Background
Despite a downward trend of mortality rates
Whereas the primary tumor can, in most cases, be efficiently treated by a combined therapeutic approach, preventing the metastatic spread of the disease is often not effective. The eradication of disseminated tumor cells present in the blood circulation and micro-metastases in distant organs therefore represents another promising approach of cancer immunotherapy. The immune system could recognize tumor-specific antigens, and eliminate cancer cells. Furthermore it has been highlighted that some cancers, such as colorectal cancer, cause direct inhibition of the host's immune response with a detrimental effect upon prognosis suggesting that stimulation of the immune system might offer a therapeutic strategy to counteract these effects
Toll-like receptors (TLRs) are important sensors of foreign microbial components as well as products of damaged or inflamed self-tissues. TLRs are a family of type I transmembrane proteins that are the major pattern recognition receptors, and using TLR proteins, host can recognize the conserved molecular structures found in pathogens called pathogen-associated molecular patterns (PAMPs). In addition to their recognition of PAMPs, several TLRs have also been shown to recognize endogenous ligands associated with inflammation, which have been termed danger-associated molecular patterns (DAMPs). Then, TLRs have an important role in maintaining tissue homeostasis by regulating the inflammatory and tissue repair in responses to injury. There are 13 TLRs described in mammals with a broad expression profile but TLR11, 12 and 13 are not expressed in the human genome. TLR1, TLR2, TLR4, TLR5 and TLR6 reside in the plasma membrane and recognize extracellular ligands
LPS are major cell wall components of Gram-negative bacteria and were found to be able to induce regression of several tumor types in animal models
OM-174 is an analogue of lipid A that exerts, in experimental models, anti-tumoral effect against different tumor types including colon and breast cancers and melanoma
Based on these experimental data showing that several administrations are needed to achieve antitumor effect, as well as on the overall favourable safety profile of lipid A analogues observed in phase I studies
This new phase one was deemed necessary as animal data suggested that repeated administration of OM-174 were needed to observe anti-tumor effect, with a maximal effect observed after 15 injections. This study also aimed to assess the pharmacokinetic profile and the biological response induced by OM-174 in the multidose administration schedule.
Methods
This was a single center phase I study conducted in the clinical research unit (Centre Georges-François Leclerc - INSERM CIC-P 803) in Dijon France. The study protocol and its amendments were approved by Ethics Committee and Health Authorities (NCT01800812 (ClinicalTrials.gov Identifier)). The trial was designed according to the current Declaration of Helsinki
Patients selection
Patients were eligible if they were ≥ 18 years, had histologically proven metastatic solid tumor with measurable progressive disease outside the central nervous system for which no standard curative measures exist. Other requirements included predicted life expectancy ≥ 3 months; a WHO performance status (PS) ≤ 2, no severe or uncontrolled medical conditions; adequate hematologic count), no severe liver impairment and adequate renal function according to international standard values.
Patients must have recovered from adverse events due to agents administered > 4 weeks earlier or ≥ 6 weeks for nitro urea or mitomycine C.
Main non-inclusion criteria were medical history of cardiac disorders; medical history of auto immune disease and corticosteroids use.
Study design and dose escalation scheme
Based on animal models suggesting that maximal anti-tumoral was achieved with 15 infusions it was decided to include the number (Nb) of infusions in the definition of Dose Levels to confirm the good tolerance of a growing number of OM-174 injections. A standard phase I design was thus used with 3 to 6 patients to be included per each OM-174 infusion dose level (DL: 600, 800 and 1000 μg/m2) and each number of infusions (5, 10, 15). Patients were observed for the first 2 weeks before the next dose escalation was deemed possible. The MTD was defined as the DL at which two or more out of 3 or 6 patients of a DL developed a dose-limiting toxicity (DLT). The recommended dose (RD) for further clinical testing, defined as the DL below the MTD, had to be assessed in at least 6 patients.
Dose Limiting Toxicities (DLTs)
DLTs were defined using the Common Terminology Criteria for Adverse Events version 2.0 (CTCAE) as any of the following events deemed attributable to OM-174, i.e. occurring during the time of the deliverance of this drug and a follow-up period of 30 days: Grade 4 neutropenia lasting 5 or more consecutive days; febrile neutropenia; neutropenic infection; Grade 4 thrombocytopenia or Grade 3 thrombocytopenia with bleeding requiring platelet transfusion; Grade 4 anaemia, any Grade 3 or greater drug related non haematological toxicity (except nausea, vomiting, alopecia).
