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© 1999 American Society for Clinical Oncology Phase I Evaluation of ISIS 3521, an Antisense Oligodeoxynucleotide to Protein Kinase C-Alpha, in Patients With Advanced CancerFrom the PRN Research, Inc, and Sammons Cancer Center at Baylor, Dallas; Cancer Therapy and Research Center, Institute for Drug Development, San Antonio, TX; and ISIS Pharmaceuticals, Carlsbad, CA. Address reprint requests to John Nemunaitis, MD, Baylor University Medical Center, 3535 Worth St, Collins Building, 5th Floor, Dallas, TX 75246.
PURPOSE: To determine the maximum-tolerated dose (MTD) and pharmacologic behavior of ISIS 3521 (ISI 641A), an antisense phosphorothioate oligonucleotide to protein kinase C-alpha. PATIENTS AND METHODS: Thirty-six patients with advanced cancer received 99 cycles of ISIS 3521 (0.15 to 6.0 mg/kg/d) as a 2-hour intravenous infusion administered three times per week for 3 consecutive weeks and repeated every 4 weeks. Plasma and urine sampling was performed during the first week of treatment and subjected to capillary gel electrophoresis to determine full-length antisense oligonucleotide in addition to chain-shortened metabolites. RESULTS: Drug-related toxicities included mild to moderate nausea, vomiting, fever, chills, and fatigue. Hematologic toxicity was limited to thrombocytopenia (grade 1, four patients; grade 2, one patient; grade 3, one patient). There was no relationship between dose, maximum concentration of the drug (Cmax), or area under the plasma concentration versus time curve (AUC) and coagulation times or complement levels. Dose escalation was discontinued because of the attainment of peak plasma concentrations, which approached that associated with complement activation in primates. Two patients with non-Hodgkin's lymphoma who completed 17 and nine cycles of therapy achieved complete responses. The pharmacokinetic profile of ISIS 3521 revealed a short elimination half-life (18 to 92 minutes), as well as a dose-dependent decrease in clearance and dose-dependent increases in Cmax, AUC, and elimination half-life. CONCLUSION: No dose-limiting toxicity of ISIS 3521 was identified, and clinical activity was observed. A short elimination half-life was identified, which suggests that alternate schedules with prolonged administration may be necessary for further clinical development.
PROTEIN KINASE C (PKC) is a family of phospholipid-dependent cytoplasmic serine threonine kinases that comprises distinct isoenzymes which differ in their biochemical properties, tissue-specific expression, and intracellular localization.1,2 The PKC isoenzymes are classified in three groups: (1) group A (classical): PKC-  , ßI, ßII, and  ; (2) group B (new): PKC- ,  ,  , and µ; and (3) group C (atypical): PKC- and  . Groups A and B are activated by 1,2-diacylglycerol, which is generated by the cleavage of membrane phospholipids via phospholipases. Phospholipases are regulated by a variety of growth factors and hormones and are responsible for the activation of severalcytoplasmic PKC isoenzymes, which in turn convey signals that lead to proliferation or differentiation.2,3
Several lines of evidence suggest that modulation of PKC activity, and of PKC-
The objective of antisense therapy is to specifically inhibit expression of a particular gene product.11-15 During gene transcription, the duplex strand of DNA becomes partially uncoiled, and the two complementary strands, sense and antisense, separate. The antisense strand of DNA is used as a template to generate messenger RNA, which in turn migrates to the cytoplasm of the cell where translation occurs to produce the protein product. The cytoplasmic mRNA is in the sense orientation, and translation can be prevented by the complementary base-pair binding of short (generally 15 to 25 mer) antisense oligonucleotides. Protein inhibition may be accomplished by destruction of the mRNA via cleavage by RNase-H, which recognizes the hybridized region, or may involve steric blocking of translation of the mRNA. This process permits highly specific inhibition of expression, including a single member of multiple groups of related proteins, such as PKC-
