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Lactobionic Acid - a Novel Polyhydroxy Bionic Acid for Skincare

Barbara A. Green, R.Ph., Richard H. Wildnauer, Ph.D., Brenda L. Edison

NeoStrata Company, Inc., Princeton, NJ, USA.
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Introduction
Lactobionic acid (4-O-B-D-galactopyranosyl-D-gluconic acid) is comprised of one molecule of galactose attached to one molecule of gluconic acid via an ether like linkage1.  Gluconic acid, found naturally occurring in cells, is a polyhydroxy AHA (PHA) that is known to provide beneficial effects to skin2,3 and is currently contained in some skin care products as gluconolactone.  Galactose is a chemically neutral, endogenous hexose sugar that is utilized in glycosaminoglycan synthesis, collagen synthesis, and cell migration, which may enhance wound heaing4-8.

Lactobionic acid is a new polyhydroxy bionic acid ingredient being introduced into skin care.  Formed by oxidation of the disaccharide lactose (milk sugar), lactobionic acid is currently being used in the pharmaceutical industry as a salt form for intravenously delivered erythromycin and in mineral supplementation9.  Its largest commercial use, however, is as a major constituent of organ preservation fluids during transplantation procedures.  This is due to its ability to suppress tissue damage caused by oxygen radicals during organ storage and subsequent reperfusion, allowing organs to be preserved outside of the body for up to two days10.  This beneficial effect is reportedly mediated through inhibition of hydroxyl radical production via complexation of FeII11.

While lactobionic acid has gained recognition for some important niche uses, its benefits in skin care have yet to be fully explored.  Early investigative work by Eugene J. Van Scott, M.D. and Ruey J. Yu, Ph.D., O.M.D., suggests that there are significant skin benefits with this and other compounds in its class12,13.
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Objective
The purpose of this poster is to review the available scientific and clinical data to evaluate the potential uses for this new polyhydroxy bionic acid skin care ingredient.
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Chemical Properties - The Bionic Acids
Lactobionic acid is chemically classified as an oligosaccharide aldonic acid, in which a carbohydrate (galactose) is chemically linked to an aldonic acid (gluconic acid).  The connection between the carbohydrate and the aldonic acid molecule is an ether-like anomeric bond that can be hydrolyzed enzymatically in vivo to form galactose and gluconic acid (an AHA / PHA).  Since there is one carbohydrate monomer linked to a single aldonic acid unit, this compound is further defined as an aldobionic acid - 'bi' indicating two units.  Chemical derivatives can be formed with the oligosaccharide aldonic acids including lactobionic acid.  For example, after losing one molecule of water, an oligosaccharide aldonic acid lactone is formed, i.e., lactobionolactone.12
Specific chemical properties of lactobionic acid include:
Molecular weight 358
Chemical composition C12H22O12
pKa ~ 3.8
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Water Binding Properties
Lactobionic acid is a hygroscopic compound, which tenaciously binds atmospheric water to the extent that a natural gel matrix forms upon evaporation at room temperature. This may be in part due to its chemical structure, which structurally resembles, and may function like, a glycosaminoglycan. The gel matrix film contains approximately 14% water and may contribute to its skin conditioning benefits and occlusivity to the skin.13

Water binding studies with lactobionic acid and other chemicals with known humectant properties indicate that lactobionic acid has superior water retention and absorbing properties.

Water Retention Experiment:

  • All test compounds were prepared as 1M aqueous solutions in petri dishes to a total volume of 25mL. The solutions were dried in an oven (100°F) until one of the test materials retained no water. The samples were weighed and the weight of water retained per mole of test material was calculated
  • Result: Lactobionic acid retained the greatest amount of water per mole of substance compared to known humectants (glycerol and sorbitol), other AHAs, salicylic acid and propylene glycol.
Weight (g) of Water Retained Per Mole of Substance *
* After exposure of 1M solutions to oven drying.

Water Absorption Experiment:
  • The test compounds were prepared and oven dried as described above, and then exposed to a 100% humidity chamber for four hours. The amount of water absorbed by each test material was calculated per mole of test material.
    Result: Lactobionic acid absorbed the greatest amount of water per mole of substance compared to known humectants (glycerol and sorbitol), other AHAs, salicylic acid and propylene glycol. Salicylic acid did not absorb any water and remained as a negative number indicating water was lost during the drying procedure.
Weight (g) of Water Absorbed Per Mole of Substance *
* After exposure to 100% relative humidity for four hours.
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Anti-Oxidant Properties
Lactobionic acid is known to function as an antioxidant in tissues by inhibiting the production of hydroxyl radicals as a result of its iron chelating properties11. In addition, many PHAs and bionic acids function as classical antioxidants in that they are capable of preventing oxidation of other substances such as anthralin, hydroquinone and banana peel13.
  1. Anthralin model - Anthralin creams (0.4%) containing 0.1M concentrations of test compounds were prepared and exposed to ambient conditions. Degree of oxidation was graded visually based on color change from bright yellow (no oxidation) to the air-oxidized brown/black color.
  2. Hydroquinone model - Hydroquinone creams (2%) containing 1% concentrations of test compounds were prepared and exposed to ambient conditions. Degree of oxidation was assessed based on color change from colorless (no oxidation) to the oxidized black material.
  3. Banana peel model - 2cm X 2cm squares of fresh banana peel were exposed on both the inner and outer surface to 0.1M test compounds in aqueous solutions and evaluated for color change from yellow (no oxidation) to black (fully oxidized).
Results indicate that lactobionic acid is an effective antioxidant in comparison to other known antioxidant substances. 

