Overview of Human Phase Ia/Ib Clinical Trial.

Nineteen patients were enrolled in the phase Ia clinical trial between May 2008 and September 2010 (SI Appendix, Table S1). Dose escalation from 3 to 30 mg/m² (dose is listed as the amount of siRNA) in 15 patients was carried out between May 2008 and May 2009 with no observed dose-limiting toxic events (DLTs). Then there was an approximately 1-y enrollment gap until September 2010. Upon trial resumption, the subsequent two patients in the 30 mg/m² cohort experienced DLTs. The final two patients in the phase Ia trial were treated at the previously well-tolerated 24 mg/m² dose level; however, these doses were not well tolerated by either patient.

Consequently, the clinical protocol was amended into a phase Ib to include an additional cohort with a modified dose schedule that was predicated on the idea that initial exposure to CALAA-01 at lower dose levels may desensitize patients to the innate immune response that might be responsible for the adverse events (SI Appendix, Table S1). Patients were to receive a lower dose of CALAA-01 for cycle 1 (18 mg/m²—well tolerated by patients during phase Ia) which, if there were no safety concerns, would be followed by higher doses (27 mg/m²) in subsequent cycles. This strategy was designed to improve the tolerability of CALAA-01 to patients while maintaining its potential efficacy. Five patients were enrolled in phase Ib between September 2011 and July 2012. Two patients in this cohort experienced DLTs, and a decision was made to end the study.

CALAA-01 Clinical Trial Patient Demographics and Tumor Response.

Table 1 and SI Appendix, Tables S2–S4, summarize the patient demographics. All clinical characteristics and demographics were similar across cohorts and dose levels. Twenty-four patients received at least one dose of CALAA-01. Mean duration on treatment was 36.6 d (median 28.4 d). Mean number of cycles completed was 1.8 (median of 2 cycles) (SI Appendix, Table S5). The primary reason for discontinuation from the study was progressive disease in seven (29%) patients as evidenced by increase in tumor size (SI Appendix, Table S6). During the study, 19 (79%) patients had at least one posttreatment scan and were considered evaluable for tumor response by RECIST v1.0. No objective tumor responses were observed; the best response per RECIST v1.0 criteria reported by the investigators was stable disease (SD) for 4 mo, a change of prior course, in one melanoma patient treated at the highest dose level. The patient received a total of six cycles of CALAA-01 at 30 mg/m² before discontinuing due to progressive disease.

Dose-Limiting Toxicities in the Clinic.

In cycle 1, two patients in the 30-mg/m² dose cohort experienced DLTs. One patient had metastatic prostate adenocarcinoma to the lungs and paraaortic lymph nodes and had previously received radiation therapy and progressed on bicalutamide. This patient developed grade 3 ischemic colitis, grade 2 diarrhea, and grade 1 fever and was immediately discontinued from the study. The second DLT occurred in a patient with metastatic melanoma who had previously received radiation therapy and high-dose IFN. The patient developed grade 4 fatigue, grade 2 flu-like symptoms, grade 2 muscular spasm, and grade 1 nausea and was immediately discontinued from the study. Both patients were hospitalized and fully recovered after receiving concomitant medication.

In phase Ib, the protocol was amended to include an additional cohort with a modified dose schedule (18 mg/m² in cycle 1 followed by 27 mg/m² in subsequent cycles) to possibly reduce the innate immune response, improve the tolerability, and maintain the potential efficacy of the study drug. Two patients in phase Ib experienced DLTs. One patient with undifferentiated small-cell carcinoma of the cervix with metastasis to the lungs experienced grade 3 hypersensitivity reactions after the third and fourth doses of 18 mg/m² in cycle 1, necessitating infusion termination during dose 4. Another patient with rectal adenocarcinoma with metastasis to the lungs, who previously received radiation therapy and five systemic therapies, showed grade 3 fatigue during cycle 1, and dose was reduced to 15 mg/m² on cycle 2 to minimize adverse events (AEs). Only two of the five patients enrolled in phase Ib completed cycle 1 and progressed to the 27-mg/m² dose level.

Overall Safety Profile.