ISIS 3521 (also designated ISI 641A) is a 20-mer phosphorothioate oligodeoxynucleotide that hybridizes to the 3'-untranslated region of the human PKC- The pharmacokinetic behavior of ISIS 3521 in animal models was similar to that observed with other phosphorothioate oligonucleotides.22 ISIS 3521 was cleared rapidly from plasma. No accumulation was observed after administration of repeated doses every other day for 27 days. Clearance from plasma seemed to be related to the combined result of distribution within tissues and metabolism principally via exonuclease-induced chain shortening. The tissue half-life was approximately 5 days in the renal cortex and 1 to 3 days in other tissues.23 The preclinical toxicologic profile of ISIS 3521 consisted of a constellation of effects that are now understood to be characteristic of phosphorothioates as a chemical class.23-27 The principal toxic effect was transient complement activation, which was both dose-related and infusion rate–related.27,28 Reduced cardiac output and hypotension in association with peak production of anaphylatoxins C3a and C5a developed at a plasma threshold concentration above 40 µg/mL of intact (full-length) oligonucleotide. Other toxic effects in animals were transient thrombocytopenia and elevation of the activated partial thromboplastin time (aPTT). Splenomegaly, lymphoid hyperplasia, and mononuclear cell infiltration involving multiple organs were observed in mice only, and were believed to be due to proliferation of lymphocytes and monocyte/macrophages. Primates receiving ISIS 3521 at a dose of 1 mg/kg by 2-hour intravenous (IV) infusion experienced no apparent toxic effects.17 The principal objectives of this phase I and pharmacologic study of ISIS 3521 included the following: (1) to characterize the principal toxicities of ISIS 3521 administered as a 2-hour IV infusion three times per week for 3 weeks and repeated every 4 weeks in patients with advanced solid malignancies, (2) to determine the maximum-tolerated dose (MTD) and recommended dose for subsequent phase II/III trials of ISIS 3521, (3) to characterize the pharmacologic behavior of ISIS 3521, and (4) to seek preliminary evidence of antitumor activity in patients with advanced cancers.
Patient Selection Patients with histologically documented solid malignancies that were refractory to standard therapy or for whom no effective therapy existed were eligible for this study. Other eligibility criteria included the following: (1) age  18 years; (2) Southwest Oncology Group (SWOG) performance status of  2; (3) no chemotherapy or investigational agents administered within 4 weeks; (4) adequate hematopoietic (absolute neutrophil count > 1,500/µL, hemoglobin > 9.0 g/dL, and platelet count > 100,000/µL), hepatic (total bilirubin < 2.0 mg/dL; AST and ALT < two times the upper limit of normal [ULN] in the absence of hepatic metastases or < five times the ULN in the presence of hepatic metastases), and renal (creatinine concentration < 2.0 mg/dL) functions; (5) normal prothrombin (PT) and partial thromboplastin time (PTT); (6) no condition requiring the administration of therapeutic doses of anticoagulants; (7) no underlying disease associated with active bleeding or a past history of coagulopathy or complement abnormality; and (8) no coexisting medical problem of sufficient severity to limit full compliance with the study. Informed consent was obtained according to federal and institutional guidelines.
Study Design
Sequential cohorts of patients received ISIS 3521 administered as a 2-hour IV infusion three times a week for 3 consecutive weeks. The dose levels tested were 0.15, 0.3, 0.6, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, and 6.0 mg/kg. Each treatment cycle was 4 weeks (3 weeks of therapy followed by 1 week of rest). Dose-limiting toxicity was defined as the presence of one or more of the following: (1) World Health Organization (WHO) Toxicity Grading Scale criteria grade 4 coagulation abnormalities associated with clinically significant bleeding; (2) WHO Toxicity Grading Scale criteria
Cohorts of three patients were evaluated at each dose level. If there was no evidence of dose-limiting toxicity after one complete treatment cycle in the first two patients and no evidence of dose-limiting toxicity in the third patient after receiving the first three doses of ISIS 3521, then the next sequential dose level was initiated. If one of the three patients demonstrated evidence of treatment-related toxicity, then an additional three patients were studied at that dose level before dose escalation. Dose escalation was to be stopped if
Clinical Agent
Pretreatment and Follow-Up Studies Measurable and assessable disease was examined at baseline and after every two treatment cycles to determine tumor response. A complete response was defined as resolution of all measurable and assessable disease for at least 4 weeks without worsening of disease-related symptoms or SWOG performance status. A partial response required at least a 50% reduction in the sum of the products of the maximum perpendicular diameters of measurable lesions and at least 50% improvement in assessable disease for at least 4 weeks. Progressive disease was defined as at least 25% increase in measurable disease or the appearance of new tumor lesions. Stable disease was defined as disease status failing to meet the criteria for response or progressive disease. Patients were allowed to continue treatment in the absence of disease progression or intolerable toxicity.
