Techniques to Determine Penetration and Permeation of Actives PDF

Techniques to determine penetration and permeation of actives Mignon Cristofoli Including previous content of Dr Milica Stevic Overview IVRT How do we know whether an active has been absorbed by the skin (i.e., partitioned into the skin and then permeated through the skin)?...

Techniques to determine penetration and permeation of actives Mignon Cristofoli Including previous content of Dr Milica Stevic Overview IVRT How do we know whether an active has been absorbed by the skin (i.e., partitioned into the skin and then permeated through the skin)? In vitro permeation testing (IVPT) In vivo testing How do we detect and quantify the active using these methods? UV spectrophotometer HPLC GC (plus others) In Vitro release testing (IVRT) 3 In vitro release testing (IVRT) IVRT uses synthetic membranes infinite doses applied occlusive Advantages more “repeatable”, as less variation in membrane during development phase, changes in manufacturing process may impact the microstructure of the formulation => IVPT can be used to determine impact of these changes on delivery of the active compare batch-to-batch variability in drug release rate at the time of manufacture and for duration of shelf life (quality control) In vitro release testing (IVRT) Disadvantages not reflective of in vivo results oversimplification of the reality of in vivo delivery and permeation Can it be used to prove bioequivalence of semi-solid dosage forms? Probably best in conjunction with IVPT In Vitro permeation Testing (IVPT) 6 Guidelines for dermal absorption studies for cosmetic purposes Guidance is provided in SCCNFP (Scientific Committee on Cosmetics and Non-Food Products) adopted a first set of “Basic Criteria” (SCCS/1358/10). SCCS NoG 12th ed 2023: “in vitro dermal absorption studies of cosmetic substances is to obtain qualitative and/or quantitative information on the compounds that may enter the systemic compartment of the human body under in-use conditions. These amounts can then be taken into consideration to calculate the MoS during risk characterisation” Considerations include: - design of diffusion cell - choice of receptor fluid - membrane - skin integrity - skin temperature - test substance - dose - sampling times - analytical techniques - mass balance studies Definition In Vitro Permeation Testing is a technique to help quantify how much of an ingredient is able to partition into the skin and permeate through the skin from a topically applied product. A heated aluminium block 10 diffusion cells 8 IVPT In Vitro Permeation Testing provides information about the efficacy of the cosmetic formulation in delivering the active. It is also used to comply with safety legislation. It is conducted during product development, quality control and safety assessment phases. Skin is a very effective barrier against the external environment. The following factors influence the delivery of an active: Solubility Partition coefficient Molecular weight Melting point Molecular structure Its ability to leave the formulation and partition into the skin 9 Diffusion cells – Open top design The assembled cell comprises two major parts: 1. A donor chamber containing a sample to be tested. 2. A receptor chamber containing a receptor medium (e.g. phosphate buffer). 1 0 Diffusion cells – Closed top design (occlusive testing) A Teflon sample Glass disc chamber ring A donor chamber containing a sample to be tested. A membrane A receptor chamber containing a receptor medium (e.g. phosphate buffer). A membrane Mini stirrer bar: required in any diffusion cell 1 1 What kinds of formulations can be tested using diffusion cells? Used in obtaining a release profile of the semi-solid formulations and patches. COSMETICS COSMETICS Anti-aging cream with Vitamin E ‘Puffy-eye attack’ patch Anti-cellulite cream with Caffeine Lidocaine patches MEDICINE MEDICINE Transdermal: Transdermal: LiDORx (to numb tissue ) Nicotine patch ColciGel (to treat arthritis) Contraceptive patch 12 What kinds of formulations can be tested using diffusion cells? Don’t forget serums! COSMETICS Caffeine under eye cream Salicylic acid serum Niacinamide serum Azaleic acid Membranes used in cosmetic testing Membranes for IVPT Ex vivo human skin is the gold standard to confirm the barrier properties of the skin are intact, we test the impedance of the skin by applying a current Ex vivo animal skin can still be used in cosmetic testing in certain circumstances best animal model is porcine skin - skin taken from the ear of a pig For safety studies - use either human or porcine skin for dermal or transdermal testing of cosmetics Other membranes: IVRT Polydimethylsilloxane (PDMS) membranes Polysulfone (Tuffryn) Cellulose Acetate Membrane Strat M 3D printed skin 14 Why porcine skin? “similarity between the epidermis compounds found in human and porcine skin as a function