Pharmacodynamic characterization of rytvela, a novel allosteric anti-inflammatory therapeutic, to prevent preterm birth and improve fetal and neonatal outcomes.

BACKGROUND: Preterm birth is the leading cause of neonatal morbidity and mortality. Studies have shown that interleukin 1 plays a major role in the pathophysiology of preterm birth by inducing the production of proinflammatory mediators and uterine activation proteins leading to labor. More importantly, uteroplacental inflammation, associated with preterm birth parturition pathways, is detrimental to fetal tissues and leads to long-term sequelae. Our group has developed an allosteric antagonist of the interleukin 1 receptor, rytvela, found to be potent and safe in preventing preterm birth by suppressing inflammation via the inhibition of the mitogen-activated protein kinase pathway while preserving the Nuclear factor kappa B pathway (important in immune vigilance). Rytvela has been shown to inhibit inflammatory up-regulation and uterine activation while preserving fetal development. OBJECTIVE: This study aimed to further the preclinical development of rytvela by evaluating its optimal dose and minimal duration of treatment to inhibit the inflammatory cascade, prolong gestation, and promote neonatal outcomes. STUDY DESIGN: Pregnant CD-1 mice were administered with lipopolysaccharide (10 µg, intraperitoneal administration) or interleukin 1 (1 µg/kg, intrauterine administration) on gestational day 16 to induce preterm labor. Rytvela was administered at different doses (0.1, 0.5, 1.0, 2.0, 4.0 mg/kg/d subcutaneously) from gestational days 16 to 18.5. To evaluate the minimal duration of treatment, the mice were administered with rytvela (2 mg/kg/d subcutaneously) for 24, 36, or 48 hours. The rate of prematurity (gestational day <18.5) and neonate survival and weight were evaluated. Gestational tissues were collected at gestational day 17.5 to quantify cytokines, proinflammatory mediators, and uterine activating proteins by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The neonatal lungs and intestines were collected from postnatal days 5 to 7 and analyzed by histology. RESULTS: Rytvela exhibited a dose-response profile and achieved maximum efficacy at a dose of 2 mg/kg/d by reducing 70% of lipopolysaccharide-induced preterm births and 60% of interleukin 1ß-induced preterm births. In addition, rytvela attained maximum efficacy at a dose of 1 mg/kg/d by increasing neonate survival by up to 65% in both models of preterm birth. Rytvela protected fetuses from inflammatory insult as of 24 hours, preserving lung and intestinal integrity, and prevented preterm birth and fetal mortality by 60% and 50%, respectively, as of 36 hours of treatment. CONCLUSION: The maximum efficacy of rytvela was achieved at 2 mg/kg/d with improved birth outcomes and prevented inflammatory up-regulation upon 36 hours (only) of treatment. Rytvela exhibited desirable properties for the safe prevention of preterm birth and fetal protection.

Inflammatory Amplification: A Central Tenet of Uterine Transition for Labor.

In preparation for delivery, the uterus transitions from actively maintaining quiescence during pregnancy to an active parturient state. This transition occurs as a result of the accumulation of pro-inflammatory signals which are amplified by positive feedback interactions involving paracrine and autocrine signaling at the level of each intrauterine cell and tissue. The amplification events occur in parallel until they reach a certain threshold, 'tipping the scale' and contributing to processes of uterine activation and functional progesterone withdrawal. The described signaling interactions all occur upstream from the presentation of clinical labor symptoms. In this review, we will: 1) describe the different physiological processes involved in uterine transition for each intrauterine tissue; 2) compare and contrast the current models of labor initiation; 3) introduce innovative models for measuring paracrine inflammatory interactions; and 4) discuss the therapeutic value in identifying and targeting key players in this crucial event for preterm birth.

Landscape of Preterm Birth Therapeutics and a Path Forward.

