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aestheticsjournal.com @aestheticsgroup Aesthetics Journal Aesthetics one point A Review of Vitamin A Roger Bloxham and Antony Wakeford explore vitamin A’s role as a treatment for ageing and photodamaged skin Introduction Vitamins are organic compounds that the human body requires to effectively function, thus they have a direct affect on our health and wellbeing. They are derived from the diet in small amounts, and, as such, are often referred to as micronutrients.1 An organic compound is considered a vitamin if a lack of it results in clinical symptoms, known as vitamin deficiency. Summary of vitamins · Vitamin A is a term used for a group of fat-soluble compounds known as retinoids; these include retinol, retinal and retinoic acid. Their primary action is to affect gene expression and the production of Messenger RNA (mRNA), resulting in the production of proteins and the differentiation of epithelial cells and tissues, which develop from the ectoderm. Vitamin A therefore plays a key role in the development and integrity of the skin (the epidermis in particular), and the outer membranes of sensory organs such as the eye and neural membranes.1,2, · Vitamin C, or ascorbic acid, is an electron donor and is the primary and highly potent water-soluble non-enzyme antioxidant present in plasma and tissues throughout the body. It also acts as an electron donor for key enzymes involved in catecholamine synthesis (hormones involved in collagen hydroxylation) and subsequent collagen structure stabilisation as well as carnitine synthesis and subsequent mitochondrial activity.1,3 · Vitamin E describes a group of fat soluble compounds known as tocopherols. Vitamin E is a very effective antioxidant, which prevents or helps to slow down lipid oxidation and subsequent damage to cell membranes and membrane structures and lipoproteins that transport fats through the blood stream.1,3 · Vitamin D is formed from cholesterol in the skin through a photolysis reaction stimulated by exposure to UV light. This fat-soluble compound plays a role in inducing protein synthesis and calcium absorption in the intestine and associated optimum calcium levels in the kidneys and bones.1,2 · Vitamin K is involved in the carboxylation of glutamic acid in a number of vitamin K dependent proteins; during the process it is converted to an epoxide form that can be regenerated to vitamin K within the body. Many of the blood clotting factor proteins are vitamin K dependent.1,2 · The B-complex vitamins are water soluble coenzymes: Thiamine (B1) is involved in enzyme functions associated with carbohydrate metabolism.1,2 Riboflavin (B2) is a precursor of coenzymes that are involved in protein metabolism.1,2 Niacin is a coenzyme of dehydrogenases that plays a key role in the metabolism of proteins and carbohydrates.1,2 Vitamin B6, of which the most stable form is pyridoxal, is a coenzyme involved in protein metabolism.1,2 Pantothenic Acid (B5) is the main component of coenzyme-A and a key requirement for cell metabolism, the synthesis of essential fats, cholesterol and hormones, including melatonin.1,2 Biotin is the active site of five carboxylating enzymes required for the biosynthesis of fatty acids and gluconeogenesis.1,2 · Folates, including folic acid, are cofactors to enzymes that facilitate the transfer of carbon units in transfer reactions, particularly associated with the metabolism of DNA. Its effects are most prevalent in rapidly dividing cells such as red blood cells. It is also key to a process known as methylation and the associated gene expression responsible for the processing of proteins, phospholipids and cell differentiation.1,2,3 · Vitamin B12 plays a key role in the metabolism of folate and coenzyme A; it is therefore closely linked to DNA metabolism and expression.1,2 Given the primary role of vitamin A in the production and differentiation of epithelial cells, which includes the skin and in particular the epidermis, as well as the fact that a lack of vitamin A causes observable changes in skin epithelial tissue, including epidermal thickening and hyperkeratosis,2 it is no surprise that retinoids became, and still are, a topic of dermatological research for skin conditions and treatments. This is the focus of the discussion below. Vitamin A: How does our body get it and use it? The retinoid compounds that the term vitamin A describes are all lipid soluble, and are related to the preformed vitamin A compound all-trans-retinol. This is the active bioavailable form of vitamin A. There are four main physiologic functions of retinoids in the body:4 1. To ensure normal embryonic development. 