Polynucleotides and polydeoxyribonucleotides in dermatology – A narrative review

INTRODUCTION

Polynucleotides (PNs) and polydeoxyribonucleotides (PDRN) represent an exciting advancement in pharmacotherapy with a robust regenerative potential as they can modulate gene expression. They are deoxyribonucleic acid (DNA)-derived drugs with tissue repair, therapeutic angiogenesis, anti-inflammatory, and immune-modulating properties. Their sourcing, molecular structure, and weight place them as therapeutic agents, which can be used for numerous indications due to their unique mechanism of action. The recent significant attention they are receiving and their involvement in various dermatological indications is also due to their safety profile and biocompatibility.

BIOLOGY OF PN AND PDRN

Nucleotides are chains of organic molecules containing monomeric units of nitrogen and phosphate bases and a sugar molecule. The chains may be oligonucleotides (short chains with mostly <30 monomeric units) or PNs (longer chains).

These form the building blocks of the genetic material, DNA or ribonucleic acid (RNA), which are also PNs.

The sugar molecule in DNA is deoxyribose, whereas, in RNA, it is ribose, both being pentose sugars. The nitrogenous bases may be purines (adenine and guanine) or pyrimidines (thymine, cytosine or uracil). The pairing of one purine with a pyrimidine base forms the genetic code. The sugar and nitrogenous bases together form a nucleoside, to which one or more phosphate groups are attached to form a nucleotide [Figure 1].

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Figure 1:

Monomeric unit of polynucleotide.

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PNs are biopolymers which are present in every cell. They are chains of nucleotides which provide the genetic code to a living organism, modulate gene expressions and bring about downstream effects such as modulation of inflammation and immune responses, wound healing, and regeneration by stimulating cell proliferation and differentiation.

Nucleotides are only sparingly soluble in water. Formulation methods to make them more soluble must be done in a controlled way to prevent degrading the molecule. They are thermoresistant and absorb ultraviolet (UV) light in the region of 260 nm.¹ The addition of hyaluronic acid (HA) to PNs has been shown to increase the proliferative capacity of PN.²

CHEMISTRY OF PNS AND PDRNS

The nucleotides present in PN may have ribose or deoxyribose as the sugar moiety, as opposed to PDRN, where it is only deoxyribose. PN may be sourced from DNA or RNA, but usually are DNA-derived and are longer fragments of the same. The PDRN extracted from salmon fish have shorter, linear, and single-stranded chains with a double helix spatial structure, and the length of the molecule has 50–2000 base pairs (bp).^3,4

PN is a broader category under which PDRN is a subset. The molecular weight of PN and PDRN is typically between 50 and 1500 kilo Daltons (kDa); however, recent studies have included DNA fragments from 1 kDa up to 10,000 kDa.⁵ Although PN and PDRN have similar molecular effects, and the terms are used interchangeably in scientific literature, it is to be understood that they are different polymers and bioequivalent drugs. To retain their individual identity, the “Marques Polynucleotide Cutoff ” was proposed to differentiate PN having molecular weights ≥1500 kDa and PDRN <1500 kDa.⁵ Differences in their physicochemical characteristics due to different molecular weights can lead to differences in product properties. Both are highly biocompatible due to their origin from natural sources.

PN “cells” or “shapes” are pentagonal or hexagonal with a slight difference in the sizes. The diameter of these is around 3–5 um.⁶ The substance of PN is shown to have intricately placed cells, with thin walls as seen on scanning electron microscopy. The cells are arranged in a tessellated pattern, without any gaps. These ultrastructural patterns place PNs uniquely, as their thin walls make them amenable to be used as slow-release encapsulated drugs, and the ability to modulate the elasticity of the extracellular matrix (ECM) due to their efficient perimeter-area ratio and unique mechanical properties.⁶ Defibrotide, another natural DNA drug, is sourced from porcine intestinal mucosa and has a similar mechanism of action to PNs. However, due to having different isolation sources, molecular weights and manufacturing techniques, it has different pharmacological properties.⁷ An example in this regard is that PDRN stimulates angiogenesis, whereas defibrotide inhibits it. Regadenoson is another DNA drug approved by the US Food and Drug Administration as a pharmacological stress agent used for myocardial perfusion imaging, and just like PDRN, which also binds to adenosine 2A (A2A) receptors and has similar but not same pharmacological properties.