All AEs are summarized in SI Appendix, Table S7. The most common treatment-related AEs of any grade with incidence in greater than 15% of patients were fatigue (n = 12), chills (n = 12), and fever (n = 10). Grade 3/4-related AEs occurring in multiple patients included lymphopenia (n = 3) and fatigue (n = 2). Grade 3/4-related AEs during CALAA-01 infusions included hypersensitivity, ischemic colitis, diarrhea, and fever (n = 1 each). Another treatment-related grade 3 AE included hyponatremia (n = 1). Possibly related serious AEs included grade 2 sinus bradycardia in one patient, grade 2 hematuria, and grade 1 pericardial effusion in one patient. Overall five (21%) patients discontinued study participation due to an adverse event. The ability to conclusively associate siRNA plasma concentration data with adverse events in this study was limited. Four patients experienced DLTs in cycle 1; the plasma concentrations observed in these patients were in the same range as others who did not experience DLTs. Furthermore, there was no correlation between elevated plasma cytokine levels and severity of patient AEs.

Pharmacokinetics of CALAA-01 in Humans and Animals.

Human pharmacokinetic (PK) data are illustrated in Fig. 2. We observed a rapid decline of the plasma concentrations of the siRNA component of CALAA-01 to below the limit of detection by 30 min after the end of infusion in almost all patients (Fig. 2A). These findings are consistent with the preclinical data in mice (13), rats, and monkeys (SI Appendix, Fig. S2A). Plasma siRNA concentration data after CALAA-01 infusion was obtained in multiple treatment cycles from seven subjects and appeared to be consistent within subjects across cycles. Fig. 2B shows the plasma siRNA concentration after four individual doses (across three cycles) of CALAA-01 in a single patient. The rate of plasma siRNA concentration decline after each dose was similar. There was no apparent accumulation of CALAA-01 siRNA upon multiple dosing in any patient.

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Fig. 2.

PK assessment of CALAA-01 in patients and across species. (A) Time course of average plasma concentration of the siRNA component of CALAA-01 following the end of infusion for all dosing cohorts. (B) Time course of plasma CALAA-01 siRNA following the end of infusion from cycles 1–3 of CALAA-01 from one patient who received 30 mg/m² CALAA-01. Plasma concentration curves are similar at each cycle. Lines connecting data points are guides for the eye only. (C) AUC and C_max relationship to dose in humans. (D) Scaling of CALAA-01 C_max across four different species.

Data shown in Fig. 2C demonstrate the relationship between the dose of CALAA-01 to the plasma siRNA area under the curve (AUC) and the maximal concentration (C_max) after i.v. infusion. AUC and C_max increased with dose, consistent with the preclinical data in rats and monkeys. C_max and AUC appeared to be linearly related in all species (SI Appendix, Fig. S2B). Fig. 2D illustrates the C_max dose scaling of CALAA-01 across four species (rat, dog, monkey, and human). These data were best fit when C_max was plotted against dose in weight (mg/kg) rather than dose in body-surface area (mg/m²). Conversion on mg/m² bases was carried out as proposed by Reagan-Shaw et al. (14).

CALAA-01 Causes Minimal Liver and Kidney Toxicity in Humans.

The primary toxicities observed in preclinical studies with CALAA-01 were liver and kidney toxicities as evidenced by dose-dependent elevations in liver enzymes, creatinine, and blood urea nitrogen (BUN) in rats and monkeys (SI Appendix, Figs. S3 and S4). The polymer delivery components of CALAA-01 (vial 1) when administered without siRNA (vial 2) induced similar elevations in liver enzymes, creatinine, and BUN; however, in monkeys, the full CALAA-01 formulation (vial 1 + vial 2) trended toward increased liver and kidney toxicity compared with the polymer delivery components alone. Postmortem tissue examination of monkeys and rats treated with CALAA-01 or polymer delivery components alone revealed similar pathologic changes in liver (increased monocytes/Kupffer cell hypertrophy) and kidney (tubular necrosis). These data suggest that the polymer delivery components of CALAA-01 were primarily responsible for the liver and kidney toxicity observed. To mitigate the possibility of kidney toxicity in patients, the treatment protocol included a pretreatment i.v. hydration bolus of 500 mL of 5% (wt/vol) dextrose in water before CALAA-01 infusions, and the patients were encouraged to drink 2–3 L of fluids per day while participating in the study.

The human serum chemistry data are presented in Fig. 3A. CALAA-01 treatment resulted in minimal and clinically insignificant dose elevations in serum aspartate transaminase (AST) and alanine amino transferase (ALT) (several patients had elevated liver enzymes at pretreatment), and no elevation in BUN or serum creatinine over pretreatment values was observed. When followed longitudinally over five cycles of therapy, serum AST and ALT were noted to increase at the start of each treatment cycle and return to baseline in the interim whereas serum creatinine remained stable (Fig. 3B).

