Identification of Anti-Inflammatory and Anti-Aging Bioactive Compounds

In pharmaceutical companies that specialize in prescription dermatology drugs,  research scientists use state-of-the-art molecular biology screening methods and in vitro cell culture models to identify and characterize bioactive compounds that alter skin cell function in beneficial ways. Bioactive compound "candidates" are evaluated using a rigorous multi-step screening program that determines:

  • The types of skin cells affected by the bioactive compound.
  • The specific cellular and molecular signaling pathways and cellular events in each cell type that are regulated by the compound.
  • The effect of each bioactive compound on the expression of skin specific genes (over 5000 genes can be screened at one time) to determine any NEGATIVE as well as POSITIVE effects on skin cells.
  • The effect of each compound on the production and secretion of inflammatory hormones and on proteins involved in skin structure.
  • The potency of each compound in regulating cellular and molecular processes.

Different screening strategies are utilized by different laboratories to determine the effects of a compound on either: 1) inflammation pathways, or 2) anti-aging events.

Skin Inflammation and Anti-Inflammatory Compound Screening Strategy

Scientists in pharmaceutical companies utilize various screening programs to identify novel synthetic chemical compounds that might prove effective in treating various diseases such as cancer, cardiovascular, arthritis or other inflammatory diseases. Such screening always starts with looking at the effect of a given compound on some biological response in cultured human or animal cells.  For example a compound being studied for anti-cancer activities, might be tested on cancer cells in culture for its ability to halt cell proliferation or, alternatively, to trigger the cancer cell to  undergo apoptosis (cell death). For dermatology applications, a chemical compound candidate might be tested for its ability to block the production of PGE-2 in human fibroblasts treated with ultraviolet radiation.

In studies conducted several years ago in the Department of Biochemistry and Molecular Biology at the University of Oklahoma Health, scientists tested the ability of a variety of natural antioxidant compounds, such as curcumin, resveratrol, quercetin, gallic acid,  vitamin C, retinol, and EGCG (from green tea) to inhibit either the production of or action of a variety of inflammatory markers (cytokines) produced by skin and immune cells and which are known to be important in the development of various inflammatory skin problems, such as psoriasis and atopic dermatitis. A typical cutaneous inflammatory response and the key cytokines and adhesion molecules expressed during this reaction are illustrated in the cartoon below.

he inflammatory reaction begins with an initial insult to the skin, which results in the release of inflammatory cytokines by keratinocytes and fibroblasts. These cytokines then stimulate endothelial cells to express adhesion molecules, which aid in the attachment and transmigration of immune cells into the area of inflammation. Once the immune cells have entered the area of insult, the inflammatory response is perpetuated until the insult and damage caused is resolved. The inhibition of the expression of one or more of these cytokines or adhesion molecules during an inflammatory response can result in a reduction or inhibition of inflammation.

Depending on the laboratory doing the work, the screening of a candidate molecule’s ability to reduce the production of these key inflammatory markers may be based on the following assays:

  1. The effect of a candidate compound on the production of inflammatory cytokines and adhesion molecules produced by keratinocytes, fibroblasts and monocytes (ELISA) is usually assessed first.
  2. The effect of the candidate on the gene expression of key inflammatory cytokines in keratinocytes, fibroblasts and monocytes (RT-PCR) is determined.
  3. The ability of the compound to alter the gene expression of key adhesion molecules produced by endothelial cells is determined using RT-PCR technology.
  4. The effect of a drug candidate on the production of other important inflammatory cytokines is determined by use of Antibody Arrays.

This anti-inflammatory screening strategy is illustrated here.

By carrying out this type of rigorous analysis for anti-inflammatory effects of natural compounds, it is possible to identify those botanically based antioxidants which have beneficial anti-inflammatory activities. This type of screening is being used by many research laboratories with excellent success in identifying safe and gentle naturally occurring antioxidants that are widely distributed in foods and which have beneficial health properties. In fact, the increasing amount of published reports discussing which fruits and vegetables to eat to improve health and reduce the risk of  disease, is largely based on results from this type of screening. The various inflammatory processes that one particular natural  antioxidant compound, TH-211, can inhibit, either directly (blue X) or indirectly (red X) are shown in the skin cartoon below. Interestingly, this particular compound is not only an antioxidant, but an approved food flavoring additive that has been used in foods worldwide for over 50 years.

 

Anti-Aging Screening Strategy

The dermis of normal (wrinkle-free) skin is composed of abundant amounts of type I collagen and type VII collagen, as well as elastin, which provide tissue strength, resiliency and recoil. An illustration of the role of these key proteins in the skin is shown below.

During innate (normal) aging, aged dermal fibroblasts produce increased amounts of enzymes called matrix metalloprotienases (MMPs), which degrade collagen and elastin. To make matters worse, the amount of collagen and elastin produced by dermal fibroblasts decreases as the cells age. Thus, the overall loss of collagen and elastin results in skin laxity and fragility, which is visible in the form of wrinkles. The ability of a compound to reduce senescence (cell aging) or to stimulate aged fibroblasts to produce increased levels of Col I/VII and elastin, as well as inhibit the production of MMPs, would lead to an effective decrease, if not reversal, of the aging process. An illustration of the molecular changes that occur during aging is shown here.

During photoaging, exposure of dermal fibroblasts to ultraviolet radiation results in the increased expression of MMP’s and elastin. The overall loss of collagen, due to degradation by MMP’s, leads to skin laxity. Further, overexpression of elastin results in irregularly formed "knots" of elastin, called elastosis. Therefore, inhibition of the overexpression of MMPs and an increase in the production of collagen by a potential compound would result in an overall reduction, and possibly reversal, of photoaging. An illustration of the molecular changes that occur during photoaging is shown below.

As was the case  for searching for compounds that can inhibit inflammatory processes, a similar type of cell culture based screening program must be developed when searching for chemical compounds that can improve skin in either intrinsically-aged or photoaged skin. The screening process for any candidate molecule typically involves a three step process which includes:

  1. Assessing the effects of a candidate compound on the expression of key aging proteins (e.g. collagen, elastin) under normal and UV-induced aging conditions (RT-PCR and Western analysis).
  2. Determining the effect of a candidate anti-aging compound on the expression of MMP’s (enzymes that destroy the dermal matrix) and TIMPS ( proteins that block MMPs) using an Antibody Array method.
  3. Examining the effects of a candidate compound on cell senescence by use of the b-galactosidase assay.

A typical anti-aging screening strategy is summarized in the flow chart below.

 

This screening strategy allows for the selection of a bioactive that can cause a marked improvement in the appearance of skin regardless of whether or not the skin has undergone photodamaged.