Pharmacokinetic Sampling and Assay Drug analysis was performed by CGE32 by Covance Laboratories (Madison, WI) using a previously described method on aliquots of each sample of plasma and urine. Samples were prepared by strong anion-exchange solid-phase extraction (SAX), followed by two desalting steps: elution from a reverse-phase solid-phase extraction column, then membrane dialysis. The preparation differed from the published method in that the SAX loading buffer used for the present study contained 0.25 mol/L KCl in lieu of 0.5 mol/L KCl. A phosphorothioate oligonucleotide composed of 27 thymidine nucleotides (T27) was added to both plasma and urine as an internal standard. CGE was performed with a Beckman P/ACE Model 5010 instrument (Beckman, Fullerton, CA) with a 27-cm column. Oligonucleotides eluting from the column were detected by ultraviolet absorption at a wavelength of 260 nm. The linear range of concentrations of oligonucleotides detectable by this method is 10 nmol/L to 20 µmol/L in plasma, and 2 nmol/L to 2 µmol/L in urine. Plasma and urine assays were validated to meet acceptance criteria for precision and reproducibility (percentage relative SD < 20%). Concentrations of full-length ISIS 3521 and major metabolites (shortened oligonucleotides of 19, 18, and 17 nucleotides in length) were calculated. To do this, the corrected area (area under the urine volume absorbency curve divided by migration time) of the appropriate electropherogram peak was determined, divided by the corrected area of the internal standard peak, and multiplied by the known starting concentration of the internal standard added. An additional correction was made for differences between the extinction coefficient of ISIS 3521 and those of the internal standard or 19-mer, 18-mer, or 17-mer metabolites.
Pharmacokinetic Analysis
Thirty-six patients received 99 cycles (775 infusions) of ISIS 3521 at doses ranging from 0.15 to 6 mg/kg/d for 3 days per week for 3 weeks and repeated every 4 weeks. All patients were assessable for toxicity. Patient characteristics are listed in Table 1. Thirty-four of the 36 patients previously received chemotherapy, immunotherapy, radiotherapy, or combinations of these modalities. The dose escalation schema, the number of patients treated at each dose level, and the number of cycles administered at each dose level are listed in Table 2. One patient required dose reduction from 5.0 to 4.0 mg/kg due to grade 2 thrombocytopenia with epistaxis. This patient had been heavily pretreated for non-Hodgkin's lymphoma. One patient's dose was escalated from 0.6 to 1.0 mg/kg (one cycle) and again to 1.5 mg/kg (two cycles) before being reduced again to 1.0 mg/kg at the patient's request due to grade 2 fatigue at the 1.5 mg/kg dose. Three patients were enrolled at each dose level, except for the 6.0 mg/kg dose level, which was expanded to six patients to gain further experience at the highest dose level studied. The median number of cycles administered per patient was two (range, one to 17 cycles).
