Open Flow Microperfusion
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Open flow microperfusion (OFM) is a sampling method for clinical and preclinical drug development studies and
biomarker In biomedical contexts, a biomarker, or biological marker, is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, p ...
research. OFM is designed for continuous sampling of analytes from the
interstitial fluid In cell biology, extracellular fluid (ECF) denotes all body fluid outside the cells of any multicellular organism. Total body water in healthy adults is about 60% (range 45 to 75%) of total body weight; women and the obese typically have a lower ...
(ISF) of various tissues. It provides direct access to the ISF by insertion of a small, minimally invasive, membrane-free probe with macroscopic openings. Thus, the entire biochemical information of the ISF becomes accessible regardless of the analyte's molecular size, protein-binding property or
lipophilicity Lipophilicity (from Greek λίπος "fat" and φίλος "friendly"), refers to the ability of a chemical compound to dissolve in fats, oils, lipids, and non-polar solvents such as hexane or toluene. Such non-polar solvents are themselves lip ...
. OFM is capable of sampling lipophilic and hydrophilic compounds, protein bound and unbound drugs, neurotransmitters,
peptide Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. ...
s and
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
s, antibodies,
nanoparticle A nanoparticle or ultrafine particle is usually defined as a particle of matter that is between 1 and 100 nanometres (nm) in diameter. The term is sometimes used for larger particles, up to 500 nm, or fibers and tubes that are less than 10 ...
s and nanocarriers,
enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
s and
vesicles Vesicle may refer to: ; In cellular biology or chemistry * Vesicle (biology and chemistry), a supramolecular assembly of lipid molecules, like a cell membrane * Synaptic vesicle ; In human embryology * Vesicle (embryology), bulge-like features o ...
.


Method

The OFM probes are perfused with a physiological solution (the perfusate) which equilibrates with the ISF of the surrounding tissue. Operating flow rates range from 0.1 to 10 μL/min. OFM allows unrestricted exchange of compounds via an open structure across the open exchange area of the probe. This exchange of compounds between the probe’s perfusate and the surrounding ISF is driven by convection and diffusion, and occurs non-selectively in either direction (Figure 1). The direct liquid pathway between the probe’s perfusate and the surrounding fluid results in collection of ISF samples. These samples can be collected frequently and are then subjected to bioanalytical analysis to enable monitoring of substance concentrations with temporal resolution during the whole sampling period. See also: The concentric OFM probe (Figure 2) works according to the same principle. The perfusate is pumped to the tip of the OFM probe through the inner, thin tubing and exits behind the Open Exchange Area. There it mixes with exogenous substances present in the ISF and is withdrawn through the outer, thick tubing.


History

The first OFM sampling probe to be used as an alternative to
microdialysis Microdialysis is a minimally-invasive sampling technique that is used for continuous measurement of free, unbound analyte concentrations in the extracellular fluid of virtually any tissue. Analytes may include endogenous molecules (e.g. neurotra ...
was described in an Austrian patent application filed by Falko Skrabal in 1987, where OFM was described as a device, which can be implanted into the tissue of living organisms. In 1992, a US patent was filed claiming a device for determining at least one medical variable in the tissue of living organisms. In a later patent by Helmut Masoner, Falko Skrabal and Helmut List a linear type of the sampling probe with macroscopic circular holes was also disclosed. Alternative and current OFM versions for
dermal The dermis or corium is a layer of skin between the epidermis (with which it makes up the cutis) and subcutaneous tissues, that primarily consists of dense irregular connective tissue and cushions the body from stress and strain. It is divided ...
and
adipose tissue Adipose tissue, body fat, or simply fat is a loose connective tissue composed mostly of adipocytes. In addition to adipocytes, adipose tissue contains the stromal vascular fraction (SVF) of cells including preadipocytes, fibroblasts, vascular ...
application were developed b
Joanneum Research
and were patented by Manfred Bodenlenz et al. Alternative materials featuring low absorption were used to enable manufacturing of probes with diameters of 0.55 mm and exchange areas of 15 mm in length. For cerebral application, special OFM probes were patented by Birngruber et al. Additionally, a patent was filed to manage the fluid handling of the ISF by using a portable peristaltic pump with a flow range of 0.1 to 10 µL/min that enables operation of up to three probes per pump.


