Chemokines and Netosis, cornerstones of our immunology approach

LDX0219

A Single Dose Study About the Influence of Food on the Oral Bioavailability of Ladarixin Capsule in Healthy Volunteers

Primary objective:

• to investigate the effect of food on the bioavailability of DF 2156Y after single dose administration of 400 mg of ladarixin to healthy male and female volunteers under fed and fasting conditions.

Secondary objectives:

• to investigate the effect of gender on the bioavailability of DF 2156Y and its metabolites (DF 2108Y and DF 2227Y) after single dose administration of 400 mg of ladarixin to healthy male and female volunteers
• to evaluate safety and tolerability of a single dose administration of ladarixin 400 mg to healthy male and female volunteers.

This is a Single center, single dose, open label, randomized, two-way, crossover, food effect on bioavailability study.

More precisely, a single oral dose of 400 mg of ladarixin (two 200 mg capsules) was administered to healthy male and female volunteers under fed (Test treatment) and fasting (Reference treatment) conditions in two consecutive study periods, according to a two-way crossover design, with a wash-out interval of at least 14 days between the two administrations.

LDX0119

Ladarixin in obese pre-diabetic patients eligible to bariatric surgery

Effect of oral ladarixin 400 mg twice a day on insulin sensitivity. A phase 2, randomized, double-blind, placebo-controlled explorative study in obese patients with pre-diabetes eligible to bariatric surgery.

Effect of the experimental drug ladarixin administered orally at a dose of 400 mg twice a day on insulin sensitivity. A phase 2, randomized, double-blind, placebo-controlled explorative study in obese patients with pre-diabetes eligible to bariatric surgery.

LDX0419

A Study to Assess Efficacy/Safety of Ladarixin in Type 1 Diabetes Patients With Preserved ß-cell Function at Baseline

The objectives of this clinical trial are:

• to evaluate whether a 12 month treatment with ladarixin is effective to improve glycemic control in newly diagnosed T1D adult patients with preserved beta-cell function.
• to evaluate the safety of ladarixin in the specific clinical setting

The study is a phase 2, multicenter, double-blind, placebo-controlled study. It will randomize approximately 75 adult patients with new-onset type 1 diabetes (T1D) and preserved beta-cell function (fasting C-peptide >0.205 nmol/l) at baseline. Patients will be assigned (2:1) to receive either oral ladarixin treatment (400 mg b.i.d. for 13 cycles of 14 days on/14 days off - treatment group) or placebo (control group).

Recruitment will be competitive among the study sites, until the planned number of patients is randomized.

Ladarixin and placebo will be both administered for 1 year. All patients will be followed-up for 18 months from the 1st administration of the study medication. The study database will be locked and data analyzed when the last patient randomized has completed month 12 follow-up visit. After this time point, the follow-up will continue under open-label conditions up to month 18.

LDX0319

A Study of Oral Ladarixin in New-onset Type 1 Diabetes and a Low Residual β-cell Function

The objective of this clinical trial is to assess whether ladarixin treatment is effective in preserving beta-cell function and delaying the progression of type 1 diabetes (T1D) in adolescent and adult patients. The safety of ladarixin in the specific clinical setting will be also evaluated.

This is a phase 3, multicenter, double-blind, placebo-controlled study. It has been designed to further evaluate whether ladarixin is effective in preserving beta-cell function and slowing-down the progression of T1D) in patients with a more severe disease presentation.

The study is planned to be performed at about 40 study centers in EU, US and in other countries, if appropriated. At each study center, the Principal Investigator (PI) will be responsible for ensuring that the investigation is conducted according to the signed Investigator agreement, the protocol, GCP guidelines, and local regulations.

The study is planned to involve -327 patients with new-onset T1D, to include about 200 adolescents (14-17 years). Patients will be randomly (2:1) assigned to receive either ladarixin treatment (400 mg b.i.d. for 13 cycles of 14 days on/14 days off - treatment group) or matched placebo (control group). The two groups will be balanced within centers.

MEX0114

A Phase 2, Multicentre, Randomized, Double-blind, Placebo-controlled Study in Patients With New-onset Type 1 Diabetes

The objective of this clinical trial is to investigate whether ladarixin has sufficient activity (preservation of β-cell function and slow-down of the progression of T1D) to warrant its further development (proof of concept trial). The safety of ladarixin in the specific clinical setting will be also evaluated.