PN acts by providing building blocks for nucleic acid synthesis needed for cellular proliferation and repair. It thus provides slower but long-lasting effects. PDRN acts directly on regeneration and repairing damaged DNA. It increases fibroblast activity, induces angiogenesis, and is anti-inflammatory. PN provides long-lasting structural support thus providing a broader skin rejuvenation. Its actions are more robust, cause enhanced collagen synthesis and have a stronger inflammatory response. PDRN causes more targeted action [Table 1].^6,8

HISTORY OF PN

An early publication in 1963 highlighted the possible therapeutic effect of synthetic PNs on mammalian cells, which were synthesized by PN phosphorylase isolated from Micrococcus lysodeikticus.⁹

PN/PDRN were first used as therapeutics before their role was explored in medical esthetics. PDRN is a registered DNA-derived propriety drug and was used for indications such as wound healing, chronic venous ulcers, and osteoarthritis knee.¹⁰ In the 1990s, there were published studies on the benefits of the use of PDRN in cervical ectropion, post-cauterization re-epithelialization, and wound healing due to its ability to cause fibroblast proliferation.^5,8 This property led to its use as an anti-aging drug.

Initial studies on human placenta extracted PNs were conducted on human knee skin fibroblasts. Research and development by the company Albert David Limited led to an injectable drug called Placentex^®. This is now also available as a gel.

Newer sources and indications are now being explored.

PHARMACOKINETICS OF PDRN

Following an intraperitoneal injection of 8 mg/kg of PDRN in a rat, a bioavailability of 90% was documented with peak levels reaching in 1-h post-injection. The half-life was measured to be 3 h and not affected by the dose of administration. However, the pharmacodynamic transduction effects last longer. The drug is found free in plasma and is distributed to those organs which have greater vascularity. It is a prodrug that is broken down by plasma and membrane-bound DNA nucleases into oligo and mononucleotides, which bring about pharmacological activity. The liver does not metabolize it. Around 65% of the drug is eliminated in the urine and some amount in the feces. This profile matches the pharmacokinetics seen in humans after an intramuscular injection of PDRN.¹¹

The shelf life of PN/PDRN products is 3 years from the date of manufacture when stored at 2–8°C. Since it is a biodrug, immediate use of the opened vial and discarding of the remaining product is recommended.¹²

SOURCING OF PN AND PDRN

Although PDRN has been sourced from the human placenta, PNs for medicinal purposes are now commonly sourced from salmon fish. PN is obtained from a broader source, the gonads of the salmon fish, whereas PDRN is sourced from sperm cells. The testicles of adult male salmon are filled with sperm and the DNA content is 7.5% of its wet weight.⁴ Salmon spermatozoa as a source is unique as it is immune-privileged due to high purity without the additives of proteins or lipids and has a high content of DNA which is similar in structure to human DNA, thus being inert and biocompatible. The fish commonly used are the salmon or rainbow trout (Oncorhynchus mykiss) or chum salmon (Oncorhynchus keta).¹³ Acipenser sinensis (Chinese Sturgeon) is another source for PDRN and PN.

PDRN and PN sourced from plants, phyto PDRN, have also been found to have similar pharmacologic effects with the advantage of sustainable and ethical sourcing. The adventitious roots of Panax ginseng (Korean ginseng) are one such source of PDRN, which was purified using microfluidization after suspension-cultivation through tissue culture.¹⁴ PDRN has also been sourced from red alga (Porphyra spp.) and shown to have proliferative effects on dermal fibroblasts.¹⁵

EXTRACTION PROCESS

A good manufacturing practice (GMP) facility and following strict guidelines and regulations for extraction are mandatory. Strict control measures to isolate and purify the drug must be in place to prevent contamination. Rigorous purification methods including the use of high temperatures, are employed to isolate DNA from the sources allowing for a recovery of 95% or more of the purified drug.^13,14 It is essential that the extraction process ensures a safe, biocompatible and efficacious product with the least immunological potential.

