Keywords

CIGB-300; Refractory tumors; Tolerability; Drug accumulation ratio; Survival

Introduction

Chemoradiotherapy is the widely deemed gold standard care for cancer patients since many years ago albeit a relative limited therapeutic index is observed in solid tumors [1]. Particularly, intrinsic drug resistance and relapse constitute the mayor problem to envision a rationale therapeutic regime [2]. Therefore, the use of the dubbed molecularly targeted drugs combined to standard chemoradiotherapy in cancer patients could be a right path to outreach tumor complexity ensuing better clinical outcome [3,4]. Among the newcomer oncology targets, experimental validation of CK2 has experienced tangible progress by using specific inhibitors comprising small molecules [5-7], antisense oligonucleotides [8] and synthetic peptides [9,10]. As CK2 is responsible about near 24% of the cellular phosphoproteoma and it is linked to physiopathology from many solid and hematopoietic tumors, the CK2 inhibitors could be appraised as promising anticancer drug candidates. However, only two of them, the CX-4945 and CIGB-300 are being tested in the clinical ground in cancer patients.

CX-4945 (Silmitasertib) is an ATP-competitive inhibitor of both CK2α and CK2α’ catalytic subunits [7] whose Maximum Tolerated Dose (MTD) has been investigated in combination with gemcitabine plus cisplatin in a Phase I trial (ClinicalTrials.gov Identifier: NCT02128282). A subsequent Phase II study is currently carried out to investigate the antitumor activity of CX-4945 combined with gemcitabine and cisplatin in cholangiocarcinoma patients at the combination previously determined. Furthermore, CIGB-300 is a synthetic peptide that impairs the CK2-mediated phosphorylation by targeting the substrate phosphoaceptor domain outcompeting the enzyme [9,11] and elicits antitumor activity when tested in preclinical animal models by either intratumor injections [9,12] or systemic delivery [12]. Importantly, inhibition of metastasis and tumor angiogenesis has been also documented in vivo by using systemic administration of CIGB-300 in a mouse model [13]. Molecularly, CIGB-300 chiefly binds and inhibits the B23/nucleophosmin (B23/NPM) phosphorylation with subsequent cellular events indicating nucleolar disassembly [11]. However, recent data in chronic lymphocytic leukemia (CLL) have shown that an inhibition of the CK2-mediated phosphorylation is also observed in substrates other than B23/NPM like AKT, PTEN and GSK-3 proteins [14]. Importantly, synergistic interactions between CIGB-300 and conventional chemotherapeutic drugs like cisplatin and paclitaxel and other cancer targeted therapies like Erlotinib have been documented at the preclinical stage [15,16]. In the clinical ground, CIGB-300 has been tested in Phase I clinical trials in cervical malignancies [17-19] where a thorough optimization of the intralesional delivery allowed the design of an appropriate protocol to be further accomplished in a Phase II trial. Briefly, injection of 70 mg of CIGB-300 in two sites into tumors leads to the highest tumor uptake. Also, a systemic allergic-like syndrome, well-correlated with plasmatic histamine levels, was the most relevant systemic side effect reported after intralesional injection of CIGB-300. However, no previous clinical studies using the intravenous route for the CIGB-300 delivery had been performed so far.

We designed a phase I study of CIGB-300 intravenously administered in patients with relapsed/refractory solid tumors with an Eastern Cooperative Oncology Group (ECOG) performance status of 3-4 and life expectancy ≤ 6 months. The primary objectives of this study were to test safety and tolerability of CIGB-300. Secondary objectives were to explore the drug accumulation ratio in plasma and clinical benefit was defined as any disease stabilization and increase of survival beyond 6 months.

Materials and Methods

Patient selection

Sixteen patients with progressive disease and refractory or relapsed solid tumors (≥ 1 cycle of chemotherapy) were recruited and verified for eligibility in the oncology service from Hermanos Ameijeiras Hospital, Celestino Hernández Hospital and Center for Medical-Surgical Research. Patients were included if they were 18 to 75 years old and gave their written, informed consent to participate and if their relapse was at least 4 weeks after the last chemotherapy treatment. All of recruited patients exhibited ECOG 3-4 with ≤ 6 months of life expectancy. Exclusion criteria were concomitant ingestion of immunosuppressive or immunomodulatory therapy, psychiatric disease, pregnancy, breastfeeding, uncontrolled chronic diseases such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, epilepsy, diabetes or hypertension, autoimmune disease, coagulation dysfunction and severe allergy with hives, dermatitis, bronchitis and persistent bronchial asthma. Patients entered into other clinical trials in the last eight weeks were also excluded.