The study is a phase 2, multicentre, double-blind study. 72 patients with new-onset type 1 diabetes (T1D) were planned to be involved, randomly (2:1) assigned to receive either ladarixin treatment (400 mg b.i.d. for 3 cycles of 14 days on/14 days off - treatment group) or placebo (control group).

Recruitment was competitive among the study sites, until the planned number of patients was enrolled. A total of 76 patients were actually recruited.

T1D is an organ-specific autoimmune disease in which the immune system attacks the insulin-producing β-cells. The onset of the disease typically occurs before adulthood and seriously affects a person's quality of life.

T1D is treated with life-long daily exogenous insulin injections and monitoring of blood glucose levels. However, even optimization of glucose control through the most recent technologies cannot adequately substitute for the finely tuned normal balance of the glucose levels. Therefore, despite marked improvements in diabetes care in recent years, insulin-dependent diabetes results in secondary long-term complications and is one of the leading causes of end-stage renal disease, blindness and amputation. Additionally, hypoglycaemia unawareness is a serious consequence of recurrent hypoglycaemia often requiring emergency care.

Maintenance of residual β-cell function (as measured by C-peptide response) was demonstrated to be associated with reduced rate of microvascular complications and hypoglycaemia, improved quality of life, and overall reduction in morbidity and associated management costs. Therefore, pharmacological approaches aimed at controlling the autoimmune response and restoring self-tolerance to pancreatic β-cells had attracted the clinical/scientific interest.

Among these, rituximab, CD3-specific monoclonal antibodies, GAD65, DiaPep277 have progressed to phase III clinical trials. Other agents, including cytokines modulators such as anti-TNF or anti-IL1, are under clinical evaluation. Unfortunately, even if safe preservation of β-cell function and improvement of glycaemic control have been evidenced for some of the pharmacological approaches evaluated so far, none has been definitely approved for the "treatment" of diabetes onset. New strategies are being evaluated which combine agents targeting sequential arms of the immune and inflammatory response involved in β-cell disruption. In this regard, IL-8 appears to be an important mediator in the progression of type 1 diabetes. Production and secretion of pro-inflammatory IL-8 has been demonstrated from human pancreatic islets upon enterovirus infections, and LPS-induced production of IL-8 by neutrophils is increased in type 1 pre-diabetic and diabetic patients. In parallel, circulating levels of IL-8 were elevated in children with T1D compared to non-diabetic controls. Specifically, levels of IL-8 correlate with glycaemic control, higher level being associated to poorer or unfavorable glucose control.

As a result of these findings, the modulation or inhibition of IL8 activity is considered a valid target for the development of innovative treatments aimed to control the progression of T1D.

Results obtained with ladarixin in mouse models of T1D, and particularly reversal of "diabetes" in the NOD mice, clearly showed the ability of this CXCR1/2 inhibitor to protect β-cells and either prevent or delay the progression of hyperglycaemia. The positive effects of ladarixin, coupled with the safety shown in phase 1 studies, provided a sound rationale for a clinical study aimed at evaluating the effect of ladarixin in patients with new onset diabetes and supported the conduct of the present study.

REP0220

Study on Efficacy and Safety of Reparixin in the Treatment of Hospitalized Patients With Severe COVID-19 Pneumonia

The study objective is to assess Efficacy and safety of Reparixin treatment as compared to placebo (both on top of standard treatment) in adult patients with severe COVID-19 pneumonia.

This is a phase 3 clinical trial designed as a randomized, double-blind, placebo-controlled, multicentre study to evaluate the efficacy and safety of Reparixin in hospitalized adult patients with severe COVID-19 pneumonia.

Patients will be screened for the participation in the study and eventually randomized based on an unbalanced randomization scheme (2:1) to Reparixin oral tablets (2 x 600 mg TID) for up to 21 days or to placebo.

An unequal randomization is justified by the need to gain experience and more safety data with the investigated treatment and by an expected better acceptability of the trial by patients.

The placebo control arm is justified by the unavailability of a well-defined standard of care for subjects with COVID-19 pneumonia who are candidates for this study.

All patient will receive the standard supportive care based on the patient's clinical need. Follow-up information on the patient's clinical condition will be collected until day 90.