The method of extraction from fish includes (a) collection of sperm from the salmon fish – adopting sterile conditions for isolation from mature, male fish. (b) Lysis of cells – involves using gentle buffers to break the sperm cells to release intact nucleotides. (c) Filtration and centrifugation – to remove cellular debris and contaminants. (d) Removal of endotoxins – using resins. (e) Chromatography – high-performance liquid chromatography is used to purify the contents further and separate the PDRNs according to their size, hydrophobicity or charge. (f) Sterilization – to prevent contamination is done using methods such as heat irradiation or filtration. (g) Freeze-drying – to enable long-term storage of the product by converting it into a stable powder form.³

MECHANISM OF ACTION

Both PN and PDRN may have overlapping mechanisms of action, but as stated earlier, PN acts by providing the nucleotides for DNA/RNA synthesis, whereas PDRN acts by directly causing healing and tissue regeneration [Figure 2].

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Figure 2:

Pharmacological actions of polydeoxyribonucleotides pertaining to dermatology. PDRN: Polydeoxyribonucleotides.

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The multiple pharmacological effects brought about by PDRN are due to the binding of adenosine in the oligo and mononucleotides of PDRN to A2A receptors and its participation in the salvage pathway.^11,13 Binding to A2A receptors is unique to this natural DNA-derived molecule of this specific molecular weight, between 50 and 1500 kDa. Other PDRN bioequivalent drugs or DNA-derived molecules which have molecular weights outside of this range, do not bind to this receptor, and should not be called PDRN.¹¹ There are many in vitro studies on cultured fibroblasts, chondrocytes, and osteoblasts, that support the different pharmacological actions of PDRN.^2,16 The A2A receptor antagonist, 3,7 – Dimethyl-1-propargyl xanthine, negates the effects of PDRN, supporting this evidence of the molecule preferentially acting on this receptor.¹¹ The A2A receptor agonist CGS-21680 has shown to have similar effects to PDRN on collagen synthesis in vitro.¹³ PDRN thus acts as a prodrug, which acts by releasing its components, the nucleotides, nucleosides, and nitrogenous bases, which bind to the A2A receptor. The salvage pathway is an energy-saving one that ensures the synthesis of DNA by providing the building blocks in the form of nucleotides and bases when de novo synthesis is not possible. This situation arises when during periods of extreme stress due to reactive oxygen species (ROS) or ischemia.¹⁷

The mechanisms of action are outlined in Table 2. Phyto PDRN also acts through this receptor.¹⁴

A2A receptors are present in the cell membrane of cells of several human tissues; however, they are highly concentrated in the immune cells, which highlights their role in inflammation.¹⁸ Adenosine receptors are of three types A1, A2A, A2B, and A3. Signal transduction through A2A receptor binding is responsible for modulating inflammation, ischemia, cell proliferation, and differentiation and angiogenesis.¹⁴ Receptor-binding causes the release of cyclic adenosine monophosphate through stimulation of Gs protein, which inhibits signaling pathways such as mitogen-activated protein kinase and nuclear factor kappa beta (NF -κB) and stimulates the transforming growth factor-beta/suppressor of mothers against decapentaplegic signaling pathway.¹⁹ The mechanisms of action of PN are similar.⁶ The net effect of these interactions reverses the actions of ROS generated through UV ray exposure or mitochondrial aerobic metabolism and leads to a decrease in the pro-inflammatory cytokines and matrix metalloproteinases.

The following mechanisms of action have been elucidated with both, in vitro research and confirmed in vivo on humans as well [Figure 3].

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Figure 3:

An outline of the two main pathways of action of polydeoxyribonucleotides. PDRN: Polydeoxyribonucleotides.

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MODES OF DELIVERY

PDRN is obtained as a white powder or a fibrous solid. It is reconstituted and delivered through various modes.⁴

The drug is mostly injected intradermally or delivered into the skin through microneedling. Topical anesthesia can be used to numb the skin. Injections are made using 32–33 G needles using serial puncture technique usually, mesoguns or cannulae. Injections allow for a deeper penetration. The depth of microneedling is dependent on the indication and anatomical site where the drug needs to be delivered.

Delivery of PDRN/PN post ablative lasers such as carbon dioxide or erbium can also be done.¹³ This method may be used to help with lesser downtime and faster healing, or as a mode of drug delivery through the channels created, combining the two treatments.

Topical formulations of PDRN/PN are gaining attention due to their non-invasiveness. The different formulations available are creams, serums, lotions, essences, and masks. PDRN healing gels are available for use after an ablative procedure.¹⁸ Hydrogel dressings have been used for diabetic ulcers.²⁶ Topicals, however, may have limited penetration and, hence, are less efficacious than injections. They may be useful as maintenance treatments and in improving skin hydration.