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Fig. 3.

Minimal evidence for liver or kidney toxicity of CALAA-01 in humans. (A) Human serum liver enzymes and kidney markers plotted against dose level. Data are from various cycle dates 24 h post CALAA-01 infusion. Pretreatment values are presented to the right of the dotted lines. (B) Liver enzymes and kidney markers over 98 d in a patient that received six cycles of CALAA-01 at 30 mg/m². Lines connecting data points are guides for the eye only. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen.

CALAA-01 Treatment Resulted in Dose-Dependent Platelet Count Decreases in Both Animals and Humans.

SI Appendix, Figs. S5 and S6, provide data that summarize the effects of CALAA-01 treatment on monkey and rat coagulation parameters and platelet count. CALAA-01 treatment in both rats and monkeys resulted in transient prolongation of activated partial thromboplastin time (APPT) and a marked drop in platelet count. Fibrinogen levels were depressed following CALAA-01 treatment in monkeys, but were elevated following treatment in rats. Prothrombin time (PT) was unaffected in either species. Treatment with the CALAA-01 polymer delivery components alone induced identical changes in these coagulation parameters, except for PT in monkeys, which was elevated following polymer delivery component treatment but not CALAA-01 treatment. These data suggest that the polymer delivery components of CALAA-01 were likely responsible for alterations in coagulation parameters and platelet counts following CALAA-01 infusion.

Fig. 4 A and B summarize the effects of CALAA-01 treatment on human coagulation parameters and platelet count. As in the preclinical animal models, CALAA-01 treatment resulted in marked dose-dependent, but clinically insignificant, decline in platelet counts; however, other coagulation parameters (APPT, PT, fibrinogen) where unaffected. When followed longitudinally over four cycles in a single patient (30 mg/m² doses), platelet count initially declined over cycles 1 and 2; however, it then recovered over the subsequent two treatment cycles. No long-term effects on APPT, PT, or fibrinogen levels were observed in this patient.

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Fig. 4.

Effects of CALAA-01 on human coagulation and complement. (A) Coagulation values plotted against dose level of CALAA-01. Data are from various cycle dates 6 h post CALAA-01 infusion for PT and APPT and 24 h post infusion for platelets and fibrinogen. Pretreatment values are presented to the right of the dotted lines. (B) Coagulation values over 65 d in a patient that received four cycles of CALAA-01 at 30 mg/m². Lines connecting data points are guides for the eye. (C) Complement levels plotted against dose level of CALAA-01. APPT, activated partial thromboplastin time; CH₅₀, total hemolytic complement capacity in serum; PT, prothrombin time.

CALAA-01 Treatment Did Not Alter Serum-Complement Function in Animals and Humans.

SI Appendix, Table S8, provides data that summarizes the effects of CALAA-01 treatment on monkey serum complement activity. Plasma Bb (activated complement factor B) is a specific and sensitive marker for activation of the alternative complement pathway, which has been shown to be most appropriate for use in detecting complement activation induced by oligonucleotides, as this class of molecules has been shown to specifically induce alternative pathway activation (15). No appreciable elevation in serum Bb was observed over baseline increases (baseline increases result from restraining the animals for treatment). CH₅₀ reflects total hemolytic complement capacity and is measured ex vivo; it reflects the residual capacity of the entire system to form membrane attack complexes when triggered ex vivo, and the occurrence of a complement activation event in vivo will therefore result in a decrease in CH₅₀ (i.e., due to consumption of complement factors). A marked reduction in CH₅₀ for most of the polymer delivery component-treated monkeys was observed at the end of infusion with complete or nearly complete recovery by 6 h postinfusion. There were also reductions in CH₅₀ for several CALAA-01–treated animals, but there was no clear dose dependence in terms of the incidence of animals with distinct decreases in CH₅₀ or the group mean values. These data suggest that the polymer delivery components’ ability to activate complement is mitigated when formulated as the CALAA-01 nanoparticle, consistent with previously published in vitro complement activation studies (10).

Fig. 4C shows data that summarize the effect of CALAA-01 treatment on human complement activity. No appreciable change in level of Bb split product or CH₅₀ activity was observed following CALAA-01 dosing in humans. Of note, a single patient in the 24-mg/m² dose cohort had CH₅₀ pretreatment values at near 0 and had a large relative drop in CH₅₀ after the day 22 dose; however, the absolute change was minimal.