REP0114

A Double-blind Study of Paclitaxel in Combination With Reparixin or Placebo for Metastatic Triple-Negative Breast Cancer (FRIDA)

The Objectives of this study:

The primary objective of the study was to evaluate progression-free survival (PFS) (defined as the number of days between the date of randomization and the date of clinical disease progression (PD) according to Response Evaluation Criteria in Solid Tumors (RECIST) criteria version 1.1, as assessed by Independent Radiology Review, or death for any cause, whichever occured first) in patients with metastatic triple-negative breast cancer (TNBC) treated with the combination of paclitaxel and orally administered reparixin compared to paclitaxel alone.

The secondary objectives were:

• To determine overall survival (OS).
• To evaluate objective response rates (ORR).
• To determine median PFS (mPFS).
• To assess the safety of the combination of paclitaxel and orally administered reparixin (referred to as combination treatment).

The study is a two arm, phase 2 study to evaluate the efficacy of the combination of paclitaxel and reparixin compared to paclitaxel and placebo in metastatic TNBC patients.

In the study two groups There were two groups:

Group 1: paclitaxel 80 mg/m2 intravenous (i.v.) (Days 1, 8, and 15 of 28-day cycle) + reparixin oral tablets 1200 mg three times a day (t.i.d.) continuing from Day 1 to Day 21.

Group 2: paclitaxel 80 mg/m2 i.v. (Days 1, 8, and 15 of 28-day cycle) + placebo oral tablets 1200 mg t.i.d. continuing from Day 1 to Day 21.

Study drug (reparixin/placebo) was administered with water prior to the start of the i.v. paclitaxel infusion on Cycle 1, Day 1 and then administered approximately every eight hours (six to ten hours) for 21 consecutive days during each cycle with seven days off-treatment between each cycle. It was preferable that reparixin was taken with food. However, if the patient was unable to eat, study drug was allowed to be administered. When in combination with paclitaxel (Day 1, 8 and 15 of each cycle), reparixin or placebo was administered every approximately eight hours with about 250 mL water and a light meal or snack. Paclitaxel was administered in combination with study drug (reparixin/placebo) as an i.v. infusion on Days 1, 8 and 15 of each 28-day cycle.

On Cycle 1, Day 1, paclitaxel was administered at the clinic after the administration of study drug (reparixin/placebo). From that point forward, study drug (reparixin/placebo) was self-administered t.i.d. for 21 days. Combination treatment (three weeks on and one week off) continued until PD according to RECIST criteria version 1.1, withdrawal of consent or unacceptable toxicity, whichever occurred first.

The next clinic visits were on Days 8 and 15 when a paclitaxel infusion was administered to the patient. The patients returned to the clinic again on Day 29/Day 1 of the next cycle.

Tumor response and/or progression assessments were performed and documented every eight weeks according to RECIST criteria version 1.1. Metastatic tissue samples were analyzed for evaluation of CD24-CD44+ and aldehyde dehydrogenase positive (ALDH+) CSCs.

REP0210

Pilot Study to Evaluate Safety & Biological Effects of Orally Administered Reparixin in Early Breast Cancer

This is a pilot "window of opportunity" clinical study in patients with operable breast cancer investigating use of reparixin as single agent in the time period between clinical diagnosis and surgery.

The primary objectives of this study were:

1- to evaluate the effects of orally administered reparixin on CSCs in the primary tumor and the tumoral microenvironment in an early breast cancer population:

A. CSC were measured in tissue samples by techniques that could include: ALDEFLUOR assay and assessment of CD44/CD24 by flow cytometry, or examination of RNA transcripts by RT-PCR, aldehyde dehydrogenase-1, CD44/CD24 and epithelial mesenchymal transition markers (Snail, Twist, Notch) by immunohistochemistry (IHC). CSC were defined as ALDEFLUOR positive (ALDH-1+) and/or CD44 high/CD24 low by flow cytometry or RT-PCR and IHC and by the detection of ALDH-1+ cells with or without epithelial mesenchymal transition (EMT) transcription factor in IHC assays.

B. Serine-threonine protein kinase (AKT), focal adhesion kinase (FAK), phosphatase and tensin homolog (PTEN) and chemokine receptor-1 (CXCR1) levels were measured in tissue samples by IHC.