Clinical Toxicity At the 5.0 mg/kg dose level, one patient each developed grade 2 and grade 3 thrombocytopenia. The patient who developed grade 2 thrombocytopenia had a prior history of extensive chemotherapy for low-grade non-Hodgkin's lymphoma. This patient began the study with a normal platelet count (187,000/µL) which remained above baseline level throughout cycle 1. The platelet count had declined to 129,000/µL by the start of cycle 2 and further declined to 74,000/µL (grade 2) on day 14 of cycle 2. The episode was associated with epistaxis, requiring dose reduction to 4.0 mg/kg. The patient was then treated at 4.0 mg/kg for seven additional cycles (nine cycles in all), during which time the platelet count nadir was 60,000/µL (cycle 3, day 14), but the platelet count was between 77,000 and 130,000/µL for the remainder of the treatment course, without further clinical sequelae. The patient who developed grade 3 thrombocytopenia entered the study with a platelet count of 131,000/µL, with a decline to 85,000/µL (grade 1) on day 14 of cycle 1. Platelets declined further to 45,000 and 39,000/µL on days 0 and 7, respectively, of cycle 2. On day 11 of cycle 2, this patient, who had sarcoma with multiple lung lesions, suffered hemoptysis and death attributed to tumor progression. Whether thrombocytopenia contributed to the episode is unclear, as a repeat platelet count was not obtained. PT and aPTT in this patient had not been elevated with ISIS 3521 administration. No other drug-related toxicity was observed. Adverse events that were judged as potentially related to ISIS 3521 are listed in Table 3.
Pharmacokinetics All 11 patient cohorts (n = 36) were studied for pharmacokinetics of ISIS 3521. Complete pharmacokinetic profiles were obtained on the first day of dosing, so the analysis represents only the first dose. A summary of pharmacokinetic parameters by dose level is included in Table 4.
Plasma concentrations of intact and total (shortmers plus intact) ISIS 3521 generally increased with dose over the range studied. The maximum concentration (Cmax) was observed at the end of the infusion or shortly thereafter. The clearance of ISIS 3521 decreased with increasing dose, leading to a dose-dependent increase in the elimination half-life and AUC. The maximum observed plasma concentration (Cmax) of intact ISIS 3521 increased approximately 50- to 60-fold over a 40-fold increase in dose (0.15 to 6 mg/kg) consistent with a nonlinear decrease in plasma clearance (Fig 1). The average Cmax after a single 2-hour infusion of 6 mg/kg of intact ISIS 3521 was 29.4 ± 8.33 µg/mL (Table 4), and the mean volume of distribution (VSS) was 141 mL/kg.
Four of six patients treated at 6 mg/kg exhibited Cmax values of intact ISIS 3521 that exceeded 30 µg/mL and approached the concentration associated with complement activation in primates (40 µg/mL). At the highest dose tested, 6 mg/kg, mean Cmax values ranged from 14 to 36 µg/mL (approximately 2 to 5 µmol/L). No accumulation of oligonucleotide was noted in the plasma, as indicated by the lack of pre-dose plasma levels of ISIS 3521 and the absence of increases in Cmax with repetitive dosing (data not shown). The principal oligonucleotide species detected in the plasma was full-length intact ISIS 3521, which predominated at all time points and represented between 45% and 65% of the total measurable oligonucleotide at the end of the 2-hour infusion (Fig 2). The primary metabolite was ISIS 3521 shortened by one nucleotide (referred to as n-1). Other chain-shortened metabolites were also detected and were present in decreasing concentrations in the order of the number of nucleotides deleted, ie, n-1 more than n-2 more than n-3, down to a minimum of n-12. The pattern of metabolite formation and proportion of chain-shortened oligonucleotides did not change with increasing dose or with repetitive dosing. Less than 1% of the administered dose was excreted in urine after 24-hour collection (data not shown).
