How can we advance drug delivery options using extracellular vesicles for pregnant women to reduce preterm birth?

1.0. Introduction

1.1. Background:

For the past several decades, the global preterm birth (PTB, birth before 37 weeks gestation) rate has remained steady, at approximately 10.5%¹. PTB remains a significant contributor to neonatal and maternal morbidity and mortality. Advancements made in perinatal medical care, knowledge of pathophysiologic pathways, and development of biomarkers to identify high-risk pregnancies have not reduced preterm birth risk and complications associated with prematurity (< 2,500 grams birth weight). Regardless of the subset of preterm births, spontaneous (idiopathic), clinically induced (iatrogenic, or indicated), interventions to reduce the risks are limited. Current interventions using tocolytics delay preterm delivery for a short duration; this window provides clinicians an opportunity to augment therapeutics to enhance lung maturation (corticosteroids) or provide neuroprotection (MgSO₄) to improve neonatal outcomes. Despite the rise in maternal and neonatal morbidity and mortality, the unique physiologic characteristics of pregnancy, pregnant women, and their fetuses remain therapeutic orphans, as they are excluded from the vast majority of clinical drug development and therapeutic trials.^2–4 The following sections will discuss some potential futuristic approaches to addressing the current crisis in reproductive health, specifically pregnancy, based on our own experience, recent reports, and personal views.

2.0. Expert Opinion:

2.1. Fetus as a patient:

Premature activation of fetal inflammatory response in response to exogenous or endogenous risk factors is associated with PTB, specifically spontaneous PTB. Many other fetal signals (endocrine, immune, and mechanical) are also reported with PTB, suggesting that the fetus as a patient contributes immensely to this syndrome. Therefore, the fetus is a patient, and fetal responses should be regulated to reduce the risk of PTB. Current interventions are primarily to reduce maternal indications and terminal events (myometrial contraction and cervical ripening). A solid emphasis on treating the fetus, along with the mother, is required. When the fetus is a patient, treating the whole fetus is not meant here. Although this can be considered, addressing the fetal membranes and the placenta, two major fetal-derived intrauterine organs that support pregnancy, pathologically contribute to adverse outcomes. Targeting these organs and reducing their pathobiological changes may directly or indirectly support the fetus and fetal growth in utero.

2.2. Feto-Maternal Interface (FMi) as a barrier for drug delivery:

Treating the fetus along with the mother poses challenges. The feto-maternal interface (FMi) barriers present several challenges in drug development and delivery. The drugs that can be used to treat both the mother and her fetus must be able to cross FMi. In both cases, the challenges in drug development are exacerbated by the paucity of models that can be used to comprehensively assess both pharmacokinetics (PK) and pharmacodynamics (PD) across the complex components of FMi; the latter is a largely unmet clinical need in developing medicines for combating the increased incidence of adverse pregnancy outcomes. Unfortunately, current preclinical models have many limitations: 1) 2D^5–7 and explant^8,9 human cell cultures, typically grown in transwells, neither embody the intrauterine environment nor maintain the inter-cellular interactions seen in utero. 2) Animal models, for example, a mouse model of FMi,¹⁰, do not structurally mimic human FMi. Specifically, the chorionic trophoblast, which is a barrier from maternal immune cell infiltration to the fetus, is rudimentary in rodents. 3) Non-human primates mimic human pregnancy the best; however, testing drug efficacy and safety is cost prohibitive. 4) Explant perfusion^11,12 studies in “at term placentas” are restricted to the placenta-decidua interface, the fetal membrane-decidua interface of FMi is not tested. Also, perfusion models do not replicate disease states, and drug efficacy testing in them is impossible.

Need for New Approach Methods (NAMs) in drug delivery and testing during pregnancy:

Major factors to be considered when developing drugs and delivery approaches for successful preclinical to clinical trials are: (1) passage through both FMi barriers. i.e., placenta/decidua and fetal membrane/decidua, (2) efficacy to control both maternal and fetal pathophysiologic pathways, (3) lack of teratogenicity, and (4) do not cause long-term consequences in the mother and neonate. The increasing number of feto-maternal mortalities and morbidities has raised the need for better “New Approach Methods “(NAMs) for preclinical trials that are translatable to clinical trials.