Study drug administration
In this open-label, nonrandomised phase I study, with double dose escalation protocol, OM-174 was administered intravenously as a 15 minutes infusion twice weekly at repeated intervals of 3 and 4 days. OM-174 was manufactured by OM Pharma Pharmaceuticals, Inc (Geneva, Switzerland).
Based on the results of a previous unpublished phase I study of single administration of OM-174 to cancer patients where only one serious adverse event, a severe transient hypoxia, occurred at the dose level of 800 μg/m2, with no other safety concerns with OM-174 dose up to 1300 μg/m2, it was decided that all patients would have to receive a first administration of 600 μg/m2 to reduce the risk of severe reaction. The dose escalation protocol and the dose levels are depicted in Table
Pre-treatment evaluation and follow up
At baseline, physical examination and routine laboratory tests were performed and WHO PS determined. Complete blood cell count was obtained on a weekly basis. Biochemical profile was performed at inclusion and each drug administration. All toxicities were graded using the CTCAE version 2.0. Each patient had to receive the full course of treatment, except in case of early progression, unacceptable toxicity, serious intercurrent illness, or patient refusal. Tumor measurements were performed at baseline, at the end of the treatment (i.e 3, 5 and 8 weeks after the beginning of treatment in the cohorts with 5, 10 and 15 infusions respectively). In case of control of disease, tumor assessment was performed on a monthly basis. Responses were assessed using RECIST
Pharmacokinetic assessment
Blood samples for pharmacokinetic (PK) of OM-174 were drawn at pre-dose, 1 h, 2 h, 4 h, 24 h for the first infusion and at pre-dose, 1 and 2 h for the 2nd, 3rd, 5th, 9th and 15th infusions, when appropriate. For all other interim injections, two blood samples were collected, before and at the end of IV-infusion.
PK parameters assessed were Cmax, Tmax, area under the curve (AUC) Last, and AUC0-inf., systemic clearance (Cl), volume of distribution (Vdβ) and elimination half-life (t1/2_lambda_z).
Cytokines measurement
Blood samples were collected for cytokine dosage (IL-1β, IL-6, IL-8, IL-10, IL-12, TNF-α) at the same time as samples for pharmacokinetic analysis. Cytokine measurements were determined by Cytometric Bead Array (CBA; BD Biosciences). Serum of patients were incubated with labeled capture beads and detection reagent for 3 h in the dark at room temperature and were analyzed with a flow cytometer (FACSCalibur; BD Biosciences) using the respective CBA Analysis software (BD Biosciences) and Bender MedSystems software. Each experiment was performed in duplicate to ensure for reproducibility of results, as previously described
Natural Killer activity analysis
Blood samples for NK cell activity analysis were collected before and 2 h after the first and the 2nd, 3rd, 9th and 15th injections. NK cells were isolated with lymphocytes from blood by density gradient centrifugation (Unicep, Novamed, Jerusalem) and purified with CD56 coated magnetic microbeads (Miltenyi Biotec). They were incubated with or without recombinant IL-2 for 40 h, then incubated in 96-well plates for 4 h with K562 human leukemia cells or Daudi cells which were used as NK and LAK target cells (TC), to yield a NK :TC ratio of 10. NK cytotoxicity was classically calculated as in chrome test assay.
TLR 4 polymorphism analysis
The analysis of 2 single nucleotide polymorphisms of TLR TLR4D299G and TLR4T399I was performed as previously described by Ducloux et al.
Prior to polymorphism analysis, gDNA was extracted from serum and amplified using the Whole Genome Amplification method Polymorphisms analysis was then performed using restriction fragment length polymorphism method and direct PCR sequencing for TLR4 (2 polymorphisms).
Statistical analysis
Because of the nature of this study, no formal statistical analysis was planned. Evaluation of the data consisted primarily on summary displays (i.e., descriptive statistics and graphs). Qualitative data were summarized by frequency and percentages, while quantitative data were summarized by descriptive statistics.
Results
Patient characteristics
From November 2004 to July 2007, 17 patients were enrolled. Their characteristics are described in Table
Drug delivery
A total of 138 infusions were administered with a median number of 5 injections (range: 3 to 15). Protocol was amended after 15 patients have been included, to suppress dose level 6 and 7 to facilitate patient enrollment. The investigator had the choice to continue treatment as up to a total of 15 infusions if a benefit was observed for the patient.