OFM System

Two types of OFM probes are currently available: Linear OFM probes for implantation into superficial tissues such as skin (dermal OFM, dOFM) and subcutaneous adipose tissue (adipose OFM, aOFM) as well as concentric probes for implantation into various regions of the
brain A brain is an organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. It is located in the head, usually close to the sensory organs for senses such as vision. It is the most complex organ in a ve ...
(cerebral OFM, cOFM).


Areas of application

OFM is routinely applied in pharmaceutical research in preclinical (e.g. mice, rats, pigs, primates) and in clinical studies in humans (Figure 3). OFM-related procedures such as probe insertions or prolonged sampling with numerous probes are well tolerated by the subjects.


Dermal OFM (dOFM)

dOFM (Figure 4) allows the investigation of transport of drugs in the dermis and their penetration into the dermis after local, topical or systemic application, and dOFM is mentioned by the
U.S. Food and Drug Administration The United States Food and Drug Administration (FDA or US FDA) is a federal agency of the Department of Health and Human Services. The FDA is responsible for protecting and promoting public health through the control and supervision of food ...
as a new method for assessment of bioequivalence of topical drugs. dOFM is used for: * conduct tissue-specific pharmacokinetic (PK) and pharmacodynamic (PD) studies of drugs. * perform head-to-head comparison of novel topical drug formulations * assess dermal
bioavailability In pharmacology, bioavailability is a subcategory of absorption and is the fraction (%) of an administered drug that reaches the systemic circulation. By definition, when a medication is administered intravenously, its bioavailability is 100%. Ho ...
. * investigate high molecular weight compounds, e.g. antibodies Head-to-head settings with OFM have proven particularly useful for the evaluation of topical generic products, which need to demonstrate
bioequivalence Bioequivalence is a term in pharmacokinetics used to assess the expected in vivo biological equivalence of two proprietary preparations of a drug. If two products are said to be bioequivalent it means that they would be expected to be, for all ...
to the reference listed drug product to obtain market approval. Applications of dOFM include ''
ex vivo ''Ex vivo'' (Latin: "out of the living") literally means that which takes place outside an organism. In science, ''ex vivo'' refers to experimentation or measurements done in or on tissue from an organism in an external environment with minimal ...
'' studies with tissue explants and preclinical and clinical ''
in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...
'' studies.


Adipose OFM (aOFM)

aOFM (Figure 4) allows continuous on-line monitoring of metabolic processes in the subcutaneous adipose tissue, e.g.
glucose Glucose is a simple sugar with the molecular formula . Glucose is overall the most abundant monosaccharide, a subcategory of carbohydrates. Glucose is mainly made by plants and most algae during photosynthesis from water and carbon dioxide, u ...
and lactate, as well as larger analytes such as insulin (5.9 kDa). The role of polypeptides for metabolic signaling ( leptin,
cytokine Cytokines are a broad and loose category of small proteins (~5–25 kDa) important in cell signaling. Cytokines are peptides and cannot cross the lipid bilayer of cells to enter the cytoplasm. Cytokines have been shown to be involved in autocrin ...
IL-6, TNFα) has also been studied with aOFM. aOFM allows the quantification of proteins (e.g. albumin size: 68 kDa) in adipose tissue and thus opens up the possibility to investigate protein-bound drugs directly in peripheral target tissues, such as highly protein-bound insulin analogues designed for a prolonged, retarded insulin action. Most recently, aOFM has been used to sample agonists to study
obesity Obesity is a medical condition, sometimes considered a disease, in which excess body fat has accumulated to such an extent that it may negatively affect health. People are classified as obese when their body mass index (BMI)—a person's ...
,
lipid metabolism Lipid metabolism is the synthesis and degradation of lipids in cells, involving the breakdown or storage of fats for energy and the synthesis of structural and functional lipids, such as those involved in the construction of cell membranes. In anim ...
and immune-inflammation. Applications of aOFM include ''ex vivo'' studies with tissue explants and preclinical and clinical ''in vivo'' studies.