C. Measurement of markers of inflammation (interleukin-1beta [IL-1β], interleukin-6 [IL-6], interleukin-8 [IL-8], tumor necrosis factor-alpha [TNF-α], granulocyte macrophage colony stimulating factor [GM-CSF], vascular endothelial growth factor [VEGF], basic fibroblast growth factor [b-FGF] and high-sensitivity C-reactive protein [hsCRP]) in plasma, leukocyte subsets (enumerate T subsets, B, and natural killer/natural killer T [NK/NKT] cells) and study polymorphonuclear leukocyte [PMN] biology in peripheral blood samples. D. Measurement of markers of angiogenesis (CD31 staining), tumor-infiltrating leukocytes (CD4, CD8, NK and macrophages), autophagy (P62 and LC3 by IHC), EpCAM and EMT markers (CD326, CD45, Twist1, SNAIL1, SLUG, ZEB1, FOXC2, TG2, Akt2, P13k and CK19 by RT-PCR) and tissue cellularity (residual disease characterization in tumor bed) in tumor tissue samples.

2. To evaluate the safety of oral reparixin administered three times daily (t.i.d.) for 21 consecutive days.

The secondary objective was to define the pharmacokinetic (PK) profile of orally administered reparixin.

According to the cancer stem cell (CSC) model, tumors are organized in a cellular hierarchy maintained by a subpopulation of cells displaying stem cell properties. These properties include self-renewal (which drives tumorigenesis) and differentiation (which generates the tumor bulk and contributes to cellular heterogeneity).

CSCs were first observed in hematological malignancies but have also been identified in solid tumors of breast, prostate, brain, colon and pancreas. CSCs are thought to be resistant to conventional chemotherapies and this may be why relapse occurs in many patients and this might explain the failure to develop therapies that are consistently able to eradicate solid tumors. Although currently available drugs can shrink metastatic tumors, these effects are usually transient and often do not appreciably extend the life of patients. One reason for the failure of these treatments is the acquisition of drug resistance by the cancer cells as they evolve; another possibility is that existing therapies fail to kill CSCs effectively. Existing therapies have been developed largely against the bulk population of tumor cells because they are often identified by their ability to shrink tumors. Because most cancer cells have limited proliferative potential, an ability to shrink a tumor mainly reflects an ability to kill these cells. It seems that normal stem cells from various tissues tend to be more resistant to chemotherapeutics than mature cell types from the same tissues. The reasons for this are not clear, but may relate to high levels of expression of anti-apoptotic proteins or adenosine triphosphate-binding cassette transporters such as the multidrug resistance gene. If the same were true of CSCs, then one would predict that these cells would be more resistant to chemotherapeutics than tumor cells with limited proliferative potential. Even therapies that cause complete regression of tumors might spare enough CSCs to allow re-growth of the tumors. Therapies that are more specifically directed against CSCs might result in much more durable responses and even cures of metastatic tumors.

There are limited data on the impact of treatment tailoring based on CSC detection. Gene profiling of CSCs could lead to identification of therapeutic targets on CSCs (e.g. hormone receptors (HR), human epidermal growth factor receptor-2 [HER-2] expression, epidermal growth factor receptor [EGFR] expression), and could represent tumor biopsy in "real time". Several groups showed frequent discordance of HER-2 status between primary tumor and CSCs, and case reports showed clinical utility for the use of trastuzumab-based therapy based on HER-2 CSCs status. Similarly, the hormonal status of CSCs could be different from that of the primary tumor, which could lead to increase the number of patients suitable for endocrine therapy, but also could explain why endocrine therapy fails in a subset of HR positive (HR+) patients. More specifically, a recent observation from Ginestier et al. demonstrated that over expression of chemokine receptor 1 (CXCR-1) is associated with the aldehyde dehydrogenase positive (ALDH+) cells. In breast carcinomas, the ALDEFLUOR+ phenotype shows partial overlap with the CD44+CD24-Lin-CSC phenotype. Cellular hierarchies have been identified in a series of molecularly characterized breast cancer cell lines and it has been demonstrated that these lines contained ALDEFLUOR+ components that were both tumorigenic and metastatic in NOD/SCID mice. Furthermore, previous observations demonstrated that the addition of recombinant interleukin-8 (IL-8) increased the CSC population as well as increasing its propensity for invasion. Moreover, tissue damage induced by chemotherapeutic agents may induce IL-8 as part of the injury response. This suggests that strategies aimed at interfering with the IL 8/CXCR-1 axis may be able to target CSCs, increasing the efficacy of current therapies. This experimental data provides another therapeutic target in breast cancer.

Reparixin seems to be a good candidate for use in breast cancer patients because of its very acceptable toxicity profile shown in the Phase I and II clinical trials conducted so far, along with its observed activity in vitro against breast cancer cell lines and in vivo in tumor xenografts in mice. A phase 1 study is currently underway to study the effects of reparixin in combination with paclitaxel in metastatic breast cancer.

