In Vitro Transporter Assays for Nutrient Absorption and Drug Nutrient interaction studies
Interaction of membrane transporters with ppcps
Importance of transporters in dermatology
Solvo's offer
Applications of transporter assays
How to benefit from our assays?
Solvo: the transporter company
Partnering with Solvo
Cooperation / service business models on drug transporters

By now, it is a common knowledge that ABC transporters play a key role in the function of the blood-brain barrier (BBB), the blood-cerebrospinal fluid barrier, the blood-testis barrier, the intestinal epithelia (absorption), the airway epithelia, the liver epithelia, the kidney epithelia and probably in the skin (?). SOLVO products can be used to study the transport properties of these pharmacological barriers. SOLVO offers drug discovery products and services to pharmaceutical, biotechnology and consumer goods industries, relevant to many therapeutic areas and product categories.

In Vitro Transporter Assays for Nutrient Absorption and Drug Nutrient interaction studies

 Figure 1: The bloodstream
(Source: Nutrition, Second Edition, Chapter 3: Digestion and Absorption, Paul Insel, R. Elaine Turner, Don Ross)

Digestion

Digestion involves the mixing of food, its movement through the digestive tract, and the chemical breakdown of the large molecules of food into smaller ones (Table 1).
Digested food molecules, water and minerals are absorbed from the cavity of the upper small intestine and then carried off in the bloodstream to other parts of the body for storage or further chemical change (Figure 1).
Nutrients are substances contained in foods which provide energy and raw materials for the synthesis and maintenance of living matter. Human nutrients are proteins, carbohydrates, fat, minerals, vitamins and water.

All cells acquire the molecules and ions they need from their surrounding extracellular fluid (ECF). There is an unceasing traffic of molecules and ions:

In and out of the cell through the plasma membrane; In eukaryotic cells, there is also transport in and out of membrane-bounded intracellular compartments such as the nucleus, endoplasmic reticulum, and mitochondria.

Transport of nutrients

Nutrients can be transported through the cell membrane in 3 ways (Figure 2):
Passive diffusion consists of the transport of water and water-soluble substances and small lipids through the lipid bilayer with a concentration gradient.
In the case of a facilitated diffusion, transporter proteins create a water-filled pore through which ions and small hydrophilic molecules can pass by diffusion. Fructose, riboflavin and vitamin B12 (in combination with intrinsic factor) are among the substances absorbed by facilitated diffusion.
During active transport, transmembrane proteins, called transporters, use the energy of ATP to force ions or small molecules through the membrane against their concentration gradient. These active transport mechanisms have been identified for intestinal absorption of many substances including glucose, galactose, amino acids, calcium, iron, folic acid, ascorbic acid, thiamin and bile acids.

Figure 2: Passive diffusion, facilitated diffusion and active transport of nutrients
(Source: Nutrition, Second Edition, Chapter 3: Digestion and Absorption, Paul Insel, R. Elaine Turner, Don Ross)

Transport proteins, embedded in lipid membranes, facilitate the import of nutrients into cells or the release of toxic products into the surrounding medium. The most important family of membrane transport proteins are the ATP-binding cassette (ABC) transporters. These ABC proteins play a central role in all living cells in the nutrient uptake, protein, drug and antibiotic excretion, osmoregulation, antigen presentation, signal transduction and other important cellular functions.

Importance of Transporters in Nutrients Absorption, Distribution, Metabolism and Elimination (ADME)

Some studies suggest that dietary constituents regulate the expression of ABC Transporters. Changes in ABC Transporter expression may represent an important physiological response to foods containing toxins and an important component of the acute phase immune response. It has been shown that dietary phytochemicals have inhibitory effects on P-glycoprotein (MDR1) and potentially cause drug-food interactions.
Moreover, a number of studies indicated that membrane transporters, i.e. Multidrug Resistant Protein 1 (MRP1), Multidrug Resistant Protein 2 (MRP 2), Breast Cancer Resistance Protein BCRP (MXR) and P-glycoprotein (MDR1) play important roles in the cellular accumulation, transport and distribution of many nutrients.
Since some of these nutrients are found in fruits and vegetables, their effect on MRP1, MRP2, MXR and MDR1 may be a mechanism relevant to carcinogenesis and the observed lowered cancer risk in humans with higher dietary intake of fruits and vegetables (Table 1).