2. To facilitate normalised epithelial differentiation in varying tissue types, including the skin. 3. To maintain healthy vision through the production of rhodopsin, an 11-cis-retinaldehyde containing eye pigment that enables night vision. 4. To enable adequate immune function and lymphocyte survival. Vitamin A and nutrition Vitamin A levels within the body depend on levels of nutrition. Preformed vitamin A, predominantly in the form of retinyl esters (esterified retinol, mainly retinyl palmitate), is available from animalbased foods – liver, butter, cheese, egg yolks and oily fish are known to have a high content. It is also possible to derive vitamin A from vegetables and fruit via a relatively small sub-section of the many carotenoid compounds present in plants, particularly alpha and beta-carotenes. These are known as pro vitamin A Reproduced from Aesthetics | Volume 2/Issue 6 - May 2015 @aestheticsgroup compounds.3,5 In addition, some food items, such as margarine and low-fat spreads, are fortified with vitamin A. The different sources of vitamin A have different levels of potency available in food items. For example, retinyl esters are membranebound within storage cells within the animal tissue. The pro-vitamin A carotenoids are not only bound to lipids but are embedded in complex cellular structures within the plant material. Therefore the amount of the retinyl ester or carotenoid does not directly reflect the amount of vitamin A (retinol) available. Because of this, standardised units known as International Units (IU) or Retinol Equivalents (RE) are often used to express the levels available per compound, and to help provide a comparison across the different food sources.2,3 There can be a wide variability in levels contained in the different food sources, for example 100g of pork liver has been shown to have 30,000 μg RE, whereas pork muscle has 6 μg RE and oily fish has 40 μg RE.2 There are risks associated with taking too much vitamin A. The UK National Health Service (NHS) has advised that having more than an average of 1.5mg a day vitamin A, over many years, may affect bones, making them more likely to fracture.6 They point out that this is particularly important for older people, especially women, who are already at risk of osteoporosis. This is when bone density reduces and there is a higher risk of fractures. Thus, women who have been through the menopause and older men, who have a higher risk of osteoporosis, are advised to avoid having more than 1.5mg of vitamin A per day from food and supplements.6 Eating liver or liver pâté more than once a week may result in too high a vitamin A intake, and it is advised that if liver is eaten every week, supplements containing vitamin A should not be taken. Additionally, if the dietary intake of vitamin D is too low, it is possible that there will be more risk of the harmful effects of too much vitamin A.6 In addition to the osteoporosis risks associated with taking too much vitamin A detailed earlier, there are additional risks in pregnancy.6 Having large amounts of vitamin A can harm an unborn baby. It is advised that women who are pregnant, or who are thinking about becoming pregnant, should not eat liver or liver products, and should not take supplements that contain vitamin A. It is recommended that a GP or midwife be consulted for more information.6 Vitamin A and bioavailability Once the food has been consumed, digestion frees the retinol from the retinyl esters. The lipid soluble retinol is then inside a predominantly aqueous environment in the intestines and gut, with fatty acids and phospholipids and bile secretions. It then has to pass through the lipid-rich intestinal mucosal cells. Enzymes either cleave vegetable derived carotenoids as they pass through the