Coagulation/Complement Studies
Antitumor Activity Case report of patient no. 106. Patient no. 106 is male, 68 years old at the time of study entry, and was diagnosed with low-grade well-differentiated lymphocytic lymphoma when he presented with diffuse adenopathy in July 1990. He had a partial response to six cycles of combination chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (two courses) and cyclophosphamide, methotrexate, vincristine, procarbazine, and prednisone (four courses), then he was treated with pulse chlorambucil/prednisone until June 1991. Disease remained stable until July 1993, then slowly progressed over the ensuing 15 months. From October 1994 until May 1995, he was treated with fludarabine, mitoxantrone, and dexamethasone. However, his disease progressed, and in August 1996 he entered the present trial, with thoracic and abdominal adenopathy but no marrow involvement at study entry. After five cycles at 0.6 mg/kg, improvement in thoracic adenopathy was observed, and the dose was escalated to 1.0 mg/kg for cycle 6 and to 1.5 mg/kg for cycles 7 and 8. At the patient's request (due to grade 2 fatigue), the dose was reduced to 1.0 mg/kg for cycle 9 and remained at that level through cycle 17 (February 1998). At that time, adenopathy had resolved (Fig 4), and bone marrow aspirate and biopsy indicated no evidence of lymphoma. Treatment was discontinued. Reevaluation 3 months later revealed no clinical or radiologic evidence of relapse, and the patient has remained free of clinically evident relapse for an additional 12+ months.
Case report of patient no. 124. Patient no. 124 is a 68-year-old female who was diagnosed in February 1980 with nodular poorly differentiated lymphocytic lymphoma involving thoracic and abdominal nodes and bone marrow. She received multiple treatment regimens from February 1980 to March 1997, including cyclophosphamide, doxorubicin, vincristine, and prednisone plus bleomycin, interferon alfa-2a, vincristine, etoposide, chlorambucil, cytarabine, radiation therapy to local adenopathy, cisplatin, methotrexate, radiolabeled antibody therapy, autologous peripheral-blood stem-cell transplantation, fludarabine, and the etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin regimen. Marrow involvement (15% to 30%) with lymphoma was persistent through prior therapy, and at study entry (March 1997), the patient also had thoracic, cervical, abdominal, and pelvic adenopathy. She received two cycles of ISIS 3521 at 5.0 mg/kg before requiring dose reduction to 4.0 mg/kg for grade 2 thrombocytopenia with epistaxis during cycle 3. She remained at the lower dose through a total of nine cycles of treatment. Computed tomography scans of the neck, chest, abdomen, and pelvis did not reveal abnormal adenopathy, and bone marrow aspirate and biopsy showed no evidence of lymphoma. Further therapy was withheld, and 4 months later, a new subcutaneous lesion (1.3 cm x 2.3 cm) was resected, revealing recurrent lymphoma. No further disease has been clinically evident, and no additional therapy has been administered for an additional 4+ months.
Clinical development of ISIS 3521 was pursued on the basis of the ability of the compound to specifically inhibit PKC- , in addition to its broad spectrum of antitumor activity and tolerable toxicity profile in preclinical studies. The choice of PKC- as a target is supported by preclinical studies, which have demonstrated a relationship between cytoplasmic serine/threonine kinases such as PKC- and the processes of tumor promotion and carcinogenesis.8-10,15,17-21 Selection of the thrice-weekly administration schedule was made on the basis of the plasma and tissue half-life data from animal studies and the observation of superior antitumor activity in human tumor xenografts with repeated dosing.