Table 1: Nutrient-transporter interactions

Solvo technology in nutritional sciences

The screening of the interaction of nutrients with transporters is an excellent way to determine the transport of nutrients and also to study drug-food interactions. It is highly recommended to perform screens for MRP1, MRP2, MXR (BCRP) and MDR1. It is also an option to screen for other transporters as well.

Interaction of membrane transporters with ppcps

Figure 3: Effects of pollution
(Source: http://www.claybennett.com)

During the past 30 years, the impact of chemical pollution has focused almost exclusively on the conventional "priority" pollutants, especially those acutely toxic/carcinogenic pesticides and industrial intermediates displaying persistence in the environment. Another diverse group of bioactive chemicals receiving comparatively little attention as potential environmental pollutants includes the pharmaceuticals and active ingredients in personal care products (PPCPs), both human and veterinary, including not just prescription drugs and biologics, but also diagnostic agents, "nutraceuticals", fragrances, sun-screen agents and numerous others.

Some PPCPs are extremely persistent and introduced to the environment in very high quantities and perhaps have already gained ubiquity worldwide, others could act as if they were persistent, simply because their continual infusion into the aquatic environment serves to sustain perpetual life-cycle exposures for aquatic organisms.

ABC transporter proteins are mostly known for their role in multidrug resistance (MDR). However, the physiological function of ABC transporters is protection of cells from xenobiotics, a phenomenom also referred to as Multixenobiotic Resistance (MXR). This function of the efflux transporters has not been explored substantially, though studies exist on aquatic organisms (Smital et al., 2004).

Figure 4: Destroying the environment
(Birds I view: Noel O. Pama, Sr,
Source: http://www.ncca.gov.ph)

Inhibition or saturation of MXR in aquatic organisms is a cause for concern. Aquatic organisms are continually exposed to pollutants present in water. When ABC transporters, and hence, the natural defense system of these organisms is saturated or inhibited, accumulation of toxins might occur. This might lead to dramatic changes in aquatic wildlife but also to introduction and accumulation of certain pollutants in the food chain.
Little is known about the interaction of human ABC transporters with common pollutants like polycyclic aromatic hydrocarbons (PAHs), heavy metals, chloroalkanes, phthalates or agrochemicals.
Information whether a widely used chemical (like di(2-ethylhexyl)phthalate, DHEP) is a substrate, inhibitor or activator of an ABC transporter, however, could be useful in determining the toxicity of this compound and analyzing its hazard.
Moreover, screening compounds might reveal structure/activity relationships of interacting compounds (see Bain et al., 1997; Van Zanden et al., 2005), inhibition studies might give structural information on ABC transporters (see Annereau et al., 2003) reveal combinatory effects (see Luckenbach and Epel, 2005) and, finally, ABC transporters might serve as indicators for toxicity in water or soil samples.

Importance of transporters in dermatology

In the past decade research into skin pharmacology has developed dramatically with new and promising drugs and therapeutic concepts being introduced regularly. The skin:

Protects the body against injury, heat and light radiation. Helps the penetration of chemical agents and invasion of microbes and microorganism. Regulates body temperature Eliminate a number of harmful substances resulting from the metabolic activities of the intestine and the liver. Secretes hormones and enzymes. Plays an immunological role, by cooperation with Langerhans cells.

The surface of the skin is made of a conglomeration of dead cells. Underneath the surface, there are very thin and distinct layers: the epidermis, the dermis and the hypodermis.