intestinal mucosa to form retinol, or they remain as they are. Retinol is then re-esterified or is bound to a specific protein retinol Aesthetics Journal Aesthetics aestheticsjournal.com binding protein (RBP), synthesised within the tissue, and passes into the blood stream. Any carotenoids or retinol that has become re-esterified in the intestinal cell wall is packaged in to lipoprotein particles and enters into the blood stream via lymph channels.3 The next step is storage, predominantly within the liver, in parenchymal cells. Retinol is detached from the RBP or the retinyl ester is removed from the circulating lipoprotein particle and then hydrolysed within the liver to produce retinol. The resulting retinol is then esterified for storage. The liver can also take up carotenoids from the lipoprotein particles for storage.3 Stored retinyl esters in the liver are mobilised by hydrolytic release of retinol and binding to a RBP, produced in the liver, enabling it to be transported in the aqueous environment of the plasma in the blood stream to target tissues and cells. The levels of plasma vitamin A are maintained at approximately 2μmol/L. The circulating retinol RBP combination forms a further complex with a larger protein transthyretin, to ensure the retinol is not filtered out through the kidney.7 The bound retinol enters into the tissue through the cellular membrane, where the retinol binds to an intracellular binding protein (CRBP). Within the cell, retinol can be esterified, most likely to form a localised store, and more importantly it can be metabolised to form retinal (also known as retinaldehyde) and then retinoic acid. The conversions of retinol to ester forms or retinal are all reversible which, consequently, can regulate the levels of retinol and retinoic acid in the cells.2,9 Vitamin A and biochemical activity The cellular conversion of retinol to retinal and retinoic acid is fundamental to its biological role and ultimately its role in the treatment of ageing and photodamaged skin. In ocular tissue and specifically the retina, retinol is converted to retinal and then rhodopsin – the pigment required for low light vision. For the control of cell growth and differentiation in all other epithelial tissue retinol is converted to retinal and then to retinoic acid (of which there are two primary isomers). These bind to receptors in the nucleus (known as Retinoic Acid Receptor (RAR) and Retinoid X Receptor (RXR) receptors), which in turn bind to specific elements of DNA and consequently regulate gene expression and transcription.8 As such, vitamin A is essential for the biological functions previously mentioned.4,9 Vitamin A deficiency is rare in the western world but can be a major problem in the developing world with specific symptoms including night blindness and xerophthalmia, which can lead to permanent blindness. Other significant negative effects associated with deficiency include growth retardation in children, skin disorders and impaired immune function. Deficiency in pregnancy can lead to congenital malformation of the eyes, lung, cardiovascular and urinary systems.5 In the environment of cosmetic dermatology and aesthetics, we do not usually have to deal with such issues of deficiency. The Table 1: Table comparing pre-formed and pro vitamin A compounds: Molecule Quantity Retinol Equivalents (RE) International Units (IU) Retinol 1μg 1.00 3.3 Retinyl Palmitate 1μg 0.55 1.8 Beta-carotene 1μg 0.17 0.6 (data adapted)1,2,3 Reproduced from Aesthetics | Volume 2/Issue 6 - May 2015 aestheticsjournal.com @aestheticsgroup Aesthetics Journal clinical focus with respect to vitamin A is for use in the treatment of physical and anatomical impairment of the epithelial tissues and cells that occurs i.e in the skin, as part of the ageing process and photodamage, and to treat other cosmetically impairing skin conditions such as pigmentation and acne. Vitamin A and the clinical enhancement of aged and photodamaged skin During skin ageing, the production and replacement of collagen, elastin and hyaluronic acid slows down as there are fewer dermal fibroblasts, and they operate less efficiently. Skin not damaged by photo radiation will age as a result of this, and become thinner, more