ACRT Journal

Introduction

Recent developments in regenerative medicine highlight the surprisingly powerful therapeutic potential of amniotic fluid (AF) and its extracellular vesicles (also called exosomes). This review examines four articles, [1–4] covering treatments for complex conditions like severe lower back pain (due to spondylitis) [1], recalcitrant diabetic ulcers [2], second- and third-degree burns [3], and pyoderma gangrenosum [4]. The studies focused on the regenerative capabilities of AF-derived products to not only control pain and inflammation, but to also stimulate tissue repair (regeneration), and improve patient outcomes in otherwise refractory cases when standard of care treatments are unproductive.

Although these studies were each single patient case reports, the consistency of the impact of AF on pain, inflammation, and tissue repair observed in all studies, was supportive of a bonafide biological impact rather than a simple placebo effect. Indeed, the impact on severe pain was almost instantaneous; however, onset of anti-inflammatory effects took in some cases a couple of hours and other cases a couple of days, and impact on tissue repair manifested after days to weeks post treatment initiation. In three of the four cases—lower back pain due to spondylitis, recalcitrant diabetic ulcers, and severe burn lesions—there was complete resolution of the problem [1–3].

ViX001 was approved by the Food and Drug Administration for clinical research involving patients. ViX001 is a fraction of human purified amniotic fluid obtained under aseptic conditions from thoroughly screened volunteers at the time of planned c-section at the term of normal pregnancies [4]. Our product ViX001 was generated through a proprietary process and kept in frozen one or two milliliter (1 or 2 mL) vials (protein content was ~1 gram/ liter) [4]. Vials were thawed prior to administration–intravenous injection in the case of spondylitis and direct (topical) application to lesions in the case of burns, diabetic ulcer, and pyoderma gangrenosum–and kept at 34^oF between uses (shelf life at 34^oF of at least two weeks) [1-4]. ViX001 therapy has received Food and Drug Administration (FDA) approval for an Investigational New Drug (IND) phase I/II human clinical trial to treat the inflammatory component of post-COVID syndrome with intravenous injections.

Brief case summaries

1. Lower Back Pain: A 65-year-old man with debilitating low thoracic spondylitis (T12-L1) and end plate inflammation experienced significant relief from bi-weekly intravenous injections of ViX001 (one ml/injection) [1]. The patient reported a 50% reduction in pain within the first month and resolution of the pain and inflammation at the end of three months (confirmed by MRI scans showing decreased inflammation of the T12-L1 joint). The patient experienced a notable eighteen-month improvement in symptoms before resurgence of pain; However, after followup, reported that the pain was managed without further need for ViX001 treatment. This case highlights the potential for ViX001based therapies to alleviate chronic pain and inflammation in conditions resistant to standard treatments.

2. Diabetic Ulcers: A recalcitrant diabetic ulcer in a 79-year-old patient responded remarkably to twice-daily topical applications of ViX001, leading to complete healing within 60 days [2]. The rapid resolution of pain and inflammation, as well as the concentric healing process, suggests that ViX001 extracellular vesicles can accelerate wound healing, particularly for cases where conventional treatments have failed.

We had the opportunity to reproduce this successful treatment with a larger and deeper diabetic ulcer that was resistant to conventional therapy and was threatening the patient to cause osteomyelitis, and per his physician report the patient was at risk for below-the-knee amputation. The patient was a 60-year-old man who had developed, two years prior to his accident, type-2 diabetes mellitus confirmed by fasting glycemia above 125 mg/dl and hemoglobin A1c results repeatedly above 6.4%. The patient was moving a refrigerator that fell on him, nailing him to the ground with the refrigerator on top of him. The corner of the refrigerator injured the external aspect of his left ankle (Figure 1).

After signing an informed consent, applications of 200 microliters of ViX001 on top of the crater of the ulcer were initiated every other day, without the need for prior debridement of the ulcer. The ViX001 product was left in place while the patient was holding his ankle strictly still for 15 minutes. Next the ulcer was covered with a sterile Band-Aid. Immediately after the initial application, the ankle discomfort resolved. A dried blood clot that was covering the ulcer partially detached and came off with removal of the Band-Aid (Figure 1). Surrounding inflammation regressed, tissue regeneration was detected at the bottom of the ulcer crater and the edge of the ulcer started contracting. After one month of applications, we could detect small patches of capillaries (neoangiogenesis) at the bottom of the ulcer while cells continued to fill the crater.