of depth. Since porcine skin is a widely used model for permeation testing this result has clinical relevance” SCCS NoG 12th ed 2023 : human skin in the first instance (gold standard) or porcine skin for skin absorption studies Membranes PDMS is a semi permeable synthetic membrane with the following properties: hydrophobic polymeric ± 80 μm thick Properties are not stable when in contact with certain organic solvents eg. ethanol, triethylamine, pentane Polydimethylsiloxane repeating unit (n) 16 Membranes Tuffryn is a synthetic membrane with the following properties: relatively inert hydrophilic polymeric having a pore size of 0.2 μm or 0.45 μm based on polysulfone Polysulfone repeating unit 17 Membranes The Strat-M Membrane Strat-M membrane is designed for: a relatively new, synthetic, active pharmaceutical transdermal test model ingredients predicts diffusion in human cosmetic actives skin formulations can be used without the personal care wetting, safety and storage products limitations pesticides and has multiple layers with chemicals varied diffusivity. 18 Membranes The Strat-M Membrane It is constructed of two layers: polyether-sulfone layer - more resistant to diffusion (epidermis like) polyolefin layer - more open and diffusive to create a porous structure (dermis like). The porous structure is impregnated with a blend of synthetic lipids enabling: a strong correlation to human skin reduces the high test variability associated with biological models. 19 IVPT protocol 20 IVPT protocol 21 IVPT protocol 22 Application of IVPT in Cosmetic Science – New Skin Type Franz Cells 23 Application of IVPT using active “DF” 14 Cumulative permeation of DF 12 10 (µg /cm2) 8 6 4 2 0 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours) S 1% V 1% O 1% Cumulative permeation of DF (μg/cm2) from three different formulations containing 1% DF. Finite doses (10 μL) were applied to porcine skin (n ≥ 4; mean ± SD). 24 Next step: Mass balance study When using human skin or porcine skin, you would perform a mass balance study What is a mass balance study? Amount remaining It is simply accounting for your active after We know exactly how much of your active study has been applied Amount We know how much has permeated after the in the time period of the study membrane We can remove any of the remaining Amount that formulation from the top of the membrane permeated and quantify it into Finally, we extract any of the formulation receptor from the membrane to determine how much Amount applied = amount in receptor has partitioned into the tissue, but not plus amount remaining on the surface + permeated any further amount in the membrane. 25 Next step: Mass balance study for DF Mass balance is important as it allows you to determine a number Percentage of amount of DF of things: 100 80 If you do not get almost 100% applied 60 recovery, why? - stability 40 - skill 20 - human/ instrumental error 0 Washing Membrane Cumul ative Total recovery permeation You can see if an active partitions S 1% V 1% O 1% into the skin, but is unlikely to reach the bloodstream. The total DF recovery is displayed as percentages for permeation, membrane surface retrieval and membrane extraction (n ≥ 4, mean ± SD). 26 IVPT - cont In vitro permeation experiments are very useful Diffusion cells are hand blown glass, very fragile and very expensive Expensive to train students 27 Application of IVPT in Cosmetic Science – 3D Printed Franz Diffusion Cells 28 Application of IVPT in Cosmetic Science – 3D Printed Franz Diffusion Cells 29 In Vivo Testing 3 0 In vivo skin sample collection using Tape Stripping CUDERM D-Squame discs Pressure instrument Eppendorf tubes 70% ethanol Scissors Forceps Gloves Syringes 20 Tape Stripping 32 Detection and quantification of your active 33 Detection and quantification of actives Experiments are of no value unless you can accurately detect and quantify your active ingredient ICH provide guidelines on the validation of your methods to detect and quantify your active (ICH Q2(R2)) Validation of analytical procedures) Guidelines that apply to the pharmaceutical industry - but are relevant to the cosmetic industry But what methods can you use? Most commonly used instruments are UV spectrophotometer, HPLC and GC Confocal raman spectroscopy is frequently used, but equipment is VERY expensive Detection and quantification of actives: a practical approach Step 1: what method? Do a literature search on your active and see if there is an HPLC method that looks appropriate for your requirements (do you have the correct/ appropriate equipment, correct columns, and solvents). Adaptation will almost always be necessary. In the lab: using the same active ingredient you add to your formulation, obtain an absorption spectrum. This is most frequently done using a UV spectrophotometer. UV-Vis spectrophotometer Double-beam spectrophotometer Single-beam spectrophotometer