lax and finely wrinkled.10,11 External factors, including UV radiation and smoking, accelerate the natural ageing process. In the 1990s, research showed that photodamaged skin had premature damage of the collagen bundles and amorphous amounts of elastotic material, which was likely due to increased synthesis of matrix metalloproteinases (MMPs) and subsequent collagen destruction.12,13 It was also demonstrated that photodamaged skin has reduced capability to produce new collagen.14 This prematurely reduces the elasticity, firmness, and smoothness of the skin, resulting in coarser lines and wrinkles. Photodamaged skin is also differentiated from naturally aged skin by a thickened and rougher appearance. With an understanding of the role of vitamin A within biological processes, and the mechanisms and effects of skin ageing and photodamage, it comes as no surprise that vitamin A is often considered an essential component of an evidence-based antiageing skin management programme. The role of oral retinoids The consumption of vitamin A via food substances or supplements could be considered to be a logical route to try and combat the effects of intrinsic skin ageing and photodamage. However, vitamin A deficiency is rare in the western world and it is known that average daily intakes of preformed retinol within the EU are well above the recommended reference intake.5 Despite this, aesthetic consultations and treatments for skin ageing and photodamage continue to rise, implying that dietary levels of vitamin A may have little correlation with the impact of chronological or photo-induced ageing. A review of clinical studies on PubMed reveals oral vitamin A supplement studies relating to skin are relatively low in number and the data that was sourced relates primarily to skin cancer. This data is briefly reviewed here, as it can reasonably be considered to have some correlation to the impact of UV in photodamaged skin. A recently published review of vitamin A in photocarcinogenesis stated that relatively high oral doses of prescription only medicines (POMs), isotretinoin and acitretin, resulted in a significant reduction of new skin cancers in high-risk patients.15 Whilst many medical aesthetic treatments crossed over from their initial use in more clinical areas, it would be difficult (based on a risk/ benefit assessment) to attempt to adapt this approach with these medications for skin ageing treatments. One study showed daily supplementation of 25,000 IU of retinol, more than 10 times the recommended daily dose, for a median period of 3.8 years, did significantly reduce the incidence of squamous cell carcinoma.16 However a four-year follow up prospective study of 73,336 women showed that their diet, including vitamin A intake, or supplementation with vitamins including vitamin A and multivitamins, had no significant impact on the incidence of basal cell carcinoma.17 Aesthetics Similarly, a study of men taking 50mg of beta-carotene on alternative days for 12 years, again more than 10 times the recommended daily intake, showed that it had no effect on the incidence of nonmelanoma skin cancer when compared to the placebo group and there was no beneficial link associated with plasma levels of betacarotene, vitamin A or vitamin E.18 Based on this, it is logical to conclude that oral intake of vitamin A is going to play a minimal role in treating ageing and photodamaged skin and, therefore, for an evidence-based approach, we need to focus on topical retinoids. The role of topical pharmaceutical retinoids As early as 1962, the effect of topical vitamin A in the form of tretinoin (all-trans-retinoic acid) was shown to affect epidermal differentiation and keratinization, and was used clinically in the treatment of ichthyoses.19 In 1969, Kligman published the first clinical paper clearly demonstrating its clinical efficacy in the treatment of acne20 and in the early 1970s tretinoin was approved by the Food and Drugs Administration (FDA) in the USA as a prescription only medicine, the first retinoid approved as a medicine, for the treatment of mild to moderate acne.19 However, it took some time before the skin physiology-changing attributes of tretinoin were appreciated in the treatment