PDRN/PN are also delivered through electroporation and iontophoresis. These methods may have the advantage of having no downtime. An oral spray formulation has been used for oral mucositis.²⁷

PN-containing devices are used as temporary fillers (intradermal gels) due to the viscoelastic properties of PNs. These act as bioreactivators and improve the skin health and cause tissue regeneration.²⁸ The PN Highly Purified Technology ^TM (PN-HPT ^TM) device contains 15 mg/2 mL of intradermal gel.²⁸

A study by Morganti et al. concluded the role of PN, a biodegradable fiber containing non-woven tissue, as a delivery system for cosmeceutical delivery. It was seen to improve the bioavailability, skin penetration, and controlled release of the cosmeceutical ingredients and inhibit any allergic reactions to the product. Many topical products such as moisturizers, antioxidants, and vitamins have been delivered using PN.⁶

PROTOCOL FOR ADMINISTRATION

There is no standardized protocol for administering PN/PDRN. Most studies have revealed a treatment plan wherein the drug is delivered once in 2–4 weeks and 3–4 sessions of the initial phase treatment are done. Maintenance sessions are scheduled periodically. Results are apparent in 4–8 weeks and improve over 3–6 months.¹³

COMBINATION TREATMENTS

PDRN treatments can be combined with other esthetic treatments depending on the indication that they are being used for. Lasers, light-based treatments, and energy-based devices are all used in combinations both for skin rejuvenation as well as for hair restoration.¹³ HA injectables are also combined with PDRN for use in volume restoration or skin health boosting. Interestingly, it has been found that the addition of even as low as 1mg/ml of HA to PDRN increases its activity by up to 20%.² Medifacials incorporating PDRN skin masks may add to the benefit of the procedure. Home care skin products containing PDRN can be prescribed with any of the above treatments.

SAFETY PROFILE OF PN/PDRN

Individuals with hypersensitivity to fish or fish products should be cautious with the use of these DNA drugs sourced from salmon as they may lead to an allergic reaction.³ However, not all patients with fish allergy develop reactions to PN/PDRN. These products are purified by filtration during extraction. Prior patch testing with the drug may help identify such patients.

A few studies that have elucidated the safety profile of PDRN have shown that it is a well-tolerated and safe drug, with no related complications. Several studies have shown that this biodrug is non-toxic. No data however is present on the long-term evaluation of the drug. No mutagenic potential of the drug has been reported so far. However, since it is a biodrug and it increases VEGF and hypoxia-inducible factor-1, some concerns about its theoretical mutagenic potential have been raised.²⁹

Most of the side effects are localized due to the mode of delivery. A minimal downtime with redness and swelling may be present after intradermal injections or microneedling for 1–3 days. Mild discomfort may be noted during the injectable treatment which can be alleviated with the use of ice packs and topical numbing. A few bumps may be present post-treatment, lasting a few hours to a day or two.³ The use of intradermal gel devices may cause burning for up to 12 h after the injections, bruising, and small hematomas rarely.²⁸ In a consensus statement, it has been recommended to avoid exposure to sun lamps and sunlight for 48 h post-PN/PDRN treatment.²⁸

The procedure should be performed with aseptic precautions in place to avoid infection of the treated area.

Both, PN, and PDRN can cause temporary histamine reactions. The use of PN/PDRN in pregnancy and lactation is not recommended due to a lack of studies.

REGULATORY CHALLENGES

Analytical methods for PDRN testing should be in place. Optimal operating conditions and quality control must be maintained from isolation to the final product, using a GMP facility. Stability reports with stability data must be in place to ensure that the drug remains stable in different storage conditions. All regulatory compliance documents complying with international and regional regulations should be present. Challenges arise due to inadequate operating protocols and structured guidelines for sourcing and manufacturing.³⁰

INDICATIONS OF PDRN IN DERMATOLOGY

PDRN and PN are products that typically fit into the realm of medical esthetics, as they are increasingly being used for therapeutic indications.^31,32 It is a biodrug, hence with more potent mechanisms of action. Patients are counseled about the cost:

INDICATIONS FOR THE USE OF PN IN DERMATOLOGY

PN has also been used for similar indications like PDRN, for both esthetic and therapeutic indications.