Partial List of Antioxidants for Food & Drug Substances
  Anthralin Hydroquinone Banana Peel
Most Effective oxalic acid oxalic acid oxalic acid
ascorbic acid
citric acid
gluconolactone
Very Effective ascorbic acid ascorbic acid lactobionic acid
citric acid citric acid tartaric acid
gluconolactone gluconolactone
lactobionic acid lactobionic acid
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Wound Healing
Wound healing is a complex process of biochemical and cellular events in which a wound defect is replaced by granulation tissue, covered by epithelial cells and then strengthened through the incorporation of collagen. During this process, efficient cell migration, growth of granulation tissue and synthesis / aggregation of collagen are essential. In wound healing models, these processes have been shown to be positively influenced by galactose4-8. Galactose is a component of lactobionic acid and is presumably liberated after enzymatic hydrolysis, which may in part account for the following wound healing observations.
Preliminary clinical findings12:
  1. Scratch test - a sterile 22 gauge needle was used to create two wounds 1cm in length and 0.5mm deep on a male forearm.  The wounds were treated with 2% lactobionic acid (aq.) and 0.9% sodium chloride control, and covered with an occlusive tape for five days.  At the end of one week, the wound treated with lactobionic acid was fully epithelialized in comparison to the control which was not. Erythema was present on both sites for nine days.
  2. Punch biopsy test - a 4mm skin biopsy punch was used to create two wounds 0.5mm in depth on a male forearm.  The wounds were treated with 2% lactobionic acid (aq.) and 0.9% sodium chloride control, and covered with an occlusive tape for five days.  At the end of one week, the wound treated with lactobionic acid was fully epithelialized in comparison to the control which was not re-epithelialized.  Erythema disappeared from both sites after nine days.
  3. Improvement of fissures - a male subject having skin fissures 2-3mm in length on the finger tips applied lactobionic acid cream (10%) to one target fissure and vehicle to a control fissure for one week.  The fissure treated with the lactobionic acid cream healed quickly and the pain disappeared after a few days.  In one week the treated fissure was completely healed and the control fissure remained unhealed.
Based on these preliminary experiments, observations suggest that topical lactobionic acid may be useful in enhancing wound healing. Further studies need to be conducted to validate these findings.
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Safety / Toxicity Studies
Routine safety and toxicity tests were conducted to establish baseline safety for this compound and can be summarized as follows:

Safety Profile of Lactobionic Acid14
Test Test Material Result
1. Ames II Assay 10% lactobionic acid Non-mutagenic: no base pair or frame shift mutations in the absence or presence of the S9 fraction
2. Cell Viability:
(EPI-100)		 8% lactobionic acid cream in contact with synthetic skin model for 1,4, and 24 hours.

negative control: water

positive control: Triton-X 100 (1%), a mild irritant		 Test material was classified as innocuous and non-irritating
2a. PGE2 assay
(EPI-100)		 (above) No inflammatory prostaglandin release.  Test material was equivalent to water control
2b. Lactate
Dehydrogenase
(LDH) (EPI-100)		 (above) No increase in cellular lysis.  Test material was approximately equivalent to water control and less than positive control
2c. Interleukin-1a
(EPI-100)		 (above) Insignificant effect on cytokines.  Test material was somewhat higher than water control and less than positive control
3. Cumulative 4% lactobionic acid + 8% gluconolactone cream, pH 3.8 Non-irritating: test material equivalent to saline control (see graph)

Cumulative Irritation Study (14 days)
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Cosmetic Improvements to Skin
The alpha-hydroxy acids and polyhydroxy acids (AHAs / PHAs) are known to provide significant cosmetic benefits to photoaged and hyperkeratotic skin.  Preliminary test results showed that lactobionic acid may function like other traditional AHAs since it shares the same basic AHA structure.  Furthermore, it may be expected that lactobionic acid can provide additional benefits to skin as a result of its polyhydroxy acid structure2,3.  Some preliminary studies are reported to show potential cosmetic benefits to skin.

Exfoliation / Normalization of Cell Turnover

  • As with other AHAs, early efficacy screening models by Drs. Van Scott and Yu evaluated the effect of lactobionic on lamellar ichthyosis, a hereditary condition causing extreme scaling and flaking. Lactobionic acid was found to produce nearly complete clearing of ichthyotic skin, but at a moderate rate compared to the AHA standard, glycolic acid15.