of photodamaged and aged skin. In 1986 Kligman published a study showing that in photoaged skin, topical tretinoin 0.05% induced a normalisation of keratinocyte exfoliation and proliferation, along with improvement in keratinocyte morphology and function, extracellular matrix structure and formation, and angiogenesis.21 A review of clinical studies available for tretinoin summarised that the shorter-term studies of four to 16 weeks demonstrated physiological changes in the epidermis and significant enhancements in appearance but with minimal dermal changes. Longer-term studies of approximately six months showed that these clinically significant improvements in fine and coarse wrinkles, sallowness, hyperpigmentation, roughness and epidermal structure continued, and that these improvements were maintained after the study periods. Interestingly, it was the studies of 12 months or more that showed clinical changes in the dermis, with new collagen production and the regulation of damaged tissue.9,22 Topical tretinoin presented some potential adverse events, such as erythema and inflammation in the areas of application, as well as increased photosensitisation. It was for this reason that lower strength tretinoin was assessed and showed good results. 0.025% and 0.1% tretinoin produced statistically equivalent results after 48 weeks’ use, with significantly less adverse events in the lower strength group.22 In the USA, the FDA review of products containing tretinoin has, to date, designated them to be POMs.24 The same is true in the UK with respect to the Medicines and Healthcare products Regulatory Agency (MHRA)25, and the EU Cosmetics Regulation26 lists tretinoin as a substance prohibited in all cosmetic products. The electronic medicines compendium (eMC) website27 shows that there are two topical medicinal products containing tretinoin available in the UK. Both are combined with an antibiotic and indicated for the treatment of acne. There are currently no forms of tretinoin suitable for use in the rejuvenation of aged and photodamaged skin with an MHRA approved marketing application (MA). If a tretinoin product is made available within the UK without an MA it must be done under the section of the Human Medicines Regulations 2012 (SI 2012 No. 1916)28 concerning unlicensed medicinal products for individual patient use. Regulation 167 of the Regulations Reproduced from Aesthetics | Volume 2/Issue 6 - May 2015 @aestheticsgroup requires that there is a bona fide unsolicited order, that the product is formulated in accordance with the requirement of a doctor or dentist, nurse independent prescriber, pharmacist independent prescriber or supplementary prescriber registered in the UK, and the product is for use by one of their individual patients (on the basis of ‘special need’) on the practitioner’s direct personal responsibility. In addition, guidance from the prescriber’s governing body should be considered. For example, the Nursing and Midwifery Council (NMC) provide such guidance accessible via their website.29 The most recent generation of medicinal retinoids are synthetically produced. They were developed to have a more selective mechanism of action and better tolerability. Adapalene, the first synthetic retinoid, is readily taken up by the pilosebaceous unit (hair shaft, hair follicle, sebaceous gland) and was shown to directly bind to RAR receptors and regulate keratinisation and inflammation.9 There are cream and gel formulation POMs approved by the MHRA for the treatment of mild to moderate acne.14 Their short-term use over four weeks and longer-term use of up to nine months, have shown significant improvements in photoaged skin,30 although such use is not in accordance with the approved indications, and would be considered to be ‘off-label’ with respect to the currently available and approved medicines. Tazarotene is another synthetically produced retinoid approved in the UK in a gel form for the treatment of psoriasis.14 Aesthetics Journal Aesthetics aestheticsjournal.com Specific retinoids are also available for use as cosmetic ingredients in compliance with the Cosmetic Product Regulations and the EU Cosmetic Directive. With respect to their use in ageing and photodamaged