http://www.keison.co.uk/jenway_6315spectrophotometer.shtml https://www.agilent.com/en-us/products/uv-vis-uv-vis-nir/uv-vis-uv-vis-nir-systems/cary-60-uv-vis 36 How does a UV spectrophotomer work? - Your dilute sample is exposed to either a single wavelength of light or a range of wavelengths that you will determine - Initially you will choose a large range eg 200 - 800 nm i.e both UV and visible light to obtain an absorption spectrum - The result will tell you at what wavelengths the sample is able to absorb light - Find the lamda max, ie that wavelength at which your substance has the strongest photon absorption - in this diagram it is at 270 nm. - As UV spectrophotometers follow Beer-Lambert’s law, in theory a UV spectrophotometer could be used for the analysis of your in vitro or in vivo work. - In reality, we normally use an HPLC. It is considered more accurate, more precise, you can use HPLC for separation, for more than one wavelength, it is more sensitive and less time consuming. Detection and quantification of actives: a practical approach Step 2: refine your method You have an HPLC method and the equipment, you know the lamda max you will be using, you have an appropriate column and the solvents you need for your HPLC method Start testing and adapting to your specific requirements eg if you want your elution time to increase and your substance is polar, reduce the polarity of the mobile phase slightly HPLC is used for separating, identifying and quantifying Solvents Degasser Pump Autosampler Autosampler & Injector Column oven Detector Column into column oven 39 https://www.youtube.com/watch?v=kz_egMtdnL4 How does an HPLC work? HPLC uses a stationary phase (column) and a mobile phase (solvent system) to separate components within a mixture. Solvent or mobile phase is delivered to the system via a high-pressure pump The sample, which is in solution, is injected into the system The mobile phase plus sample interact with the stationary phase (aka the column) Compounds with a higher affinity for the mobile phase, will elute more quickly than compounds that have a greater affinity for the stationary phase (column) Once these compounds leave the column they pass through a detector. You will have chosen a wavelength specific to your analyte. Data acquisition is in the form of chromatograms. We are interested in: a sharp peak with no shoulders, the time of elution Many HPLC systems are based on reverse-phase (the retention time), the area under the curve chromatography: i.e column/ stationary phase= non-polar (AUC) Mobile phase: polar AUC is proportional to concentration Can adjust, flow rate of solvent, oven temperature, Retention time is specific to a given set of wavelength, mobile phase, column, sample size conditions and can be compared to a standard Detection and quantification of actives: a practical approach Step 3: Create a calibration curve (CC) How: Make up range of concentrations of the analyte (minimum of 5 concentrations per CC). Run these on the HPLC. Concentration Concentration Average Average AUC AUC (μg Calibration curve for DF (μg // mL) mL) (absorption) (absorption) 1200 n n == 3 3 y = 21.801x + 4.3238 1000 R² = 0.9998 50 1091 50 1091 800 Absorption 25 551 25 551 600 10 235 400 10 235 5 113 200 5 113 1 22 0 1 22 0 10 20 30 40 50 60 0.5 12 Concentration of DF in µg /mL 0.5 12 0.1 2 0.1 2 Why do we need a CC? It enables us to detect and quantify our compound of interest. Not only for testing how the active partitions and permeates, but also to confirm quantity in formulation, stability etc Detection and quantification of actives: a practical approach Step 4: you have a calibration curve and a method Agilent 1100 series including: G1379A Degasser that appears robust. You now need to validate your HPLC details G1311A Quaternary Pump G1313A Autosampler G1316 Column Compartment method in accordance with the ICH guidelines. You Column Shiseido C18, 250 x 4.6 mm Acetonitrile 70% Mobile phase (v/v) will need to show that your method is suitable for Temperature ° C 0.1% Trifluoroacetic acid in water (H2O) 30% 25 Flow rate (mL/ min) 1 mL / min its intended purpose Wavelength (nm) Run time (min) 277 6 Injection volume (µL) 10 Your method outline should include the aim or purpose of the method, details of the equipment, operating parameters, reagents, standards, details of sample preparation and storage, and any other procedural descriptions (US Food and Drug Administration, 2015). Detection and quantification of actives: a practical approach Step 4: method validation continued There are eight steps of method validation including specificity, linearity, accuracy, precision, range, quantitation limit, detection limit and robustness. Specificity: Refers to the ability to analyse the compound in question despite the presence of other components in the sample. It is a 2-step process: identification and accurate quantification of the