After two months, the ulcer was less than half of its original size, and after 2.5 months the ulcer was healed, applications were discontinued. Two months later, the ulcer scar remained intact.

3. Severe burn lesions: A 24-year-old woman was referred to our group after sustaining full-thickness burns on her right posterior thigh and partial-thickness burns on her lower legs and back [3]. Her injuries resulted from an explosion while attending a 4th of July party where fireworks were ignited. One rocket flew at a horizontal angle into the crowd, hitting the patient and exploded. She was promptly rushed to the local emergency room. The injuries to the right leg were cleaned and bandaged and she was sent home with an antibiotic cream and wrap. Pain management was with hydrocodone. The next day, the patient was unable to move her leg due to severe pain (score 6-10/10 and 10/10 during cleaning of the burns).

On day three following the accident, the patient was referred to the burn unit where her legs were debrided. Physicians of the burn unit estimated her total recovery time to be greater than 2 months and skin grafting options were entertained for the full thickness burn lesions. Instead, at the request of the patient, she was treated with ViX001 (4 ml) administered topically over 2 days. Forty-five minutes following application, her pain decreased from 10/10 to 2/10, making it easier to clean the affected area, improving sleep, and allowing more range of motion and autonomy, without the use of opioids. Accelerated recovery time allowed the patient to return to work sooner, avoiding painful and costly skin grafting.

4. Pyoderma Gangrenosum: A 78-year-old patient with advanced pyoderma gangrenosum, a rare and dreadful inflammatory skin condition, showed significant improvement after receiving every third-day applications of ViX001 [4]. Within 30 minutes of the first application, the patient’s pain decreased dramatically, and visible healing of the ulcers occurred over several weeks. This case demonstrates the anti-inflammatory and regenerative potential of ViX001, even for the worst kind of skin lesions.

Figure 1: Evolution of the diabetic ulcer for our case report with every other day applications of an aseptic fraction of our human purified amniotic fluid product, ViX001. The upper left picture shows the wound after three months of standard treatment that did not promote healing of the ulcer. The next picture is 19 days later, the day of the first application of ViX001 (200 microliters) showing that if anything, the crater had further enlarged. The next six pictures show the progressive shrinking of the ulcer, which is filling with cells fed by capillaries indicating skin regeneration and contraction of the wound. After 76 days of treatment (11 weeks) the wound had closed, and treatment was discontinued. The last picture obtained two months after treatment discontinuation shows further progression of the skin healing.

Conclusion

The collective evidence from these five case reports highlights the transformative potential of purified amniotic fluid (ViX001) in the treatment of challenging situations where current therapies are limited in their effectiveness: chronic inflammatory disorders like pyoderma gangrenosum [4] diabetic ulcers [2] and spondylitis [1], or acute injuries like severe burns [3]. Further research and clinical trials are warranted to validate these findings, optimize dosing strategies, and explore broader applications of purified amniotic fluid-based therapies in regenerative medicine.

In all cases, ViX001 demonstrated potent anti-inflammatory effects, reducing pain nearly instantly, and inflammation in conditions such as pyoderma gangrenosum, diabetic ulcers, severe burns, and lower back pain [1–4]. ViX001 also promotes tissue regeneration, as evidenced by the healing of diabetic ulcers and severe burn lesions. Perhaps most importantly, whether ViX001 was applied topically or injected intravenously, ViX001 was welltolerated across all case reports, with no significant adverse events reported, even in patients with complex medical histories.

Is it surprising that the ViX001 fraction of purified amniotic fluid has these exceptional anti-inflammatory and regenerative properties? Not really. Human amniotic fluid has been refined over at least seven million years of evolution [5]. We have described pregnancy as a unique case of heterochronic parabiosis, where the fetus is basically “plugged-in” to the maternal body via the placenta that is anchored into the uterus of the mother, with the umbilical cord connecting the placenta to the baby to allow for the formidable pregnancy machinery to happen and function [5]. Not only does amniotic fluid support the development of the fetus, but it is also utilized by the mother to support and enhance her physiology in response to the rigors of pregnancy and childbirth. 