skin, retinol has had established evidence-based cosmetic use since 1984, and is accorded ‘generally recognized as safe’ (GRAS) status by the FDA. Retinaldehyde (retinal) has generated clinical data supporting its use and the retinol derivatives such as retinyl palmitate are widely used due to their stability and ease of formulation, although less clinical evidence of their efficacy as a monotherapy is available.9 All of the retinoids referred to above require a biological conversion within the skin to form retinoic acid.9,31 The advantage of the cosmetic retinoids is that they are more widely available for use than medicinal products, and their skin tolerance has been shown to be significantly greater than tretinoin.32 The fact that they are also cosmetics allows greater flexibility with respect to formulation. The evidence of the clinical efficacy of retinol is compelling. Kang et al showed that 1.6% retinol induced similar effects to 0.025% retinoic acid, without similar measurable skin irritation.33 The retinol caused epidermal thickening and enhanced expression of retinol binding protein mRNA, similar to that of retinoic acid. However, the erythema produced by retinol application was significantly less. Interestingly, they discovered that the retinoic acid levels found in the epidermis were approximately a thousand times less in the retinol treated skin compared to the retinoic acid treated skin, despite seeing similar clinical effects and markers. They postulated that the retinol was being converted to retinoic acid in a tightly controlled manner at physiologically relevant sites; the right amount at the right location within the skin. Further studies have shown that retinol is safe and effective in the treatment of photodamaged skin.31,34,35 It is acknowledged, however, that formulation is key as retinol-based products can be unstable, particularly on exposure to light and air.9 Because of the clinical value of retinol and the fact that its efficacy Figure 1: Figure 2: The role of topical cosmetic retinoids Before Before After A 42-year-old female before (left) and after (right) eight weeks of treatment with Retinol (0.5%) aqeuous protein-rich suspension. Images courtesy of Dr Michael Gold. Figure 3: Before After After A 69-year-old female before (top) and after (bottom) eight weeks of treatment with Retinol (1.0%) aqeuous protein-rich suspension. Images courtesy of Dr Vivian Bucay. Visia brown spot analysis of a 41-year-old female before (left) and after (right) eight weeks of treatment with Retinol (0.5%) aqueous protein-rich suspension. Images courtesy of Dr Michael Gold. Reproduced from Aesthetics | Volume 2/Issue 6 - May 2015 aestheticsjournal.com @aestheticsgroup Aesthetics Journal depends greatly on its stability and bioavailability, research and formulation developments continue. Gold et al demonstrated in 2013 the efficacy and tolerability of such a product, reporting a pilot study of a serum with retinol 1% in a novel oil-free aqueous protein rich suspension, showing improved visible signs of photodamage after eight and 12 weeks of daily use. Their subsequent study using 0.5% retinol in the same formulation showed that hyperpigmentation, telangiectasias, skin laxity, roughness and actinic keratosis showed significant improvement from baseline after four and eight weeks daily use of the formulation. Skin tolerability was reported to be good.33 Conclusion When it comes to aged skin, and skin whose ageing process, structure and appearance has been worsened by photodamage, topical vitamin A plays a key role in its treatment and visible and physical enhancement. There are robust double blind placebo controlled clinical studies regarding the use of tretinoin (retinoic acid) in the treatment of aged and photodamaged skin.21,22,23,32 Whilst clinically visible effects can be seen after four weeks, the greatest physiological effects are seen after daily use of 12 months or more, and the studies show the treatment has a good safety profile over such periods. This indicates that on-going long-term treatment is desirable, although skin tolerability issues were often reported particularly in the early phases of treatment. In the UK, the availability of tretinoin in