substance in the presence of other substances eg you could extract it from the formulation containing other excipients Linearity and range: Linearity requires proportionality between the concentration of the analyte and the response. A minimum of 5 concentrations are required. The R2 value will indicate correlation. Your range of concentrations should take into account the tests you will be performing. Accuracy and precision: Accuracy: How close the experimental value is to the true value. Test various concentrations of your active on its own and in the formulation. Precision: Test if your ability to detect and quantify are consistent intra-day and inter-day. This also includes system suitability - the ability of the equipment to consistently produce the same results. Detection and quantification of actives: a practical approach Step 4: method validation continued Robustness: is your method able to withstand small, deliberate variations eg changes to to wavelength, injection volume, solvent ratios, temperature of column and flow rate (not all at once!). Limit of detection (LOD) and quantification (LOQ): LOD is the smallest amount you can detect using your method. LOQ is the smallest amount you can actually quantify - and use in your data and experimental results I.e. you can’t just use your CC! It is subject to these limitations Now you are ready to proceed with your experiments! What if your analyte does not absorb light in the UV Vis range? You can’t use an HPLC or a UV spectrophotometer You may be able to use Gas chromatography It is generally used for samples that do not absorb light in the UV or visible regions Samples must be volatile i.e. capable of being converted into a gas at the temperature range used by the particular GC eg up to 400 ˚C Should be stable at high temperatures How does an GC work? GC uses a stationary phase (column) and a mobile phase (inert gas) to separate components within a mixture. The sample, which is in solution or undiluted, is injected into the GC inlet In the inlet the sample is vapourised => gas The mobile phase Eg helium or nitrogen carries the sample through the column (stationary phase) Compounds present in the sample interact differently with the stationary phase (column), depending on their chemical structure, causing them to move through at different speeds Once these compounds leave the column they pass through a detector. Data acquisition is in the form of chromatograms, which shows the peak size, indicating the amount of each component reaching the detector. https://blog.perkinelmer.com/posts/gas-chromatography-explained-what-it-is-and-how-it-works/ AUC is proportional to concentration Position of each peak indicates the retention A CC would be obtained to detect and quantify your volatile compound time for individual compounds. Method validation would follow the same approach as before Other experimental methods include PAMPA parallel artificial membrane permeability assay an in vitro model using a 96-well plate containing an artificial membrane mimicking human skin very rapid when compared with standard diffusion studies barrier function is not as effective as human skin use HPLC to detect and quantify the active https://www.pion-inc.com/solutions/applications/transdermal- drug-delivery Raman spectroscopy An excellent in vivo method, not destructive, not invasive Can operate up to 100 μm below the surface of the skin Often used in combination with in vitro studies Not all compounds are Raman-active Not all compounds can be well separated https://www.riverd.com/ Conclusion We have considered two experimental procedures to determine the partition and permeation of an active We have looked at the importance of developing an appropriate method for the detection and quantification of an active These are useful to understand in both product development and safety assessments References: INTERNATIONAL CONFERENCE ON HARMONISATION EXPERT WORKING GROUP. Validation of analytical procedures: text and methodology Q2 (R1). International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2005 Geneva, Switzerland. UNITED STATES PHARMACOPOEIAL CONVENTION 2017. First Supplement to USP 40 - NF 35. Validation of Compendial Prodecures. Rockville: United States Pharmacopeial Convention. US FOOD AND DRUG ADMINISTRATION 2015. Analytical procedures and methods validation for drugs and biologics : Guidance for Industry. Silver Spring: US Department of Health and Human Services. https://www.riverd.com/raman-spectroscopy/ https://www.pion-inc.com/solutions/products/pampa https://www.youtube.com/watch?v=kz_egMtdnL4 (RSC video on HPLC - old but good) https://www.thermofisher.com/uk/en/home/industrial/chromatography/chromatography-learning-center/liquid- chromatography-information/hplc-basics.html - watch the video on HPLC SCCS Notes of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation-12th Revision- SCCS/1647/22.