Such a connection is complicated because 50% of the genes of the fetus are inherited from the father of the baby, and the resulting antigens theoretically are exposed to rejection by the immune system of the mother. The maternal and fetal circulations are selectively connected and interact primarily through the exchange of plasma and exosomes, but not cells (nearly insignificant quantities of intact cells are exchanged between mother and fetus). These exosomes, produced by the placenta, amnion, and fetal kidneys, carry essential molecules such as lipids, proteins, complex carbohydrates, microRNAs (miRs), and Klotho that support fetal development and maternal adaptation [6].

The amniotic fluid is a pink opalescent liquid that surrounds and protects the developing fetus during pregnancy [7]. It is produced early in pregnancy from maternal (plasma) and fetal sources that include the placenta, the coelomic fluid, and fluid filtered through the amniotic membrane [6,8,9]. Later in pregnancy, it is further modified though swallowing by the fetus and absorption through the gastrointestinal tract and excretion through the kidneys [6,10]. It is also aspirated through the airways where it mixes with lung secretions. It plays several essential roles in fetal development and overall pregnancy health [6,7,11,12]. It prepares the lungs for birth and initial contact with ambient oxygen in air. It also fills the intestine, covers the skin, the eyes, fills upper airways, and thus it is no surprise that it possesses unique protective properties for these tissues [6,13].

Another critical role of the amniotic fluid is to surround and support the formation of the nervous system, from differentiation of the ectoplasm to neurulation and maturation of the brain and spinal cord [14]. It is also the source of the original cerebrospinal fluid [15]. Hence, it will be instructive to study the impact of amniotic fluid on the development of neurodegenerative disorders and perhaps their prevention or regression. We recently had the opportunity to evaluate the impact of exosomes produced by Schwann cells (SCE) obtained from healthy donors on the progression of amyotrophic lateral sclerosis (ALS) in a severely affected patient [16] using a protocol approved by the FDA. It will be instructive to compare the effectiveness of SCE versus amniotic fluid exosomes because of the involvement of the latter exosomes in the development of the nervous system.

Across these studies, a recurring theme is the ability of exosomes, whether derived from amniotic fluid or other sources, to mediate significant therapeutic effects through modulation of pain, inflammation, promotion of tissue repair, and stabilization of disease progression or regression [5,16–20] Notably, evidence indicates that the therapeutic effects of amniotic fluid are primarily mediated by exosomes [18,20–23]. Antounians et al. demonstrated that, when compared to the rest of the secretome from rat amniotic fluid stem cells, only small EVs (exosomes) specifically mediated the beneficial effects, highlighting their pivotal role in regenerative processes [20].

These vesicles appear to exert their effects both locally (e.g., paracrine effect at sites of tissue injury) [19,21,22]. and systemically (e.g., endocrine effect via intravenous circulation) [22,24] [23], underscoring their versatility in treating a wide range of conditions. These myriad uses of exosomes coupled with low immunogenicity and ease of obtainment necessitate further study investigating their efficacy, optimal dosing, and long-term outcomes [21].

To further unlock the therapeutic potential of exosomes of amniotic fluid (ViX001), comprehensive multi-omic studies are essential to elucidate their signature molecular contents and mechanisms of action. By employing proteomic, genomic, and metabolomic analyses, researchers can identify the specific bioactive molecules within exosomes that are responsible for their formidable antiinflammatory and regenerative effects. Understanding these molecular signatures will not only shed light on the pathways through which ViX001 exosomes exert their effects but also pave the way for the development of targeted therapies with enhanced efficacy.

Acknowledgements: We extend our sincere gratitude to our patients and their families for their unwavering support and participation in these studies. Their contribution was invaluable to our research. We also thank Neobiosis LLC for their exceptional work in manufacturing the proprietary purified amniotic fluid fraction (ViX001) used in our case studies. Additionally, we are grateful to the United States Army for supporting and funding our team member, Brock Sevilla, through the Career Skills Program.

References

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