topical formulations suitable for use in treating aged and photodamaged skin is currently limited to unlicensed medicinal products. These may only be supplied in accordance with the Human Medicines Regulations concerning unlicensed medicines for individual patient use and by prescribers able to prescribe such medicines, and in accordance with their governing body / council such as the General Medical Council. There is also compelling data for the efficacy of retinoids, available in cosmetics for the clinical improvement of aged and photodamaged skin.33, 34, 35, 36 Cosmetics containing these ingredients are more widely available and generally display better skin tolerability than prescription-only tretinoin-based topical medicines. Within this category, retinolbased formulations are supported by a high level of clinical data, although formulations need to address the inherent instability of retinol as an ingredient.9,31,33,34,36 On-going research and novel formulation enhancements means that retinol-based cosmetics can and will continue to play a significant role in the clinical management of aged and photodamaged skin. Roger Bloxham is the managing director of Ferndale Pharmaceuticals Ltd and AesthetiCare, with a BSc (Hons) in Applied Biology and over 28 years of commercial, technical and regulatory experience in the pharmaceutical, medical device and cosmetic industries. In the last 18 years, his focus has been on the dermatology sector. Antony Wakeford is a chartered chemist and member of the Royal Society of Chemistry. As the owner and managing director of QP-Services UK Ltd., an independent provider of technical, quality, safety and regulatory services to the pharmaceutical, medical device and cosmetic industry, he has over 32 years experience in the industry. Aesthetics REFERENCES 1. Oregon Sate University Linus Pauling Institute Micronutrient Research for Optimum Health, Micronutrient Information Center (US: Oregon State University, 2015) <http://lpi.oregonstate.edu/ infocenter> [Accessed 14 April 2015] 2. Belitz HD et al. Food Chemistry, 4th Revised and Extend Version (Berlin: Springer, 2009) p. 403-419 3. Food and Agriculture Organization of the United Nations. Human Vitamin and Mineral Requirements. Report of a joint Food and Agricultural Organisation of the United Nations (FAO) and World Health Organization (WHO) Expert Consultation (Italy: foa.org, 2001) < http:// www.fao.org/docrep/004/y2809e/y2809e00.htm> [Accessed 14 April 2015] 4. Kang S, ‘The mechanism of action of topical retinoids’, Cutis, 75(suppl 2) (2005), p. 10-13. 5. European Commission, Scientitifc Committee on Food. Opinion of the Scientific Committee on Food on the Tolerable Upper Intake Level of Preformed Vitamin A (retinol and retinyl esters) (Brussels: European Commission – Europa, 2002) <http://ec.europa.eu/food/fs/sc/scf/ out145_en.pdf> [Accessed 14 April 2015] 6. NHS Choices, Vitamins and Minerals – Vitamin A (UK: NHS Choices, 2015) <http://www.nhs.uk/ Conditions/vitamins-minerals/Pages/Vitamin-A.aspx> [Accessed 14 April 2015] 7. Blomhoff, R. ‘Vitamin A metabolism: new perspectives on absorption, transport, and storage’, Physiol Revs, 71(1991), p. 951-990. 8. Allenby et al, ‘Retinoic acid receptors and retinoid X receptors: Interactions with endogenous retinoic acids’, Proc Nati Acad Sci, 90 (1993), p.30-34. 9. Mukherjee et al, ‘Retinoids in the treatmentof skin ageing: an overview of clinical efficacy and safety’, Clinical interventions in Aging, 1(4) (2006), p.327-348. 10. Fisher et al, ‘Mechanisms of photoaging and chronological skin aging’, Arch dermatolo, 138 (2002), p.1462-1470. 11. Varani et al, ‘Decreased Collagen Production in Chronologically Aged Skin:,Roles of Age- Dependent Alteration in Fibroblast Function and Defective Mechanical Stimulation’, Am J Path, 168 (2006). 12. Fisher et al, ‘The molecular basis of sun induced premature ageing and retinoid antagonism’, Nature, 379 (1996) p. 335-338. 13. Fisher et al, ‘Pathophysiology of premature skin ageing induced by ultraviolet light’, N. Engl J med, 337 (1997), p. 1419-28. 14. Griffiths et al, ‘An in-vivo experimental model for topical retinoid effects on human skin’, Bt J Dermatol, 29 (1993), p. 389-99. 