This trial followed the principles of Good Clinical Practices, in accordance with the Declaration of Helsinki and its amendments. The protocol was approved by the Ethic Committees of the involved hospitals and by the Cuban Regulatory Authority, the State Center for the Control of Drugs, Equipment & Medical Devices (CECMED): reference number: 06.027.11.B. Cuban Public Registry of Clinical Trials: RPCEC00000112.

Study design and treatment plan

A randomized, open-label, uncontrolled, dose-escalating, sequential trial was performed. In the stage 1, an escalation protocol for determining the Maximum Tolerated Dose (MTD) was performed by the Continual Reassessment Method, starting from 0.2 mg/kg until 1.6 mg/kg of CIGB-300. Treatment allocation was planned from a dose-toxicity model that sets the subsequent dose to use in terms of the toxicity observed in the previous dose level ensuring that probability of severe toxicity did not exceed the maximum allowable dimension (33%). In the stage 2 of this study, a pharmacokinetic analysis was performed after infusions #1 and #5 during the first CIGB-300 cycle in four patients treated with the highest dose (1.6 mg/kg).

CIGB-300 administration was performed by 15 min intravenous infusions using appropriated pumps once daily on days 1-5 in alternate weeks until completing 3 cycles (15 doses). CIGB- 300 was synthesized and supplied by the Peptide Synthesis Unit, Center for Genetic Engineering and Biotechnology (CIGB), Havana, in 5, 15 or 35 mg vials, as lyophilized powder. Vials were reconstituted in 50 mL of saline solution to provide the appropriated dose in all the cases. Premedication with diphenhydramine and hydrocortisone 30 min prior to each CIGB- 300 administration was performed, in order to mitigate the expected allergic-like events, previously reported by intralesional route [17-19]. If necessary, other interventions were indicated for the management of adverse events according to established clinical practices.

Safety evaluation

Pretreatment evaluation included a detailed history and physical examination. In addition, hematological counts, blood chemistry, coagulation and imagenogical studies were performed. Systemic toxicity was evaluated during 24 h after each CIGB- 300 administration and further followed during 3, 6, 9 and 12 months after completing CIGB-300 treatment. Severity of adverse events was classified according to the Cancer Therapy Evaluation Program, Common Terminology Criteria [20] and they were subsequently classified by the WHO Adverse Reaction Terminology [21]. Causal relationship was classified as very probable (definitive), probable, possible or remote (doubtful) [22]. MTD was defined as the highest dose explored with <33% of severe toxicity. The CIGB-300-induced histamine levels were measured in plasma by a commercially available EIA kit (Labor Diagnostika, GmBH, Oberursel, Germany) before, 15 min and 24 h after the first infusion.

Drug accumulation ratio

To verify whether CIGB-300 accumulates after successive intravenous infusions, blood samples were taken immediately, 5, 15, 30 min and 1, 2, 3, 5, 7 and 12 h after the first and the fifth infusions in the first CIGB-300 cycle. Heparinized plasma was collected and CIGB-300 was quantified by an in-house validated immune competition-based enzyme immunoassay (EIA) [23]. Drug disposition evaluation was performed individually by non-compartmental analysis (NCA) and the pharmacokinetic parameters were calculated using the WinNonlin professional software (Version 5.1, Pharsight Inc., 2005, NC, USA). To calculate the drug accumulation ratio, both Cmax and AUC_0-12 values from infusions #1 and #5 were taken into account.

Clinical activity

Signs of CIGB-300 clinical activity were defined as any increase in the patient survival beyond their life expectancy (≤ 6 months). Survival time was followed up since the beginning of CIGB-300 treatment until patients die.