Preterm birth (PTB) remains the leading cause of infant morbidity and mortality. Despite 50 years of research, therapeutic options are limited and many lack clear efficacy. Tocolytic agents are drugs that briefly delay PTB, typically to allow antenatal corticosteroid administration for accelerating fetal lung maturity or to transfer patients to high-level care facilities. Globally, there is an unmet need for better tocolytic agents, particularly in low- and middle-income countries. Although most tocolytics, such as betamimetics and indomethacin, suppress downstream mediators of the parturition pathway, newer therapeutics are being designed to selectively target inflammatory checkpoints with the goal of providing broader and more effective tocolysis. However, the relatively small market for new PTB therapeutics and formidable regulatory hurdles have led to minimal pharmaceutical interest and a stagnant drug pipeline. In this review, we present the current landscape of PTB therapeutics, assessing the history of drug development, mechanisms of action, adverse effects, and the updated literature on drug efficacy. We also review the regulatory hurdles and other obstacles impairing novel tocolytic development. Ultimately, we present possible steps to expedite drug development and meet the growing need for effective preterm birth therapeutics.

Cooperative effects of sequential PGF2α and IL-1ß on IL-6 and COX-2 expression in human myometrial cells†.

The change from the state of pregnancy to the state of parturition, which we call uterine transitioning, requires the actions of inflammatory mediators and results in an activated uterus capable of performing the physiology of labor. Interleukin (IL)-1ß and prostaglandin (PG)F2α are two key mediators implicated in preparing the uterus for labor by regulating the expression of uterine activation proteins (UAPs) and proinflammatory cytokines and chemokines. To investigate this process, primary human myometrial smooth muscle cells (HMSMC) isolated from the lower segment of women undergoing elective cesarean sections at term (not in labor) were used to test the inflammatory cytokine and UAP outputs induced by PGF2α and IL-1ß alone or in sequential combinations. PGF2α and IL-1ß regulate mRNA abundance of the PGF2α receptor FP, the IL-1 receptor system, interleukin 6, and other UAPs (OXTR, COX2), driving positive feedback interactions to further amplify their own proinflammatory effects. Sequential stimulation of HMSMC by PGF2α and IL-1ß in either order results in amplified upregulation of IL-6 and COX-2 mRNA and protein, compared to their effects individually. These profound increases were unique to myometrium and not observed with stimulation of human fetal membrane explants. These results suggest that PGF2α and IL-1ß act cooperatively upstream in the birth cascade to maximize amplification of IL-6 and COX-2, to build inflammatory load and thereby promote uterine transition. Targeting PGF2α or IL-1ß, their actions, or intermediates (e.g. IL-6) would be an effective therapeutic intervention for preterm birth prevention or delay.

Maternal and fetal intrauterine tissue crosstalk promotes proinflammatory amplification and uterine transition†.

Birth is a complex biological event requiring genetic, cellular, and physiological changes to the uterus, resulting in a uterus activated for completing the physiological processes of labor. We define the change from the state of pregnancy to the state of parturition as uterine transitioning, which requires the actions of inflammatory mediators and localized paracrine interactions between intrauterine tissues. Few studies have examined the in vitro interactions between fetal and maternal gestational tissues within this proinflammatory environment. Thus, we designed a co-culture model to address this gap, incorporating primary term human myometrium smooth muscle cells (HMSMCs) with human fetal membrane (hFM) explants to study interactions between the tissues. We hypothesized that crosstalk between tissues at term promotes proinflammatory expression and uterine transitioning for parturition. Outputs of 40 cytokines and chemokines encompassing a variety of proinflammatory roles were measured; all but one increased significantly with co-culture. Eighteen of the 39 cytokines increased to a higher abundance than the sum of the effect of each tissue cultured separately. In addition, COX2 and IL6 but not FP and OXTR mRNA abundance significantly increased in both HMSMCs and hFM in response to co-culture. These data suggest that synergistic proinflammatory upregulation within intrauterine tissues is involved with uterine transitioning.