2.3. Extracellular vesicles (EV) based drug delivery approaches:

EVs (exosomes 30–200 nm particles) are considered next-generation therapeutics or vehicles of therapeutics. These natural nanoparticles are immunologically inert, safe, stable, and specific and, most importantly, can cross the FMi barrier. EVs are tested successfully as a drug¹³ or as a drug delivery vehicle¹⁴ to delay PTB. Many of the challenges identified above are addressed during the preclinical trials of EV based drugs. Several challenges still lie ahead of EVs being considered for therapeutic options during pregnancy: (1) EV source (native cells vs engineered cells), methods of loading the drug in EVs, and the efficiency of loading (2) Target specific delivery of drug and the mechanism of action (3) Knowledge of EVs and their interactions with various cells (4) Properties (pharmacologic, immune, metabolic) that define the function of the drug (5) Manufacturing and regulatory challenges.

2.4. Drug Development Tools (DDT):

Logistical and operational challenges in conducting research in pregnancy also include a lack of proper preclinical trial models. To address this limitation, a biomimetic, microphysiologic (MPS), interconnected, organ-on-a-chip platform (OOC) that integrates critical feto/maternal organ systems supporting pregnancy can be used. An integrated model of systems that include the placenta, fetal membrane (fetal), decidua, and cervix (maternal) will enable recapitulation of pregnancy physiology and FMi barriers¹⁵. The MPS models are being developed as drug development tools in pregnancy for preclinical trials. Our own laboratory has reported the utility of this device in generating pharmacokinetics and dynamics data of drugs that are currently FDA approved and newly developed drugs¹⁶. This report determined how a multiorgan FMi platform on a chip can reproduce reported data from human clinical trials validating the utility of this platform for preclinical trials. 3D bioprinted FMi platforms and placental organoids have also now been developed to accelerate clinical trials of new and repurposed drugs^17–19. These microfluidic platforms are also useful for biomarker discovery and testing. OOCs are also used to develop noninvasive perinatal testing using fetal-specific EVs or fetal organ-specific cells as biomarkers of fetal indications contributing to adverse pregnancy outcomes.

2.5. Replace, reduce, and refine animal use in pregnancy research:

The FDA Modernization Act 2.0 bill requires the Food and Drug Administration to establish a process that supports nonclinical testing methods for drug development that do not involve the use of animals. Animal models have taught us several mechanistic mediations in pregnancy. However, the use of pregnant animal models that do not resemble human pregnancy physiology has not been beneficial in generating preclinical trial data translatable into a clinical trial during pregnancy. The 3R concepts coined by William Russell and Rex Burch in 1959 on replacement, reduction, and refinement have been the guiding principles for the ethical use of animals in research ^20,21 The DDT proposed above using the organ-on-chip platform is a proposed alternative to animal use. The translational research centers using microphysiologic systems (TraCe-MPS) established by the National Center for Advanced Translational Science (NCATS) (https://ncats.nih.gov/research/research-activities/tissue-chip/projects#microphysiological) has taken a major initiative in linking DDT researchers to have a pathway for approval of their devices for accelerating clinical trials based on the principles of 3Rs. TraCe-MPS will support the widespread adoption and use of tissue chip technology in drug discovery and development. The TraCe MPS program, in partnership with the FDA, is seeking to qualify tissue chips with a defined context of use as FDA-designated drug development tools. We do acknowledge that replacing animal models in pregnancy research is highly unlikely as the OOC models are restricted in generating some end phenotypes, the most obvious being the delivery of the fetus. Even if OOCs reproduce almost all the reproductive organs on a tissue chip platform, several endpoints including the short- and long-term effects of intrauterine exposure to a drug during the growth of the neonates or transgenerational effects still need to be tested in animal models.

2.6. The NAMs are not just EVs and OOCs, as mentioned above. Innovative in silico (AI/ML platforms) and in chemico approaches are being proposed to translate scientific advancements to reach the much-needed vulnerable population of pregnant women.

Achieving the ultimate goal of PTB rate reduction will depend on technological advancements and the use of innovative approaches. Initiatives like Complement Animal Research in Experimentation by the National Institute of Health (NIH, USA) to reduce, refine, and replace unreliable animal models with more humanized models have been launched. The development and testing of natural, non-toxic human-derived products (e.g., EVs to cargo natural compounds as drugs) can be the future of the therapeutic field. Strategic planning of research agencies like NIH and other agencies has been emphasizing the concerns of regulatory agencies (drugs, drug testing models, testing platforms, etc.). A collaborative network of specialists in perinatal medicine and reproductive biology and pharmacology researchers is needed to address the unmet need for therapies during pregnancy to reduce the incidences of PTB.