Statistical analysis

Clinical data were double entered and validated on MS InfoPath and then imported into SPSS for Windows (version 15.0, IBM Analytics 2006, Armonk, North Castle, NY, USA) for further analysis. Assumptions of normality and the homogeneity of variance were verified using Shapiro-Wilks test and Levene’s test, respectively. The probability of severe toxicity was estimated in each dose group conditioned to the number of subjects included sequentially. Also the probability of exceed the maximum level of acceptable toxicity (30%) and the upper bound of toxicity were estimated. Pharmacokinetic parameters obtained from plasma profiles after first and fifth infusions were compared by Student's t-test. Correlation between histamine and CIGB-300 levels in plasma and between histamine levels and severity of the allergic reaction were performed by Spearman rank correlation analyses. The survival time was estimated from the Kaplan Meier curve.

Results

Patient characteristics and dosing history

Sixteen enrolled cancer patients, 9 (lung cancer), 4 (breast), 1 (ovary), 1 (thymus sarcoma), 1 (skin squamous cell carcinoma), fulfilled the selection criteria. Baseline characteristics are shown in Table 1. Most of the patients were men and white and mean age was 56 years, ranging from 41 to 73 year-old. Mean weight was 70 kg, ranging from 46 to 108 kg. Fourteen patients completed the whole CIGB-300 treatment and patients HHA-03 and HHA-15 definitively interrupted it. Patient HHA-03 received just eleven administrations of CIGB-300 because of disease progression and patient HHA-15 got infection after the second CIGB-300 administration at the site of implantable system for drug delivery. During stage 1 two individuals were included sequentially in each dose level (0.2, 0.4 and 0.8 mg/kg) and 10 patients were allocated to the highest dose level (1.6 mg/kg) for the stage 2 of this trial.

Data are reported as number of patients (%) or mean ± standard deviation (range)

Table 1: Demographic and baseline characteristics of the cohorts.

Safety and tolerability

During the stage 1, neither MTD nor dose-limiting toxicity (DLT) were observed using this CIGB-300 monotherapy regimen. A total of 294 adverse events were recorded. Among them, mild side effects were the most common (61.6%), moderate (37.1%) and severe (1.4%). For moderate and severe adverse events, a dose-dependent effect was observed in terms of frequency. Otherwise, mild effect rate behave irrespectively to the CIGB-300 dose level. Near all the patients exhibited allergic-like reactions at some extent except patient HHA-07 from the highest dose level group (1.6 mg/kg) who curiously did not experience any adverse event. Particularly, itching (62.5%), allergic reaction (50%) and flushing (25%) were the most common adverse events (Table 2). Duration of all side effects was about 15 min with spontaneous resolution. The only two drug-related severe events (facial edema and itching) were also classified as allergic reactions in two patients allocated to the 1.6 mg/kg CIGB-300 dose. Both patients experienced spontaneous recovery so they did not interrupt the CIGB-300 treatment. However, two patients, CH-09 and HHA- 11, from the same cohort, had a fever episode and a drug nonrelated alimentary intoxication, respectively, which required treatment and temporary interruption of the CIGB-300 delivery. Patient CH-09 discontinued just one day when he started second cycle, whereas patient HHA-11 interrupted it seven days just after the first infusion, first cycle. Overall, rates and severity of adverse events decreased as successive CIGB-300 infusions were delivered during the entire regime (data not shown).

As previously reported for CIGB-300, the adverse events classified as “allergic-like reactions" (18) had a significant correlation (p<0.0001) with plasma histamine levels which in turn did correlate (p<0.0001) with the plasma CIGB-300 levels, 5 min after infusions. Overall, vital signs remained within normal ranges during treatment and it did no modify neither blood counts nor serum chemistry at any dose level (data not shown).

Table 2: Treatment-emergent adverse events.

Drug accumulation ratio

Similar clearance curves were observed on days 1 and 5 during the first cycle of CIGB-300 1.6 mg/kg (Figures 1A and 1B). Pharmacokinetic data summarized in Table 3 indicated no significant differences (p>0.05) respect CIGB-300 infusions from the first and fifth day. Accordingly, the RCmax and RAUC values near to 1.0 indicated no relevant accumulation of successive CIGB-300 i.v. deliveries from day 1 to 5. Furthermore, both t1/2 and total CL values did not experience significant changes among both CIGB- 300 infusions.

Figure 1: CIGB-300 average values after 15 min intravenous infusion of CIGB-300 1.6 mg/kg during the first cycle of treatment. Peptide levels were quantified by ELISA in plasma. (A): First administration (N=4); (B): Fifth administration (N=3). Standard deviations are showed.