Number of patients and total of delivered infusions in each DLs are depicted in Table
The majority of the subjects (82%) received all the administrations planed in each DL. At DL3 (15 injections of 600 μg/m2), a 63-year-old woman with a metastatic breast cancer, received only 6 infusions due to a rapid alteration of her general status in relation to a progressive disease; at DL 5 (10 injections of 800 μg/m2), a 46-year-old woman with ovarian cancer and a 68-year-old man with liver cancer received respectively only 4 and 3 out of 10 OM-174 infusions because of a progressive disease in the first case and a severe adverse event, bronchospasm, in the other one.
Safety results
All patients were evaluable for safety analysis. No haematological disorders were observed after OM-174 administration, in any patient.
Non haematological adverse events are shown in Table
Gastro-intestinal disorders were seen in 7 patients. Four of them had nausea or vomiting and 3 diarrhea. Four patients experienced fatigue and three reported having headache after OM-174 administration.
All other adverse events, hypertension not requiring treatment, dizziness, somnolence, and dysphagia, were graded one and were experienced by a single patient.
Grade 1 hepatic cytolysis and cholestasis were observed in a patient who had a progressive disease, and it was concluded that this abnormalities were unlikely related to the drug.
No death related to the drug was observed during the study.
Determination of the MTD
All the 17 patients enrolled were evaluable for the determination of the MTD, but since no DLT was observed its determination was not feasible.
However, a patient included in the study for progressive hepatocarcinoma and scheduled to be treated at dose level 5 (10 infusions of 800 μg/m2 of OM-174) experienced a serious adverse event after the third administration. The patient exhibited a severe bronchospasm with oxygen desaturation, hypoxemia immediately at the end of the treatment. He fully recovered after an IV-infusion of corticosteroids but did not receive any further administration of the study drug. This was not considered as a dose-limiting toxicity since it was not related to the dose and was more likely to be of immunological origin, as patients receiving the next dose of 1000 μg/m2 and the 5 additional patients who received the same dose of OM-174 (800 μg/m2) for at least five administrations did not experience such grade 3 or 4 toxicity.
Pharmacokinetics
All 17 patients were evaluable for PK.
After the first IV infusion at the single dose of 600 μg/m2, OM-174 pharmacokinetic profile showed a Tmax longer than the end of the infusion, a Cmax reaching 394 ng/ml and a two phases elimination process (an initial phase of only a few minutes and a second phase lasting about 22 hr). Clearance and volume of distribution were low, 0.11 L/h (66 mL/hr/m2) and 4.6 L (2643 mL/m2), respectively. No accumulation occurred at the multiple dosages of 600 μg/m2 twice a week, whereas a small accumulation was evidenced for the higher 800 and 1000 μg/m2 dosages.
Cytokines dosage
All patients were evaluable for this analysis. Cytokine changes induced by OM-174 infusions are summarized in Table
A Increase of IL-8 concentration after each injection of OM-174 with a peak at second hour, independently of dose of OM-174 and number of injections
a Increase of IL-8 concentration after each injection of OM-174 with a peak at second hour, independently of dose of OM-174 and number of injections. Black, doted and white boxes represent the value of the cytokine of interest 1, 2 and 4 hours after the injection of interest. Analyses were conducted on all 17 patients according to their number of OM-174 injections. 1b and 1c Both concentration of TNF-α and IL-6 decreased after each injection of OM-174 suggesting a tolerance. The peak of secretion is observed respectively 1 and 2 hours after the first injection of OM-174 for TNF-α and IL-6. Black, doted and white boxes represent the value of the cytokine of interest 1, 2 and 4 hours after the injection of interest. Analyses were conducted on all 17 patients according to their number of OM-174 injections.
Natural killer activity analysis
When all patients with available data were considered, neither the number nor the activity of NK cells were increased. Nevertheless in patients receiving 15 injections of OM-174, irrespective of the dose, NK cell number was increased compared with baseline value, from 0.67 ± 0.25 × 106 to 2.21 ± 0.17 × 106 NK cells (p<0.005). Furthermore, patient 17, who received the highest dose of OM-174 (1000 μg/m2) and the highest number of injections (15) showed a significant progressive increase in NK cell activity as OM-174 infusions were repeated (Figure
Increase of NK activity after each injection of 1000 μg/m2 OM-174 and in association with IL-2
Increase of NK activity after each injection of 1000 μg/m2 OM-174 and in association with IL-2.
TRL 4 polymorphism analysis
All but one (16 out of 17) patients of this cohort carried the wild type alleles for TLR4D299G and all for TLR4T399I polymorphisms, making us unable to assess the relationship between TLR4 polymorphisms and response to OM-174, either in terms of biological or clinical response.
