that these abnormalities are non–sequence-specific effects of the phosphorothioate oligonucleotide molecule. The mechanism underlying this toxicity is not completely understood but seems to result from the polyanionic structure of these molecules, being inhibited in vitro by the polycationic drug protamine sulfate.26 The potential for sequence-specific toxicity as a result of downregulation of Bcl-2 expression in normal tissues is perhaps of greater concern. Combination of bcl-2 antisense oligonucleotides with chemotherapeutic agents may potentiate this effect, as the increased induction of apoptosis could produce significant toxicity. Bcl-2 is normally expressed in adults in a relatively restricted distribution.27 Transgenic mice with deletion of both copies of bcl-2 are viable but display a variety of abnormalities, including polycystic kidneys, growth retardation, hair hypopigmentation in the second hair follicle cycle, and impaired intestinal epithelial turnover.28,29 Hematopoiesis is normal in Bcl-2–deficient mice, and although Bcl-2 is normally expressed in megakaryocytes, both megakaryocyte number and platelet count were normal in these animals. Fundamentally, the bcl-2 gene is not essential for survival of the pluripotent stem cell, and as such, Bcl-2 downregulation will not lead to a loss of renewal potential for the normal cell. Bcl-x, a homolog of bcl-2, seems more important in the regulation of apoptosis in hematopoietic precursors,30 and indeed Bcl-x–deficient mice undergo massive cell death of immature hematopoietic cells and neurons and die around 13 days of gestation.31 Early lymphoid development was also normal in bcl-2 knockout mice, but the animals subsequently developed lymphopenia and displayed fulminant apoptosis of lymphocytes in the thymus and spleen, suggesting a dependence of mature B and T lymphocytes on Bcl-2 for survival.28 The only clinical or laboratory abnormality observed in our study likely to have arisen from downregulation of Bcl-2 was lymphopenia. There have been no other reports of lymphopenia in clinical trials of phosphorothioate oligonucleotides, but nine patients had a transient reduction in lymphocyte count during the 2-week period of treatment with G3139. Of these, Bcl-2 expression was assessable in five patients’ PBMCs and reduced in three. An alternative explanation for the occurrence of lymphopenia in these patients with advanced NHL is progressive bone marrow involvement by disease. However, this would not explain the temporal relationship to treatment we observed, and lymphopenia could therefore be a reversible sequence-specific effect of G3139. The Css level of G3139 observed in patient no. 8, who showed a complete response, was consistent with that measured in the mouse after continuous infusion at therapeutic doses.12 Indeed, the pharmacologically active plasma level, associated with a reduction in target protein expression, seems to be consistent between mice and humans at approximately 1 µg/mL (Fig 5). 12 This suggests that the therapeutic window for this molecule is wide in comparison with conventional cytotoxic agents, as DLT was only observed when plasma levels exceeded 4 µg/mL. At a given dose level, up to 6.9-fold variations in Css were observed (eg, 0.96 for patient no. 11 and 6.67 for patient no. 17, both treated at 147.2 mg/m2/d). However, patient no. 11 required multiple changes in infusion site because of skin toxicity that could potentially have affected the Css levels. Furthermore, no patient treated at a dose of 110.4 mg/m2/d or below achieved a plasma level of G3139 in the range associated with toxicity, which suggests that treatment with these doses should be consistently safe. Although it was difficult to evaluate absorption in our pharmacokinetic analysis because of the complexity of the administration and elimination, the pharmacokinetics were linear with increasing doses suggesting that the absorption was consistent whatever the dose. This is contrary to what has been described in the rat, in which the bioavailability of a 20-mer phosphorothioate after subcutaneous administration has been shown to increase with dose.32 In a study in which G3139 was infused intravenously, Css plasma levels after intravenous administration were very similar to those observed in our study, which indicates that G3139 is well absorbed by the subcutaneous route.14 The t1/2 of G3139 (7.2 hours) was different from that reported for other phosphorothioate oligonucleotides in the clinical setting, where t1/2s of 30 minutes and 26 hours have been reported.24,33,34 However, these studies were performed using short intravenous infusions, and it is possible that both the long infusion and the subcutaneous route affected the terminal t1/2, as was observed in the preclinical model.32 The variation in t1/2 observed between patients was unrelated to dose or renal function and may have reflected differences in rate of absorption from the subcutaneous compartment after the end of the infusion. The overall plasma clearance, however, correlates well with what has been reported in other studies with short intravenous infusion.33
There is great potential for the further development of bcl-2 antisense oligonucleotides in the treatment of malignant disease. One of the most interesting possibilities is their use as chemosensitizing agents, both in NHL and in other malignancies characterized by Bcl-2 overexpression. This principle has been demonstrated in vitro and in vivo with several human tumor models, including lymphoma,9,35 breast cancer,36 small-cell lung cancer,37 prostate cancer,38 and melanoma.16 Clinical trials are now underway in several malignancies besides NHL, using G3139 alone or in combination with cytotoxic agents. Our results suggest that advanced-low grade lymphoma is amenable to bcl-2 antisense oligonucleotide therapy. Based on the results from this phase I study, a phase II trial is now in progress at the Royal Marsden Hospital using G3139 in combination with standard cytotoxic regimens for patients with relapsed, chemotherapy-resistant NHL.
Supported in part by Genta Incorporated, Lexington, MA. P.A.C. and F.R. are funded by Cancer Research Campaign grant no. SP2330/0201. F.E.C. is funded by grants from the Leukaemia Research Fund and the Medical Research Council.
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ABSTRACT
INTRODUCTION
; patients no. 2, 5, and 7).
), in patients with refractory cancer. Proc Am Soc Clin Oncol 16:246a, 1997 (abstr 870)