This small pilot study aims at exploring the effects on breast CSC markers as well as the safety and PK profile of orally administered single agent reparixin in HER-2 negative (HER-2-) early breast cancer patients in the 3 weeks prior to surgery.

The study will be performed in the interval between disease diagnosis and planned surgery and may lead to a minimal delay in surgery. This is balanced by the potential benefits of the study by evaluating CSCs and their prognostic importance as well as obtaining information about the impact of reparixin therapy.

REP0111

Pilot Study to Evaluate Reparixin With Weekly Paclitaxel in Patients With HER 2 Negative Metastatic Breast Cancer (MBC)

This is a phase I study to evaluate the safety and define the pharmacokinetic (PK) profile of orally administered reparixin in combination with paclitaxel in HER 2 (Human epidermal growth factor receptor-2) negative metastatic breast cancer patients.

The primary objective of this study was to evaluate the safety and define the pharmacokinetic (PK) profile of orally administered reparixin in combination with paclitaxel in HER-2 negative MBC patients.

The secondary objectives were to:

1. Evaluate the effects of orally administered reparixin on cancer stem cell (CSC) markers, the tumoral microenvironment and markers of cytokine inflammation;
2. Evaluate peripheral blood samples for enumeration of circulating tumor cells (CTCs), molecular characterization as CSCs and perform epithelial-mesenchymal transition (EMT) biomarker profiling;
3. Assess disease response for indication of efficacy.

The CSC (Cancer stem cell) concept has important implications for understanding carcinogenesis as well as for the development of cancer therapeutics. According to this concept, tumors are initiated and maintained by a cellular subcomponent that displays stem cell properties. These properties include self-renewal, which drives tumorigenesis, and differentiation (albeit aberrant), which contributes to tumor cellular heterogeneity. The existence of CSCs has been described in a variety of hematologic and solid tumors including those of the breast, brain, colon, pancreas, lung, liver, and head and neck. In addition to driving tumorigenesis, CSCs may contribute to tumor metastasis as well as to tumor recurrence after treatment.

One of the therapeutic strategies being pursued to target CSCs involves inhibition of self renewal or survival pathways in these cells. These pathways include NOTCH (Notch signaling pathway), Hedgehog, and WNT (Wnt signaling pathway). Such strategies may be limited by the role of these pathways in normal stem cell function, which could result in systemic toxicities from pathway inhibition. In addition to intrinsic pathways regulating stem cell functions, normal and malignant stem cells are regulated by extrinsic signals generated in the microenvironment or CSC niche. In the breast, this niche is composed of immune cells, mesenchymal elements that include fibroblasts, endothelial cells, adipocytes, and extracellular matrix components. These components play an important role in normal breast development and carcinogenesis. If the cellular microenvironment plays an important role in the regulation of CSC growth and survival, then strategies aimed at interfering with these interactions represent a rational approach to target breast CSCs.

There are limited data on the impact of treatment tailoring based on CSCs detection. Gene profiling of CSCs could lead to identification of therapeutic targets on CSCs (e.g. hormone receptors, HER-2 [Human epidermal growth factor receptor-2] expression, EGFR [Epidermal growth factor receptor] expression), and could represent tumor biopsy in "real time". Several groups showed frequent discordance of HER-2 status between primary tumor and CSCs, and case reports showed clinical utility to use of trastuzumab-based therapy based on HER-2 CSCs status. Similarly, the hormonal status of CSCs could be different from that of the primary tumor, which could lead to increase the number of patients suitable for endocrine therapy, but also could explain why endocrine therapy fails in a subset of hormone receptor-positive patients. The study provided the in vivo demonstration that CXCR-1 (Chemokine receptor 1) targeting with specific blocking antibodies or reparixin is associated with reduced systemic metastases. The experimental data provides another therapeutic target in metastatic disease and warrants a pilot study investigation in humans to further explore effects of reparixin on breast CSCs and the tumoral microenvironment.

Reparixin seems to be a good candidate for use in breast cancer patients because of its very acceptable toxicity profile shown in the Phase I and II clinical trials conducted so far, along with its observed activity in vitro against breast cancer cell lines and in vivo in tumor xenografts in mice. It potentially addresses another therapeutic target in metastatic disease. The current pilot study thus aims at exploring the safety and PK profile of orally administered reparixin in HER-2 negative metastatic breast cancer patients and its effects on breast CSC markers.