Complement activation by phosphorothioate oligonucleotides, observed and in some cases associated with death in primates,22,24-26 raised concern about the potential for similar events in humans. Therefore, a conservative starting dose was chosen for the present trial. However, the toxicities associated with ISIS 3521 in this trial were mild, occurred predominantly in the peritreatment period, and consisted of nausea, vomiting, fever, and chills. These were similar to toxicities observed in clinical trials of other phosphorothioate oligonucleotides.29,30,33 Increases in the aPTT and the complement split product C3a were observed in the absence of clinical sequelae or effects on the PT or the C5a complement split product. The highest dose studied, 6 mg/kg, did not exceed the MTD. A conventional MTD was not achieved in this study because of the attainment of plasma concentrations at the 6 mg/kg dose level (> 30 µg/mL) that approached those associated with complement activation in primates ( To characterize the pharmacokinetic profile of ISIS 3521, an analytic assay was used that consisted of an initial two-phase extraction procedure followed by CGE.32 This method allows for the detection of intact oligonucleotide and is also able to quantify the chain-shortened metabolic products. The previous method for determining pharmacokinetic analysis of phosphorothioate oligonucleotides relied on radiolabeled tracer analysis.22,34-36 Whereas oligonucleotides with phosphodiester backbones were subject to rapid degradation by 3'-exonucleases present in human serum, the phosphorothioate modification has improved stability without sacrificing activity.15 Our results indicate an initial plasma elimination half-life of 92 minutes at the 6.0 mg/kg dose level, with 45% to 65% of full-length ISIS 3521 present at 2 hours. The short plasma half-life has been shown to be related to rapid tissue uptake followed by slow clearance from tissue due primarily to continued nuclease metabolism in nonhuman primates.37 The slow clearance from tissue predicts that ISIS 3521 will accumulate in some tissues upon repeated administration. The similarity between the human and primate data suggests that a saturable distribution process, suggested by the primate results, may be present in humans. In addition, the ability to achieve similar plasma concentrations according to body weight alone suggests that the plasma disposition may be driven by a weight-linear process (eg, organ distribution), rather than a typical surface-area disposition, such as renal excretion or metabolism. Elimination of ISIS 3521 seemed principally due to tissue distribution and metabolism. Preclinical studies of oligonucleotides support tissue metabolism by exonucleases, without measurable recirculation of intact oligonucleotide after initial tissue distribution. No plasma accumulation was noted during repeat administration, and the metabolite formation pattern was similar across doses. Although more prolonged infusions of ISIS 3521 could be beneficial, antitumor activity was observed with the intermittent schedule used in this trial. Despite the advanced disease stage of patients entered, it was encouraging that responses were observed in two patients with heavily pretreated non-Hodgkin's lymphoma who are free of clinically evident disease more than 1.5 and 2 years after therapy was initiated. Antitumor activity has also been observed in a phase 1 trial of ISIS 3521 administered by continuous infusion.38 These results are in contrast to prior trials of antisense oligonucleotides in cancer, which had not demonstrated objective responses, although evidence of biologic activity had been reported in a trial of an oligonucleotide against bcl-2 in non-Hodgkin's lymphoma.39 This phase I trial was not designed to investigate potential mechanisms of antitumor activity of ISIS 3521. Although oligonucleotides with a CpG motif have been reported to have immunostimulatory effects, the sequence of ISIS 3521 does not include the palindromic sequence that has been reported as being required for maximal immunostimulation.40 Further, studies assessing the effect of ISIS 3521 on murine B and NK cells showed little stimulatory effect,41 and immunostimulation with ISIS 3521 in vivo was limited to mice, with little or no evidence of immunostimulation being observed in primate toxicology studies.37 Finally, clinical signs or symptoms suggestive of immune stimulation were absent in the patients treated on the present trial. Thus we believe that the antitumor effects noted in the present trial were unlikely to be due to a nonspecific biologic effect and were more likely due to a specific antisense effect. However, further clinical studies will be needed to elucidate the mechanism of action of ISIS 3521 in patients. The results of this phase I trial demonstrate that, despite prior concerns surrounding the use of antisense oligonucleotides to treat cancer, such an approach is in fact feasible and promising. By the schedule used in this trial, a dose of 6 mg/kg is recommended for future trials. Because interference with the signal transduction pathways by agents such as ISIS 3521 may not result in tumor shrinkage as the predominant effect, future clinical development strategies should include assessment of end points such as time to tumor progression, which may be helpful in assessing this agent's potential efficacy. In addition, combining antisense targeting of a signal transduction pathway with standard cytotoxic chemotherapy is an attractive strategy, with the potential to enhance the activity of standard therapy, inhibiting proliferative signals or disrupting pathways that are downstream of DNA damage and resulting in conditions that favor apoptosis. Finally, we cannot rule out the possibility of using combinations of antisense molecules (at safe total oligonucleotide doses), which may be used in the future to target more than one gene product without increasing toxicity.
We thank Ana Petrovich for manuscript preparation.
ISIS 3521 (ISI 641A) is developed jointly by Isis Pharmaceuticals, Inc, and Novartis Pharma AG, Basel, Switzerland.
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Copyright © 1999 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