Parameter	1st dose (N=4)	5th dose (N=4)	p (student t-test)
Tmax (h)	0.12 ± 0.09	0.083 ± 0.0	0.44
Cmax (µg/mL)	14.8 ± 2.7	14.6 ± 3.3	0.95
l  (h-1)	0.92 ± 0.17	0.92 ± 0.17	1
RCmax	--	1	--
t1/2 (h)	0.75 ± 0.12	0.77 ± 0.13	0.77
AUC0-12 (µg.h/mL)	11.4 ± 3.52	11.8 ± 4.22	0.91
RAUC	--	0.9997 ± 0.0006	--
MRT (h)	0.92 ± 0.50	1.06 ± 0.49	0.72
CL (L/h)	12.4 ± 7.4	12.4 ± 9.0	1
CL/kg (L/h.kg)	0.15 ± 0.05	0.15 ± 0.06	1
Vss (L)	8.95 ± 1.95	10.2 ± 1.32	0.38
Vss/kg (L/kg)	0.13 ± 0.05	0.14 ± 0.04	0.62

Abbreviations: Tmax: Time to Peak; Cmax: Maximum Observed Plasma Concentration; AUC: Area under Plasma Concentration-Time Curve from time zero to the last measurable time point; λ: Elimination Rate Constant; t_1/2: Terminal Half-Life; MRT: Mean Residence Time; CL: Total Plasma Clearance; Vss: Volume of Distribution at Steady State; RCmax: Accumulation Ratio of C_max; R_AUC: Accumulation Ratio of AUC
Data are expressed as mean ± standard deviation.

Table 3 :Main pharmacokinetics parameters estimated by NCA using plasmatic concentration data obtained from ELISA after first and fifth infusion of 1.6 mg/kg of CIGB-300, cycle 1.

Clinical response

On the intend-to-treat basis, 75% (12/16) and 43.7% (7/16) of patients were alive after 6 and 12 months respectively (Table 4). Lung cancer patients had a median survival of 11.4 months whereas for those patients who received 1.6 mg/kg survival reached 15.2 months. Interestingly, one patient with Lung Epidermoid Carcinoma lived >39 months post-CIGB-300 treatment with stable disease according to RECIST criteria [24]. Of note, the CIGB-300 regime significantly improved the respiratory symptoms and recurrent pneumonitis in a patient with thymic carcinoma exhibiting lung and pleura baseline metastasis who lived 27.0 months. CIGB-300 long-term toxicity was not observed in any patient (data not shown).

Patient	Target	Stage	CIGB-300	Survival after CIGB-300 start (months)
			Dose level (mg/kg)
HHA-01	Breast lobular carcinoma	IV	0.2	2.67
HHA-02	Undifferentiated NSCLC	IV	0.2	6.8
HHA-03	NSCLC	IV	0.4	1.97
HHA-04	Infiltrating ductal carcinoma of the breast	IV	0.4	12.97
HHA-05	NSCLC	IV	0.8	8.37
CH-06	Infiltrating breast lobular carcinoma with lymph node infiltration	IV	0.8	18.53
HHA-07	NSCLC	IV	1.6	10.47
CH-08	Skin epidermoid carcinoma	IV	1.6	14.97
CH-09	Moderately differentiated Lung Epidermoid Carcinoma	IV	1.6	39.3
HHA-10	Adenocarcinoma of ovary	IV	1.6	5.57
HHA-11	SCLC	IV	1.6	7.77
HHA-12	Mucoproductive adenocarcinoma of the lung	IV	1.6	4.7
HHA-13	Lung Epidermoid Carcinoma	IV	1.6	10.93
HHA-14	Carcinoma of Thymus with pericardium and lung infiltration	IV	1.6	27
HHA-15	Lung Epidermoid Carcinoma	IV	1.6	20.23
HHA-16	Breast lobular carcinoma	IV	1.6	12

Abbreviations: NSCLC: Non-Small Cell Lung Carcinoma; SCLC: Small Cell Lung Cancer

Table 4: Therapeutic response: Survival time after CIGB-300 monotherapy.