Its effectiveness in skin rejuvenation of the face and body such as neck aging by improving skin thickness, improving skin elasticity, reducing pore siz e, improving skin tone, skin sagging, and wrinkling has been sustantiated.^6,49 It has been used in cocktails with PRP, HA, and growth factors for skin boosting.⁶

Similar results are seen in Asian skins, which have thinner epidermis.⁵⁰ In a Korean study, the top six indications for the use of PN were (in order of decreasing popularity), fine lines on the cheek, infraorbital fine lines, fine lines in the periorbital area, uneven texture of the facial skin, skin dryness, skin glow, and fine lines on the forehead.⁶

PN is increasingly being used as a filler material as it provides a more natural-looking result without any major risks or complications as it is bio inert. Proper patient selection is necessary for optimal results.⁵¹ PN-HPT ^TM is used intradermally for skin rejuvenation, revitalization and evening skin tone for both the face, and body. The neck, décolleté, and hands are the body areas that have been treated. These treatments have also been used in the scalp, eyebrows, and beard for normalization of follicular activity and hair growth.²⁸ A concept called the “PN-HPT ^TM priming” is the use of PN to prepare the tissues for a stronger and faster response to a second revitalization stimulus. It prepares the tissues before combination with other treatments for both, esthetic treatments as well as wound healing.^28,41

PN filler when compared with HA filler for treating periocular crow’s feet wrinkles showed the same result, but was found to last longer.⁶ When compared to polycaprolactone for the same indication, both showed improvement, but PN was faster to act and lasted longer with fewer side effects such as bruising and swelling.⁵²

Hair growth and hair follicle revival is shown to increase with the use of PN-HPT ^TM, injected into the scalp once in 7–14 days for 4 weeks, followed by 4 more sessions 21– 30 days apart.²⁸

A randomized, double-blinded controlled trial revealed that an adjuvant treatment with PN along with fractional laser led to a favorable outcome in wound healing. PN-based therapy has shown to improve scar visibility and improved skin texture across cases with post-traumatic, burn, or postoperative scars, including scars after open thyroidectomy.^53,54

A prospective controlled study was conducted in a small population to study the effect of highly purified PN for the treatment of moderate to severe acne scars, and it concluded its effectiveness in the treatment.⁵⁵ PN has also been useful in treating post-surgical atrophic scars.

Facial erythema due to inflammatory facial dermatoses such as rosacea, eczemas, or photoaging has been treated with PN in combination with lasers or microneedling radiofrequency and phototherapy. It has been found effective in protection of the skin barrier, improve skin hydration, vascular stabilization, and anti-inflammation.^6,56

PN-HPT ^TM has been found useful for stretch mark treatment in combination with PN along with HA vials for injection.²⁸ A medical device containing polyphosphoric acid, mannitol, HA, and PN-HPT was applied which intradermally showed improvement in 85% of patients with stretch marks.⁵⁷

PN is a promising alternative for the treatment of periorbital rejuvenation with significant improvement in the under-eye area. It helps with laxity, wrinkling, and pigmentation. PN filler has been used and compared with non-cross-linked HA fillers for periorbital rejuvenation. The improvement in skin elasticity and hydration was better seen in the PN group, and so was the improvement in the pore volume and skin roughness. There were no adverse effects seen.⁵⁸

Several studies have highlighted the effectiveness of PN for vulvo-vaginal rejuvenation. It ameliorates vaginal atrophy, vaginal dryness, and itching. It has been used as a filler with HA for labia majora rejuvenation.^59,60

CONTRAINDICATIONS TO THE USE OF PN/PDRN

A patch test is warranted in patients who give a history of hypersensitivity to fish. Other contraindications to be mindful of are as mentioned [Table 4].

CONCLUSION

PN/PDRN are biomaterials with proven efficacy in various regenerative and dermatological indications. This review presents developments that are pivotal in broadening the accessibility and acceptance of PDRN and PN-based therapies in various medical and esthetic fields. Although it highlights the strong regenerative potential of the biomaterials, more controlled studies are needed to bridge the gaps in the evidence-based use of these PNs. Thus, the present positioning of PN/PDRN use as a drug in the regenerative treatment segment is promising yet needs attention regarding certain critical issues.