Effects of CALAA-01 Treatment on Other Hematologic Parameters in Animals and Humans.

SI Appendix, Figs. S7 and S8, provides data that summarize the effects of CALAA-01 treatment on monkey and rat hematologic parameters. In both species, dose-dependent decreases in serum hemoglobin and red blood cell count were observed following CALAA-01 treatment. These effects were similar in groups dosed with the polymer delivery components alone. In monkeys, treatment with both CALAA-01 and polymer delivery components alone resulted in reduced reticulocyte counts (a measure of new red blood cell production). These data suggest that, in monkeys, the polymer delivery components of CALAA-01 interfere with new red blood cell production, thereby preventing replenishment of the red cells lost following the repeated phlebotomy necessary to obtain the serum toxicology specimens. Dose-dependent white blood cell count elevations were also observed in monkeys and rats treated with either CALAA-01 or polymer delivery components alone.

SI Appendix, Fig. S9, shows data that summarizes the effects of CALAA-01 treatment on human hematologic parameters. No dose-dependent effects on serum hemoglobin, red blood cell count, or lymphocyte counts were observed. A single patient receiving 24 mg/m² of CALAA-01 was found to have severe anemia that was possibly related to CALAA-01 treatment. Two subjects in the phase Ib cohort were found to have lymphopenia that was deemed probably treatment related.

Mild, Dose-Dependent Elevations in Serum Cytokines Following CALAA-01 Treatment Were Observed in Animals and Humans.

The siRNA component of CALAA-01 is not chemically modified. Much preclinical work has demonstrated that nonchemically modified siRNAs can be immunostimulatory under certain conditions (16). Specifically, systemic administration of siRNA with lipid carriers can cause significant immunostimulation. However, when administered intravenously alone, nonchemically modified siRNAs do not necessarily result in immunostimulation in mice (17). Additionally, other cationic polymer-based siRNA/nanoparticle systems have been shown to not elicit a pronounced immune response in vivo (18). Preclinical data characterizing the immune response to CALAA-01 dosing in monkeys has previously been reported (19). Elevated levels of IL-6 were observed at the highest dosing group (27 mg/kg). Increasing amounts of IL-12 and IFN-γ were observed in one monkey at 9 and 27 mg/kg, respectively. However, the increases in these cytokines were not quantitatively impressive or suggestive of intensive immunostimulation.

Fig. 5A shows data that summarize the assessment of CALAA-01 treatment-induced immune responses in humans. Plasma and serum were obtained from study patients at prescribed time points and analyzed for cytokines (IL-2, IL-4, IL-6, IL-10, IL-12p40, IL-12p70, TNF-α, and IFN-γ). These results indicate apparent treatment-related elevations in some cytokines—namely IL-6, IL-10, TNF-α, and IFN-γ. No other cytokines showed any apparent trends or changes in response to CALAA-01 administration. These elevations were all transient in nature, with cytokine levels returning to baseline by 24 h after the end of infusion in all cases (SI Appendix, Fig. S10). Fig. 5B shows the serum IL-6, IL-10, TNF-α, and IFN-γ levels post dose over six cycles of treatment in a single patient (30 mg/m² doses). IL-6 response attenuated with each dose whereas IL-10 response increased upon continued dosing. IL-6 and TNF-α are each indicative of an inflammatory response; the fact that both were transiently elevated in some patients suggests that CALAA-01 induces an inflammatory response in some patients at some of the higher dose levels. IL-10 is indicative of a T helper 2 (Th2)-mediated immune response; however, IL-4 is indicative of the same type of response, and CALAA-01–dependent changes in IL-4 levels were not observed. Overall, the results suggest that CALAA-01 may induce a Th2-mediated immune response in some subjects at some of the higher dose levels examined in the clinical study.

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Fig. 5.

CALAA-01 treatment resulted in mild dose-dependent immunostimulation. (A) Various cytokine levels, expressed as ratio over baseline, plotted against CALAA-01 dosing level. (B) Cytokine levels over six cycles of CALAA-01 treatment at 30 mg/m² in a single patient. Data from each cycle were collected immediately before CALAA-01 infusion and 2, 6, and 24 h post infusion. The x axis is labeled as cycle number (C#), overall time since treatment initiation (D#), and hours post infusion as appropriate.