The keratinocyte is the major cell type of the epidermis, making up about 90% of epidermal cells. The epidermis is divided into four layers based on keratinocyte morphology:

basal layer (at the junction with the dermis) stratum granulosum stratum spinosum stratum lucidum stratum corneum

A large number of ABC Transporters have been found in keratinocytes:

It is well known that various personal health and beauty care products contain numerous substances that are substrates of ABC transporters.
For example, the ointment is a medication preparation that is applied topically (onto the skin). Ointments combine oil (80%) and water (20%). This combination generally forms a more effective barrier against moisture loss than creams and lotions so ointments tend to be better moisturizers.

The following table summarizes ABC Transporter Substrate Drugs Applied in Ointments:

There are many opportunities of transporter related studies in dermatology, since skin is the major barrier with the largest surface area between the human body and the environment. There are potential applications as the skin is the interface for:

Drugs Detergents/surfactants Toxic compounds entering the human body.

Although there are many relevant transporters in dermatology studies, many unresolved issues still exist.
We need to determine if the keratinocytes in the skin are polarized as well as the localization of the transporters on the keratinocyte membranes.
We also need to develop specific skin assay systems to study the transporter-compound interactions relevant in the skin.

Solvo's offer

SOLVO Biotechnology provides four different solutions for ABC efflux (xenobiotic) and uptake transporter assays to be used in compound discovery and development:

Membrane Preparations - to be used for ATPase Assay and Vesicular Transport Assay (direct uptake) measurements Fee-For-Service Screening Sublicense for the Calcein Assay Custom-Made Expression and Screening Systems

Applications of transporter assays

Figure 5: The human cells
(Source: http://www.biocenter.hu)

The three major applications of the ABC Transporter assays are:

Predicting the penetration of test compounds through the pharmacological barriers (blood-brain barrier, intestinal mucosa, kidney, liver skin etc.) = in vitro predictive ADME, Predicting the penetration of active cytostatics into the cancer cells, Testing of functional effect of MDR modulators.

How to benefit from our assays?

SOLVO's mainstream products can be used for:

Expression of a wide array of human ABC transporters (MDR1, MDR2/3, MRP1, MRP2, MRP3, MRP4, MRP5, MRP6, MXR, BSEP and others) and rodent (rat, mouse) transporters in various expression systems (e.g. baculovirus-based insect cell) Functional characterization of proteins (ATPase, transport and binding data) Identification of new drug targets on ABC transporters. Development of various cell lines expressing a range of pharmacologically relevant uptake transporter proteins

Our unique technology platform is characterized by the following attributes:

Flexible collaboration models for Client focused Services and Reagent sales Cost-efficiency Reliability, as only one transporter is expressed in each individual reagent at one time Easy-to-use methodology with detailed HT protocols available for all reagents (including all insect and mammalian membrane assays)

A wide array of ABC transporter assays and cell lines expressing various uptake transporter proteins assays are available from SOLVO. SOLVO is constantly developing new assays, for additional ABC and for other transporters as well.
The patented Calcein Assay, ATPase Assay and some Vesicular transport assays are optimized for high-throughput screening on 96 well plates and widely used by pharmaceutical companies. SOLVO provides full technology transfer for in-house use as well as fee-for-service screening.

Solvo: the transporter company

Partnering with Solvo

Partnering is a key part of our research and business strategy. Our group is committed to collaborating with others, developing long-term partnerships that leverage each company's strengths, and bringing significant advances to researchers worldwide.

SOLVO is interested in collaborations which may take many forms:

Products- co-development, product acquisitions and/or co-promotion/co-marketing arrangements Strategic partnerships that build on complementary capabilities to develop comprehensive Technology Platforms Broad based research collaborations, including alternative discovery initiatives and new collaborative model In-licensing compounds, technologies or other intellectual property from Academic and Industrial Research laboratories and out-licensing our technology from the R&D pipeline Academic liaisons

Cooperation / service business models on drug transporters