15. Shapiro et al, ‘Vitamin A and Its Derivatives in Experimental Photocarcinogenesis: Preventive Effects and Relevance to Humans’, Journal of Drugs in Dermatology, 12(4) (2013), p. 456-458. 16. Moon et al, ‘Effect of retinol in preventing squamous cell skin cancer in moderate-risk subjects: a randomized, double-blind, controlled trial. Southwest Skin Cancer Prevention Study Group’, Cancer Epidemiol Biomarkers Prev, 6(11) (1997) p. 949-56. 17. Hunter el al, ‘Diet and risk of basal cell carcinoma of the skin in a prospective cohort of women’, Ann Epidemiol, 2(3) (1992) p. 231-9. 18. Schaumberg et al, ‘No effect of beta-carotene supplementation on risk of nonmelanoma skin cancer among men with low baseline plasma beta-carotene’, Cancer Epidemiol Biomarkers Prev, 13(6) (2004), p. 1079-80. 19. Zainglein A L, ‘Topical Retinoids in the Treatment of Acne Vulgaris. ‘Semin Cutan’’, Med Surg, 27 (2008) p.177-182. 20. Kligman et al, ‘Topical Vitamin A acid in acne vulgaris’, Arch Dermatol, 99 (1969) p.469-476. 21. Kligman et al, ‘Topical tretinoin for photoaged skin’, J Am Acad Dermatol, 15 (4 pt 2) (1986), p.836-59. 22. Kang et al, ‘Long-Term Efficacy and Safety of Tretinoin Emollient Cream 0.05% in the Treatment of Photodamaged Facial Skin: A Two-Year, Randomized, Placebo-Controlled Trial’, J Am J Clin Dermatol, 6 (4) (2005), p. 245-253. 23. Griffiths et al, ‘Two concentrations of of topical tretinoin cause similar improvement of photoageing but different degrees of irritation’, Arch Dermatol, 131 (1995), p.1037-44. 24. Foods and Drugs Administration, Drugs@FDA – FDA Approved Drug Products (Tretinoin), (US: Access Data, FDA, 2015) <http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index. cfm?fuseaction=Search.Overview&DrugName=TRETINOIN> [Accessed 14 April 2015] 25. Gov.uk, Medicines & Healthcare products Regulatory Agency, (UK: Gov.uk, 2015) <www.mhra.gov. uk> [Accessed 14 April 2015] 26. European Parliament, Council of the European Union, Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products (EU: EUR- Lex, 2009) <http://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32009R1223> [Accessed 14 April 2015] 27. electronic Medicines Compendium, eMC+ <www.medicines.org.uk/emc> (UK: electronic Medicines Compendium, 2015) [Accessed 14 April 2014] 28. UK Parliament, The Human Medicines Regulations 2012 (SI 2012 No. 1916) (UK: legislation.gov.uk, 2012) <http://www.legislation.gov.uk/uksi/2012/1916/pdfs/uksi_20121916_en.pdf> [Accessed 14 April 2015] 29. Nursing and Midwifery Council, Nurse and midwife independent prescribing of unlicensed medicines (UK: Nursing and Midwifery Council, 2010) <www.nmc.org.uk/globalassets/ sitedocuments/circulars/2010circulars/nmccircular04_2010.pdf> 30. Kang et al, ‘Assessment of adapalene gel for the treatment of actinic keratoses and lentigines: A randomized trial’, J Am Acad Dermatol, 49 (2003) p. 83-90. 31. Kang et al, ‘Improvement of naturally aged skin with vitamin A (Retinol)’, Arch Dermatol, 14395 (2007) p. 606-612. 32. Fluhr JW et al, ‘Tolerance profile of retinol, retinaldehyde and retinoic acid under maximized and long-term clinical conditions’, Dermatology, 199(Suppl 1) (1999), p. 57-60. 33. Kang et al, ‘Application of Retinol to Human Skin In Vivo Induces Edipermal Hyperplasia and Cellular Retinoid Binding Proteins Characteristic of Retinoic Acid but Without Measurable Retinoic Acid Levels or Irritation’, Soc Investig Drmatol, 105(4) (1995), p. 549-556. 34. Duell EA et al, ‘Unoccluded retinol penetrates human skin in vivo more effectively than unoccluded retinyl palmitate or retinoic acid’, J Investig Dermatol, 106 (1997) p. 316-320. 35. Tucker-Samaras et al, ‘A stabilized 0.1% retinol facial moisturizer improves the appearance of photodamaged skin in an eight-week, double-blind, vehicle-controlled study’, Journal of Drugs in Dermatology, 8(10) (2009), p. 932-936. 36. Gold et al, ‘Treatment of facial photdamage using a novel retinol formulation’, Journal of Drugs in Dermatology, 12(5) (2013) p. 533-510. Ferndale Pharmaceuticals Ltd / AesthetiCare market products containing vitamin A ingredients and QP-Services UK Ltd are technical and regulatory service providers to Ferndale Pharmaceuticals Ltd. Reproduced from Aesthetics | Volume 2/Issue 6 - May 2015