    A small pilot study was undertaken to evaluate the effects of lactobionic acid (8% cream, pH 3.5) on forearm cell turnover after staining with dansyl chloride in comparison to an untreated site. The findings show enhancement of cell turnover with lactobionic acid.
Dansyl Chloride Cell Turnover Study *
* Abbreviated model used - dansyl chloride (5% in petrolatum) was applied 3 hours under
occlusion.  Degree of flourescence was evaluated using a Wood’s lamp in a darkened room.

Skin Turgor and Youthfulness
  • In an effort to evaluate a compound's usefulness in 'plumping' photoaged forearm skin, test compounds (in solution) are applied topically twice daily, and total skin thickness measurements are made using micrometer calipers as described previously16.  Skin thickness results are expressed relative to baseline scores.
    Preliminary findings suggest that lactobionic acid causes a measurable increase in skin thickness. Similar previous findings have correlated well with reversal of histological symptoms of skin aging16,17.
Improvement in Total Skin Thickness After Topical Application of Lactobionic Acid
. Week 5 Week 11
Lactobionic acid 21.6% + 8% (n=1) + 43% (n=2)
Lactobionic acid 10% + 7% (n=3) Not available
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Conclusion
Lactobionic acid is an exciting new class of compounds that shows promise for use as a polyhydroxy bionic acid in skin care.  Its antioxidant effects have been clearly established as well as further evaluated in this poster.  Lactobionic acid strongly attracts and binds water producing a gel matrix in comparison to other known humectants.  Its film forming properties are thought to provide unique cosmetic and aesthetic attributes of lactobionic acid containing formulations.  Preliminary studies of the cosmetic benefits of lactobionic acid suggest that it provides anti-aging and cell turnover benefits to skin.  In addition, lactobionic acid may be useful in enhancing wound healing. Lactobionic acid is a safe compound and may be taken orally or intravenously as the counter ion in some vitamin supplements and antibiotics. Furthermore, studies on skin indicate that this compound is safe and non-irritating. After considerable evaluation of the significant preliminary information pertaining to this compound, lactobionic acid emerges as a new and novel skin care ingredient worthy of further investigation.
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References
  1. The Merck Index, Twelfth Edition.
  2. Berardesca E, Distante F, Vignoli GP, Oresajo C, Green B. Alpha hydroxy acids modulate stratum corneum barrier function. Br. J. Dermatol 1997;137:934-8.
  3. Green B, Tseng C, Wildnauer R, Herndon J, Rizer, R. Safety and Efficacy of a Gluconolactone (Poly Hydroxyacid) Containing Regimen on Sensitive Skin and Photodamage Following Controlled Consumer Use. Amer Acad of Derm Poster Exhibit: New Orleans, March, 1999.
  4. Kossi J, Peltonen J, Ekfors T, Niinikoski J, Laato M. Effects of hexose sugars: glucose, fructose, galactose and mannose on wound healing in the rat. Eur Surg Res 1999;31(1):74-82.
  5. Panjwani N, Ahmad S, Raizman MB. Cell surface glycoproteins of corneal epithelium.  Invest Ophthalmol Vis Sci 1995;36(2):355-63.
  6. Schmidt RJ, Spyratou O, Turner TD. Biocompatibility of wound management products: the effect of various monosaccharides on L929 and 2002 fibroblast cells in culture. J Pharm Pharmacol 1989;41(11):781-4.
  7. Latha B, Ramkishnan M, Jayaraman V, Babu M. Physicochemical properties of extracellular matrix proteins in post-burn human granulation tissue. Comp Biochem Physiol B Biochem Mol Biol 1999;124(3):241-9.
  8. Wilhelmi BJ, Blackwell SJ, Mancoll JS, Phillips LG. Creep vs. stretch: a review of the viscoelastic properties of skin. Ann Plast Surg 1998;41:215-219.
  9. Facts and comparisons. A Wolters Kluwer Company, St. Louis, MO.
  10. Southard JH, Belzer FO. Organ preservation. Annu Rev Med 1995;46:235-47.
  11. Charloux C, Paul M, Loisance D, Astier A. Inhibition of hydroxyl radical production by lactobionate, adenine, and tempol. Free Radical Bio & Med 1995;19(5):699-704.
  12. U.S. Patents Nos 5,554,652; 5,554,654; 5,554,597; 5,561,158; 5,561,157 and patent pending.
  13. Yu RJ, Van Scott EJ. 'Hydroxyacids and their topical use in the elderly' in 'Skin Diseases in the Elderly' to be published in 2000 by Marcel Dekker, Inc.
  14. Data on file, NeoStrata Company, Inc.
  15. Unpublished clinical observation, Dr. Van Scott.
  16. Ditre CM, Griffin TD, Murphy GF, Sueki H, Telegan B, Johnson WC, Yu RJ, Van Scott EJ.  Effects of a-hydroxy acids on photoaged skin: A pilot clinical, histologic, and ultrastructural study.  J Am Acad Dermatol 1996:34:187-95.
  17. Bernstein EF, Underhill CB, Lakkakorpi J, Ditre CM, Uitto J, Yu RJ, Van Scott EJ. Citric Acid increases viable epidermal thickness & glycosaminoglycan content of sun-damaged skin.  Dermatol Surg 1997; 23: 689-94.
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Products containing Lactobionic Acid
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