Discussion

Patients with relapsed/refractory solid tumors are very difficult to treat and have considerable unmet need for novel treatments. This dose-escalation Phase 1 study is the first in describing safety, tolerability, pharmacokinetics, and clinical effect of CIGB-300 using the i.v. route in such a kind of patients. Of note, seven patients (43.7%) experienced survival increase >12 months and two patients lived 39.3 and 27.0 months after CIGB-300 monotherapy, respectively.

Despite the unbalanced amount of patients included in each clinical niche, lung cancer patients trended to have a better clinical response profile to CIGB-300 in terms of survival. Interestingly, in patients included in the expansion phase who received the highest CIGB-300 dose, the median of survival time was about 15 months. Such increase beyond the expected limit (≤ 6 months) is an encouraging finding for these kinds of patients who had already failed prior regimens and exhibited ECOG 3-4. Of note, 3/4 breast cancer patients lived 12 months or more.

Importantly, the safety profile for the CIGB-300 intravenous delivery in both the dose-escalation and expansion phases were manageable up to 1.6 mg/kg and consistent with previous clinical observations reported for this experimental drug by intralesional delivery [17-19]. In both phases of the study the most common adverse events were itching, allergic-like reaction and flushing. Neither DLT nor MTD were observed despite the dose-dependent effect in terms of frequency and magnitude of moderate/severe events (grade ≤ 3). Otherwise, mild effects were irrespective to the CIGB-300 dose level. The previously reported correlation between transient CIGB-300-induced histamine and peptide levels in plasma [18,19] was similarly observed in this study supporting the appearance of the above-mentioned histamine related events. Interestingly, the C_max and AUC values indicated no relevant accumulation ratio from days 1 to 5 which suggest that CIGB-300 can be also tested in more intensive regimens based on twice daily administrations. As expected for nude peptides, the CIGB-300 exhibited appropriate volume of distribution and a relatively short half-life after intravenous delivery (45 min). However, considering that CIGB-300 internalizes tumor cells as soon as 3 min [25], we would expect that the CIGB-300 intravenously administered can efficiently target circulating tumor cells in blood stream thus preventing implantation and growth of new metastasis in these patients. Supporting this notion, in two different murine metastasis models the i.v. administration of CIGB-300, it significantly reduced the tumor cell dissemination and lung colonization [13]. Likewise, the antiproliferative effect of CIGB-300 has been also confirmed and molecularly characterized on in vitro models [26,27].

Conclusion

In conclusion, the CIGB-300 intravenous delivery as monotherapy was safe, well tolerated and exhibited efficacy signs in patients with relapsed/refractory solid tumors using this clinical protocol described here. Particularly, the 1.6 mg/kg dose of this peptidebased drug merits to be tested in Phase 2-3 trials in NSCLC patients where either once- or twice-daily administrations can be taken into account.

Acknowledgment

The following are members of the clinical trial team: Ivelisse Nuvia-Fleites (“Hermanos Ameijeiras” Hospital), Leovaldo Álvarez- Falcón, Lissandra Méndez-Sánchez (CIGB-300 Research and Development Group), Ubaldo Vásquez-Estévez, Tania Bartomeu Villamil, Luis J López-Carrazana (“Celestino Hernández” Hospital), Mauricio Catalá-Ferrer, Marlén Izquierdo-González (Center for Medical-Surgical Research).

We thank to Drs. Sergio Martínez, Jorge Rodriguez and Luis Castillo and the nurses Yorinko Chacón-Díaz and Ada I Zamora- López, for their valuable contribution in the clinical work. Also we thank to Yunia Delgado, Cimara Bermúdez, Grettel Melo, Ketty Cruz, María A Delgado, Silvia Barcelona, Marisol Cruz, Lourdes Hernández for their logistic assistance and Matilde López, Osvaldo Acosta, Hilda Garay for their contribution in the drug characterization and formulation. This study was supported by HeberBiotec S.A, Havana (product, insurance, reagents), by Biorec and by the Ministry of Public Health of Cuba (hospital facilities and general medical care of the patients).

Competing Interests

Authors LGM, IGG, IRC, CMVS, VRN, YPN, BEAC, VLMG and SEPR are employees of the Center for Genetic Engineering and Biotechnology (CIGB), Havana network, where CIGB-300 peptide was conceived and their formulation developed. DFA and RG are employed by Biorec. SEPR is the main author of the patent WO 03/054002 and US patent # 7374767 that sustain this project. The remaining authors declare no conflict of interests.

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