Cerebral OFM (cOFM)

cOFM (Figure 5) is used to conduct PK/PD preclinical studies in the animal brain. Access to the brain includes monitoring of the blood-brain barrier function and drug transport across the intact blood-brain barrier. cOFM allows taking a look behind the blood-brain barrier and assesses concentrations and effects of
neuroactive Neuroactive is a Synthpop, Futurepop, EBM music group from Finland Finland ( fi, Suomi ; sv, Finland ), officially the Republic of Finland (; ), is a Nordic country in Northern Europe. It shares land borders with Sweden to the nort ...
substances directly in the targeted brain tissue. The blood-brain barrier is a natural shield that protects the brain and limits the exchange of nutrients, metabolites and chemical messengers between blood and brain. The blood-brain barrier also prevents potential harmful substances from entering and damaging the brain. However, this highly effective barrier also prevents neuroactive substances from reaching appropriate targets. For researchers that develop neuroactive drugs, it is therefore of major interest to know whether and to what extent an active pharmaceutical component can pass the blood-brain barrier. Experiments have shown that the blood-brain barrier has fully reestablished 15 days after implantation of the cOFM probe in the brain of rats. The cOFM probe has been specially designed to avoid a reopening of the blood-brain barrier or causing additional
trauma Trauma most often refers to: * Major trauma, in physical medicine, severe physical injury caused by an external source * Psychological trauma, a type of damage to the psyche that occurs as a result of a severely distressing event *Traumatic i ...
to the brain after implantation. cOFM enables continuous sampling of cerebral ISF with intact blood-brain barrier cOFM and thus allows continuous PK monitoring in brain tissue.


Quantification of ISF compounds

ISF compounds can be quantified either indirectly from merely diluted ISF samples by using OFM and additional calibration techniques, or directly from undiluted ISF samples which can be collected with additional OFM methods. Quantification of compounds from diluted ISF samples requires additional application of calibration methods, such as Zero Flow Rate, No Net Flux or Ionic Reference. Zero Flow Rate has been used in combination with dOFM by Schaupp et al. to quantify
potassium Potassium is the chemical element with the symbol K (from Neo-Latin ''kalium'') and atomic number19. Potassium is a silvery-white metal that is soft enough to be cut with a knife with little force. Potassium metal reacts rapidly with atmosph ...
,
sodium Sodium is a chemical element with the symbol Na (from Latin ''natrium'') and atomic number 11. It is a soft, silvery-white, highly reactive metal. Sodium is an alkali metal, being in group 1 of the periodic table. Its only stable ...
and glucose in adipose ISF samples. No Net Flux has been applied to quantify several analytes in OFM studies in subcutaneous adipose, muscle and dermal ISF: the absolute lactate concentrations and the absolute glucose concentrations in adipose ISF, the absolute
albumin Albumin is a family of globular proteins, the most common of which are the serum albumins. All the proteins of the albumin family are water-soluble, moderately soluble in concentrated salt solutions, and experience heat denaturation. Albumins ...
concentration in muscle ISF and the absolute insulin concentration in adipose and muscle ISF have been successfully determined. Dragatin et al. used No Net Flux in combination with dOFM to assess the absolute ISF concentration of a fully human therapeutic antibody. Ionic Reference has been used in combination with OFM to assess the absolute glucose concentration and the absolute lactate concentration in adipose ISF. Dermal OFM has also been used to quantify the concentrations of human insulin and an insulin analogue in the ISF with inulin as exogenous marker. Additional OFM methods, such as OFM recirculation and OFM suction can collect undiluted ISF samples from which direct and absolute quantification of compounds is feasible.{{cite journal , last1=Hummer , first1=Joanna , last2=Schwingenschuh , first2=Simon , last3=Raml , first3=Reingard , last4=Boulgaropoulos , first4=Beate , last5=Schwagerle , first5=Gerd , last6=Augustin , first6=Thomas , last7=Sinner , first7=Frank , last8=Birngruber , first8=Thomas , title=OFM-recirculation and OFM-suction: advanced in-vivo open flow microperfusion (OFM) methods for direct and absolute quantification of albumin in interstitial fluid , journal=Biomedical Physics & Engineering Express , date=1 November 2020 , volume=6 , issue=6 , pages=065031 , doi=10.1088/2057-1976/abc3a7 , pmid=33843658 , s2cid=226336167 OFM recirculation to collect undiluted ISF samples recirculates the perfusate in a closed loop until equilibrium concentrations between perfusate and ISF are established. Using albumin as analyte, 20 recirculation cycles have been enough to reach equilibrium ISF concentrations. OFM suction is performed by applying a mild vacuum, which pulls ISF from the tissue into the OFM probe.


References


External links


Joanneum Research - HEALTH – Institute for Biomedicine and Health Sciences
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