Antitumor activity
Responses were assessed by the investigator in every patient.
One patient with hepatocellular carcinoma who was withdrawn from the study after the third administration because of grade 3 toxicity (bronchospam) was not available for tumor response.
Stabilization of disease was documented in 3 patients (17%) and seen at DL 3, DL4 and DL8. Characteristics of these patients are summarized in Table
Discussion
Our study strengthens data on the overall very favourable safety profile of OM-174, as administered for up to 15 injections as a biweekly infusion and up to 1000 μg/m2 which is the recommended dose. Chills (70%) and fever (41%) were the most common adverse advents, with no grade 3 or 4, explaining that the MTD could not be determined, and were reversible with acetaminophen, NSAID or steroid treatments. Furthermore no increase in severity occurred as injections were repeated. It might be argued that giving acetaminophen or NSAIDs to patients with grade 1 or 2 fever might impair antitumoral effect as it has been suggested that response to IL-2 plus interferon-alpha might be altered by acetaminophen in patients with metastatic melanoma
Others adverse events included nauseas, vomiting, fatigue, headache and diarrhoea and were generally of mild to moderate intensity. The more severe adverse event was an episode of bronchospasm which occurred for a patient with a progressive hepatocarcinoma after the third infusion of 800 μg/m2.
The safety profile of OM-174 we describe here is similar to that observed in others studies with other lipids A
OM-174 PK parameters appeared to be independent of dose within the dose range of 600 – 1000 μg/m2. A trend toward higher OM-174 concentrations was observed when administered doses increased, but the difference was not significant. OM-174 is a drug with a low systemic clearance (0.11 L/h) and a small distribution volume (4.6 L). Our results are in agreement with those of de Bono et al.
Pharmacodynamic studies showed OM-174 IV-infusion induced a significant increase in IL-8, IL-10, IL-6 and TNF-α concentrations. IL-8 and IL-10 concentrations increased after each OM-174 injection whereas TNF-α and IL-6 concentrations were highest after the first OM-174 injection and declined thereafter as OM-174 injections were repeated. This response was not correlated to the dose of OM-174 and is known as tolerance. The occurrence of tolerance or host desensitization in animals and humans administered multiple doses of microbial products has long been recognized
Interestingly, our study also suggests that a sufficient number of OM-174 injections has to be administered to reach a biological activity since a trend toward an increase in NK cells number and activity was observed only in patients receiving 15 infusions. As only three patients received 15 infusions these findings deserve further studies to be confirmed.
As OM-174 has been shown, in mice, to induce TNF-α production via TLR4 receptors, a family of receptors which recognized pathogen-associated microbial structure and are involved in innate immune cell activation and antitumor immune response inducing tumor cell death
We did not observed objective tumour responses, as it was the case in the Vosika
It has to be agreed that lipid A analogues, such as OM-174, do not appear very promising when used alone in cancer patients. Nevertheless our team has been working extensively in animal model and our knowledge of underlying mechanism or tumor growth inhibition led us to assess the possible synergy of OM-174 with different drugs. Like this, we have shown (unpublished data) that whereas treatment by either oxaliplatin or OM-174 alone doesn’t lead to regression of voluminous peritoneal tumors, induced in BDIX rats by intraperitoneal injection of syngeneic PROb colon cancer cells. Their combination induces the disappearance of carcinomatosis.
Conclusion
We have shown that the lipid A analog, OM-174, administered twice weekly, for up to 15 injections and 1000 μg/m2 offers a predictable and overall favourable safety profile and good tolerability at biologically active concentrations. Clinical and biological activities deserve further experiments, likely in conjunction with chemotherapy, as preclinical experiments conducted in our unit suggest that this combination might be synergistic. To confirm this hypothesis, we have designed a phase Ib study in association with a derivate of platine that will soon be enrolling patients.
Competing interest
All the authors except Jacques Bauer declare that they have no competing interests. Jacques Bauer is an employee of OM-Pharma which has been integrated in Vifor Pharma in 2009.
Authors’ contributions
Conceived and designed: JB, JFJ, MB. Acquisition of data: NI, PF, SZ, MB. Analysis and interpretation of data: NI, PF, CP, KR, GL, JFJ, MB. Wrote the paper: NI, CP, JB, JFJ, MB. Read and approved the final version of the manuscript: NI, PF, CP, SZ, JB, KR, GL, JFJ, MB. All authors read and approved the final manuscript.